Search Results (323)

Bacterial–fungal interactions are prevalent in microbial communities, and fungi often facilitate bacterial dispersal along networks created by fungal hyphae. Using a microfluidic device, we examined how diverse bacterial species disperse in monoculture versus travel in coculture with Aspergillus flavus. Most of the bacteria traveled further when in coculture, although this was not absolute. Two bacteria showing significant dispersal rates only in coculture were the human pathogens Listeria monocytogenes and Staphylococcus aureus. Mechanistically, L. monocytogenes dispersal required flagella, with dispersal impaired in flagellar mutants but enhanced in ∆mogR strains that upregulate flagellar expression. In contrast, the non-flagellar bacterium S. aureus exhibited a unique, wave-like dispersal pattern along the hyphae, a phenomenon that was abolished in agr quorum-sensing mutants deficient in phenol-soluble modulins (PSMs). In a triculture of L. monocytogenes, S. aureus, and A. flavus, L. monocytogenes limited S. aureus dispersal along the fungal hyphae; however, this inhibition was dependent on an intact L. monocytogenes quorum system. Our findings reveal that bacterial motility on fungal networks arises from diverse, species-specific mechanisms, including flagella, transcriptional regulation, potential quorum-sensing-mediated interactions, as well as other notable dispersal phenomena that warrant further investigation. Full article

Particles moving in a fluid interact through the flow field they generate, which can lead to complex nonlinear dynamics. One important example is the synchronization of oscillatory motion in biological systems, such as the coordinated beating of cilia or flagella. In this work, we investigate the synchronization of two oscillators interacting through a Newtonian fluid using numerical simulations based on the lattice Boltzmann method. The oscillators are modeled as solid particles undergoing periodic motion, while hydrodynamic interactions are resolved explicitly through the surrounding flow. We analyze how synchronization depends on key physical parameters, including the fluid viscosity, the distance between the oscillators, the natural oscillation frequency, and the initial phase difference. The results are compared with predictions from the Kuramoto model in order to relate the hydrodynamic interaction to an effective phase coupling. We find that the coupling strength required for synchronization increases with both the oscillation frequency and the fluid viscosity, while it decreases with the distance between the oscillators. These results provide insight into the mechanisms underlying fluid-mediated synchronization and help bridge microscopic hydrodynamic models with reduced phase-oscillator descriptions. Full article

Background/Objectives: Spotty liver disease (SLD), caused by Campylobacter hepaticus, is an emerging disease that leads to substantial production losses in the egg industry. The shift toward antibiotic-free and cage-free production systems has further intensified the impact of SLD. The current control measures largely rely on autogenous killed vaccines; however, their use is constrained by the slow and fastidious growth of C. hepaticus and inconsistent efficacy. To overcome these limitations, this study aimed to identify immunogenic mimotopes as vaccine candidates and express them on the surface of an avian pathogenic Escherichia coli (APEC) vector. Methods: To identify immunogenic mimotopes, Ph.D.-12 phage display peptide library was screened using the hyperimmune serum raised against killed whole-cell C. hepaticus in specific pathogen-free chickens. Subsequently, the outer membrane protein C (OmpC) of E. coli was used as a scaffold for constructing a surface display library. A single restriction site, PstI, located in the seventh external loop of OmpC, was strategically utilized to insert each 12-amino-acid mimotope with a six-histidine (6xHis) tag sequence at its N-terminus, generating ompC + mimotope fusion constructs. These constructs were cloned into the inducible expression vector pTrc and electroporated into an E. coli DH5α ∆ompC strain, which lacked ompC. The surface expression of the mimotopes was confirmed in vitro. The verified ompC + mimotope constructs were subsequently subcloned into the pYA3422 constitutive expression vector and electroporated into the APEC PSUO78 ∆aroA ∆asd vaccine vector strain. A chicken vaccination–challenge trial was conducted using nine groups of chickens, including an unvaccinated challenged control and an unvaccinated–unchallenged negative control. Each experimental group received a mixture of two recombinant E. coli strains carrying different mimotopes at a dose of 1 × 10⁹ CFU, which were administered orally twice at 16 and 18 weeks of age. Results: Fourteen immunogenic mimotopes corresponding to 13 different C. hepaticus proteins were identified as potential vaccine candidates. The expression of these mimotopes on the surface of the E. coli was successfully demonstrated using the OmpC-mediated surface display system. Of the 14 mimotopes tested, two flagellar-related peptides and one major outer membrane protein (MOMP)-derived peptide elicited significant immune responses and conferred protection against the C. hepaticus challenge. Conclusions: We successfully developed a functional E. coli surface display system that was capable of expressing 12-amino-acid mimotopes of C. hepaticus, providing a robust platform for evaluating vaccine candidates against SLD. Immunogenicity and efficacy studies in chickens demonstrated that three identified mimotopes conferred protection against C. hepaticus colonization of the bile and liver. Future in vivo investigations are necessary to develop and evaluate the immunogenicity and protective efficacy of a multivalent mimotope vaccine consisting of three identified mimotopes against both C. hepaticus and APEC, utilizing the ΔaroA Δasd APEC PSU078 strain as the vaccine vector. Full article

