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Topical Collection Information

Dear Colleagues,

This Topical Collection “Feature Papers in Molecular Microbiology” aims to collect high-quality research articles, short communications, and review articles in the molecular biology of microbes with a focus on prokaryotes (bacteria and archaea) and eukaryotic microorganisms. Since the aim of this Topical Collection is to illustrate, through selected works, frontier research in molecular microbiology, we encourage Editorial Board Members of the Molecular Microbiology Section of the International Journal of Molecular Sciences to contribute papers reflecting the latest progress in their research field or to invite relevant experts and colleagues to do so.

Prof. Dr. Andreas Burkovski
Collection Editor

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Published Papers (101 papers)

2024

Jump to: 2023, 2022, 2021, 2020, 2019

15 pages, 5465 KiB  
Article
The Response of Growth and Transcriptome Profiles of Tea Grey Blight Disease Pathogen Pestalotiopsis theae to the Variation of Exogenous L-Theanine
by Yuqian Zhang, Feiyan Wang, Lijie Wang, Lingyun Zhang, Richard V. Espley, Kui Lin-Wang and Fanrong Cao
Int. J. Mol. Sci. 2024, 25(6), 3493; https://doi.org/10.3390/ijms25063493 - 20 Mar 2024
Viewed by 398
Abstract
Tea grey blight disease is one of the most destructive diseases that infects tea and is caused by the pathogen Pestalotiopsis theae (Sawada) Steyaert. L-theanine is a unique non-protein amino acid of the tea plant. Different concentrations of L-theanine exhibit significant inhibitory effects [...] Read more.
Tea grey blight disease is one of the most destructive diseases that infects tea and is caused by the pathogen Pestalotiopsis theae (Sawada) Steyaert. L-theanine is a unique non-protein amino acid of the tea plant. Different concentrations of L-theanine exhibit significant inhibitory effects on the growth and sporulation ability of the pathogen causing tea grey blight disease. To understand the effect mechanism of L-theanine on P. theae, transcriptome profiling was performed on the pathogenic mycelium treated with three different concentrations of L-theanine: no L-theanine treatment (TH0), 20 mg/mL theanine treatment (TH2), and 40 mg/mL theanine treatment (TH4). The colony growths were significantly lower in the treatment with L-theanine than those without L-theanine. The strain cultured with a high concentration of L-theanine produced no spores or only a few spores. In total, 2344, 3263, and 1158 differentially expressed genes (DEGs) were detected by RNA-sequencing in the three comparisons, Th2 vs. Th0, Th4 vs. Th0, and Th4 vs. Th2, respectively. All DEGs were categorized into 24 distinct clusters. According to GO analysis, low concentrations of L-theanine primarily affected molecular functions, while high concentrations of L-theanine predominantly affected biological processes including external encapsulating structure organization, cell wall organization or biogenesis, and cellular amino acid metabolic process. Based on KEGG, the DEGs of Th2 vs. Th0 were primarily involved in pentose and glucuronate interconversions, histidine metabolism, and tryptophan metabolism. The DEGs of Th4 vs. Th0 were mainly involved in starch and sucrose metabolism, amino sugar, and nucleotide sugar metabolism. This study indicated that L-theanine has a significant impact on the growth and sporulation of the pathogen of tea grey blight disease and mainly affects amino acid metabolism, carbohydrate metabolism, and cellular structure-related biosynthesis processes of pathogenic fungi. This work provides insights into the direct control effect of L-theanine on pathogenic growth and also reveals the molecular mechanisms of inhibition of L-theanine to P. theae. Full article
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24 pages, 9939 KiB  
Article
Bioinformatic Analysis of Sulfotransferases from an Unexplored Gut Microbe, Sutterella wadsworthensis 3_1_45B: Possible Roles towards Detoxification via Sulfonation by Members of the Human Gut Microbiome
by Lauryn Langford and Dhara D. Shah
Int. J. Mol. Sci. 2024, 25(5), 2983; https://doi.org/10.3390/ijms25052983 - 04 Mar 2024
Viewed by 707
Abstract
Sulfonation, primarily facilitated by sulfotransferases, plays a crucial role in the detoxification pathways of endogenous substances and xenobiotics, promoting metabolism and elimination. Traditionally, this bioconversion has been attributed to a family of human cytosolic sulfotransferases (hSULTs) known for their high sequence similarity and [...] Read more.
Sulfonation, primarily facilitated by sulfotransferases, plays a crucial role in the detoxification pathways of endogenous substances and xenobiotics, promoting metabolism and elimination. Traditionally, this bioconversion has been attributed to a family of human cytosolic sulfotransferases (hSULTs) known for their high sequence similarity and dependence on 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as a sulfo donor. However, recent studies have revealed the presence of PAPS-dependent sulfotransferases within gut commensals, indicating that the gut microbiome may harbor a diverse array of sulfotransferase enzymes and contribute to detoxification processes via sulfation. In this study, we investigated the prevalence of sulfotransferases in members of the human gut microbiome. Interestingly, we stumbled upon PAPS-independent sulfotransferases, known as aryl-sulfate sulfotransferases (ASSTs). Our bioinformatics analyses revealed that members of the gut microbial genus Sutterella harbor multiple asst genes, possibly encoding multiple ASST enzymes within its members. Fluctuations in the microbes of the genus Sutterella have been associated with various health conditions. For this reason, we characterized 17 different ASSTs from Sutterella wadsworthensis 3_1_45B. Our findings reveal that SwASSTs share similarities with E. coli ASST but also exhibit significant structural variations and sequence diversity. These differences might drive potential functional diversification and likely reflect an evolutionary divergence from their PAPS-dependent counterparts. Full article
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19 pages, 12890 KiB  
Article
Selection and Validation of Reference Genes for Gene Expression in Bactericera gobica Loginova under Different Insecticide Stresses
by Hongshuang Wei, Jingyi Zhang, Mengke Yang, Yao Li, Kun Guo, Haili Qiao, Rong Xu, Sai Liu and Changqing Xu
Int. J. Mol. Sci. 2024, 25(4), 2434; https://doi.org/10.3390/ijms25042434 - 19 Feb 2024
Viewed by 472
Abstract
Insecticide resistance has long been a problem in crop pest control. Bactericera gobica is a major pest on the well-known medicinal plants Lycium barbarum L. Investigating insecticide resistance mechanisms of B. gobica will help to identify pesticide reduction strategies to control the pest. [...] Read more.
Insecticide resistance has long been a problem in crop pest control. Bactericera gobica is a major pest on the well-known medicinal plants Lycium barbarum L. Investigating insecticide resistance mechanisms of B. gobica will help to identify pesticide reduction strategies to control the pest. Gene expression normalization by RT-qPCR requires the selection and validation of appropriate reference genes (RGs). Here, 15 candidate RGs were selected from transcriptome data of B. gobica. Their expression stability was evaluated with five algorithms (Delta Ct, GeNorm, Normfinder, BestKeeper and RefFinder) for sample types differing in response to five insecticide stresses and in four other experimental conditions. Our results indicated that the RGs RPL10 + RPS15 for Imidacloprid and Abamectin; RPL10 + AK for Thiamethoxam; RPL32 + RPL10 for λ-cyhalothrin; RPL10 + RPL8 for Matrine; and EF2 + RPL32 under different insecticide stresses were the most suitable RGs for RT-qPCR normalization. EF1α + RPL8, EF1α + β-actin, β-actin + EF2 and β-actin + RPS15 were the optimal combination of RGs under odor stimulation, temperature, developmental stages and both sexes, respectively. Overall, EF2 and RPL8 were the two most stable RGs in all conditions, while α-TUB and RPL32 were the least stable RGs. The corresponding suitable RGs and one unstable RG were used to normalize a target cytochrome P450 CYP6a1 gene between adult and nymph stages and under imidacloprid stress. The results of CYP6a1 expression were consistent with transcriptome data. This study is the first research on the most stable RG selection in B. gobica nymphs exposed to different insecticides, which will contribute to further research on insecticide resistance mechanisms in B. gobica. Full article
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16 pages, 2220 KiB  
Article
Unearthing a Cryptic Biosynthetic Gene Cluster for the Piperazic Acid-Bearing Depsipeptide Diperamycin in the Ant-Dweller Streptomyces sp. CS113
by Coral García-Gutiérrez, Ignacio Pérez-Victoria, Ignacio Montero, Jorge Fernández-De la Hoz, Mónica G. Malmierca, Jesús Martín, José A. Salas, Carlos Olano, Fernando Reyes and Carmen Méndez
Int. J. Mol. Sci. 2024, 25(4), 2347; https://doi.org/10.3390/ijms25042347 - 16 Feb 2024
Viewed by 1156
Abstract
Piperazic acid is a cyclic nonproteinogenic amino acid that contains a hydrazine N-N bond formed by a piperazate synthase (KtzT-like). This amino acid, found in bioactive natural products synthesized by non-ribosomal peptide synthetases (NRPSs), confers conformational constraint to peptides, an important feature for [...] Read more.
Piperazic acid is a cyclic nonproteinogenic amino acid that contains a hydrazine N-N bond formed by a piperazate synthase (KtzT-like). This amino acid, found in bioactive natural products synthesized by non-ribosomal peptide synthetases (NRPSs), confers conformational constraint to peptides, an important feature for their biological activities. Genome mining of Streptomyces strains has been revealed as a strategy to identify biosynthetic gene clusters (BGCs) for potentially active compounds. Moreover, the isolation of new strains from underexplored habitats or associated with other organisms has allowed to uncover new BGCs for unknown compounds. The in-house “Carlos Sialer (CS)” strain collection consists of seventy-one Streptomyces strains isolated from the cuticle of leaf-cutting ants of the tribe Attini. Genomes from twelve of these strains have been sequenced and mined using bioinformatics tools, highlighting their potential to encode secondary metabolites. In this work, we have screened in silico those genomes, using KtzT as a hook to identify BGCs encoding piperazic acid-containing compounds. This resulted in uncovering the new BGC dpn in Streptomyces sp. CS113, which encodes the biosynthesis of the hybrid polyketide–depsipeptide diperamycin. Analysis of the diperamycin polyketide synthase (PKS) and NRPS reveals their functional similarity to those from the aurantimycin A biosynthetic pathway. Experimental proof linking the dpn BGC to its encoded compound was achieved by determining the growth conditions for the expression of the cluster and by inactivating the NRPS encoding gene dpnS2 and the piperazate synthase gene dpnZ. The identity of diperamycin was confirmed by High-Resolution Mass Spectrometry (HRMS) and Nuclear Magnetic Resonance (NMR) and by analysis of the domain composition of modules from the DpnP PKS and DpnS NRPS. The identification of the dpn BGC expands the number of BGCs that have been confirmed to encode the relatively scarcely represented BGCs for depsipeptides of the azinothricin family of compounds and will facilitate the generation of new-to-nature analogues by combinatorial biosynthesis. Full article
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15 pages, 2949 KiB  
Article
SARS-CoV-2 Infection Alters the Phenotype and Gene Expression of Adipocytes
by Paola Quaranta, Gaia Scabia, Barbara Storti, Alessia Dattilo, Lara Quintino, Paola Perrera, Cristina Di Primio, Mario Costa, Mauro Pistello, Ranieri Bizzarri and Margherita Maffei
Int. J. Mol. Sci. 2024, 25(4), 2086; https://doi.org/10.3390/ijms25042086 - 08 Feb 2024
Viewed by 818
Abstract
Epidemiological evidence emphasizes that excess fat mass is associated with an increased risk of severe COVID-19 disease. Nevertheless, the intricate interplay between SARS-CoV-2 and adipocytes remains poorly understood. It is crucial to decipher the progression of COVID-19 both in the acute phase and [...] Read more.
Epidemiological evidence emphasizes that excess fat mass is associated with an increased risk of severe COVID-19 disease. Nevertheless, the intricate interplay between SARS-CoV-2 and adipocytes remains poorly understood. It is crucial to decipher the progression of COVID-19 both in the acute phase and on long-term outcomes. In this study, an in vitro model using the human SGBS cell line (Simpson-Golabi-Behmel syndrome) was developed to investigate the infectivity of SARS-CoV-2 in adipocytes, and the effects of virus exposure on adipocyte function. Our results show that SGBS adipocytes expressing ACE2 are susceptible to SARS-CoV-2 infection, as evidenced by the release of the viral genome into the medium, detection of the nucleocapsid in cell lysates, and positive immunostaining for the spike protein. Infected adipocytes show remarkable changes compared to uninfected controls: increased surface area of lipid droplets, upregulated expression of genes of inflammation (Haptoglobin, MCP-1, IL-6, PAI-1), increased oxidative stress (MnSOD), and a concomitant reduction of transcripts related to adipocyte function (leptin, fatty acid synthase, perilipin). Moreover, exogenous expression of spike protein in SGBS adipocytes also led to an increase in lipid droplet size. In conclusion using the human SGBS cell line, we detected SARS-CoV-2 infectivity in adipocytes, revealing substantial morphological and functional changes in infected cells. Full article
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22 pages, 4905 KiB  
Article
Disparate Effects of Two Clerodane Diterpenes of Giant Goldenrod (Solidago gigantea Ait.) on Bacillus spizizenii
by Zoltán Bozsó, Virág Lapat, Péter G. Ott and Ágnes M. Móricz
Int. J. Mol. Sci. 2024, 25(3), 1531; https://doi.org/10.3390/ijms25031531 - 26 Jan 2024
Viewed by 613
Abstract
New substances with antimicrobial properties are needed to successfully treat emerging human, animal, or plant pathogens. Seven clerodane diterpenes, previously isolated from giant goldenrod (Solidago gigantea) root, were tested against Gram-positive Bacillus subtilis, Bacillus spizizenii and Rhodococcus fascians by measuring [...] Read more.
New substances with antimicrobial properties are needed to successfully treat emerging human, animal, or plant pathogens. Seven clerodane diterpenes, previously isolated from giant goldenrod (Solidago gigantea) root, were tested against Gram-positive Bacillus subtilis, Bacillus spizizenii and Rhodococcus fascians by measuring minimal bactericidal concentration (MBC), minimal inhibitory concentration (MIC) and half-maximal inhibitory concentration (IC50). Two of them, Sg3a (a dialdehyde) and Sg6 (solidagoic acid B), were proved to be the most effective and were selected for further study. Bacillus spizizenii was incubated with the two diterpenes for shorter (1 h) or longer (5 h) periods and then subjected to genome-wide transcriptional analyses. Only a limited number of common genes (28 genes) were differentially regulated after each treatment, and these were mainly related to the restoration of cell membrane integrity and to membrane-related transports. Changes in gene activity indicated that, among other things, K+ and Na+ homeostasis, pH and membrane electron transport processes may have been affected. Activated export systems can be involved in the removal of harmful molecules from the bacterial cells. Inhibition of bacterial chemotaxis and flagellar assembly, as well as activation of genes for the biosynthesis of secondary metabolites, were observed as a general response. Depending on the diterpenes and the duration of the treatments, down-regulation of the protein synthesis-related, oxidative phosphorylation, signal transduction and transcription factor genes was found. In other cases, up-regulation of the genes of oxidation–reduction processes, sporulation and cell wall modification could be detected. Comparison of the effect of diterpenes with the changes induced by different environmental and nutritional conditions revealed several overlapping processes with stress responses. For example, the Sg6 treatment seems to have caused a starvation-like condition. In summary, there were both common and diterpene-specific changes in the transcriptome, and these changes were also dependent on the length of treatments. The results also indicated that Sg6 exerted its effect more slowly than Sg3a, but ultimately its effect was greater. Full article
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27 pages, 1003 KiB  
Review
Targeting of Specialized Metabolites Biosynthetic Enzymes to Membranes and Vesicles by Posttranslational Palmitoylation: A Mechanism of Non-Conventional Traffic and Secretion of Fungal Metabolites
by Juan F. Martín and Paloma Liras
Int. J. Mol. Sci. 2024, 25(2), 1224; https://doi.org/10.3390/ijms25021224 - 19 Jan 2024
Viewed by 990
Abstract
In nature, the formation of specialized (secondary) metabolites is associated with the late stages of fungal development. Enzymes involved in the biosynthesis of secondary metabolites in fungi are located in distinct subcellular compartments including the cytosol, peroxisomes, endosomes, endoplasmic reticulum, different types of [...] Read more.
