Search Results (37)

Short linear motifs (SLiMs), such as PDZ-binding motifs (PDZbms), are compact interaction modules that mediate transient, specific protein–protein interactions. While PDZbms are well characterized in viral pathogenesis, subverting host protein functions, their role in bacterial systems requires further study. Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that mainly infects macrophages. The type VII secretion system (T7SS) of Mtb secretes a subset of effector proteins (Esx) involved in virulence. By using molecular docking and support vector machine-based prediction, we analyzed PDZbm occurrence in T7SS Esx effector proteins and their ability to bind human PDZ domain-containing proteins. We identified PDZbms in most of the Esx proteins studied, with EsxA and EsxG showing the best PDZ-dependent interaction with syntenin-1, a host scaffold protein involved in vesicular trafficking and immune signaling. Additional Esx proteins were predicted to engage other host PDZ proteins. Proteome-wide analysis of Mtb H37Rv revealed that 23.1% of expressed proteins with ≥50 amino acids contained a C-terminal PDZbm. Gene Ontology and Reactome pathway enrichment revealed their involvement in processes related to bacterial and bacterial–host interactions, including redox balance, immunomodulation, and membrane localization, at various stages of infection. Our results support the existence of a PDZbm-mediated interface between Mtb and the human host, extending the PDZbm mimicry hypothesis beyond viruses to bacterial systems as an immune evasion strategy. This work may open multiple research lines focused on experimental validation and the development of a comparative PDZbm catalogue to uncover conserved virulence mechanisms that may guide the design of host-directed therapeutics. Full article

Intravesical Bacillus Calmette–Guérin (iBCG) immunotherapy is the standard adjuvant treatment of non-muscle-invasive bladder cancer (NMIBC). Among the potential complications, cases of mycotic aneurysms and acute respiratory distress syndrome (ARDS) are rare but can be life-threatening. Because prior reports have not included whole-genome sequencing (WGS) of clinical Mycobacterium bovis BCG (M. bovis BCG) isolates to assess whether the infecting strain acquires mutations in vivo, we performed WGS in a severe disseminated iBCG-related infection. A 72-year-old man with bladder cancer underwent iBCG instillation. Twelve months after the final instillation, the patient developed an abdominal aortic aneurysm, which was detected and treated with endovascular aneurysm repair (EVAR). Two months later, the patient presented with fever, abdominal pain, and septic shock. Contrast-enhanced computed tomography (CT) and ¹⁸F-fluorodeoxyglucose positron emission tomography/CT (FDG-PET/CT) showed rapid aneurysm enlargement. Ziehl–Neelsen staining and PCR of aortic material identified M. bovis BCG. Direct PCR on BAL fluid and urine was negative; however, BAL and urine culture subsequently grew M. bovis BCG, and PCR performed on the culture isolate confirmed M. bovis BCG. Despite combined antituberculosis triplet therapy (isoniazid, rifampicin, and ethambutol), the patient developed ARDS, which gradually improved after surgical management. WGS (with >96% genome coverage) showed the isolate was highly concordant with the vaccine strain and lacked additional virulence-associated mutations, including in esxM. This case illustrates that severe systemic iBCG-related complications can occur without detectable in vivo acquisition of virulence-enhancing mutations; however, interpretation is limited by the single-case design and the absence of host genetic susceptibility testing. Our findings underscore the need for prolonged vigilance regarding late-onset vascular and pulmonary complications after iBCG, and highlight the importance of early multidisciplinary management. Full article

Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant clinical challenge due to its multidrug resistance, particularly to β-lactam antibiotics. This study comprehensively evaluated the natural compound emodin for its anti-MRSA activity, mechanisms of action, and potential for synergy with β-lactam antibiotics. Our findings demonstrate that emodin dose-dependently inhibits MRSA growth and abrogates biofilm formation at 2× MIC. Mechanistic studies revealed that emodin compromises cell membrane and wall integrity, induces oxidative stress, and downregulates the virulence factors SPA and EsxA. Furthermore, emodin acted synergistically with β-lactam antibiotics: it enhanced the ability of cefalexin to block bacterial adhesion and invasion of HaCat cells, and potentiated the efficacy of amoxicillin in clearing MRSA from infected macrophages. In conclusion, emodin employs a multi-target mechanism against MRSA and can resensitize the bacterium to conventional β-lactam antibiotics, presenting a promising strategy for combination therapy that may help curb antibiotic use and resistance development. Full article