Acidithiobacillus caldus is perpetually exposed to multiple extreme environmental stresses. CsrA, functioning as a post-transcriptional regulator of physiological metabolism, acts as a differential modulator, facilitating more economical and efficient adaptation to extreme environments. The csrA expression recombinant strain was constructed in A. caldus MTH-04 by conjugative transfer technology pJD215. Physiological characterization revealed enhanced acid tolerance, significantly elongated flagella, elevated extracellular secretion, and altered biofilm composition. Notably, intracellular concentrations of free glutamate and aspartate increased to 24.18 mg/L and 16.07 mg/L, respectively. The secondary structure of CsrA protein was determined in vitro through circular dichroism spectroscopy and size-exclusion chromatography. Electrophoretic Mobility Shift Assay (EMSA) successfully demonstrated in vitro binding activity of CsrA to the rpoS leader mRNA. CsrA suppresses rpoS mRNA translation by competing with ribosomes for binding sites, thereby negatively regulating rpoS expression. Critical binding sites were further validated through site-directed mutagenesis. Through EMSA, RT-qPCR and the translation reporter system, it was also found that CsrA has a dual regulatory function for nearby flagella- and motility-related gene clusters (flgC, 07035, motD, 15040), which also implies the global regulatory role of CsrA. In summary, a potential overall post-transcriptional regulatory mechanism based on CsrA and rpoS by extremophile A. caldus was proposed. Finally, the efficiency of bioleaching application by csrA overexpression strain was improved by 20.81%. Full article

Background and Objectives: Pseudomonas aeruginosa is a versatile, opportunistic pathogen responsible for a wide spectrum of infections, particularly in immunocompromised patients and those with disrupted epithelial barriers and chronic respiratory conditions. Its clinical significance is amplified by intrinsic and acquired antibiotic resistance, contributing to high mortality rates and treatment challenges. The bacterium’s pathogenic success stems from a multifaceted repertoire of virulence factors, including adhesins, pili, fimbriae, flagella, exopolysaccharides, biofilm-associated proteins, secreted toxins, proteases, lipases, phospholipases, rhamnolipids and redox-active metabolites. These factors are tightly regulated through complex networks, such as quorum sensing and c-di-GMP signaling, enabling dynamic adaptation to host environments and modulation of acute and chronic infection phenotypes. Biofilm formation and nutrient acquisition strategies further support survival in resource-limited conditions and protect against immune clearance and antibiotic pressure. Antibiotic resistance in P. aeruginosa limits therapeutic options. In addition, it may indirectly enhance virulence through modulation of stress responses and quorum sensing. P. aeruginosa’s pathogenicity emerges from the synergy between traditional virulence determinants and adaptive survival strategies, supporting long-term persistence, chronic infection, and resistance to host immunity and therapy. Materials and Methods: This narrative review is based on a comprehensive analysis of recent peer-reviewed literature focusing on virulence regulation, biofilm formation, nutrient acquisition strategies, and the interplay between antibiotic resistance and pathogenicity. Results: The reviewed evidence indicates that virulence expression in P. aeruginosa is highly dynamic and context-dependent, with regulatory networks integrating environmental signals to fine-tune pathogenic responses. A consistent finding across studies is the central role of biofilm-associated adaption in promoting persistence and antimicrobial tolerance. Moreover, the interaction between resistance mechanisms and global regulatory pathways appears to enhance bacterial fitness and long-term survival within the host. Conclusions: A deeper understanding of these interconnected mechanisms may facilitate the development of more effective anti-virulence and therapeutic strategies. Full article