In nature, the formation of specialized (secondary) metabolites is associated with the late stages of fungal development. Enzymes involved in the biosynthesis of secondary metabolites in fungi are located in distinct subcellular compartments including the cytosol, peroxisomes, endosomes, endoplasmic reticulum, different types of vesicles, the plasma membrane and the cell wall space. The enzymes traffic between these subcellular compartments and the secretion through the plasma membrane are still unclear in the biosynthetic processes of most of these metabolites. Recent reports indicate that some of these enzymes initially located in the cytosol are later modified by posttranslational acylation and these modifications may target them to membrane vesicle systems. Many posttranslational modifications play key roles in the enzymatic function of different proteins in the cell. These modifications are very important in the modulation of regulatory proteins, in targeting of proteins, intracellular traffic and metabolites secretion. Particularly interesting are the protein modifications by palmitoylation, prenylation and miristoylation. Palmitoylation is a thiol group-acylation (S-acylation) of proteins by palmitic acid (C16) that is attached to the SH group of a conserved cysteine in proteins. Palmitoylation serves to target acylated proteins to the cytosolic surface of cell membranes, e.g., to the smooth endoplasmic reticulum, whereas the so-called toxisomes are formed in trichothecene biosynthesis. Palmitoylation of the initial enzymes involved in the biosynthesis of melanin serves to target them to endosomes and later to the conidia, whereas other non-palmitoylated laccases are secreted directly by the conventional secretory pathway to the cell wall space where they perform the last step(s) of melanin biosynthesis. Six other enzymes involved in the biosynthesis of endocrosin, gliotoxin and fumitremorgin believed to be cytosolic are also targeted to vesicles, although it is unclear if they are palmitoylated. Bioinformatic analysis suggests that palmitoylation may be frequent in the modification and targeting of polyketide synthetases and non-ribosomal peptide synthetases. The endosomes may integrate other small vesicles with different cargo proteins, forming multivesicular bodies that finally fuse with the plasma membrane during secretion. Another important effect of palmitoylation is that it regulates calcium metabolism by posttranslational modification of the phosphatase calcineurin. Mutants defective in the Akr1 palmitoyl transferase in several fungi are affected in calcium transport and homeostasis, thus impacting on the biosynthesis of calcium-regulated specialized metabolites. The palmitoylation of secondary metabolites biosynthetic enzymes and their temporal distribution respond to the conidiation signaling mechanism. In summary, this posttranslational modification drives the spatial traffic of the biosynthetic enzymes between the subcellular organelles and the plasma membrane. This article reviews the molecular mechanism of palmitoylation and the known fungal palmitoyl transferases. This novel information opens new ways to improve the biosynthesis of the bioactive metabolites and to increase its secretion in fungi. Full article
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2023

Jump to: 2024, 2022, 2021, 2020, 2019

14 pages, 2045 KiB  
Article
Clonality and Diversity in the Soft Rot Dickeya solani Phytopathogen
by Frédérique Van Gijsegem, Perrine Portier, Géraldine Taghouti and Jacques Pédron
Int. J. Mol. Sci. 2023, 24(24), 17553; https://doi.org/10.3390/ijms242417553 - 16 Dec 2023
Viewed by 526
Abstract
Bacterial diversity analyses often suffer from a bias due to sampling only from a limited number of hosts or narrow geographic locations. This was the case for the phytopathogenic species Dickeya solani, whose members were mainly isolated from a few hosts–potato and [...] Read more.
Bacterial diversity analyses often suffer from a bias due to sampling only from a limited number of hosts or narrow geographic locations. This was the case for the phytopathogenic species Dickeya solani, whose members were mainly isolated from a few hosts–potato and ornamentals–and from the same geographical area–Europe and Israel, which are connected by seed trade. Most D. solani members were clonal with the notable exception of the potato isolate RNS05.1.2A and two related strains that are clearly distinct from other D. solani genomes. To investigate if D. solani genomic diversity might be broadened by analysis of strains isolated from other environments, we analysed new strains isolated from ornamentals and from river water as well as strain CFBP 5647 isolated from tomato in the Caribbean island Guadeloupe. While water strains were clonal to RNS05.1.2A, the Caribbean tomato strain formed a third clade. The genomes of the three clades are highly syntenic; they shared almost 3900 protein families, and clade-specific genes were mainly included in genomic islands of extrachromosomal origin. Our study thus revealed both broader D. solani diversity with the characterisation of a third clade isolated in Latin America and a very high genomic conservation between clade members. Full article
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14 pages, 1633 KiB  
Article
Unveiling Distinct Proteomic Signatures in Complicated Crohn’s Disease That Could Predict the Disease Course
by Laura A. Lucaciu, Radu Seicean, Alina Uifălean, Maria Iacobescu, Cristina A. Iuga and Andrada Seicean
Int. J. Mol. Sci. 2023, 24(23), 16966; https://doi.org/10.3390/ijms242316966 - 30 Nov 2023
Viewed by 793
Abstract
Crohn’s disease (CD) is characterized by a chronic, progressive inflammation of the gastrointestinal tract often leading to complications, such as strictures and fistulae. Currently, there are no validated tools anticipating short- and long-term outcomes at an early stage. This investigation aims to elucidate [...] Read more.
Crohn’s disease (CD) is characterized by a chronic, progressive inflammation of the gastrointestinal tract often leading to complications, such as strictures and fistulae. Currently, there are no validated tools anticipating short- and long-term outcomes at an early stage. This investigation aims to elucidate variations in protein abundance across distinct CD phenotypes with the objective of uncovering potential biomarkers implicated in disease advancement. Serum samples collected from 30 CD patients and 15 healthy age-matched controls (HC) were subjected to depletion of highly abundant proteins and to a label-free mass spectrometry analysis. Twenty-four proteins were shown to be significantly different when comparing CD with HC. Of these, WD repeat-containing protein 31 (WDR31), and proteins involved in the acute inflammatory response, leucine-rich alpha-2-glycoprotein (LRG1) and serum amyloid A1 (SAA1), were more abundant in the aggressive subgroup. Against standard biomarkers, a positive correlation between SAA1 and WDR31 and C-reactive protein (CRP) was found. In this study, a unique serum biomarker panel for aggressive CD was identified, which could aid in predicting the disease course. Full article
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12 pages, 3168 KiB  
Article
Changes in Diversity and Composition of Rhizosphere Bacterial and Fungal Community between Resistant and Susceptible Pakchoi under Plasmodiophora brassicae
by Dan-Dan Xi, Lu Gao, Li-Ming Miao, Li-Ao Ge, Ding-Yu Zhang, Zhao-Hui Zhang, Xiao-Feng Li, Yu-Ying Zhu, Hai-Bin Shen and Hong-Fang Zhu
Int. J. Mol. Sci. 2023, 24(23), 16779; https://doi.org/10.3390/ijms242316779 - 26 Nov 2023
Cited by 1 | Viewed by 742
Abstract
Plasmodiophora brassicae (P. brassicae) is a soil-born pathogen worldwide and can infect most cruciferous plants, which causes great yield decline and economic losses. It is not well known how microbial diversity and community composition change during P. brassicae infecting plant roots. [...] Read more.
Plasmodiophora brassicae (P. brassicae) is a soil-born pathogen worldwide and can infect most cruciferous plants, which causes great yield decline and economic losses. It is not well known how microbial diversity and community composition change during P. brassicae infecting plant roots. Here, we employed a resistant and a susceptible pakchoi cultivar with and without inoculation with P. brassicae to analyze bacterial and fungal diversity using 16S rRNA V3-V4 and ITS_V1 regions, respectively. 16S rRNA V3-V4 and ITS_V1 regions were amplified and sequenced separately. Results revealed that both fungal and bacterial diversity increased, and composition was changed in the rhizosphere soil of the susceptible pakchoi compared with the resistant cultivar. In the four groups of R_mock, S_mock, R_10d, and S_10d, the most relatively abundant bacterium and fungus was Proteobacteria, accounting for 61.92%, 58.17%, 48.64%, and 50.00%, respectively, and Ascomycota, accounting for 75.11%, 63.69%, 72.10%, and 90.31%, respectively. A total of 9488 and 11,914 bacteria were observed uniquely in the rhizosphere soil of resistant and susceptible pakchoi, respectively, while only 80 and 103 fungi were observed uniquely in the correlated soil. LefSe analysis showed that 107 and 49 differentially abundant taxa were observed in bacteria and fungi. Overall, we concluded that different pakchoi cultivars affect microbial diversity and community composition, and microorganisms prefer to gather around the rhizosphere of susceptible pakchoi. These findings provide a new insight into plant–microorganism interactions. Full article
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20 pages, 2492 KiB  
Review
Adenoviral Gene Therapy Vectors in Clinical Use—Basic Aspects with a Special Reference to Replication-Competent Adenovirus Formation and Its Impact on Clinical Safety
by Aleksi J. Leikas, Seppo Ylä-Herttuala and Juha E. K. Hartikainen
Int. J. Mol. Sci. 2023, 24(22), 16519; https://doi.org/10.3390/ijms242216519 - 20 Nov 2023
Viewed by 1046
Abstract
Adenoviral vectors are commonly used in clinical gene therapy. Apart from oncolytic adenoviruses, vector replication is highly undesired as it may pose a safety risk for the treated patient. Thus, careful monitoring for the formation of replication-competent adenoviruses (RCA) during vector manufacturing is [...] Read more.
Adenoviral vectors are commonly used in clinical gene therapy. Apart from oncolytic adenoviruses, vector replication is highly undesired as it may pose a safety risk for the treated patient. Thus, careful monitoring for the formation of replication-competent adenoviruses (RCA) during vector manufacturing is required. To render adenoviruses replication deficient, their genomic E1 region is deleted. However, it has been known for a long time that during their propagation, some viruses will regain their replication capability by recombination in production cells, most commonly HEK293. Recently developed RCA assays have revealed that many clinical batches contain more RCA than previously assumed and allowed by regulatory authorities. The clinical significance of the higher RCA content has yet to be thoroughly evaluated. In this review, we summarize the biology of adenovirus vectors, their manufacturing methods, and the origins of RCA formed during HEK293-based vector production. Lastly, we share our experience using minimally RCA-positive serotype 5 adenoviral vectors based on observations from our clinical cardiovascular gene therapy studies. Full article
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19 pages, 4370 KiB  
Article
Regulation of the Gene for Alanine Racemase Modulates Amino Acid Metabolism with Consequent Alterations in Cell Wall Properties and Adhesive Capability in Brucella spp.
by Mingyue Hao, Minghui Wang, Ting Tang, Danyu Zhao, Shurong Yin, Yong Shi, Xiaofang Liu, Gaowa Wudong, Yuanhao Yang, Mengyu Zhang, Lin Qi, Dong Zhou, Wei Liu, Yaping Jin and Aihua Wang
Int. J. Mol. Sci. 2023, 24(22), 16145; https://doi.org/10.3390/ijms242216145 - 09 Nov 2023
Viewed by 824
Abstract
Brucella, a zoonotic facultative intracellular pathogenic bacterium, poses a significant threat both to human health and to the development of the livestock industry. Alanine racemase (Alr), the enzyme responsible for alanine racemization, plays a pivotal role in regulating virulence in this bacterium. [...] Read more.
Brucella, a zoonotic facultative intracellular pathogenic bacterium, poses a significant threat both to human health and to the development of the livestock industry. Alanine racemase (Alr), the enzyme responsible for alanine racemization, plays a pivotal role in regulating virulence in this bacterium. Moreover, Brucella mutants with alr gene deletions (Δalr) exhibit potential as vaccine candidates. However, the mechanisms that underlie the detrimental effects of alr knockouts on Brucella pathogenicity remain elusive. Here, initially, we conducted a bioinformatics analysis of Alr, which demonstrated a high degree of conservation of the protein within Brucella spp. Subsequent metabolomics studies unveiled alterations in amino acid pathways following deletion of the alr gene. Furthermore, alr deletion in Brucella suis S2 induced decreased resistance to stress, antibiotics, and other factors. Transmission electron microscopy of simulated macrophage intracellular infection revealed damage to the cell wall in the Δalr strain, whereas propidium iodide staining and alkaline phosphatase and lactate dehydrogenase assays demonstrated alterations in cell membrane permeability. Changes in cell wall properties were revealed by measurements of cell surface hydrophobicity and zeta potential. Finally, the diminished adhesion capacity of the Δalr strain was shown by immunofluorescence and bacterial enumeration assays. In summary, our findings indicate that the alr gene that regulates amino acid metabolism in Brucella influences the properties of the cell wall, which modulates bacterial adherence capability. This study is the first demonstration that Alr impacts virulence by modulating bacterial metabolism, thereby providing novel insights into the pathogenic mechanisms of Brucella spp. Full article
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47 pages, 4959 KiB  
Review
Microbiology and Biochemistry of Pesticides Biodegradation
by José Roberto Guerrero Ramírez, Lizbeth Alejandra Ibarra Muñoz, Nagamani Balagurusamy, José Ernesto Frías Ramírez, Leticia Alfaro Hernández and Javier Carrillo Campos
Int. J. Mol. Sci. 2023, 24(21), 15969; https://doi.org/10.3390/ijms242115969 - 04 Nov 2023
Cited by 1 | Viewed by 3656
Abstract
Pesticides are chemicals used in agriculture, forestry, and, to some extent, public health. As effective as they can be, due to the limited biodegradability and toxicity of some of them, they can also have negative environmental and health impacts. Pesticide biodegradation is important [...] Read more.
Pesticides are chemicals used in agriculture, forestry, and, to some extent, public health. As effective as they can be, due to the limited biodegradability and toxicity of some of them, they can also have negative environmental and health impacts. Pesticide biodegradation is important because it can help mitigate the negative effects of pesticides. Many types of microorganisms, including bacteria, fungi, and algae, can degrade pesticides; microorganisms are able to bioremediate pesticides using diverse metabolic pathways where enzymatic degradation plays a crucial role in achieving chemical transformation of the pesticides. The growing concern about the environmental and health impacts of pesticides is pushing the industry of these products to develop more sustainable alternatives, such as high biodegradable chemicals. The degradative properties of microorganisms could be fully exploited using the advances in genetic engineering and biotechnology, paving the way for more effective bioremediation strategies, new technologies, and novel applications. The purpose of the current review is to discuss the microorganisms that have demonstrated their capacity to degrade pesticides and those categorized by the World Health Organization as important for the impact they may have on human health. A comprehensive list of microorganisms is presented, and some metabolic pathways and enzymes for pesticide degradation and the genetics behind this process are discussed. Due to the high number of microorganisms known to be capable of degrading pesticides and the low number of metabolic pathways that are fully described for this purpose, more research must be conducted in this field, and more enzymes and genes are yet to be discovered with the possibility of finding more efficient metabolic pathways for pesticide biodegradation. Full article
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23 pages, 4707 KiB  
Review
Prioritization of Critical Factors for Surveillance of the Dissemination of Antibiotic Resistance in Pseudomonas aeruginosa: A Systematic Review
by Jung Hun Lee, Nam-Hoon Kim, Kyung-Min Jang, Hyeonku Jin, Kyoungmin Shin, Byeong Chul Jeong, Dae-Wi Kim and Sang Hee Lee
Int. J. Mol. Sci. 2023, 24(20), 15209; https://doi.org/10.3390/ijms242015209 - 15 Oct 2023
Viewed by 2454
Abstract
Pseudomonas aeruginosa is the primary opportunistic human pathogen responsible for a range of acute and chronic infections; it poses a significant threat to immunocompromised patients and is the leading cause of morbidity and mortality for nosocomial infections. Its high resistance to a diverse [...] Read more.