The early secreted antigenic target of 6 kDa (ESAT-6), a main effector molecule of the ESX-1 secretion system, is identified as a virulence determinant and immunoregulatory protein of Mycobacterium tuberculosis (Mtb), affecting the interaction between host immune cells and pathogens. ESAT-6 facilitates the survival of mycobacteria and their cell-to-cell spreading through membrane-permeabilizing activity and the regulation of host immune cell functions. In this review, we first summarize the recent knowledge of the roles of ESAT-6 in the survival of bacteria, phagosomal escape, and pathogenicity during Mtb infection. Then, we focused on its complex immunomodulatory effects on different immune cells, such as macrophages, dendritic cells, neutrophils, and T cells, accentuating its capability to either facilitate or inhibit immune responses through different signaling pathways. While our review has summarized its main roles in immunopathology in the context of tuberculosis, we additionally search for emerging evidence indicating that ESAT-6 has anti-inflammatory and immunosuppressive properties. Particularly, we discuss recent preclinical studies showing its capability to suppress transplant rejection and alloimmunity, probably via the induction of regulatory T cells. Nevertheless, the potential clinical use of ESAT-6 remains uncertain and needs further verification by comprehensive preclinical and clinical studies. Thus, we propose that ESAT-6 may be exploited to ameliorate immunopathology in TB infection and to suppress immune-mediated inflammation or transplant rejection as well. Full article

Bacillus paranthracis, a species of the genus Bacillus, is a Gram-positive bacterium classified as an opportunistic pathogen that can cause foodborne diarrhea and other intestinal diseases in humans and various animals. To date, there has been limited research on B. paranthracis, and there are few records of this bacterium being isolated from animal intestines. In this study, a strain named Qf-1 was isolated and purified from faecal samples of mink. Through culturomics, 16S rRNA gene sequencing, whole-genome sequencing, and average nucleotide identity (ANI) analysis, the strain was confirmed to be B. paranthracis. Whole-genome sequencing revealed that the strain has a genome size of 5.27 Mb, comprising one chromosome (5,224,739 bp) and one plasmid (51,964 bp). Functional annotation of its genome identified multiple potential pathogenic factors associated with pneumonia, including the key genes AsbD and AsbF, which facilitate bacterial colonisation of the lungs and trigger inflammatory responses, as well as EsxB and EsxL, which exacerbate lung inflammation and promote infection spread. Comparative genomics analysis revealed that this strain shares a close evolutionary relationship with previously reported B. paranthracis strains. The structure and function of the bacterial genes were analyzed in depth using multi-omics methods. Through mouse pathogenicity experiments, it was found that this bacterium may cause pneumonia and enteritis in mice. We predict that it may also pose a threat to the health of the mink. These research findings contribute to the establishment of a stable experimental model between pathogens and mink hosts, laying the foundation for further elucidating their pathogenicity and pathogenic mechanisms. This is of great significance for the diagnosis and prevention of bacterial diseases in mink in the future. Full article

Mycobacterium tuberculosis (Mtb) is one of the most successful bacterial pathogens in human history. Even in the antibiotic era, Mtb is widespread and causes millions of new cases of tuberculosis each year. The ability to disrupt the host’s innate and adaptive immunity, as well as natural persistence, complicates disease control. Tuberculosis traditional therapy involves the long-term use of several antibiotics. Treatment failures are often associated with the development of resistance to one or more drugs. The development of medicines that act on new targets will expand treatment options for tuberculosis caused by multidrug-resistant or extensively drug-resistant Mtb. Therefore, the development of drugs that target virulence factors is an attractive strategy. Such medicines do not have a direct bacteriostatic or bactericidal effect, but can disarm the pathogen so that the host immune system becomes able to eliminate it. Although cell wall-associated targets are being actively studied for anti-TB drug development, other virulence factors important for adaptation and host interaction are also worth comprehensive analysis. In this review, specific Mtb virulence factors (such as secreted phosphatases, regulatory systems, and the ESX-1 secretion system) are identified as promising targets for novel anti-virulence drug development. Additionally, models for the search of virulence inhibitors are discussed, such as virtual screening in silico, in vitro enzyme inhibition assay, the use of recombinant Mtb strains with reporter constructs, phenotypic analysis using in vitro cell infection models and specific environments. Full article