The escalating global demand for large-scale, cost-effective, and sustainable high-quality protein has positioned single-cell protein (SCP) production from one-carbon (C1) gases as a highly promising solution. In this study, eight chemolithoautotrophic hydrogen-oxidizing bacteria (HOB) were isolated from mangrove sediments. Based on the 16S rRNA gene sequence analysis, they belonged to genera Sulfurimonas, Sulfurovum, Thiomicrolovo, and Marinobacterium. Among these, Thiomicrolovo sp. ZZH C-3 was identified as the most promising candidate for SCP production based on the highest biomass and protein content, and was selected for further characterization. Strain ZZH C-3 is a Gram-negative, short rod-shaped bacterium with multiple flagella. It can grow chemolithoautotrophically by using molecular hydrogen as an energy source and molecular oxygen as an electron acceptor. Genomic analysis further confirmed that ZZH C-3 harbors a complete reverse tricarboxylic acid (rTCA) cycle gene set for carbon fixation, and diverse hydrogenases (Group I, II, IV) for hydrogen oxidation. Subsequently, its cultivation conditions and medium composition for SCP production were systematically optimized using single-factor experiments and response surface methodology (RSM). Results showed that the optimal growth conditions were 28 °C, pH 7.0, and with 1 g/L (NH₄)₂SO₄ as the nitrogen source, 5–10% oxygen concentration, 9.70 mg/L FeSO₄·7H₂O, 0.17 g/L CaCl₂·2H₂O, and 1.90 mg/L MnSO₄·H₂O. Under the optimized conditions, strain ZZH C-3 achieved a maximum specific growth rate of 0.46 h⁻¹. After 28 h of cultivation, the optical density at 600 nm (OD₆₀₀) reached 0.94, corresponding to a biomass concentration of 0.60 g/L, and the protein content ranked at 73.56%. The biomass yield on hydrogen (Y_H2) was approximately 3.01 g/g H₂, with an average H₂-to-CO₂ consumption molar ratio of about 3.78. Compared to the model HOB Cupriavidus necator, strain ZZH C-3 exhibited a lower H₂/CO₂ consumption ratio, superior substrate conversion efficiency, and high protein content. Overall, this study not only validated the potential of mangrove HOB for SCP production but also offers new insights for future metabolic engineering strategies designed to enhance CO₂-to-biomass conversion efficiency. Full article

Segmented filamentous bacteria (SFB) are host-specific, immune-modulating microorganisms that colonize the small intestine of various vertebrate species, playing a crucial role in stimulating immune maturation during early life. Previous research on the genomes of SFB from humans, rats, and mice has revealed significant differences among SFB strains associated with various hosts, suggesting that their evolution is closely linked to their relationships with specific hosts. However, the genome of SFB from chickens has not been extensively investigated. In this study, we present the metagenomic reconstruction of an SFB genome derived from the ileum of layer Lohmann Select Leghorn (LSL) chickens. We utilized Hi-C sequencing techniques to assemble the LSL-SFB and annotate the avian SFB from both turkeys and chickens. Our reference-guided consensus assembly, followed by Hi-C scaffolding, produced a high-quality genome for LSL-SFB. Our pangenomic analysis revealed substantial conservation of core gene clusters among mammalian SFB strains, but we also identified a distinct repertoire of genes in chicken and turkey SFB. Furthermore, metabolic network analysis indicated a reduced capacity for biosynthesis, signifying an increased reliance on the host, as shown by the absence of key biosynthetic and utilization pathways. We also discovered a unique flagellin subunit (fliC-2) in chicken SFB from different genetic lines and confirmed its interaction with the chicken flagellin receptor, Toll-like receptor five. This study provides the first high-quality genome and annotation of LSL-SFB, alongside that of turkeys, offering valuable insights into the mechanisms of host specificity and adaptation. Understanding the interactions between host-specific SFB and their hosts, as well as their role in promoting immune maturation, is essential for improving intestinal health. Full article