Pseudomonas aeruginosa is the primary opportunistic human pathogen responsible for a range of acute and chronic infections; it poses a significant threat to immunocompromised patients and is the leading cause of morbidity and mortality for nosocomial infections. Its high resistance to a diverse array of antimicrobial agents presents an urgent health concern. Among the mechanisms contributing to resistance in P. aeruginosa, the horizontal acquisition of antibiotic resistance genes (ARGs) via mobile genetic elements (MGEs) has gained recognition as a substantial concern in clinical settings, thus indicating that a comprehensive understanding of ARG dissemination within the species is strongly required for surveillance. Here, two approaches, including a systematic literature analysis and a genome database survey, were employed to gain insights into ARG dissemination. The genome database enabled scrutinizing of all the available sequence information and various attributes of P. aeruginosa isolates, thus providing an extensive understanding of ARG dissemination within the species. By integrating both approaches, with a primary focus on the genome database survey, mobile ARGs that were linked or correlated with MGEs, important sequence types (STs) carrying diverse ARGs, and MGEs responsible for ARG dissemination were identified as critical factors requiring strict surveillance. Although human isolates play a primary role in dissemination, the importance of animal and environmental isolates has also been suggested. In this study, 25 critical mobile ARGs, 45 critical STs, and associated MGEs involved in ARG dissemination within the species, are suggested as critical factors. Surveillance and management of these prioritized factors across the One Health sectors are essential to mitigate the emergence of multidrug-resistant (MDR) and extensively resistant (XDR) P. aeruginosa in clinical settings. Full article
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13 pages, 2301 KiB  
Article
Toll and IMD Immune Pathways Are Important Antifungal Defense Components in a Pupal Parasitoid, Pteromalus puparum
by Lei Yang, Lei Yang, Xiaofu Wang, Cheng Peng, Xiaoyun Chen, Wei Wei, Xiaoli Xu, Gongyin Ye and Junfeng Xu
Int. J. Mol. Sci. 2023, 24(18), 14088; https://doi.org/10.3390/ijms241814088 - 14 Sep 2023
Cited by 2 | Viewed by 822
Abstract
Insects employ multifaceted strategies to combat invading fungi, with immunity being a promising mechanism. Immune pathways function in signal transduction and amplification, ultimately leading to the activation of antimicrobial peptides (AMPs). Although several studies have shown that immune pathways are responsible for defending [...] Read more.
Insects employ multifaceted strategies to combat invading fungi, with immunity being a promising mechanism. Immune pathways function in signal transduction and amplification, ultimately leading to the activation of antimicrobial peptides (AMPs). Although several studies have shown that immune pathways are responsible for defending against fungi, the roles of parasitoid immune pathways involved in antifungal responses remain unknown. In this study, we evaluated the roles of the Toll and IMD pathways of a pupal parasitoid, Pteromalus puparum (Hymenoptera: Pteromalidae), in fighting against Beauveria bassiana (Hypocreales: Cordycipitaceae). Successful colonization of B. bassiana on P. puparum adults was confirmed by scanning electron microscopy (SEM). AMPs were induced upon B. bassiana infection. The knockdown of key genes, PpTollA and PpIMD, in Toll and IMD signaling pathways, respectively, significantly compromised insect defense against fungal infection. The knockdown of either PpTollA or PpIMD in P. puparum dramatically promoted the proliferation of B. bassiana, resulting in a decreased survival rate and downregulated expression levels of AMPs against B. bassiana compared to controls. These data indicated that PpTollA and PpIMD participate in Toll and IMD-mediated activation of antifungal responses, respectively. In summary, this study has greatly broadened our knowledge of the parasitoid antifungal immunity against fungi. Full article
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17 pages, 2419 KiB  
Article
Probiotics Modulate Host Immune Response and Interact with the Gut Microbiota: Shaping Their Composition and Mediating Antibiotic Resistance
by Walaa K. Mousa, Sara Mousa, Rose Ghemrawi, Dana Obaid, Muhammad Sarfraz, Fadia Chehadeh and Shannon Husband
Int. J. Mol. Sci. 2023, 24(18), 13783; https://doi.org/10.3390/ijms241813783 - 07 Sep 2023
Viewed by 1727
Abstract
The consortium of microbes inhabiting the human body, together with their encoded genes and secreted metabolites, is referred to as the “human microbiome.” Several studies have established a link between the composition of the microbiome and its impact on human health. This impact [...] Read more.
The consortium of microbes inhabiting the human body, together with their encoded genes and secreted metabolites, is referred to as the “human microbiome.” Several studies have established a link between the composition of the microbiome and its impact on human health. This impact spans local gastrointestinal inflammation to systemic autoimmune disorders and neurodegenerative diseases such as Alzheimer’s and Autism. Some of these links have been validated by rigorous experiments that identify specific strains as mediators or drivers of a particular condition. Consequently, the development of probiotics to compensate for a missing beneficial microbe(s) has advanced and become popular, especially in the treatment of irritable bowel diseases and to restore disrupted gut flora after antibiotic administration. The widespread use of probiotics is often advocated as a natural ecological therapy. However, this perception is not always accurate, as there is a potential for unexpected interactions when administering live microbial cultures. Here, we designed this research to explore the intricate interactions among probiotics, the host, and microbes through a series of experiments. Our objectives included assessing their immunomodulatory effects, response to oral medications, impact on microbial population dynamics, and mediation of antibiotic resistance. To achieve these goals, we employed diverse experimental protocols, including cell-based enzyme -linked immunosorbent assay (ELISA), antibiotic susceptibility testing, antimicrobial activity assays, computational prediction of probiotic genes responsible for antibiotic resistance, polymerase chain reaction (PCR)-based validation of predicted genes, and survival assays of probiotics in the presence of selected oral medications. Our findings highlight that more than half of the tested probiotics trigger an inflammatory response in the Caco-2 cell line, are influenced by oral medications, exhibit antibacterial activity, and possess genes encoding antimicrobial resistance. These results underscore the necessity for a reevaluation of probiotic usage and emphasize the importance of establishing regulations to govern probiotic testing, approval, and administration. Full article
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16 pages, 4380 KiB  
Article
Comparative Transcriptomics of Chilodonella hexasticha and C. uncinata Provide New Insights into Adaptations to a Parasitic Lifestyle and Mdivi-1 as a Potential Agent for Chilodonellosis Control
by Xialian Bu, Weishan Zhao, Wenxiang Li, Hong Zou, Ming Li and Guitang Wang
Int. J. Mol. Sci. 2023, 24(17), 13058; https://doi.org/10.3390/ijms241713058 - 22 Aug 2023
Viewed by 853
Abstract
Chilodonella hexasticha is a harmful parasitic ciliate that can cause severe damage to fish and high mortalities worldwide. Its congeneric species, C. uncinata, is a facultative parasite that not only can be free-living but also can parasitize on fish gills and fins. [...] Read more.
Chilodonella hexasticha is a harmful parasitic ciliate that can cause severe damage to fish and high mortalities worldwide. Its congeneric species, C. uncinata, is a facultative parasite that not only can be free-living but also can parasitize on fish gills and fins. In this study, single-cell transcriptomes of these two species were assembled and characterized. Numerous enzymes related to energy metabolism and parasitic adaption were identified through annotation in the Non-Redundant (NR), Clusters of Orthologous Genes (COG), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The expression of isocitrate dehydrogenase (IDH), cytochrome c oxidase subunit 1 (Cox1) and ATP synthase F1, delta subunit (ATP5D) was up-regulated in C. hexasticha compared with C. uncinata. The oxidative phosphorylation process was also enriched in C. hexasticha. The main mitochondrial metabolic pathways in C. hexasticha were depicted and enzymes related to energy metabolism pathways were compared between these two species. More importantly, mitochondrial division inhibitor 1 (mdivi-1) proved to be very effective in killing both C. hexasticha and C. uncinata, which could be a novel drug for Chilodonellosis control. This study can help us better understand the energy metabolisms of C. hexasticha and C. uncinata and provide new insight into novel targets for chilodonellosis control. Meanwhile, the transcriptome data can also facilitate genomic studies of these two species in the future. Full article
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12 pages, 1903 KiB  
Article
Proteomic Approach to Anemonia sulcata and Its Symbiont Symbiodinium spp. as New Source of Potential Biotechnological Applications and Climate Change Biomarkers
by Ana Maria Melendez-Perez, Almudena Escobar Niño, Rafael Carrasco-Reinado, Laura Martin Diaz and Francisco Javier Fernandez-Acero
Int. J. Mol. Sci. 2023, 24(16), 12798; https://doi.org/10.3390/ijms241612798 - 14 Aug 2023
Viewed by 1305
Abstract
Marine ecosystems are among the richest in terms of biodiversity, and at present, still remain largely unknown today. In the molecular biology era, several analyses have been conducted to unravel the biological processes in this ecosystem. These systems have provided biotechnological solutions to [...] Read more.
Marine ecosystems are among the richest in terms of biodiversity, and at present, still remain largely unknown today. In the molecular biology era, several analyses have been conducted to unravel the biological processes in this ecosystem. These systems have provided biotechnological solutions to current problems, including the treatment of diseases, as well as for the development of new biotechnological tools with applications in biomedicine and/or agri-food. In addition, in the context of climate change and global warming, these studies become even more necessary for the development of molecular tools that allow a reliable follow-up of this situation to anticipate alterations and responses of bioindicator species and to create a database to prevent and predict the environmental and climatic changes before the damage is irreversible. Proteomics approaches have revealed their potential use to obtain the set of biological effectors that lead to the real biological station on a specific stage, the proteins. In addition, proteomics-based algorithms have allowed the discovery of proteins with new potential biotechnological applications from proteome data through “applied proteomics”. In this project, the first proteome analysis of the sea anemone, Anemonia sulcata, and its symbiont has been developed. These organisms present a wide distribution sea ecosystem. In Spain, it is accepted as a fishing and aquaculture species. Moreover, Anemonia sulcate has a symbiotic relation with autotroph Dinoflagellates, Symbiodinium spp., that allows the study of its relation at the molecular level. For the first characterization of A. sulcata proteome, three independent biological replicates were used, and proteins were extracted and analyzed by LC–MS/MS, allowing the quantification of 325 proteins, 81 from Symbiodinium spp. proteins and 244 from A. sulcata proteins. These proteins were subjected to gene ontology categorization by Cellular Component, Molecular Function and Biological Process. These analyzes have allowed the identification of biomarkers of gene expression as potential powerful emerging diagnostic tools to identify and characterize the molecular drivers of climate change stresses and improve monitoring techniques. In addition, through the application of novel algorithms for the detection of bioactive compounds based on the analysis of molecules of marine origin, the proteome has allowed the identification of proteins with potential applications in the fields of biomedicine and agri-food. Full article
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17 pages, 7213 KiB  
Article
Robust ParB Binding to Half-parS Sites in Pseudomonas aeruginosa—A Mechanism for Retaining ParB on the Nucleoid?
by Adam Kawalek, Aneta Agnieszka Bartosik and Grazyna Jagura-Burdzy
Int. J. Mol. Sci. 2023, 24(15), 12517; https://doi.org/10.3390/ijms241512517 - 07 Aug 2023
Viewed by 947
Abstract
Chromosome segregation in Pseudomonas aeruginosa is assisted by the tripartite ParAB–parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB) and its target parS sequence(s). ParB forms a nucleoprotein complex around four parSs (parS1–parS4) that overlaps oriC and [...] Read more.
Chromosome segregation in Pseudomonas aeruginosa is assisted by the tripartite ParAB–parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB) and its target parS sequence(s). ParB forms a nucleoprotein complex around four parSs (parS1–parS4) that overlaps oriC and facilitates relocation of newly synthesized ori domains inside the cells by ParA. Remarkably, ParB of P. aeruginosa also binds to numerous heptanucleotides (half-parSs) scattered in the genome. Here, using chromatin immunoprecipitation-sequencing (ChIP-seq), we analyzed patterns of ParB genome occupancy in cells growing under conditions of coupling or uncoupling between replication and cell division processes. Interestingly, a dissipation of ParB–parS complexes and a shift of ParB to half-parSs were observed during the transition from the exponential to stationary phase of growth on rich medium, suggesting the role of half-parSs in retaining ParB on the nucleoid within non-dividing P. aeruginosa cells. The ChIP-seq analysis of strains expressing ParB variants unable to dislocate from parSs showed that the ParB spreading ability is not required for ParB binding to half-parSs. Finally, a P. aeruginosa strain with mutated 25 half-parSs of the highest affinity towards ParB was constructed and analyzed. It showed altered ParB coverage of the oriC region and moderate changes in gene expression. Overall, this study characterizes a novel aspect of conserved bacterial chromosome segregation machinery. Full article
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14 pages, 2516 KiB  
Article
Virulence and Metabolism Crosstalk: Impaired Activity of the Type Three Secretion System (T3SS) in a Pseudomonas aeruginosa Crc-Defective Mutant
by Teresa Gil-Gil, Trinidad Cuesta, Sara Hernando-Amado, Jose Antonio Reales-Calderón, Fernando Corona, Juan F. Linares and José L. Martínez
Int. J. Mol. Sci. 2023, 24(15), 12304; https://doi.org/10.3390/ijms241512304 - 01 Aug 2023
Cited by 2 | Viewed by 994
Abstract
Pseudomonas aeruginosa is a ubiquitous nosocomial opportunistic pathogen that harbors many virulence determinants. Part of P. aeruginosa success colonizing a variety of habitats resides in its metabolic robustness and plasticity, which are the basis of its capability of adaptation to different nutrient sources [...] Read more.
Pseudomonas aeruginosa is a ubiquitous nosocomial opportunistic pathogen that harbors many virulence determinants. Part of P. aeruginosa success colonizing a variety of habitats resides in its metabolic robustness and plasticity, which are the basis of its capability of adaptation to different nutrient sources and ecological conditions, including the infected host. Given this situation, it is conceivable that P. aeruginosa virulence might be, at least in part, under metabolic control, in such a way that virulence determinants are produced just when needed. Indeed, it has been shown that the catabolite repression control protein Crc, which together with the RNA chaperon Hfq regulates the P. aeruginosa utilization of carbon sources at the post-transcriptional level, also regulates, directly or indirectly, virulence-related processes in P. aeruginosa. Among them, Crc regulates P. aeruginosa cytotoxicity, likely by modulating the activity of the Type III Secretion System (T3SS), which directly injects toxins into eukaryotic host cells. The present work shows that the lack of Crc produces a Type III Secretion-defective phenotype in P. aeruginosa. The observed impairment is a consequence of a reduced expression of the genes encoding the T3SS, together with an impaired secretion of the proteins involved. Our results support that the impaired T3SS activity of the crc defective mutant is, at least partly, a consequence of a defective protein export, probably due to a reduced proton motive force. This work provides new information about the complex regulation of the expression and the activity of the T3SS in P. aeruginosa. Our results highlight the need of a robust bacterial metabolism, which is defective in the ∆crc mutant, to elicit complex and energetically costly virulence strategies, as that provided by the T3SS. Full article
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17 pages, 3650 KiB  
Article
Xylose Isomerase Depletion Enhances Virulence of Xanthomonas citri subsp. citri in Citrus aurantifolia
by André Vessoni Alexandrino, Evandro Luis Prieto, Nicole Castro Silva Nicolela, Tamiris Garcia da Silva Marin, Talita Alves dos Santos, João Pedro Maia de Oliveira da Silva, Anderson Ferreira da Cunha, Franklin Behlau and Maria Teresa Marques Novo-Mansur
Int. J. Mol. Sci. 2023, 24(14), 11491; https://doi.org/10.3390/ijms241411491 - 15 Jul 2023
Cited by 1 | Viewed by 906
Abstract
Citrus canker, caused by the bacterium Xanthomonas citri (Xcc), is one of the most devastating diseases for the citrus industry. Xylose is a constituent of the cell wall of plants, and the ability of Xcc to use this carbohydrate may play a role [...] Read more.