The ESX-1 secretion system is critical for the virulence of Mycobacterium tuberculosis as well as for conjugation in the saprophytic model Mycolicibacterium smegmatis. EsxB (CFP-10) and EsxA (ESAT-6) are secreted effectors required for the function of ESX-1 systems. While some transcription factors regulating the expression of esxB and esxA have been identified, little work has addressed their promoter structures or other determinants of their expression. Here, we defined two promoters, one located two genes upstream of esxB and one located immediately upstream, that contribute substantially to the expression of esxB and esxA. We also defined an mRNA cleavage site within the esxB 5′ untranslated region (UTR) and found that a single-nucleotide substitution reprogramed the position of this cleavage event without impacting esxB-esxA transcript abundance. We furthermore investigated the impact of a double stem-loop structure in the esxB 5′ UTR and found that it does not confer stability on a reporter gene transcript. Consistent with this, there was no detectable correlation between mRNA half-life and secondary structure near the 5′ ends of 5′ UTRs on a transcriptome-wide basis. Collectively, these data shed light on the determinants of esxB-esxA expression in M. smegmatis as well as provide broader insight into the determinants of mRNA cleavage in mycobacteria and the relationship between 5′ UTR secondary structure and mRNA stability. Full article

PE/PPE proteins secreted by the ESX-5 type VII secretion system constitute a major protein repertoire in pathogenic mycobacteria and are essential for bacterial survival, pathogenicity, and host–pathogen interaction; however, little is known about their expression and secretion. The scarcity of arginine and lysine residues in PE/PPE protein sequences and the high homology of their N-terminal domains limit protein identification using classical trypsin-based proteomic methods. This study used endoproteinase AspN and trypsin to characterize the proteome of Mycobacterium marinum. Twenty-seven PE/PPE proteins were uniquely identified in AspN digests, especially PE_PGRS proteins. These treatments allowed the identification of approximately 50% of the PE/PPE pool encoded in the genome. Moreover, EspG5 pulldown assays retrieved 44 ESX-5-associated PPE proteins, covering 85% of the PPE pool in the identified proteome. The identification of PE/PE_PGRS proteins in the EspG5 interactome suggested the presence of PE–PPE pairs. The correlation analysis between protein abundance and phylogenetic relationships found potential PE/PPE pairs, indicating the presence of multiple PE/PE_PGRS partners in one PPE. We validated that EspG5 interacted with PPE31 and PPE32 and mapped critical residues for complex formation. The modified proteomic platform increases the coverage of PE/PPE proteins and elucidates the expression and localization of these proteins. Full article

Tuberculosis (TB) continues to be a major global health burden and kills over a million people annually. New immunization strategies are required for the development of an efficacious TB vaccine that can potentially induce sterilizing immunity. In this study, we first confirmed that a live vaccine strain of Mycobacterium smegmatis, previously designated as IKEPLUS, conferred a higher survival benefit than the Bacillus Calmette-Guerin (BCG) in a murine model of intravenous Mycobacterium tuberculosis (Mtb) infection. We have shown that there was a significant increase in the expression of the Rv0282 gene, which is encoded in the esx-3 locus, which played an important role in iron uptake when IKEPLUS was grown in both low zinc and iron-containing Sauton medium. We then confirmed using in vitro assays of biofilm formation that zinc plays a vital role in the growth and formation of M. smegmatis biofilms. IKEPLUS grown in low zinc media led to the better protection of mice after intravenous challenge with a very high dosage of Mtb. We also showed that various variants of IKEPLUS induced apoptotic cell-death of infected macrophages at a higher rate than wild-type M. smegmatis. We next attempted to determine if zinc containing ribosomal proteins such as rpmb2 could contribute to protective efficacy against Mtb infection. Since BCG has an established role in anti-mycobacterial efficacy, we boosted BCG vaccinated mice with rmpb2, but this did not lead to an increment in the protection mediated by BCG. Full article