Background/Objectives: Dynein axonemal heavy chain (DNAH) genes, including DNAH6, are implicated in male infertility, particularly multiple morphological abnormalities of the spermatozoa flagellum (MMAF). However, an underlying mechanism is unclear. Methods: This in silico study analyzed 19 previously reported DNAH6 mutations to elucidate their effects on the structural, mechanical, and microstructural aspects and axonemal assembly of flagellum and how these changes impact reproductive health, correlating with pathogenicity scores, ATP binding capacity, and protein interactions. Results: DNAH mutations were associated with CDGP (52.63%), male infertility (36.84%), and primary ovarian insufficiency (10.53%). MMAF-linked mutations exhibited higher SNAP2 scores (57.25 ± 5.68 vs. −32.58 ± 44.85, p = 0.002), reduced ATP binding affinity (−6.27 ± 4.20 vs. −8.92 ± 0.23 kcal/mol, p = 0.05), and smaller catalytic cavity size (17,646 ± 13,005 vs. 27190 ± 3485 ų, p = 0.04). These mutations showed reduced DNAH6-CLIP4 binding affinity (−303.90 ± 5.23 vs. −313.60 ± 4.28 kcal/mol, p = 0.002). Literature-based semen analysis revealed correlations between Phred scores and absent flagella (r = 0.952, p = 0.012) and inverse correlations between ATP binding capacity and absent flagella (r = −0.902, p = 0.036) or irregular width (r = −0.949, p = 0.014). A mathematical model of ATP binding kinetics predicted reduced flagellar motility in MMAF mutants due to impaired dynein function. Ultrastructural analyses indicated that high pathogenicity scores and reduced ATP binding correlate with absent inner dynein arms and radial spokes, while impaired DNAH6-CLIP4 interactions disrupt axonemal assembly. Conclusions: In silico analyses, integrated with microstructural, axonemal, and mathematical modeling data, demonstrate that DNAH6 mutations cause MMAF by impairing ATP binding, protein interactions, and axonemal assembly, leading to severe flagellar dysfunction and thereby negatively affecting reproductive health. Full article

The phage-resistant mutant (PRM) strains of Escherichia coli (E. coli) exhibited abundant genetic and phenotypic diversity. IS elements played a vital role in creating various genetic divergences and regulating gene functions under phage infection stress. Genetic variations of PRM strains derived from E. coli MG1655 and mutation frequencies of coevolved E. coli populations with phages were explored by high-throughput sequencing and resequencing. Infrequent-restriction-site PCR (IRS-PCR) and carbon utilization test revealed the genetic and phenotypic diversity of the PRM strains. Numerous and discrepant mutation sites (MSs) were observed in the PRM strains and the coevolved populations, and many MSs were related to the synthesis of flagella and LPS, which often serve as receptors in a phage invasion. The insertions of various IS elements in key gene locations were also frequently found in the PRM strains, which indicate for the first time that IS elements played a vital role in generating genetic divergence and regulating gene functions under phage infection stress. Resequencing revealed that the coevolved populations at three evolving stages had discrepant profiles of MSs, and nearly all detected MSs occurred in the coevolved populations, which led to coexisting phages that increased the mutation rates and expedited the occurrence of the defective MSs in E. coli populations. In summary, our results reveal that the widespread and abundant presence of phages may provide one important force driving bacterial genomic evolution and prompt bacterial genetic divergence via accelerated mutation and increased mutation rates in the E. coli genome. Full article

Spermiogenesis requires extensive molecular and structural remodeling to produce motile sperm. Mutations in the testis-specific RNA methyltransferase NSUN7 are associated with defective fibrous sheath, impaired sperm motility, and male infertility. However, the underlying molecular mechanisms remain poorly understood. Here, we performed proteomic profiling of sorted, elongated, and round spermatids, as well as mature spermatozoa from Nsun7 knockout mice. We showed that NSUN7 is present at all stages of spermiogenesis and is most abundant in round spermatids, which corresponds to the formation of the flagellum and fibrous sheath assembly. Loss of NSUN7 altered the abundance of proteins essential for dynein arm assembly (PIH1D3, CCDC103, CCDC40), intraflagellar transport (IFT122), and fibrous sheath organization (AKAP3, AKAP4, ROPN1L). We also showed that the previously detected impaired retention of cytoplasm in elongated spermatids may be caused by plectin accumulation. Interestingly, no statistically significant changes were found in mature sperm proteomes upon Nsun7 inactivation. Our findings support a model in which NSUN7 primarily stabilizes protein complexes and coordinates flagellar assembly. This indicates that NSUN7 is a critical regulator of spermiogenesis, and its malfunction is a contributing factor to male infertility. Full article