Citrus canker, caused by the bacterium Xanthomonas citri (Xcc), is one of the most devastating diseases for the citrus industry. Xylose is a constituent of the cell wall of plants, and the ability of Xcc to use this carbohydrate may play a role in virulence. Xcc has two genes codifying for xylose isomerase (XI), a bifunctional enzyme that interconverts D-xylose into D-xylulose and D-glucose into D-fructose. The aim of this work was to investigate the functional role of the two putative XI ORFs, XAC1776 (xylA1) and XAC4225 (xylA2), in Xcc pathogenicity. XI-coding genes of Xcc were deleted, and the single mutants (XccΔxylA1 or XccΔxylA2) or the double mutant (XccΔxylA1ΔxylA2) remained viable. The deletion of one or both XI genes (xylA1 and/or xylA2) increased the aggressiveness of the mutants, causing disease symptoms. RT-qPCR analysis of wild strain and xylA deletion mutants grown in vivo and in vitro revealed that the highest expression level of hrpX and xylR was observed in vivo for the double mutant. The results indicate that XI depletion increases the expression of the hrp regulatory genes in Xcc. We concluded that the intracellular accumulation of xylose enhances Xcc virulence. Full article
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42 pages, 19523 KiB  
Review
Fungal BGCs for Production of Secondary Metabolites: Main Types, Central Roles in Strain Improvement, and Regulation According to the Piano Principle
by Alexander A. Zhgun
Int. J. Mol. Sci. 2023, 24(13), 11184; https://doi.org/10.3390/ijms241311184 - 06 Jul 2023
Cited by 3 | Viewed by 2029
Abstract
Filamentous fungi are one of the most important producers of secondary metabolites. Some of them can have a toxic effect on the human body, leading to diseases. On the other hand, they are widely used as pharmaceutically significant drugs, such as antibiotics, statins, [...] Read more.
Filamentous fungi are one of the most important producers of secondary metabolites. Some of them can have a toxic effect on the human body, leading to diseases. On the other hand, they are widely used as pharmaceutically significant drugs, such as antibiotics, statins, and immunosuppressants. A single fungus species in response to various signals can produce 100 or more secondary metabolites. Such signaling is possible due to the coordinated regulation of several dozen biosynthetic gene clusters (BGCs), which are mosaically localized in different regions of fungal chromosomes. Their regulation includes several levels, from pathway-specific regulators, whose genes are localized inside BGCs, to global regulators of the cell (taking into account changes in pH, carbon consumption, etc.) and global regulators of secondary metabolism (affecting epigenetic changes driven by velvet family proteins, LaeA, etc.). In addition, various low-molecular-weight substances can have a mediating effect on such regulatory processes. This review is devoted to a critical analysis of the available data on the “turning on” and “off” of the biosynthesis of secondary metabolites in response to signals in filamentous fungi. To describe the ongoing processes, the model of “piano regulation” is proposed, whereby pressing a certain key (signal) leads to the extraction of a certain sound from the “musical instrument of the fungus cell”, which is expressed in the production of a specific secondary metabolite. Full article
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19 pages, 8873 KiB  
Article
TgMORN2, a MORN Family Protein Involved in the Regulation of Endoplasmic Reticulum Stress in Toxoplasma gondii
by Jinxuan Li, Qianqian Xiao, Qianqian Tan, Junpeng Chen, Lingyu Sun, Xiao Chen, Ziyu Chu, Hongxia Wu, Zhenzhao Zhang, Hongmei Li, Xiaomin Zhao and Xiao Zhang
Int. J. Mol. Sci. 2023, 24(12), 10228; https://doi.org/10.3390/ijms241210228 - 16 Jun 2023
Cited by 1 | Viewed by 927
Abstract
MORN proteins play a key role in the cytoskeletal structure of eukaryotes and are essential for the close arrangement of the endoplasmic reticulum and plasma membrane. A gene with nine MORN motifs (TGGT1_292120, named TgMORN2) was identified in the Toxoplasma gondii [...] Read more.
MORN proteins play a key role in the cytoskeletal structure of eukaryotes and are essential for the close arrangement of the endoplasmic reticulum and plasma membrane. A gene with nine MORN motifs (TGGT1_292120, named TgMORN2) was identified in the Toxoplasma gondii genome; it was presumed to belong to the MORN protein family and to have the function of forming the cytoskeleton, which affects the survival of T. gondii. However, the genetic deletion of MORN2 did not noticeably affect parasite growth and virulence. Using adjacent protein labeling techniques, we identified a network of TgMORN2 interactions, which mainly included endoplasmic reticulum stress (ER stress)-related proteins. In exploring these data, we found that the pathogenicity of the KO-TgMORN2 strain was significantly reduced in the case of tunicamycin-induced ER stress. Reticulon TgRTN (TGGT1_226430) and tubulin β-Tubulin were identified as interaction proteins of TgMORN2. Collectively, TgMORN2 plays a role in ER stress, which lays a foundation for further research on the function of the MORN protein in T. gondii. Full article
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11 pages, 1530 KiB  
Article
A Pilot Study to Detect Viable Salmonella spp. in Diarrheal Stool Using Viability Real-Time PCR as a Culture-Independent Diagnostic Tool in a Clinical Setting
by Surangi H. Thilakarathna and Linda Chui
Int. J. Mol. Sci. 2023, 24(12), 9979; https://doi.org/10.3390/ijms24129979 - 10 Jun 2023
Viewed by 1440
Abstract
Frontline laboratories are adopting culture-independent diagnostic testing (CIDT) such as nucleic acid amplification tests (NAATs) due to numerous advantages over culture-based testing methods. Paradoxically, the viability of pathogens, a crucial factor determining active infections, cannot be confirmed with current NAATs alone. A recent [...] Read more.
Frontline laboratories are adopting culture-independent diagnostic testing (CIDT) such as nucleic acid amplification tests (NAATs) due to numerous advantages over culture-based testing methods. Paradoxically, the viability of pathogens, a crucial factor determining active infections, cannot be confirmed with current NAATs alone. A recent development of viability PCR (vPCR) was introduced to mitigate this limitation associated with real-time PCR (qPCR) by using a DNA-intercalating dye to remove residual and dead cell DNA. This study assessed the applicability of the vPCR assay on diarrheal stools. Eighty-five diarrheal stools confirmed for Salmonellosis were tested via qPCR and vPCR using in-house primers and probe targeting the invA gene. vPCR-negative stools (Ct cut off > 31) were enriched in mannitol selenite broth (MSB) to verify low bacterial loads. vPCR assay showed ~89% sensitivity (qPCR- and vPCR-positive stools: 76/85). vPCR-negative stools (9/85; qPCR-positive: 5; qPCR-negative: 4) were qPCR- and culture-positive post-MSB-enrichment and confirmed the presence of low viable bacterial loads. Random sampling error, low bacterial loads, and receiving stools in batches could contribute to false negatives. This is a pilot study and further investigations are warranted to explore vPCR to assess pathogen viability in a clinical setting, especially when culture-based testing is unavailable. Full article
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14 pages, 2089 KiB  
Article
The Small RNA-Binding Protein CcaF1 Promotes Formation of Photosynthetic Complexes in Rhodobacter sphaeroides
by Julian Grützner, Janek Börner, Andreas Jäger and Gabriele Klug
Int. J. Mol. Sci. 2023, 24(11), 9515; https://doi.org/10.3390/ijms24119515 - 30 May 2023
Cited by 1 | Viewed by 1018
Abstract
In natural habitats, bacteria frequently need to adapt to changing environmental conditions. Regulation of transcription plays an important role in this process. However, riboregulation also contributes substantially to adaptation. Riboregulation often acts at the level of mRNA stability, which is determined by sRNAs, [...] Read more.
In natural habitats, bacteria frequently need to adapt to changing environmental conditions. Regulation of transcription plays an important role in this process. However, riboregulation also contributes substantially to adaptation. Riboregulation often acts at the level of mRNA stability, which is determined by sRNAs, RNases, and RNA-binding proteins. We previously identified the small RNA-binding protein CcaF1, which is involved in sRNA maturation and RNA turnover in Rhodobacter sphaeroides. Rhodobacter is a facultative phototroph that can perform aerobic and anaerobic respiration, fermentation, and anoxygenic photosynthesis. Oxygen concentration and light conditions decide the pathway for ATP production. Here, we show that CcaF1 promotes the formation of photosynthetic complexes by increasing levels of mRNAs for pigment synthesis and for some pigment-binding proteins. Levels of mRNAs for transcriptional regulators of photosynthesis genes are not affected by CcaF1. RIP-Seq analysis compares the binding of CcaF1 to RNAs during microaerobic and photosynthetic growth. The stability of the pufBA mRNA for proteins of the light-harvesting I complex is increased by CcaF1 during phototrophic growth but decreased during microaerobic growth. This research underlines the importance of RNA-binding proteins in adaptation to different environments and demonstrates that an RNA-binding protein can differentially affect its binding partners in dependence upon growth conditions. Full article
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11 pages, 1741 KiB  
Article
Involvement of Lipid Rafts in the Invasion of Opportunistic Bacteria Serratia into Eukaryotic Cells
by Yuliya Berson, Sofia Khaitlina and Olga Tsaplina
Int. J. Mol. Sci. 2023, 24(10), 9029; https://doi.org/10.3390/ijms24109029 - 20 May 2023
Cited by 1 | Viewed by 1008
Abstract
Cell membrane rafts form signaling platforms on the cell surface, controlling numerous protein–protein and lipid–protein interactions. Bacteria invading eukaryotic cells trigger cell signaling to induce their own uptake by non-phagocytic cells. The aim of this work was to reveal the involvement of membrane [...] Read more.
Cell membrane rafts form signaling platforms on the cell surface, controlling numerous protein–protein and lipid–protein interactions. Bacteria invading eukaryotic cells trigger cell signaling to induce their own uptake by non-phagocytic cells. The aim of this work was to reveal the involvement of membrane rafts in the penetration of the bacteria Serratia grimesii and Serratia proteamaculans into eukaryotic cells. Our results show that the disruption of membrane rafts by MβCD in the three cell lines tested, M-HeLa, MCF-7 and Caco-2, resulted in a time-dependent decrease in the intensity of Serratia invasion. MβCD treatment produced a more rapid effect on the bacterial susceptibility of M-HeLa cells compared to other cell lines. This effect correlated with a faster assembly of the actin cytoskeleton upon treatment with MβCD in M-HeLa cells in contrast to that in Caco-2 cells. Moreover, the 30 min treatment of Caco-2 cells with MβCD produced an increase in the intensity of S. proteamaculans invasion. This effect correlated with an increase in EGFR expression. Together with the evidence that EGFR is involved in S. proteamaculans invasion but not in S. grimesii invasion, these results led to the conclusion that an increase in EGFR amount on the plasma membrane with the undisassembled rafts of Caco-2 cells after 30 min of treatment with MβCD may increase the intensity of S. proteamaculans but not of S. grimesii invasion. Thus, the MβCD-dependent degradation of lipid rafts, which enhances actin polymerization and disrupts signaling pathways from receptors on the host cell’s surface, reduces Serratia invasion. Full article
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11 pages, 576 KiB  
Opinion
Repurposing Amphotericin B and Its Liposomal Formulation for the Treatment of Human Mpox
by Daniela Peruzzu, Katia Fecchi, Giulietta Venturi and Maria Cristina Gagliardi
Int. J. Mol. Sci. 2023, 24(10), 8896; https://doi.org/10.3390/ijms24108896 - 17 May 2023
Cited by 3 | Viewed by 1524
Abstract
Mpox (monkeypox) is a zoonotic viral disease caused by the mpox virus (MPXV). Recently in 2022, a multi-country Mpox outbreak has determined great concern as the disease rapidly spreads. The majority of cases are being noticed in European regions and are unrelated to [...] Read more.
Mpox (monkeypox) is a zoonotic viral disease caused by the mpox virus (MPXV). Recently in 2022, a multi-country Mpox outbreak has determined great concern as the disease rapidly spreads. The majority of cases are being noticed in European regions and are unrelated to endemic travel or known contact with infected individuals. In this outbreak, close sexual contact appears to be important for MPXV transmission, and an increasing prevalence in people with multiple sexual partners and in men who have sex with men has been observed. Although Vaccinia virus (VACV)-based vaccines have been shown to induce a cross-reactive and protective immune response against MPXV, limited data support their efficacy against the 2022 Mpox outbreak. Furthermore, there are no specific antiviral drugs for Mpox. Host-cell lipid rafts are small, highly dynamic plasma-membrane microdomains enriched in cholesterol, glycosphingolipids and phospholipids that have emerged as crucial surface-entry platforms for several viruses. We previously demonstrated that the antifungal drug Amphotericin B (AmphB) inhibits fungal, bacterial and viral infection of host cells through its capacity to sequester host-cell cholesterol and disrupt lipid raft architecture. In this context, we discuss the hypothesis that AmphB could inhibit MPXV infection of host cells through disruption of lipid rafts and eventually through redistribution of receptors/co-receptors mediating virus entry, thus representing an alternative or additional therapeutic tool for human Mpox. Full article
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27 pages, 6584 KiB  
Article
Yeast Chaperone Hsp70-Ssb Modulates a Variety of Protein-Based Heritable Elements
by Lina M. Jay-Garcia, Joseph L. Cornell, Rebecca L. Howie, Quincy L. Faber, Abigail Salas, Tatiana A. Chernova and Yury O. Chernoff
Int. J. Mol. Sci. 2023, 24(10), 8660; https://doi.org/10.3390/ijms24108660 - 12 May 2023
Viewed by 1263
Abstract
Prions are transmissible self-perpetuating protein isoforms associated with diseases and heritable traits. Yeast prions and non-transmissible protein aggregates (mnemons) are frequently based on cross-β ordered fibrous aggregates (amyloids). The formation and propagation of yeast prions are controlled by chaperone machinery. Ribosome-associated chaperone Hsp70-Ssb [...] Read more.