DNA quantification is a crucial step in the STR typing workflow for human identification purposes. Given the reaction’s nature, qPCR assays may be subjected to the same stochastic effects of traditional PCR for low-input concentrations. The study aims to evaluate the precision of the PowerQuant^® (Promega) kit assay measurements and the degree of variability for DNA templates falling below the optimal threshold of the PowerPlex^® ESX-17 Fast STR typing kit (Promega). Five three-fold dilutions of the 2800 M control DNA (Promega) were set up. Each dilution (concentrations: 0.05, 0.0167, 0.0055, 0.00185, and 0.000617 ng/µL) was quantified and amplified in four replicates. Variability for qPCR results, STR profile completeness, and EPGs’ peak height were evaluated. The qPCR-estimated concentration of casework samples was correlated with profile completeness and peak intensity, to assess the predictive value of qPCR results for the successful STR typing of scarce samples. qPCR was subjected to stochastic effects, of which the degree was inversely proportional to the initial input template. Quantitation results and the STR profile’s characteristics were strongly correlated. Due to the intrinsic nature of real casework samples, a qPCR-derived DNA concentration threshold for correctly identifying probative STR profiles may be difficult to establish. Quantitation data may be useful in interpreting and corroborating STR typing results and for clearly illustrating them to the stakeholders. Full article

The Esx-1 family proteins of the Type VII secretion systems of Mycobacterium bovis and Mycobacterium tuberculosis have been assessed and are frequently used as candidates for tuberculosis (TB) diagnosis in both humans and animals. The presence of ESAT-6 and CFP 10 proteins, which are the most immunogenic proteins of the Esx-1 system and have been widely investigated for the immunodiagnosis of tuberculosis, in some Mycobacteriaceae and in Mycobacterium leprae, poses limitations for their use in specific diagnoses of TB. As such, to improve the specificity of the ESAT-6/CFP 10-based cell-mediated immunity (CMI) assays, other proteins encoded by genes within and outside the RD 1 region of the esx-1 locus have been evaluated as candidate antigens for CMI, as well as to investigate humoral responses in combination with ESAT-6 and or CFP 10, with varying specificity and sensitivity results. Hence, in this study, we evaluated various non-tuberculous mycobacteria (NTM), Mycolicibacterium, Mycolicibacter and Mycobacteroides species genomes available on the NCBI database for the presence and composition of the RD1 region of the esx-1 locus. In addition, we also assayed by polymerase chain reaction (PCR) and sequencing of Mycobacteriaceae available in our culture collection for the presence and sequence diversity of esxA and esxB genes encoding ESAT-6 and CFP 10, respectively. Whole genome sequence (WGS) data analysis revealed the presence of RD 1 gene orthologs in 70 of the over 100 published genomes of pathogenic and non- pathogenic Mycobcteriaceae other than tuberculosis. Among species evaluated from our culture collection, in addition to earlier reports of the presence of esxA and esxB in certain Mycolicibacterium, Mycolicibacterium septicum/peregrinum, Mycolicibacterium porcinum and Mycobacterium sp. N845T were also found to harbour orthologs of both genes. Orthologs of esxA only were detected in Mycobacterium brasiliensis, Mycolicibacterium elephantis and Mycolicibacterium flouroantheinivorans, whereas in Mycolicibacter engbackii, Mycolicibacterium mageritense and Mycobacterium paraffinicum, only esxB orthologs were detected. A phylogenetic analysis based on esxA and esxB sequences separated slow-growing from rapidly growing bacteria. These findings strengthen previous suggestions that esxA and esxB may be encoded in the majority of Mycobacteriaceae. The role of the Esx-1 system in both pathogenic and non-pathogenic Mycobacteriaceae needs further investigation, as these species may pose limitations to immunological assays for TB. Full article

The antimicrobial application of carbon nanomaterials, such as carbon nanotubes (CNTs), capped CNTs, CNT_2–5, C₆₀, C₇₀, HO-C₆₀, [C₆₀]₂, and [C₆₀]₃ fullerenes, is increasing, owing to their low cytotoxicity properties compared to other nanomaterials such as metallic nanoparticles. Enhanced mechanical properties and antibacterial activity can be caused by the incorporation of CNTs in 3-dimensional (3D) printed nanocomposites (NCs). The interruption of the bacterial membrane resulting from the cylindrical shape and high aspect ratio properties has been found to be the most prominent antibacterial mechanism of CNTs. However, the unraveling interaction of CNTs, capped CNTs, CNT_2–5, C₆₀, C₇₀, HO-C₆₀, [C₆₀]₂, and [C₆₀]₃ fullerenes with virulence factors of the main bacterial pathogenesis has not yet been understood. Therefore, in the present study, interactions of these carbon-based nanomaterials with the eight virulence factors, including protein kinase A and (ESX)-secreted protein B of Mycobacterium tuberculosis, pseudomonas elastase and exotoxin A of Pseudomonas aeruginosa, alpha-hemolysin and penicillin-binding protein 2a of Staphylococcus aureus, and shiga toxin 2a and heat-labile enterotoxin of Escherichia coli, were evaluated with the molecular docking method of AutoDock Vina. This study disclosed that the binding affinity was highest for CNT_2–5 and [C₆₀]₃ toward alpha-hemolysin, with binding energies of −32.7 and −26.6 kcal/mol, respectively. The stability of the CNT_2–5–alpha-hemolysin complex at different times was obtained according to the normal mode analysis of ElNémo and iMOD servers. Full article

