Journal of Bacteriology最新文献

{"title":"Analysis of novel zinc-binding proteins in the cell wall of <i>Corynebacterium diphtheriae</i>.","authors":"Eric D Peng, Lindsey R Lyman, Michael P Schmitt","doi":"10.1128/jb.00239-25","DOIUrl":"10.1128/jb.00239-25","url":null,"abstract":"<p><p>Zinc is a critical nutrient for all living organisms, including bacterial pathogens such as <i>Corynebacterium diphtheriae</i>, the causative agent of the severe human respiratory disease diphtheria. As such, zinc acquisition is essential for many pathogens to cause disease. We previously showed that the zinc-regulated ABC transporter encoded by the <i>znu</i> locus is one of several zinc uptake systems that support the growth of <i>C. diphtheriae</i> in zinc-limited medium. In this study, we examine the function and cellular localization of components encoded by the <i>znu</i> gene cluster, which includes the ZnuABC transporter and the novel membrane and cell wall proteins ZnuE, ZnuF, and ZnuG. Deletion of <i>znuE</i> results in reduced growth in zinc-limited media, suggesting a role in zinc uptake. While the growth of <i>znuF</i> and <i>znuG</i> mutants was not affected in zinc-limited medium, deletion of these two genes in a mutant also lacking the <i>znuABC</i> transporter restored growth to wild-type levels, suggesting that ZnuF and ZnuG exert an unusual modulating effect on zinc import. ZnuE, ZnuF, and ZnuG possess a unique motif that is associated with Zn binding as demonstrated by thermal shift assays, targeted mutagenesis, and structural analysis. Although ZnuF and ZnuG are both present in the cell wall, only ZnuG contains a sortase recognition signal that is critical for localizing proteins to the cell wall. Furthermore, ZnuF localization does not require any of the six known sortase enzymes in <i>C. diphtheriae</i>, suggesting a novel localization mechanism.</p><p><strong>Importance: </strong>Zinc is a critical nutrient required by many bacterial pathogens. While the function of multiple zinc importer systems has been previously characterized in <i>Corynebacterium diphtheriae</i>, the transporter encoded by the <i>znu</i> gene cluster includes components not found in other metal transport systems. In this report, we examined the roles of three components of the <i>znu</i> gene cluster, ZnuE, ZnuF, and ZnuG, and show that these proteins all possess a putative zinc-binding domain and have varying effects on growth in zinc-limited medium. Additionally, ZnuF uses a novel mechanism for cell wall localization. This study further expands our understanding of <i>C. diphtheriae</i> zinc import and points to a potentially novel mechanism for the localization of cell wall proteins.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0023925"},"PeriodicalIF":3.0,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12445094/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144873333","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>Staphylococcus aureus</i> phenol-soluble modulins have dispersal and anti-aggregation activity towards corynebacteria.","authors":"Joshua T Huffines, Megan R Kiedrowski","doi":"10.1128/jb.00183-25","DOIUrl":"10.1128/jb.00183-25","url":null,"abstract":"<p><p><i>Staphylococcus aureus</i> is a common upper respiratory tract (URT) pathobiont with high carriage rates in the upper airway disease chronic rhinosinusitis (CRS). CRS is associated with increased prevalence and abundance of <i>S. aureus</i>, and <i>S. aureus-</i>secreted toxins are implicated in CRS pathogenesis. Conversely, in CRS disease, the abundance of non-pathogenic commensal species has been observed to decline, leading to microbial dysbiosis that contributes to persistent inflammation. Here, we investigated possible mechanisms <i>S. aureus</i> could employ to outcompete commensal organisms and contribute to establishing the <i>S. aureus-</i>dominant microbiome found in individuals with CRS. We performed a targeted screen to identify <i>S. aureus-</i>secreted factors that affect the growth and aggregation of a URT commensal bacterium, <i>Corynebacterium pseudodiphtheriticum</i>, which is negatively correlated with <i>S. aureus</i> in CRS. <i>S. aureus</i> cell-free conditioned media prevented <i>C. pseudodiphtheriticum</i> aggregation; however, anti-aggregation activity was significantly reduced in <i>S. aureus</i> mutants lacking a functional accessory gene regulator (agr) quorum-sensing system, phenol-soluble modulin (PSM) transporters, and the PSM toxin δ-toxin. Addition of purified recombinant δ-toxin peptide or a related PSM, PSMα3, inhibited <i>C. pseudodiphtheriticum</i> aggregation and induced dispersal of aggregates. Recombinant δ-toxin also reduced <i>C. pseudodiphtheriticum</i> adherence and aggregation on human nasal epithelial cells. PSMs are known to play a role in biofilm structure and remodeling in staphylococci, and here, we demonstrate that PSMs have activity against other bacteria. These results identify a novel mechanism by which <i>S. aureus</i> can disrupt the commensal lifestyle of microbes that inhabit the same upper respiratory niche via secreted PSM toxins.IMPORTANCEIncreased <i>Staphylococcus aureus</i> abundance and microbial dysbiosis are associated with the pathogenesis of chronic rhinosinusitis disease. Here, we show that <i>S. aureus</i> δ-toxin, a secreted phenol-soluble modulin (PSM) toxin, can inhibit the ability of commensal <i>Corynebacterium</i> species to aggregate, adhere to, and grow in association with human nasal epithelial cells. PSMs are known to play a key role in the <i>S. aureus</i> biofilm life cycle, regulating <i>S. aureus</i> biofilm structure and detachment; however, a role for these toxins in modifying biofilm and aggregate structures of other bacteria has not been previously demonstrated. These results suggest a potential mechanism for <i>S. aureus</i> to establish dominance in the upper respiratory tract microbiome in disease through direct antagonism of commensal microbes with PSM toxins.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0018325"},"PeriodicalIF":3.0,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12445096/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144855242","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A <i>ptsH</i> mutation suppresses growth defects and antibiotic sensitivity in a <i>cpgA</i> mutant defective in metabolite proofreading.","authors":"Ankita J Sachla, Ahmed Gaballa, Diana Herrera, John D Helmann","doi":"10.1128/jb.00162-25","DOIUrl":"10.1128/jb.00162-25","url":null,"abstract":"<p><p><i>Bacillus subtilis</i> CpgA (<u>c</u>ircularly <u>p</u>ermuted <u>G</u>TPase) is a ribosome assembly GTPase that has a secondary function as a metabolite proofreading enzyme. CpgA hydrolyzes 4-phosphoerythronate, a toxic metabolite produced from erythrose-4-phosphate by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In a ∆<i>cpgA</i> strain, carbon sources that feed into the pentose phosphate pathway trigger metabolic intoxication. This results in poor growth and increased sensitivity to antibiotics that block peptidoglycan synthesis, a process reliant on sugars from central metabolism. Here, we describe a mutation in <i>ptsH</i> (<i>ptsH</i>-G54D) that improves growth of a ∆<i>cpgA</i> strain on media containing both glucose and gluconate. The <i>ptsH</i> gene encodes the histidine-containing phosphocarrier protein (HPr) that functions in phosphotransferase system sugar import and gene regulation. Prior studies of HPr suggested three possible mechanisms to account for the ability of HPr-G54D to increase fitness of the ∆<i>cpgA</i> strain: (i) restricting HPr-dependent uptake of glucose, (ii) reducing the GAPDH-dependent production of 4-phosphoerythronate, or (iii) decreasing expression of genes required for uptake and catabolism of gluconate. Here, we present evidence consistent with the third model: HPr-G54D improves fitness of a ∆<i>cpgA</i> strain by increasing catabolite repression of the gluconate operon. Consistently, genetic suppression by HPr-G54D requires Ser46, a site of regulatory phosphorylation important for carbon catabolite repression. In addition, we demonstrate that the metabolic proofreading function of CpgA is conserved among related gram-positive bacteria.IMPORTANCEMetabolism relies on the concerted action of hundreds of enzymes, many of which have some activity with non-canonical substrates. The resulting reactions constitute an often-ignored underground metabolism. Glyceraldehyde-3-phosphate