Prions are transmissible self-perpetuating protein isoforms associated with diseases and heritable traits. Yeast prions and non-transmissible protein aggregates (mnemons) are frequently based on cross-β ordered fibrous aggregates (amyloids). The formation and propagation of yeast prions are controlled by chaperone machinery. Ribosome-associated chaperone Hsp70-Ssb is known (and confirmed here) to modulate formation and propagation of the prion form of the Sup35 protein [PSI+]. Our new data show that both formation and mitotic transmission of the stress-inducible prion form of the Lsb2 protein ([LSB+]) are also significantly increased in the absence of Ssb. Notably, heat stress leads to a massive accumulation of [LSB+] cells in the absence of Ssb, implicating Ssb as a major downregulator of the [LSB+]-dependent memory of stress. Moreover, the aggregated form of Gγ subunit Ste18, [STE+], behaving as a non-heritable mnemon in the wild-type strain, is generated more efficiently and becomes heritable in the absence of Ssb. Lack of Ssb also facilitates mitotic transmission, while lack of the Ssb cochaperone Hsp40-Zuo1 facilitates both spontaneous formation and mitotic transmission of the Ure2 prion, [URE3]. These results demonstrate that Ssb is a general modulator of cytosolic amyloid aggregation, whose effect is not restricted only to [PSI+]. Full article
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13 pages, 1358 KiB  
Article
Dengue Virus Capsid Protein Facilitates Genome Compaction and Packaging
by Priscilla L. S. Boon, Ana S. Martins, Xin Ni Lim, Francisco J. Enguita, Nuno C. Santos, Peter J. Bond, Yue Wan, Ivo C. Martins and Roland G. Huber
Int. J. Mol. Sci. 2023, 24(9), 8158; https://doi.org/10.3390/ijms24098158 - 02 May 2023
Viewed by 1821
Abstract
Dengue virus (DENV) is a single-stranded (+)-sense RNA virus that infects humans and mosquitoes, posing a significant health risk in tropical and subtropical regions. Mature virions are composed of an icosahedral shell of envelope (E) and membrane (M) proteins circumscribing a lipid bilayer, [...] Read more.
Dengue virus (DENV) is a single-stranded (+)-sense RNA virus that infects humans and mosquitoes, posing a significant health risk in tropical and subtropical regions. Mature virions are composed of an icosahedral shell of envelope (E) and membrane (M) proteins circumscribing a lipid bilayer, which in turn contains a complex of the approximately 11 kb genomic RNA with capsid (C) proteins. Whereas the structure of the envelope is clearly defined, the structure of the packaged genome in complex with C proteins remains elusive. Here, we investigated the interactions of C proteins with viral RNA, in solution and inside mature virions, via footprinting and cross-linking experiments. We demonstrated that C protein interaction with DENV genomes saturates at an RNA:C protein ratio below 1:250. Moreover, we also showed that the length of the RNA genome interaction sites varies, in a multimodal distribution, consistent with the C protein binding to each RNA site mostly in singlets or pairs (and, in some instances, higher numbers). We showed that interaction sites are preferentially sites with low base pairing, as previously measured by 2′-acetylation analyzed by primer extension (SHAPE) reactivity indicating structuredness. We found a clear association pattern emerged: RNA-C protein binding sites are strongly associated with long-range RNA–RNA interaction sites, particularly inside virions. This, in turn, explains the need for C protein in viral genome packaging: the protein has a chief role in coordinating these key interactions, promoting proper packaging of viral RNA. Such sites are, thus, highly consequential for viral assembly, and, as such, may be targeted in future drug development strategies against these and related viruses. Full article
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19 pages, 1327 KiB  
Review
The Role of Microbiota-Derived Metabolites in Colorectal Cancer
by Coco Duizer and Marcel R. de Zoete
Int. J. Mol. Sci. 2023, 24(9), 8024; https://doi.org/10.3390/ijms24098024 - 28 Apr 2023
Cited by 1 | Viewed by 2109
Abstract
The impact of bacterial members of the microbiota on the development of colorectal cancer (CRC) has become clear in recent years. However, exactly how bacteria contribute to the development of cancer is often still up for debate. The impact of bacteria-derived metabolites, which [...] Read more.
The impact of bacterial members of the microbiota on the development of colorectal cancer (CRC) has become clear in recent years. However, exactly how bacteria contribute to the development of cancer is often still up for debate. The impact of bacteria-derived metabolites, which can influence the development of CRC either in a promoting or inhibiting manner, is undeniable. Here, we discuss the effects of the most well-studied bacteria-derived metabolites associated with CRC, including secondary bile acids, short-chain fatty acids, trimethylamine-N-oxide and indoles. We show that the effects of individual metabolites on CRC development are often nuanced and dose- and location-dependent. In the coming years, the array of metabolites involved in CRC development will undoubtedly increase further, which will emphasize the need to focus on causation and mechanisms and the clearly defined roles of bacterial species within the microbiota. Full article
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16 pages, 8771 KiB  
Article
Characterization of PcSTT3B as a Key Oligosaccharyltransferase Subunit Involved in N-glycosylation and Its Role in Development and Pathogenicity of Phytophthora capsici
by Tongshan Cui, Quanhe Ma, Fan Zhang, Shanshan Chen, Can Zhang, Jianjun Hao and Xili Liu
Int. J. Mol. Sci. 2023, 24(8), 7500; https://doi.org/10.3390/ijms24087500 - 19 Apr 2023
Viewed by 1085
Abstract
Asparagine (Asn, N)-linked glycosylation is a conserved process and an essential post-translational modification that occurs on the NXT/S motif of the nascent polypeptides in endoplasmic reticulum (ER). The mechanism of N-glycosylation and biological functions of key catalytic enzymes involved in this process are [...] Read more.
Asparagine (Asn, N)-linked glycosylation is a conserved process and an essential post-translational modification that occurs on the NXT/S motif of the nascent polypeptides in endoplasmic reticulum (ER). The mechanism of N-glycosylation and biological functions of key catalytic enzymes involved in this process are rarely documented for oomycetes. In this study, an N-glycosylation inhibitor tunicamycin (TM) hampered the mycelial growth, sporangial release, and zoospore production of Phytophthora capsici, indicating that N-glycosylation was crucial for oomycete growth development. Among the key catalytic enzymes involved in N-glycosylation, the PcSTT3B gene was characterized by its functions in P. capsici. As a core subunit of the oligosaccharyltransferase (OST) complex, the staurosporine and temperature sensive 3B (STT3B) subunit were critical for the catalytic activity of OST. The PcSTT3B gene has catalytic activity and is highly conservative in P. capsici. By using a CRISPR/Cas9-mediated gene replacement system to delete the PcSTT3B gene, the transformants impaired mycelial growth, sporangial release, zoospore production, and virulence. The PcSTT3B-deleted transformants were more sensitive to an ER stress inducer TM and display low glycoprotein content in the mycelia, suggesting that PcSTT3B was associated with ER stress responses and N-glycosylation. Therefore, PcSTT3B was involved in the development, pathogenicity, and N-glycosylation of P. capsici. Full article
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11 pages, 2483 KiB  
Review
Changes in K+ Concentration as a Signaling Mechanism in the Apicomplexa Parasites Plasmodium and Toxoplasma
by Benedito M. Dos Santos, Jude M. Przyborski and Célia R. S. Garcia
Int. J. Mol. Sci. 2023, 24(8), 7276; https://doi.org/10.3390/ijms24087276 - 14 Apr 2023
Viewed by 1287
Abstract
During their life cycle, apicomplexan parasites pass through different microenvironments and encounter a range of ion concentrations. The discovery that the GPCR-like SR25 in Plasmodium falciparum is activated by a shift in potassium concentration indicates that the parasite can take advantage of its [...] Read more.
During their life cycle, apicomplexan parasites pass through different microenvironments and encounter a range of ion concentrations. The discovery that the GPCR-like SR25 in Plasmodium falciparum is activated by a shift in potassium concentration indicates that the parasite can take advantage of its development by sensing different ionic concentrations in the external milieu. This pathway involves the activation of phospholipase C and an increase in cytosolic calcium. In the present report, we summarize the information available in the literature regarding the role of potassium ions during parasite development. A deeper understanding of the mechanisms that allow the parasite to cope with ionic potassium changes contributes to our knowledge about the cell cycle of Plasmodium spp. Full article
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17 pages, 4432 KiB  
Article
HIV-Differentiated Metabolite N-Acetyl-L-Alanine Dysregulates Human Natural Killer Cell Responses to Mycobacterium tuberculosis Infection
by Baojun Yang, Tanmoy Mukherjee, Rajesh Radhakrishnan, Padmaja Paidipally, Danish Ansari, Sahana John, Ramakrishna Vankayalapati, Deepak Tripathi and Guohua Yi
Int. J. Mol. Sci. 2023, 24(8), 7267; https://doi.org/10.3390/ijms24087267 - 14 Apr 2023
Cited by 2 | Viewed by 2089
Abstract
Mycobacterium tuberculosis (Mtb) has latently infected over two billion people worldwide (LTBI) and caused ~1.6 million deaths in 2021. Human immunodeficiency virus (HIV) co-infection with Mtb will affect the Mtb progression and increase the risk of developing active tuberculosis by 10–20 [...] Read more.
Mycobacterium tuberculosis (Mtb) has latently infected over two billion people worldwide (LTBI) and caused ~1.6 million deaths in 2021. Human immunodeficiency virus (HIV) co-infection with Mtb will affect the Mtb progression and increase the risk of developing active tuberculosis by 10–20 times compared with HIV- LTBI+ patients. It is crucial to understand how HIV can dysregulate immune responses in LTBI+ individuals. Plasma samples collected from healthy and HIV-infected individuals were investigated using liquid chromatography–mass spectrometry (LC-MS), and the metabolic data were analyzed using the online platform Metabo-Analyst. ELISA, surface and intracellular staining, flow cytometry, and quantitative reverse-transcription PCR (qRT-PCR) were performed using standard procedures to determine the surface markers, cytokines, and other signaling molecule expressions. Seahorse extra-cellular flux assays were used to measure mitochondrial oxidative phosphorylation and glycolysis. Six metabolites were significantly less abundant, and two were significantly higher in abundance in HIV+ individuals compared with healthy donors. One of the HIV-upregulated metabolites, N-acetyl-L-alanine (ALA), inhibits pro-inflammatory cytokine IFN-γ production by the NK cells of LTBI+ individuals. ALA inhibits the glycolysis of LTBI+ individuals’ NK cells in response to Mtb. Our findings demonstrate that HIV infection enhances plasma ALA levels to inhibit NK-cell-mediated immune responses to Mtb infection, offering a new understanding of the HIV–Mtb interaction and providing insights into the implication of nutrition intervention and therapy for HIV–Mtb co-infected patients. Full article
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15 pages, 1346 KiB  
Article
Natural Isoforms of Listeria monocytogenes Virulence Factor Inlb Differ in c-Met Binding Efficiency and Differently Affect Uptake and Survival Listeria in Macrophage
by Yaroslava M. Chalenko, Daria A. Slonova, Olga I. Kechko, Egor V. Kalinin, Vladimir A. Mitkevich and Svetlana A. Ermolaeva
Int. J. Mol. Sci. 2023, 24(8), 7256; https://doi.org/10.3390/ijms24087256 - 14 Apr 2023
Cited by 2 | Viewed by 1273
Abstract
Listeria monocytogenes virulence factor InlB specifically interacts with the receptors c-Met and gC1q-R. Both receptors are present in non-professional and professional phagocytes, including macrophages. Phylogenetically defined InlB isoforms differently support invasion into non-professional phagocytes. This work deals with the effects of InlB isoforms [...] Read more.
Listeria monocytogenes virulence factor InlB specifically interacts with the receptors c-Met and gC1q-R. Both receptors are present in non-professional and professional phagocytes, including macrophages. Phylogenetically defined InlB isoforms differently support invasion into non-professional phagocytes. This work deals with the effects of InlB isoforms on L. monocytogenes uptake and intracellular proliferation in human macrophages. Three isoforms of the receptor binding domain (idInlB) were derived from phylogenetically distinct L. monocytogenes strains belonging to the highly virulent CC1 (idInlBCC1), medium-virulence CC7 (idInlBCC7), and low-virulence CC9 (idInlBCC9) clonal complexes. The constant dissociation increased in the order idInlBCC1 << idInlBCC7 < idInlBCC9 for interactions with c-Met, and idInlBCC1 ≈ idInlBCC7 < idInlBCC9 for interactions with gC1q-R. The comparison of uptake and intracellular proliferation of isogenic recombinant strains which expressed full-length InlBs revealed that the strain expressing idInlBCC1 proliferated in macrophages twice as efficiently as other strains. Macrophage pretreatment with idInlBCC1 followed by recombinant L. monocytogenes infection disturbed macrophage functions decreasing pathogen uptake and improving its intracellular multiplication. Similar pretreatment with idInlBCC7 decreased bacterial uptake but also impaired intracellular multiplication. The obtained results demonstrated that InlB impaired macrophage functions in an idInlB isoform-dependent manner. These data suggest a novel InlB function in L. monocytogenes virulence. Full article
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15 pages, 4697 KiB  
Article
Strain-Specific Interactions between the Viral Capsid Proteins VP4, VP7 and VP6 Influence Rescue of Rotavirus Reassortants by Reverse Genetics
by Roman Valusenko-Mehrkens, Ashish K. Gadicherla, Reimar Johne and Alexander Falkenhagen
Int. J. Mol. Sci. 2023, 24(6), 5670; https://doi.org/10.3390/ijms24065670 - 16 Mar 2023
Cited by 2 | Viewed by 1569
Abstract
Rotavirus A (RVA) genome segments can reassort upon co-infection of target cells with two different RVA strains. However, not all reassortants are viable, which limits the ability to generate customized viruses for basic and applied research. To gain insight into the factors that [...] Read more.
Rotavirus A (RVA) genome segments can reassort upon co-infection of target cells with two different RVA strains. However, not all reassortants are viable, which limits the ability to generate customized viruses for basic and applied research. To gain insight into the factors that restrict reassortment, we utilized reverse genetics and tested the generation of simian RVA strain SA11 reassortants carrying the human RVA strain Wa capsid proteins VP4, VP7, and VP6 in all possible combinations. VP7-Wa, VP6-Wa, and VP7/VP6-Wa reassortants were effectively rescued, but the VP4-Wa, VP4/VP7-Wa, and VP4/VP6-Wa reassortants were not viable, suggesting a limiting effect of VP4-Wa. However, a VP4/VP7/VP6-Wa triple-reassortant was successfully generated, indicating that the presence of homologous VP7 and VP6 enabled the incorporation of VP4-Wa into the SA11 backbone. The replication kinetics of the triple-reassortant and its parent strain Wa were comparable, while the replication of all other rescued reassortants was similar to SA11. Analysis of the predicted structural protein interfaces identified amino acid residues, which might influence protein interactions. Restoring the natural VP4/VP7/VP6 interactions may therefore improve the rescue of RVA reassortants by reverse genetics, which could be useful for the development of next generation RVA vaccines. Full article
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24 pages, 5823 KiB  
Article
Genome-Wide Analysis of Antigen 43 (Ag43) Variants: New Insights in Their Diversity, Distribution and Prevalence in Bacteria
by Valentin Ageorges, Ivan Wawrzyniak, Philippe Ruiz, Cédric Bicep, Mohamed A. Zorgani, Jason J. Paxman, Begoña Heras, Ian R. Henderson, Sabine Leroy, Xavier Bailly, Panagiotis Sapountzis, Eric Peyretaillade and Mickaël Desvaux
Int. J. Mol. Sci. 2023, 24(6), 5500; https://doi.org/10.3390/ijms24065500 - 13 Mar 2023
Cited by 1 | Viewed by 1895
Abstract
Antigen 43 (Ag43) expression induces aggregation and biofilm formation that has consequences for bacterial colonisation and infection. Ag43 is secreted through the Type 5 subtype “a” secretion system (T5aSS) and is a prototypical member of the family of self-associating autotransporters (SAATs). As a [...] Read more.