Nanoparticles have been identified in numerous studies as effective antigen delivery systems that enhance immune responses. However, it remains unclear whether this enhancement is a result of increased antigen uptake when carried by nanoparticles or the adjuvanticity of the nanoparticle carriers. Consequently, it is important to quantify antigen uptake by dendritic cells in a manner that is free from artifacts in order to analyze the immune response when antigens are carried by nanoparticles. In this study, we demonstrated several scenarios (antigens on nanoparticles or inside cells) that are likely to contribute to the generation of artifacts in conventional fluorescence-based quantification. Furthermore, we developed the necessary assay for accurate uptake quantification. PLGA NPs were selected as the model carrier system to deliver EsxB protein (a Staphylococcus aureus antigen) in order to testify to the feasibility of the established method. The results showed that for the same antigen uptake amount, the antigen delivered by PLGA nanoparticles could elicit 3.6 times IL-2 secretion (representative of cellular immune response activation) and 1.5 times IL-12 secretion (representative of DC maturation level) compared with pure antigen feeding. The findings above give direct evidence of the extra adjuvanticity of PLGA nanoparticles, except for their delivery functions. The developed methodology allows for the evaluation of immune cell responses on an antigen uptake basis, thus providing a better understanding of the origin of the adjuvanticity of nanoparticle carriers. Ultimately, this research provides general guidelines for the formulation of nano-vaccines. Full article

Mycobacterium tuberculosis (Mtb) modulates diverse cell death pathways to escape the host immune responses and favor its dissemination, a complex process of interest in pathogenesis-related studies. The main virulence factors of Mtb that alter cell death pathways are classified according to their origin as either non-protein (for instance, lipomannan) or protein (such as the PE family and ESX secretion system). The 38 kDa lipoprotein, ESAT-6 (early antigen-secreted protein 6 kDa), and another secreted protein, tuberculosis necrotizing toxin (TNT), induces necroptosis, thereby allowing mycobacteria to survive inside the cell. The inhibition of pyroptosis by blocking inflammasome activation by Zmp1 and PknF is another pathway that aids the intracellular replication of Mtb. Autophagy inhibition is another mechanism that allows Mtb to escape the immune response. The enhanced intracellular survival (Eis) protein, other proteins, such as ESX-1, SecA2, SapM, PE6, and certain microRNAs, also facilitate Mtb host immune escape process. In summary, Mtb affects the microenvironment of cell death to avoid an effective immune response and facilitate its spread. A thorough study of these pathways would help identify therapeutic targets to prevent the survival of mycobacteria in the host. Full article

Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB), is one of the most successfully adapted human pathogens. Human-to-human transmission occurs at high rates through aerosols containing bacteria, but the pathogen evolved prior to the establishment of crowded populations. Mtb has developed a particular strategy to ensure persistence in the host until an opportunity for transmission arises. It has refined its lifestyle to obviate the need for virulence factors such as capsules, flagella, pili, or toxins to circumvent mucosal barriers. Instead, the pathogen uses host macrophages, where it establishes intracellular niches for its migration into the lung parenchyma and other tissues and for the induction of long-lived latency in granulomas. Finally, at the end of the infection cycle, Mtb induces necrotic cell death in macrophages to escape to the extracellular milieu and instructs a strong inflammatory response that is required for the progression from latency to disease and transmission. Common to all these events is ESAT-6, one of the major virulence factors secreted by the pathogen. This narrative review highlights the recent advances in understanding the role of ESAT-6 in hijacking macrophage function to establish successful infection and transmission and its use as a target for the development of diagnostic tools and vaccines. Full article