dehydrogenase catalyzes a secondary reaction that produces 4-phosphoerythronate, a toxic dead-end metabolite. <i>Bacillus subtilis</i> CpgA is a widely conserved metabolite proofreading enzyme that protects cells against metabolic intoxication, which can increase antibiotic sensitivity. Loss of CpgA can be suppressed by an altered function mutation affecting the histidine-containing phosphocarrier protein (HPr). This mutant HPr protein increases carbon catabolite repression to restrict import of intoxicating gluconate. These studies highlight the ability of mutations in HPr to rewire carbon catabolism to help avoid the toxic effects of metabolic dysregulation.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0016225"},"PeriodicalIF":3.0,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12445079/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144855243","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Host niche-specific challenges hindering the treatment of polymicrobial infections.","authors":"Caroline Black, Catherine A Wakeman","doi":"10.1128/jb.00142-25","DOIUrl":"10.1128/jb.00142-25","url":null,"abstract":"<p><p>Antimicrobial recalcitrance is a growing problem in today's world. Not only are bacteria developing resistance at an alarming pace but the antibiotic discovery pipeline has gone dry, making antimicrobial stewardship essential for preserving the activity of the antibiotics still currently available for use. In addition to resistance, bacteria also display tolerance to certain treatments as they adapt to their body site-specific niche and cooperate with other organisms in polymicrobial communities. Thus, new and existing antibiotics must contend with altered bacterial metabolism, polymicrobial synergy, increased biofilm production, and nutrient-related adaptations present within specific infectious sites. Finally, these treatments must face the challenging process of moving to the infection site and doing their job without causing harm to the patient. This minireview explores the difficulties antimicrobials face when challenging organisms at different body sites, focusing on the niche-specific dynamics present at sites of infection.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0014225"},"PeriodicalIF":3.0,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12445101/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144955219","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Investigating the molecular transmission dynamics of <i>bla</i>_NDM in antibiotic-selective environments.","authors":"Shashi Kumari, Lekshmi Narendrakumar, Meenal Chawla, Sanjib Das, Hemanta Koley, Bhabatosh Das","doi":"10.1128/jb.00133-25","DOIUrl":"10.1128/jb.00133-25","url":null,"abstract":"<p><p>Carbapenem resistance mediated by <i>bla</i>_NDM-encoded metallo-beta-lactamases is often linked to IS<i>Aba125</i>, an insertion sequence from the IS<i>30</i> family, which is widely distributed among critical and high-priority bacterial pathogens. The rapid dissemination of IS<i>Aba125</i>-linked <i>bla</i>_NDM in both nosocomial and community-acquired infections presents a serious challenge to healthcare systems and pharmaceutical industries. Despite the urgency of this issue, the factors driving <i>bla</i>_NDM spread and the molecular mechanisms governing IS<i>Aba125</i> mobility remain poorly understood. In this study, we engineered the genomes of <i>Vibrio cholerae</i> and <i>Escherichia coli</i> to investigate the mobility of <i>bla</i>_NDM under controlled conditions both with and without the genetically linked IS<i>Aba125</i>. We also examined the transmission efficiency and the stability of <i>bla</i>_NDM in environments with and without sublethal antibiotic concentrations. Our <i>in vitro</i> findings were validated in a rabbit ileal loop model. The results revealed that antibiotic pressure significantly influences the mobility of <i>bla</i>_NDM, shedding light on the molecular dynamics of its transmission. These insights are crucial for developing strategies to curb the spread of <i>bla</i>_NDM and mitigate the growing threat of carbapenem resistance in bacterial pathogens.IMPORTANCEInsertion sequences are the simplest form of mobile genetic elements that play a critical