Background/Objectives: Chagas disease, caused by Trypanosoma cruzi, remains a major neglected tropical disease with limited therapeutic options restricted to benznidazole and nifurtimox, both associated with significant toxicity and reduced efficacy during chronic infection. Seeking novel, safe, and sustainable chemotherapeutic candidates, two new saturated cardanol-derived phospholipid analogs—LDT10 and LDT119—were rationally designed based on the molecular scaffold of miltefosine and biosourced from cashew nut shell liquid (CNSL). This study aimed to evaluate the pharmacokinetic properties of these compounds in silico and assess their antiparasitic activity, cytotoxicity, and morphological and ultrastructural effects on all developmental forms of T. cruzi in vitro. Materials and Methods: In silico ADMET predictions (SwissADME, pkCSM) were performed to determine bioavailability, pharmacokinetic behavior, CYP inhibition, mutagenicity, and hepatotoxicity. Antiproliferative activity was evaluated in epimastigotes, trypomastigotes, and intracellular amastigotes using dose–response assays and flow cytometry. Cytotoxicity was assessed in HEPG2 and HFF-1 cells using resazurin-based viability assays. Morphological and ultrastructural alterations were investigated through scanning (SEM) and transmission (TEM) electron microscopy. Reactive oxygen species (ROS) generation was quantified with H₂DCFDA after 4 h and 24 h of exposure. Results: In silico analyses indicated favorable drug-like profiles, high intestinal absorption (>89%), absence of mutagenicity or hepatotoxicity, and non-penetration of the blood–brain barrier. LDT10 was not a P-gp substrate, and LDT119 acted as a P-gp inhibitor, suggesting reduced efflux and higher intracellular retention. Both compounds inhibited epimastigote proliferation with low IC₅₀ values (LDT10: 0.81 µM; LDT119: 1.2 µM at 48 h) and reduced trypomastigote viability (LD₅₀ LDT10: 2.1 ± 2 µM; LDT119: 1.8 ± 0.8 µM). Intracellular amastigotes were highly susceptible (IC₅₀ LDT10: 0.48 µM; LDT119: 0.3 µM at 72 h), with >90% inhibition at higher concentrations. No cytotoxicity was observed in mammalian cells up to 20 µM. SEM revealed membrane wrinkling, pore-like depressions, rounded cell bodies, and multiple flagella, indicating cell division defects. TEM showed Golgi disorganization, autophagic vacuoles, mitochondrial vesiculation, and abnormal kinetoplast replication, while host cells remained structurally preserved. Both compounds induced significant ROS production in trypomastigotes after 24 h in a dose-dependent manner. Conclusions: LDT10 and LDT119 exhibited potent and selective in vitro activity against all developmental stages of T. cruzi, with low micromolar to submicromolar IC₅₀/LD₅₀ values, minimal mammalian cytotoxicity, and extensive morphological and ultrastructural damage consistent with disruption of phospholipid biosynthesis pathways. Combined with favorable in silico pharmacokinetic predictions, these CNSL-derived phospholipid analogs represent promising candidates for future Chagas disease chemotherapy and warrant further in vivo evaluation. Full article

Post-translational modifications (PTMs) are critically important for protein structure and function, with glycosylation being one of the most common forms of PTM. The gastric pathogen Helicobacter pylori has a general glycosylation system, which performs complex glycosylation of lipopolysaccharide, flagella proteins, and outer membrane proteins (OMPs). One of the best-described OMPs of H. pylori is the blood group binding adhesin (BabA), which interacts with the Lewis histo-blood group antigen, Lewis b. The 3D structure for BabA has been determined, and the ligand specifically described. Although BabA is reported to be a glycoprotein, there are limited data examining the effects of glycosylation on the structure and function of this protein. This study examined the folding and thermostability of non-glycosylated recombinant BabA and used computational approaches to predict the effect of glycosylation on the protein, with a focus on its possible heterologous expression in mammalian cells. Three potential O-linked and three potential N-linked glycosylation sites were predicted. Furthermore, the effect of glycan shielding on the solvent-accessible surface area of BabA was examined. Molecular dynamics simulations highlighted local indicators, including root mean square fluctuation and the number of protein-glycan contacts that were affected by glycosylation. Taken together, the findings support a role of glycans in surface shielding and promoting local stabilization in specific areas of the BabA protein. This study helps to strengthen the understanding of the importance of glycosylation and the role it plays in the structure, function, and stability of H. pylori proteins. Full article