Antigen 43 (Ag43) expression induces aggregation and biofilm formation that has consequences for bacterial colonisation and infection. Ag43 is secreted through the Type 5 subtype “a” secretion system (T5aSS) and is a prototypical member of the family of self-associating autotransporters (SAATs). As a T5aSS protein, Ag43 has a modular architecture comprised of (i) a signal peptide, (ii) a passenger domain that can be subdivided into three subdomains (SL, EJ, and BL), (iii) an autochaperone (AC) domain, and (iv) an outer membrane translocator. The cell-surface SL subdomain is directly involved in the “Velcro-handshake” mechanism resulting in bacterial autoaggregation. Ag43 is considered to have a ubiquitous distribution in E. coli genomes and many strains harbour multiple agn43 genes. However, recent phylogenetic analyses indicated the existence of four distinct Ag43 classes exhibiting different propensities for autoaggregation and interactions. Given the knowledge of the diversity and distribution of Ag43 in E. coli genomes is incomplete, we have performed a thorough in silico investigation across bacterial genomes. Our comprehensive analyses indicate that Ag43 passenger domains cluster in six phylogenetic classes associated with different SL subdomains. The diversity of Ag43 passenger domains is a result of the association of the SL subtypes with two different EJ-BL-AC modules. We reveal that agn43 is almost exclusively present among bacterial species of the Enterobacteriaceae family and essentially in the Escherichia genus (99.6%) but that it is not ubiquitous in E. coli. The gene is typically present as a single copy but up to five copies of agn43 with different combinations of classes can be observed. The presence of agn43 as well as its different classes appeared to differ between Escherichia phylogroups. Strikingly, agn43 is present in 90% of E. coli from E phylogroup. Our results shed light on Ag43 diversity and provide a rational framework for investigating its role in E. coli ecophysiology and physiopathology. Full article
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17 pages, 3263 KiB  
Article
Participants in the Trans-Antarctic Winter Traverse Expedition Showed Increased Bacterial Load and Diversity in Saliva but Maintained Individual Differences within Stool Microbiota and Across Metabolite Fingerprints
by Simon J. S. Cameron, Arwyn Edwards, Robert J. Lambert, Mike Stroud and Luis A. J. Mur
Int. J. Mol. Sci. 2023, 24(5), 4850; https://doi.org/10.3390/ijms24054850 - 02 Mar 2023
Viewed by 1256
Abstract
Understanding the impact of long-term physiological and environmental stress on the human microbiota and metabolome may be important for the success of space flight. This work is logistically difficult and has a limited number of available participants. Terrestrial analogies present important opportunities to [...] Read more.
Understanding the impact of long-term physiological and environmental stress on the human microbiota and metabolome may be important for the success of space flight. This work is logistically difficult and has a limited number of available participants. Terrestrial analogies present important opportunities to understand changes in the microbiota and metabolome and how this may impact participant health and fitness. Here, we present work from one such analogy: the Transarctic Winter Traverse expedition, which we believe is the first assessment of the microbiota and metabolome from different bodily locations during prolonged environmental and physiological stress. Bacterial load and diversity were significantly higher during the expedition when compared with baseline levels (p < 0.001) in saliva but not stool, and only a single operational taxonomic unit assigned to the Ruminococcaceae family shows significantly altered levels in stool (p < 0.001). Metabolite fingerprints show the maintenance of individual differences across saliva, stool, and plasma samples when analysed using flow infusion electrospray mass spectrometry and Fourier transform infrared spectroscopy. Significant activity-associated changes in terms of both bacterial diversity and load are seen in saliva but not in stool, and participant differences in metabolite fingerprints persist across all three sample types. Full article
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26 pages, 4156 KiB  
Article
Polymer-Degrading Enzymes of Pseudomonas chloroaphis PA23 Display Broad Substrate Preferences
by Nisha Mohanan, Michael C.-H. Wong, Nediljko Budisa and David B. Levin
Int. J. Mol. Sci. 2023, 24(5), 4501; https://doi.org/10.3390/ijms24054501 - 24 Feb 2023
Cited by 4 | Viewed by 2144
Abstract
Although many bacterial lipases and PHA depolymerases have been identified, cloned, and characterized, there is very little information on the potential application of lipases and PHA depolymerases, especially intracellular enzymes, for the degradation of polyester polymers/plastics. We identified genes encoding an intracellular lipase [...] Read more.
Although many bacterial lipases and PHA depolymerases have been identified, cloned, and characterized, there is very little information on the potential application of lipases and PHA depolymerases, especially intracellular enzymes, for the degradation of polyester polymers/plastics. We identified genes encoding an intracellular lipase (LIP3), an extracellular lipase (LIP4), and an intracellular PHA depolymerase (PhaZ) in the genome of the bacterium Pseudomonas chlororaphis PA23. We cloned these genes into Escherichia coli and then expressed, purified, and characterized the biochemistry and substrate preferences of the enzymes they encode. Our data suggest that the LIP3, LIP4, and PhaZ enzymes differ significantly in their biochemical and biophysical properties, structural-folding characteristics, and the absence or presence of a lid domain. Despite their different properties, the enzymes exhibited broad substrate specificity and were able to hydrolyze both short- and medium-chain length polyhydroxyalkanoates (PHAs), para-nitrophenyl (pNP) alkanoates, and polylactic acid (PLA). Gel Permeation Chromatography (GPC) analyses of the polymers treated with LIP3, LIP4, and PhaZ revealed significant degradation of both the biodegradable as well as the synthetic polymers poly(ε-caprolactone) (PCL) and polyethylene succinate (PES). Full article
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28 pages, 1626 KiB  
Review
The Skin Microbiome: Current Landscape and Future Opportunities
by Paisleigh Smythe and Holly N. Wilkinson
Int. J. Mol. Sci. 2023, 24(4), 3950; https://doi.org/10.3390/ijms24043950 - 16 Feb 2023
Cited by 14 | Viewed by 11764
Abstract
Our skin is the largest organ of the body, serving as an important barrier against the harsh extrinsic environment. Alongside preventing desiccation, chemical damage and hypothermia, this barrier protects the body from invading pathogens through a sophisticated innate immune response and co-adapted consortium [...] Read more.
Our skin is the largest organ of the body, serving as an important barrier against the harsh extrinsic environment. Alongside preventing desiccation, chemical damage and hypothermia, this barrier protects the body from invading pathogens through a sophisticated innate immune response and co-adapted consortium of commensal microorganisms, collectively termed the microbiota. These microorganisms inhabit distinct biogeographical regions dictated by skin physiology. Thus, it follows that perturbations to normal skin homeostasis, as occurs with ageing, diabetes and skin disease, can cause microbial dysbiosis and increase infection risk. In this review, we discuss emerging concepts in skin microbiome research, highlighting pertinent links between skin ageing, the microbiome and cutaneous repair. Moreover, we address gaps in current knowledge and highlight key areas requiring further exploration. Future advances in this field could revolutionise the way we treat microbial dysbiosis associated with skin ageing and other pathologies. Full article
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17 pages, 826 KiB  
Review
The Human Virome and Its Crosslink with Glomerulonephritis and IgA Nephropathy
by Fabio Sallustio, Angela Picerno, Francesca Montenegro, Maria Teresa Cimmarusti, Vincenzo Di Leo and Loreto Gesualdo
Int. J. Mol. Sci. 2023, 24(4), 3897; https://doi.org/10.3390/ijms24043897 - 15 Feb 2023
Cited by 3 | Viewed by 2599
Abstract
The prokaryotic, viral, fungal, and parasitic microbiome exists in a highly intricate connection with the human host. In addition to eukaryotic viruses, due to the existence of various host bacteria, phages are widely spread throughout the human body. However, it is now evident [...] Read more.
The prokaryotic, viral, fungal, and parasitic microbiome exists in a highly intricate connection with the human host. In addition to eukaryotic viruses, due to the existence of various host bacteria, phages are widely spread throughout the human body. However, it is now evident that some viral community states, as opposed to others, are indicative of health and might be linked to undesirable outcomes for the human host. Members of the virome may collaborate with the human host to retain mutualistic functions in preserving human health. Evolutionary theories contend that a particular microbe’s ubiquitous existence may signify a successful partnership with the host. In this Review, we present a survey of the field’s work on the human virome and highlight the role of viruses in health and disease and the relationship of the virobiota with immune system control. Moreover, we will analyze virus involvement in glomerulonephritis and in IgA nephropathy, theorizing the molecular mechanisms that may be responsible for the crosslink with these renal diseases. Full article
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12 pages, 3336 KiB  
Article
Cell Sorting-Directed Selection of Bacterial Cells in Bigger Sizes Analyzed by Imaging Flow Cytometry during Experimental Evolution
by Di Tian, Caiyan Wang, Yunfei Liu, Yueyue Zhang, Adriano Caliari, Hui Lu, Yang Xia, Boying Xu, Jian Xu and Tetsuya Yomo
Int. J. Mol. Sci. 2023, 24(4), 3243; https://doi.org/10.3390/ijms24043243 - 07 Feb 2023
Cited by 2 | Viewed by 2573
Abstract
Cell morphology is an essential and phenotypic trait that can be easily tracked during adaptation and evolution to environmental changes. Thanks to the rapid development of quantitative analytical techniques for large populations of cells based on their optical properties, morphology can be easily [...] Read more.
Cell morphology is an essential and phenotypic trait that can be easily tracked during adaptation and evolution to environmental changes. Thanks to the rapid development of quantitative analytical techniques for large populations of cells based on their optical properties, morphology can be easily determined and tracked during experimental evolution. Furthermore, the directed evolution of new culturable morphological phenotypes can find use in synthetic biology to refine fermentation processes. It remains unknown whether and how fast we can obtain a stable mutant with distinct morphologies using fluorescence-activated cell sorting (FACS)-directed experimental evolution. Taking advantage of FACS and imaging flow cytometry (IFC), we direct the experimental evolution of the E. coli population undergoing continuous passage of sorted cells with specific optical properties. After ten rounds of sorting and culturing, a lineage with large cells resulting from incomplete closure of the division ring was obtained. Genome sequencing highlighted a stop-gain mutation in amiC, leading to a dysfunctional AmiC division protein. The combination of FACS-based selection with IFC analysis to track the evolution of the bacteria population in real-time holds promise to rapidly select and culture new morphologies and association tendencies with many potential applications. Full article
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11 pages, 2123 KiB  
Article
Symbiotic Bacteria System of Locusta migratoria Showed Antifungal Capabilities against Beauveria bassiana
by Shuqian Tan, Hongshuang Wei, Ibrahima Camara, Haoran Jia, Kaili Cao and Wangpeng Shi
Int. J. Mol. Sci. 2023, 24(4), 3138; https://doi.org/10.3390/ijms24043138 - 05 Feb 2023
Viewed by 1705
Abstract
The stability of symbiotic flora is an important indicator of the health of an organism. Symbiotic bacteria have been proven to be closely involved in the immune process of organisms. The pathogenicity of Beauveria bassiana was studied in relation to symbiotic bacteria on [...] Read more.
The stability of symbiotic flora is an important indicator of the health of an organism. Symbiotic bacteria have been proven to be closely involved in the immune process of organisms. The pathogenicity of Beauveria bassiana was studied in relation to symbiotic bacteria on the surface and inside of the migratory locust (Locusta migratoria). The results showed that the surface disinfection of test locusts contributed to the pathogenicity of B. bassiana to locusts. Most of the surface bacteria of L. migratoria caused some inhibition of B. bassiana growth, and LM5-4 (Raoultella ornithinolytica), LM5-2 (Enterobacter aerogenes), and LM5-13 (Citrobacter freundii) showed the highest inhibitory effect on the growth of B. bassiana. The inoculation of locusts with additional surface symbiotic bacteria reduced the virulence of B. bassiana to L. migratoria. Infection by different strains of B. bassiana caused similar changes in the symbiotic flora of migratory locusts. The inoculation of locusts with additional intestinal symbiotic bacteria (Enterobacter sp.) reduced the virulence of B. bassiana to L. migratoria. These findings illustrate the effect of bacterial communities on fungal infections in L. migratoria when seen from the perspective of ecology in a microenvironment. The active antifungal substances of such bacteria and their mechanisms of action need further study. Full article
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21 pages, 2792 KiB  
Article
Diversity and Biosynthetic Potential of Fungi Isolated from St. John’s Island, Singapore
by Madhaiyan Munusamy, Kenneth Tan, Choy Eng Nge, Martin Muthee Gakuubi, Sharon Crasta, Yoganathan Kanagasundaram and Siew Bee Ng
Int. J. Mol. Sci. 2023, 24(2), 1033; https://doi.org/10.3390/ijms24021033 - 05 Jan 2023
Viewed by 1641
Abstract
Adaptation to a wide variety of habitats allows fungi to develop unique abilities to produce diverse secondary metabolites with diverse bioactivities. In this study, 30 Ascomycetes fungi isolated from St. John’s Island, Singapore were investigated for their general biosynthetic potential and their ability [...] Read more.
Adaptation to a wide variety of habitats allows fungi to develop unique abilities to produce diverse secondary metabolites with diverse bioactivities. In this study, 30 Ascomycetes fungi isolated from St. John’s Island, Singapore were investigated for their general biosynthetic potential and their ability to produce antimicrobial secondary metabolites (SMs). All the 30 fungal isolates belong to the Phylum Ascomycota and are distributed into 6 orders and 18 genera with Order Hypocreales having the highest number of representative (37%). Screening for polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes using degenerate PCR led to the identification of 23 polyketide synthases (PKSs) and 5 nonribosomal peptide synthetases (NRPSs) grouped into nine distinct clades based on their reduction capabilities. Some of the identified PKSs genes share high similarities between species and known reference genes, suggesting the possibility of conserved biosynthesis of closely related compounds from different fungi. Fungal extracts were tested for their antimicrobial activity against S. aureus, Methicillin-resistant S. aureus (MRSA), and Candida albicans. Bioassay-guided fractionation of the active constituents from two promising isolates resulted in the isolation of seven compounds: Penilumamides A, D, and E from strain F4335 and xanthomegnin, viomellein, pretrichodermamide C and vioxanthin from strain F7180. Vioxanthin exhibited the best antibacterial activity with IC50 values of 3.0 μM and 1.6 μM against S. aureus and MRSA respectively. Viomellein revealed weak antiproliferative activity against A549 cells with an IC50 of 42 μM. The results from this study give valuable insights into the diversity and biosynthetic potential of fungi from this unique habitat and forms a background for an in-depth analysis of the biosynthetic capability of selected strains of interest with the aim of discovering novel fungal natural products. Full article
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2022

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15 pages, 2070 KiB  
Article
A Small RNA, UdsC, Interacts with the RpoHII mRNA and Affects the Motility and Stress Resistance of Rhodobacter sphaeroides
by Daniel-Timon Spanka, Julian Grützner, Andreas Jäger and Gabriele Klug
Int. J. Mol. Sci. 2022, 23(24), 15486; https://doi.org/10.3390/ijms232415486 - 07 Dec 2022
Cited by 2 | Viewed by 1165
Abstract
sRNAs have an important role in the regulation of bacterial gene expression. The sRNA, UdsC, of Rhodobacter sphaeroides is derived from the 3′ UTR of the RSP_7527 mRNA, which encodes a hypothetical protein. Here, we showed the effect of UdsC on the resistance [...] Read more.
sRNAs have an important role in the regulation of bacterial gene expression. The sRNA, UdsC, of Rhodobacter sphaeroides is derived from the 3′ UTR of the RSP_7527 mRNA, which encodes a hypothetical protein. Here, we showed the effect of UdsC on the resistance of Rhodobacter sphaeroides to hydrogen peroxide and on its motility. In vitro binding assays supported the direct interaction of UdsC with the 5′ UTR of the rpoHII mRNA. RpoHII is an alternative sigma factor with an important role in stress responses in R. sphaeroides, including its response to hydrogen peroxide. We also demonstrated that RpoHII controls the expression of the torF gene, which encodes an important regulator of motility genes. This strongly suggested that the observed effect of UdsC on TorF expression is indirect and mediated by RpoHII. Full article
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29 pages, 2039 KiB  
Review
Two-Component Systems of Streptomyces coelicolor: An Intricate Network to Be Unraveled
by Ricardo Sánchez de la Nieta, Ramón I. Santamaría and Margarita Díaz
Int. J. Mol. Sci. 2022, 23(23), 15085; https://doi.org/10.3390/ijms232315085 - 01 Dec 2022
Cited by 8 | Viewed by 2435
Abstract
Bacteria of the Streptomyces genus constitute an authentic biotech gold mine thanks to their ability to produce a myriad of compounds and enzymes of great interest at various clinical, agricultural, and industrial levels. Understanding the physiology of these organisms and revealing their regulatory [...] Read more.