The bacterial flagellum plays a crucial role in pathogenesis. However, the mechanism by which the flagellum interferes with host energy metabolism remains unclear. In this study, we confirmed that deletion of the fliC gene resulted in a 30% reduction in the virulence of Pseudomonas plecoglossicida against the large yellow croaker (Larimichthys crocea). Compared to the wild-type strain (WT) infection group, the ΔfliC infection group exhibited a 29.54% decrease in the number of vacuolar degeneration hepatocytes and a 50.83% higher liver glycogen content. Furthermore, infection led to decreased mitochondrial complex V activity, a reduced NAD+/NADH ratio, lower levels of reduced glutathione (GSH), and increased lipid peroxide levels; however, these metabolic perturbations were significantly ameliorated in the ΔfliC group compared to the WT group. Proteomic analysis revealed that the dysregulation of the complement cascade and core carbon metabolic pathways observed in the WT infection group liver was significantly alleviated in the ΔfliC infection group. Additionally, in the ΔfliC infection group, pro-inflammatory genes (such as Tlr5, Tnfα, and Il1β) were downregulated, while lipid metabolism-related genes (such as Acox1, Cpt1a, and Pparα) were upregulated, suggesting the suppression of the Tlr5/NF-κB immune signaling axis and enhanced fatty acid β-oxidation. Collectively, fliC may mediate the disruption of host glucose and lipid metabolism homeostasis through a Tlr5-triggered immunometabolic regulatory axis. In conclusion, this study demonstrates that bacterial flagella modulate host energy metabolism, expanding our understanding of flagellum-mediated pathogenesis. Full article

Background: Uropathogenic Escherichia coli (UPEC) is the number one cause of urinary tract infections (UTIs) in humans. The ability to bind to uroepithelial cells through type 1 pili and ascend the urinary tract via flagella is important in the early stages of a UTI. However, both type 1 pili and flagella can also target the bacteria for elimination via monocytes/macrophages later in a UTI. We hypothesized that the loss of both type 1 pili and flagella on the UPEC cells would make them less likely to be phagocytized by phagocytic cells. Methods: In this study, ΔfimA, ΔfliC, and ΔfimA ΔfliC mutants were compared to the wild type UPEC strain NU149 in phagocytosis assays using human and murine monocytic cell lines. Results: A ΔfimA ΔfliC double mutant was phagocytized significantly less than the wild type strain. Conclusion: The data show that the loss of both type 1 pili and flagella expression on the UPEC cells reduces phagocytosis of the bacteria by human and murine monocytes. Although type 1 pili and flagella are important for establishing a UTI and ascension into the kidneys, the loss of these proteinaceous structures may allow the UPEC cells to evade the innate immune defenses in certain environments within the human body. Full article

Helicobacter pylori uses a cluster of polar flagella for motility. H. pylori FapH forms a ring-like flagellar motor accessory associated with the outer membrane. A H. pylori ΔfapH mutant displays a motility-dependent sensitivity to bacitracin, an antibiotic that is normally excluded by the outer membrane, which suggests that FapH helps to maintain the integrity of the outer membrane during flagellar rotation. We report here that deletion of the ferric uptake regulator (fur) gene suppressed the bacitracin sensitivity of the H. pylori ΔfapH mutant. Depleting intracellular iron in the H. pylori ΔfapH mutant with the iron chelator 2,2′-dipyridyl similarly suppressed the bacitracin sensitivity of the strain. We postulate the altered expression of Fur-regulated genes as a result of deleting fur or that iron deprivation suppressed the bacitracin sensitivity of the ΔfapH mutant. We also isolated two bacitracin-resistant ΔfapH strains that had a nonsense mutation in lpxF, which encodes a lipid A 4′-phosphatase. Loss of LpxF alters the structure of the lipid A backbone in lipopolysaccharide that stabilizes the outer membrane, which we hypothesize compensated for the loss of FapH by minimizing damage to the membrane resulting from flagellar rotation. Full article