Bacteria of the Streptomyces genus constitute an authentic biotech gold mine thanks to their ability to produce a myriad of compounds and enzymes of great interest at various clinical, agricultural, and industrial levels. Understanding the physiology of these organisms and revealing their regulatory mechanisms is essential for their manipulation and application. Two-component systems (TCSs) constitute the predominant signal transduction mechanism in prokaryotes, and can detect a multitude of external and internal stimuli and trigger the appropriate cellular responses for adapting to diverse environmental conditions. These global regulatory systems usually coordinate various biological processes for the maintenance of homeostasis and proper cell function. Here, we review the multiple TCSs described and characterized in Streptomyces coelicolor, one of the most studied and important model species within this bacterial group. TCSs are involved in all cellular processes; hence, unravelling the complex regulatory network they form is essential for their potential biotechnological application. Full article
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17 pages, 4978 KiB  
Article
Comparative Genomics and Physiological Characterization of Two Aerobic Spore Formers Isolated from Human Ileal Samples
by Anella Saggese, Rosa Giglio, Nicola D’Anzi, Loredana Baccigalupi and Ezio Ricca
Int. J. Mol. Sci. 2022, 23(23), 14946; https://doi.org/10.3390/ijms232314946 - 29 Nov 2022
Cited by 2 | Viewed by 1630
Abstract
Spore formers are ubiquitous microorganisms commonly isolated from most environments, including the gastro-intestinal tract (GIT) of insects and animals. Spores ingested as food and water contaminants safely transit the stomach and reach the intestine, where some of them germinate and temporarily colonize that [...] Read more.
Spore formers are ubiquitous microorganisms commonly isolated from most environments, including the gastro-intestinal tract (GIT) of insects and animals. Spores ingested as food and water contaminants safely transit the stomach and reach the intestine, where some of them germinate and temporarily colonize that niche. In the lower part of the GIT, they re-sporulate and leave the body as spores, therefore passing through their entire life cycle in the animal body. In the intestine, both un-germinated spores and germination-derived cells interact with intestinal and immune cells and have health-beneficial effects, which include the production of useful compounds, protection against pathogenic microorganisms, contribution to the development of an efficient immune system and modulation of the gut microbial composition. We report a genomic and physiological characterization of SF106 and SF174, two aerobic spore former strains previously isolated from ileal biopsies of healthy human volunteers. SF106 and SF174 belong respectively to the B. subtilis and Alkalihalobacillus clausii (formerly Bacillus clausii) species, are unable to produce toxins or other metabolites with cytotoxic activity against cultured human cells, efficiently bind mucin and human epithelial cells in vitro and produce molecules with antimicrobial and antibiofilm activities. Full article
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18 pages, 4107 KiB  
Article
Different Effects of Different Lactobacillus acidophilus Strains on DSS-Induced Colitis
by Zheng Huang, Lei Gong, Yan Jin, Catherine Stanton, Reynolds Paul Ross, Jianxin Zhao, Bo Yang and Wei Chen
Int. J. Mol. Sci. 2022, 23(23), 14841; https://doi.org/10.3390/ijms232314841 - 27 Nov 2022
Cited by 4 | Viewed by 2077
Abstract
Inflammatory bowel disease (IBD) is a worldwide chronic intestinal inflammatory immune-related disease. In this study, mice with dextran sulfate sodium (DSS)-induced colitis were used to evaluate the effect of Lactobacillus acidophilus on colitis. The results revealed that L. acidophilus CCFM137 and FAHWH11L56 show [...] Read more.
Inflammatory bowel disease (IBD) is a worldwide chronic intestinal inflammatory immune-related disease. In this study, mice with dextran sulfate sodium (DSS)-induced colitis were used to evaluate the effect of Lactobacillus acidophilus on colitis. The results revealed that L. acidophilus CCFM137 and FAHWH11L56 show potential for relieving colitis symptoms, while L. acidophilus FGSYC48L79 did not show a protective effect. Moreover, L. acidophilus NCFM and FAHWH11L56 showed similar effects on various indicators of DSS-induced colitis, increasing the IL-10 and IL-17 in the colon, and modifying the CCL2/CCR2 axis and CCL3/CCR1 axis. For L. acidophilus CCFM137, its effects on colitis were different from the above two strains. Moreover, L. acidophilus FGSYC48L79 had negative effects on colitis by increasing the abundance of harmful bacteria in the gut microbiota and may promote the signaling of chemokines and their receptors. This may be related to its special genome compared to the other strains. Full article
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13 pages, 3155 KiB  
Article
A Bacillus licheniformis Glycoside Hydrolase 43 Protein Is Recognized as a MAMP
by Zhixiang Yuan, Ying Zhao, Zhitong Mo and Hongxia Liu
Int. J. Mol. Sci. 2022, 23(22), 14435; https://doi.org/10.3390/ijms232214435 - 20 Nov 2022
Cited by 6 | Viewed by 1646
Abstract
Glycoside hydrolases from pathogens have often been reported as inducers of immune responses. However, the roles of glycoside hydrolase from plant-growth-promoting rhizobacteria (PGPR) in the resistance of plants against pathogens is not well studied. In this study, we identified a glycoside hydrolase 43 [...] Read more.
Glycoside hydrolases from pathogens have often been reported as inducers of immune responses. However, the roles of glycoside hydrolase from plant-growth-promoting rhizobacteria (PGPR) in the resistance of plants against pathogens is not well studied. In this study, we identified a glycoside hydrolase 43 protein, H1AD43, produced by Bacillus licheniformis BL06 that can trigger defense responses, including cell death. Ion-exchange and size-exclusion chromatography were used for separation, and the amino acid sequence was identified by mass spectrometry. The recombinant protein generated by prokaryotic expression was able to elicit a hypersensitive response (HR) in Nicotiana benthamiana and trigger early defense responses, including reactive oxygen species (ROS) burst, callose accumulation, and the induction of defense genes. In addition, the protein could induce resistance in N. benthamiana, in which it inhibited infection by Phytophthora capsici Leonian and tobacco mosaic virus-green fluorescent protein (TMV-GFP) expression. H1AD43 thus represents a microbe-associated molecular pattern (MAMP) of PGPR that induces plant disease resistance and may provide a new method for the biological control of plant disease. Full article
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15 pages, 4202 KiB  
Article
In Vitro Production of Galactooligosaccharides by a Novel β-Galactosidase of Lactobacillus bulgaricus
by Alexander Arsov, Ivan Ivanov, Lidia Tsigoriyna, Kaloyan Petrov and Penka Petrova
Int. J. Mol. Sci. 2022, 23(22), 14308; https://doi.org/10.3390/ijms232214308 - 18 Nov 2022
Cited by 1 | Viewed by 1840
Abstract
β-galactosidase is an enzyme with dual activity and important industrial application. As a hydrolase, the enzyme eliminates lactose in milk, while as a trans-galactosidase it produces prebiotic galactooligosaccharides (GOS) with various degrees of polymerization (DP). The aim of the present study is the [...] Read more.
β-galactosidase is an enzyme with dual activity and important industrial application. As a hydrolase, the enzyme eliminates lactose in milk, while as a trans-galactosidase it produces prebiotic galactooligosaccharides (GOS) with various degrees of polymerization (DP). The aim of the present study is the molecular characterization of β-galactosidase from a Bulgarian isolate, Lactobacillus delbrueckii subsp. bulgaricus 43. The sequencing of the β-gal gene showed that it encodes a new enzyme with 21 amino acid replacements compared to all other β-galactosidases of this species. The molecular model revealed that the new β-galactosidase acts as a tetramer. The amino acids D207, H386, N464, E465, Y510, E532, H535, W562, N593, and W980 form the catalytic center and interact with Mg2+ ions and substrate. The β-gal gene was cloned into a vector allowing heterologous expression of E. coli BL21(DE3) with high efficiency, as the crude enzyme reached 3015 U/mL of the culture or 2011 U/mg of protein. The enzyme’s temperature optimum at 55 °C, a pH optimum of 6.5, and a positive influence of Mg2+, Mn2+, and Ca2+ on its activity were observed. From lactose, β-Gal produced a large amount of GOS with DP3 containing β-(1→3) and β-(1→4) linkages, as the latter bond is particularly atypical for the L. bulgaricus enzymes. DP3-GOS formation was positively affected by high lactose concentrations. The process of lactose conversion was rapid, with a 34% yield of DP3-GOS in 6 h, and complete degradation of 200 g/L of lactose for 12 h. On the other hand, the enzyme was quite stable at 55 °C and retained about 20% of its activity after 24 h of incubation at this temperature. These properties expand our horizons as regards the use of β-galactosidases in industrial processes for the production of lactose-free milk and GOS-enriched foods. Full article
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18 pages, 2825 KiB  
Article
The Functional Characteristics of Goat Cheese Microbiota from a One-Health Perspective
by Bruno Tilocca, Alessio Soggiu, Federica Iavarone, Viviana Greco, Lorenza Putignani, Maria Vittoria Ristori, Gabriele Macari, Anna Antonella Spina, Valeria Maria Morittu, Carlotta Ceniti, Cristian Piras, Luigi Bonizzi, Domenico Britti, Andrea Urbani, Daniel Figeys and Paola Roncada
Int. J. Mol. Sci. 2022, 23(22), 14131; https://doi.org/10.3390/ijms232214131 - 16 Nov 2022
Cited by 5 | Viewed by 2229
Abstract
Goat cheese is an important element of the Mediterranean diet, appreciated for its health-promoting features and unique taste. A pivotal role in the development of these characteristics is attributed to the microbiota and its continuous remodeling over space and time. Nevertheless, no thorough [...] Read more.
Goat cheese is an important element of the Mediterranean diet, appreciated for its health-promoting features and unique taste. A pivotal role in the development of these characteristics is attributed to the microbiota and its continuous remodeling over space and time. Nevertheless, no thorough study of the cheese-associated microbiota using two metaomics approaches has previously been conducted. Here, we employed 16S rRNA gene sequencing and metaproteomics to explore the microbiota of a typical raw goat milk cheese at various ripening timepoints and depths of the cheese wheel. The 16S rRNA gene-sequencing and metaproteomics results described a stable microbiota ecology across the selected ripening timepoints, providing evidence for the microbiologically driven fermentation of goat milk products. The important features of the microbiota harbored on the surface and in the core of the cheese mass were highlighted in both compositional and functional terms. We observed the rind microbiota struggling to maintain the biosafety of the cheese through competition mechanisms and/or by preventing the colonization of the cheese by pathobionts of animal or environmental origin. The core microbiota was focused on other biochemical processes, supporting its role in the development of both the health benefits and the pleasant gustatory nuances of goat cheese. Full article
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10 pages, 7996 KiB  
Article
On the TPPP Protein of the Enigmatic Fungus, Olpidium—Correlation between the Incidence of p25alpha Domain and That of the Eukaryotic Flagellum
by Ferenc Orosz
Int. J. Mol. Sci. 2022, 23(22), 13927; https://doi.org/10.3390/ijms232213927 - 11 Nov 2022
Cited by 3 | Viewed by 1190
Abstract
Loss of the flagellum was an important step in the evolution of fungi. The flagellated fungi of the phylum Olpidiomycota are the closest relative of the non-flagellated terrestrial fungi. There are genes encoding proteins, the occurrence of which shows a strong correlation with [...] Read more.
Loss of the flagellum was an important step in the evolution of fungi. The flagellated fungi of the phylum Olpidiomycota are the closest relative of the non-flagellated terrestrial fungi. There are genes encoding proteins, the occurrence of which shows a strong correlation with the incidence of the flagellum. One of these gene/protein families is “TPPP-like proteins” whose main feature is the presence of the p25alpha domain. The functional link between TPPP and flagellum has also been shown. Most of the phyla of flagellated fungi have been known to contain TPPP-like proteins but Olpidiomycota was an exception. This study demonstrates that Olpidium bornovanus, similarly to some fungi of Chytridiomycota and Blastocladiomycota, has a “fungal-type” TPPP characterized by the presence of two (a complete and an incomplete) p25alpha domains. Full article
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14 pages, 1765 KiB  
Review
Gut Microbiota Host–Gene Interaction
by Paola Cuomo, Rosanna Capparelli, Marco Alifano, Antonio Iannelli and Domenico Iannelli
Int. J. Mol. Sci. 2022, 23(22), 13717; https://doi.org/10.3390/ijms232213717 - 08 Nov 2022
Cited by 4 | Viewed by 2359
Abstract
Studies carried out in the last ten years have shown that the metabolites made up from the gut microbiota are essential for multiple functions, such as the correct development of the immune system of newborns, interception of pathogens, and nutritional enrichment of the [...] Read more.
Studies carried out in the last ten years have shown that the metabolites made up from the gut microbiota are essential for multiple functions, such as the correct development of the immune system of newborns, interception of pathogens, and nutritional enrichment of the diet. Therefore, it is not surprising that alteration of the gut microbiota is the starting point of gastrointestinal infection, obesity, type 2 diabetes, inflammatory bowel disease, colorectal cancer, and lung cancer. Diet changes and antibiotics are the major factors damaging the gut microbiota. Early exposure of the newborns to antibiotics may prevent their correct development of the immune system, exposing them to pathogen infections, allergies, and chronic inflammatory diseases. We already know much on how host genes, microbiota, and the environment interact, owing to experiments in several model animals, especially in mice; advances in molecular technology; microbiota transplantation; and comparative metagenomic analysis. However, much more remains to be known. Longitudinal studies on patients undergoing to therapy, along with the identification of bacteria prevalent in responding patients may provide valuable data for improving therapies. Full article
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11 pages, 1815 KiB  
Article
The Effect of Proline on the Freeze-Drying Survival Rate of Bifidobacterium longum CCFM 1029 and Its Inherent Mechanism
by Shumao Cui, Wenrui Zhou, Xin Tang, Qiuxiang Zhang, Bo Yang, Jianxin Zhao, Bingyong Mao and Hao Zhang
Int. J. Mol. Sci. 2022, 23(21), 13500; https://doi.org/10.3390/ijms232113500 - 04 Nov 2022
Cited by 9 | Viewed by 1561
Abstract
Amino acids, which are important compatible solutes, play a significant role in probiotic lyophilization. However, studies on the functions of Bifidobacterium during freeze-drying are limited. Therefore, in this study, we compared the freeze-drying survival rate of Bifidobacterium longum CCFM 1029 cultivated in different [...] Read more.
Amino acids, which are important compatible solutes, play a significant role in probiotic lyophilization. However, studies on the functions of Bifidobacterium during freeze-drying are limited. Therefore, in this study, we compared the freeze-drying survival rate of Bifidobacterium longum CCFM 1029 cultivated in different media containing different kinds of compatible solutes. We found that the addition of 21 g/L proline to the culture media substantially improved the freeze-drying survival rate of B. longum CCFM 1029 from 18.61 ± 0.42% to 38.74 ± 1.58%. Interestingly, this change has only been observed when the osmotic pressure of the external culture environment is increased. Under these conditions, we found that proline accumulation in this strain increased significantly. This change also helped the strain to maintain its membrane integrity and the activity of some key enzymes during freeze-drying. Overall, these results show that the addition of proline can help the strain resist a tough environment during lyophilization. The findings of this study provide preliminary data for producers of probiotics who wish to achieve higher freeze-drying survival rates during industrial production. Full article
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12 pages, 2026 KiB  
Article
Whole Genome Analyses Accurately Identify Neisseria spp. and Limit Taxonomic Ambiguity
by May Khoder, Marwan Osman, Issmat I. Kassem, Rayane Rafei, Ahmad Shahin, Pierre Edouard Fournier, Jean-Marc Rolain and Monzer Hamze
Int. J. Mol. Sci. 2022, 23(21), 13456; https://doi.org/10.3390/ijms232113456 - 03 Nov 2022
Cited by 2 | Viewed by 1703
Abstract
Genome sequencing facilitates the study of bacterial taxonomy and allows the re-evaluation of the taxonomic relationships between species. Here, we aimed to analyze the draft genomes of four commensal Neisseria clinical isolates from the semen of infertile Lebanese men. To determine the phylogenetic [...] Read more.
Genome sequencing facilitates the study of bacterial taxonomy and allows the re-evaluation of the taxonomic relationships between species. Here, we aimed to analyze the draft genomes of four commensal Neisseria clinical isolates from the semen of infertile Lebanese men. To determine the phylogenetic relationships among these strains and other Neisseria spp. and to confirm their identity at the genomic level, we compared the genomes of these four isolates with the complete genome sequences of Neisseria gonorrhoeae and Neisseria meningitidis and the draft genomes of Neisseria flavescens, Neisseria perflava, Neisseria mucosa, and Neisseria macacae that are available in the NCBI Genbank database. Our findings revealed that the WGS analysis accurately identified and corroborated the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) species identities of the Neisseria isolates. The combination of three well-established genome-based taxonomic tools (in silico DNA-DNA Hybridization, Ortho Average Nucleotide identity, and pangenomic studies) proved to be relatively the best identification approach. Notably, we also discovered that some Neisseria strains that are deposited in databases contain many taxonomical errors. The latter is very important and must be addressed to prevent misdiagnosis and missing emerging etiologies. We also highlight the need for robust cut-offs to delineate the species using genomic tools. Full article
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18 pages, 1711 KiB  
Review
Helminths and Bacterial Microbiota: The Interactions of Two of Humans’ “Old Friends”
by Kevin Llinás-Caballero and Luis Caraballo
Int. J. Mol. Sci. 2022, 23(21), 13358; https://doi.org/10.3390/ijms232113358 - 01 Nov 2022
Cited by 8 | Viewed by 5187
Abstract
Humans have coexisted with helminths and bacteria for the entire existence of our species. Nowadays, helminth infections affect more than 1.9 billion people worldwide, especially in underdeveloped regions that lack optimal sanitary conditions. In addition, commensal microorganisms inhabit several compartments of humans, including [...] Read more.
Humans have coexisted with helminths and bacteria for the entire existence of our species. Nowadays, helminth infections affect more than 1.9 billion people worldwide, especially in underdeveloped regions that lack optimal sanitary conditions. In addition, commensal microorganisms inhabit several compartments of humans, including the gastrointestinal tract, constituting what we know as the microbiota. Helminths and bacterial microbiota can interact in various ways. In this review, the interactions between helminths and commensal bacteria are analyzed in both animal models and humans. In developing countries, the gut microbiota exhibits high diversity, which could be linked to the high burden of helminthiasis in these areas. In fact, several studies show that helminth infections are associated with an increased gut microbiota diversity and changes in its composition. Interestingly, these changes can modify the risk for some diseases, such as asthma, colitis, viral infections, and metabolic conditions. Besides, the microbiota is necessary for the establishment of some helminth infections and can also influence the evolution of these diseases. Specific bacterial taxa can contribute to the resistance or susceptibility to certain helminths. The mechanisms underlying helminth–microbiota interactions are not completely understood. More research is necessary to address this and other unmet needs, especially considering that available studies are heterogeneous and sometimes yield conflicting results. Full article
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14 pages, 2553 KiB  
Article
Complementary Regulation of BfmRS Two-Component and AbaIR Quorum Sensing Systems to Express Virulence-Associated Genes in Acinetobacter baumannii
by Hyo-Jeong Kim, Na-Yeong Kim, Seo-Yeon Ko, Seong-Yong Park, Man-Hwan Oh, Min-Sang Shin, Yoo-Chul Lee and Je-Chul Lee
Int. J. Mol. Sci. 2022, 23(21), 13136; https://doi.org/10.3390/ijms232113136 - 28 Oct 2022
Cited by 6 | Viewed by 2086
Abstract
Acinetobacter baumannii expresses various virulence factors to adapt to hostile environments and infect susceptible hosts. This study investigated the regulatory network of the BfmRS two-component and AbaIR quorum sensing (QS) systems in the expression of virulence-associated genes in A. baumannii ATCC 17978. The [...] Read more.
Acinetobacter baumannii expresses various virulence factors to adapt to hostile environments and infect susceptible hosts. This study investigated the regulatory network of the BfmRS two-component and AbaIR quorum sensing (QS) systems in the expression of virulence-associated genes in A. baumannii ATCC 17978. The ΔbfmS mutant exhibited a significant decrease in surface motility, which presumably resulted from the low expression of pilT and A1S_0112-A1S_0119 gene cluster. The ΔbfmR mutant displayed a significant reduction in biofilm and pellicle formation due to the low expression of csu operon. The deletion of abaR did not affect the expression of bfmR or bfmS. However, the expression of abaR and abaI was upregulated in the ΔbfmR mutant. The ΔbfmR mutant also produced more autoinducers than did the wild-type strain, suggesting that BfmR negatively regulates the AbaIR QS system. The ΔbfmS mutant exhibited no autoinducer production in the bioassay system. The expression of the A1S_0112-A1S_0119 gene cluster was downregulated in the ΔabaR mutant, whereas the expression of csu operon was upregulated in this mutant with a high cell density. In conclusion, for the first time, we demonstrated that the BfmRS-AbaIR QS system axis regulated the expression of virulence-associated genes in A. baumannii. This study provides new insights into the complex network system involved in the regulation of virulence-associated genes underlying the pathogenicity of A. baumannii. Full article
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19 pages, 3245 KiB  
Article
Role of EmaSR in Ethanol Metabolism by Acinetobacter baumannii
by Hung-Yu Shu, Yu-Wen Huang, Ping-Yi Tsai, Kun-Sheng Hsieh and Guang-Huey Lin
Int. J. Mol. Sci. 2022, 23(20), 12606; https://doi.org/10.3390/ijms232012606 - 20 Oct 2022
Cited by 2 | Viewed by 1528
Abstract
Acinetobacter baumannii is a well-known nosocomial pathogen that can survive in different environments through the use of intricate networks to regulate gene expression. Two-component systems (TCS) form an important part of such regulatory networks, and in this study, we describe the identification and [...] Read more.
Acinetobacter baumannii is a well-known nosocomial pathogen that can survive in different environments through the use of intricate networks to regulate gene expression. Two-component systems (TCS) form an important part of such regulatory networks, and in this study, we describe the identification and characterization of a novel EmaSR TCS in A. baumannii. We constructed a Tn5-tagged mutagenesis library, from which an emaS sensor kinase gene and emaR response regulator gene were identified. We found that emaS/emaR single-mutants and double-mutants were unable to replicate in M9 medium with 1% ethanol as the single carbon source. Motility and biofilm formation were negatively affected in double-mutants, and transcriptomic analysis showed that mutation of emaSR dysregulated genes required for carbon metabolism. In addition, emaS/emaR single-mutants and double-mutants were unable to survive in acetic acid- and sodium acetate-containing medium, indicating that the EmaSR TCS is also important for acetate metabolism. Furthermore, virulence against Galleria mellonella was diminished in emaS/emaR single- and double-mutants. Taken together, these results show that this novel EmaSR TCS is involved in the regulation of A. baumannii ethanol metabolism and acetate metabolism, with important implications on motility, biofilm formation, and virulence if mutated. Further research on the underlying mechanisms is warranted. Full article
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14 pages, 2936 KiB  
Article
Anion-Exchange Chromatography at the Service of Gene Therapy: Baseline Separation of Full/Empty Adeno-Associated Virus Capsids by Screening of Conditions and Step Gradient Elution Mode
by Megane K. Aebischer, Hugo Gizardin-Fredon, Honorine Lardeux, Dominik Kochardt, Carsten Elger, Markus Haindl, Raphael Ruppert, Davy Guillarme and Valentina D’Atri
Int. J. Mol. Sci. 2022, 23(20), 12332; https://doi.org/10.3390/ijms232012332 - 15 Oct 2022
Cited by 7 | Viewed by 3844
Abstract
Gene therapy is opening unprecedented opportunities for novel therapeutic approaches. Based on the concept of rescuing function mutations by co-expressing the correct gene to allow biological functions to be restored, it requires the use of viral vectors to ensure the proper delivery of [...] Read more.
Gene therapy is opening unprecedented opportunities for novel therapeutic approaches. Based on the concept of rescuing function mutations by co-expressing the correct gene to allow biological functions to be restored, it requires the use of viral vectors to ensure the proper delivery of therapeutic genes. In this context, recombinant adeno-associated viruses (rAAV) are the most widely used vectors. Their biomanufacturing process requires the insertion of the therapeutic gene into the rAAV (full capsids). However, a percentage of rAAV that do not contain the desired gene (empty capsids), as well as partly filled capsids, might also be produced, potentially impacting the efficiency of the therapy. Therefore, the determination of the rAAV capsids’ full/empty ratio needs to be monitored to ensure consistent product quality and efficacy. Anion-exchange chromatography (AEX) can serve this need. In this contribution, thorough AEX method development, including a mobile phase, a stationary phase and gradient conditions, has highlighted its potential in supporting gene therapy. Taking advantage of the fact that viral capsids follow an “on/off” retention behavior, the application of a step gradient approach to the rAAV serotype 8 (rAAV8) allowed the unprecedented separation of rAAV8 full/empty capsids, with a resolution gain of 3.7 as compared to the resolution obtained with a fully optimized linear gradient. Finally, the developed analytical approach allowed a precise and accurate baseline separation and quantification of full and empty rAAV8 capsids, with the potential to be applied as a high-throughput quality control (QC) method. Full article
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13 pages, 2265 KiB  
Article
ppGpp, the General Stress Response Alarmone, Is Required for the Expression of the α-Hemolysin Toxin in the Uropathogenic Escherichia coli Isolate, J96
by Jorge Fernández-Vázquez, Juan David Cabrer-Panes, Anna Åberg, Antonio Juárez, Cristina Madrid, Tania Gaviria-Cantin, Llorenç Fernández-Coll, Andrés Felipe Vargas-Sinisterra, Carlos Jonay Jiménez and Carlos Balsalobre
Int. J. Mol. Sci. 2022, 23(20), 12256; https://doi.org/10.3390/ijms232012256 - 14 Oct 2022
Cited by 2 | Viewed by 2100
Abstract
ppGpp is an intracellular sensor that, in response to different types of stress, coordinates the rearrangement of the gene expression pattern of bacteria to promote adaptation and survival to new environmental conditions. First described to modulate metabolic adaptive responses, ppGpp modulates the expression [...] Read more.
ppGpp is an intracellular sensor that, in response to different types of stress, coordinates the rearrangement of the gene expression pattern of bacteria to promote adaptation and survival to new environmental conditions. First described to modulate metabolic adaptive responses, ppGpp modulates the expression of genes belonging to very diverse functional categories. In Escherichia coli, ppGpp regulates the expression of cellular factors that are important during urinary tract infections. Here, we characterize the role of this alarmone in the regulation of the hlyCABDII operon of the UPEC isolate J96, encoding the toxin α-hemolysin that induces cytotoxicity during infection of bladder epithelial cells. ppGpp is required for the expression of the α-hemolysin encoded in hlyCABDII by stimulating its transcriptional expression. Prototrophy suppressor mutations in a ppGpp-deficient strain restore the α-hemolysin expression from this operon to wild-type levels, confirming the requirement of ppGpp for its expression. ppGpp stimulates hlyCABDII expression independently of RpoS, RfaH, Zur, and H-NS. The expression of hlyCABDII is promoted at 37 °C and at low osmolarity. ppGpp is required for the thermoregulation but not for the osmoregulation of the hlyCABDII operon. Studies in both commensal and UPEC isolates demonstrate that no UPEC specific factor is strictly required for the ppGpp-mediated regulation described. Our data further support the role of ppGpp participating in the coordinated regulation of the expression of bacterial factors required during infection. Full article
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19 pages, 1939 KiB  
Article
Characterizing Antimicrobial Resistance in Clinically Relevant Bacteria Isolated at the Human/Animal/Environment Interface Using Whole-Genome Sequencing in Austria
by Adriana Cabal, Gerhard Rab, Beatriz Daza-Prieto, Anna Stöger, Nadine Peischl, Ali Chakeri, Solveig Sølverød Mo, Harald Bock, Klemens Fuchs, Jasmin Sucher, Krista Rathammer, Petra Hasenberger, Silke Stadtbauer, Manuela Caniça, Peter Strauß, Franz Allerberger, Markus Wögerbauer and Werner Ruppitsch
Int. J. Mol. Sci. 2022, 23(19), 11276; https://doi.org/10.3390/ijms231911276 - 24 Sep 2022
Cited by 5 | Viewed by 2583
Abstract
Antimicrobial resistance (AMR) is a public health issue attributed to the misuse of antibiotics in human and veterinary medicine. Since AMR surveillance requires a One Health approach, we sampled nine interconnected compartments at a hydrological open-air lab (HOAL) in Austria to obtain six [...] Read more.
Antimicrobial resistance (AMR) is a public health issue attributed to the misuse of antibiotics in human and veterinary medicine. Since AMR surveillance requires a One Health approach, we sampled nine interconnected compartments at a hydrological open-air lab (HOAL) in Austria to obtain six bacterial species included in the WHO priority list of antibiotic-resistant bacteria (ARB). Whole genome sequencing-based typing included core genome multilocus sequence typing (cgMLST). Genetic and phenotypic characterization of AMR was performed for all isolates. Eighty-nine clinically-relevant bacteria were obtained from eight compartments including 49 E. coli, 27 E. faecalis, 7 K. pneumoniae and 6 E. faecium. Clusters of isolates from the same species obtained in different sample collection dates were detected. Of the isolates, 29.2% were resistant to at least one antimicrobial. E. coli and E. faecalis isolates from different compartments had acquired antimicrobial resistance genes (ARGs) associated with veterinary drugs such as aminoglycosides and tetracyclines, some of which were carried in conjugative and mobilizable plasmids. Three multidrug resistant (MDR) E. coli isolates were found in samples from field drainage and wastewater. Early detection of ARGs and ARB in natural and farm-related environments can identify hotspots of AMR and help prevent its emergence and dissemination along the food/feed chain. Full article
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