role in the adaptation of bacteria, allowing them to rapidly acquire new traits like resistance genes that enhance their survival. IS<i>Aba125</i> is one such insertion sequence that facilitates the spread of <i>bla</i>_NDM, contributing to the global challenge of carbapenem resistance. In this study, we developed reporter strains that could be used as a valuable tool for investigating the dynamics of IS<i>Aba125-</i>linked <i>bla</i>_NDM<i>sh-ble</i> and evaluated the transposition frequency of IS<i>Aba125-</i>linked <i>bla</i>_NDM<i>sh-ble</i> in the presence and absence of sublethal concentration of antibiotics. Our results demonstrated that IS<i>Aba125</i> enhances the spread of <i>bla</i>_NDM<i>sh-ble</i> under sublethal concentration of antibiotics that induces SOS response.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0013325"},"PeriodicalIF":3.0,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12445084/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144816726","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Breaking the silence-role of MucR as a virulence determinant in <i>Brucella</i>.","authors":"Ian S Barton, Connor B Cribb, Beatriz Tartilán-Choya, Graham J Bitzer, Jodi Ogle, Neil Garza Hernandez, Daniel W Martin, Nieves Vizcaíno, Xindan Wang, Ilaria Baglivo, R Martin Roop","doi":"10.1128/jb.00170-25","DOIUrl":"https://doi.org/10.1128/jb.00170-25","url":null,"abstract":"<p><p>The Zn finger protein MucR is an H-NS-like protein that serves as a gene silencer and nucleoid-structuring protein in the α-proteobacteria. MucR is also an essential virulence determinant in <i>Brucella</i>, where it directly and indirectly controls the expression of many genes required for the virulence of these bacteria in their mammalian hosts, including those encoding the Type IV secretion system and its effectors, the autotransporter adhesins BtaE and BmaC, and the quorum sensing regulators VjbR and BabR. Experimental evidence suggests that one of the primary functions of the <i>Brucella</i> MucR is to ensure that virulence genes are only expressed when their corresponding gene products provide fitness benefits to these bacteria during their infectious lifecycle, and we propose that this function is central to the well-established role of MucR as a virulence determinant.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0017025"},"PeriodicalIF":3.0,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145080702","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction for Korgaonkar and Whiteley, \"<i>Pseudomonas aeruginosa</i> Enhances Production of an Antimicrobial in Response to <i>N</i>-Acetylglucosamine and Peptidoglycan\".","authors":"Aishwarya K Korgaonkar, Marvin Whiteley","doi":"10.1128/jb.00209-25","DOIUrl":"https://doi.org/10.1128/jb.00209-25","url":null,"abstract":"","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0020925"},"PeriodicalIF":3.0,"publicationDate":"2025-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145040237","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Carbohydrates and the oxidative branch of the pentose phosphate pathway modify <i>Bacteroides thetaiotaomicron</i> phage resistance by phase-variable S-layers.","authors":"Jaime J Fuentes, Shaleni Singh, Nicholas A Pudlo, Stacey L Heaver, Ruth E Ley, Eric C Martens","doi":"10.1128/jb.00178-25","DOIUrl":"10.1128/jb.00178-25","url":null,"abstract":"<p><p>The human gut microbiota consists of hundreds of bacterial species, some of which persist in the presence of lytic phage that infect them. <i>Bacteroides</i> employ numerous phase-variable strategies to survive in the presence of phage, including capsular polysaccharides (CPS) and S-layer lipoproteins. We previously reported that a <i>Bacteroides thetaiotaomicron</i> strain lacking CPS exhibits almost complete resistance to multiple phages when forced to express the S-layer protein BT1927. However, this strain was only resistant after certain growth conditions, suggesting nutritional variables alter infection and resistance. We grew this strain on various simple sugars and polysaccharides, finding that some substrates (fructose, glucose) promote strong resistance to a single phage (ARB25), while others like <i>N</i>-acetylgalactosamine (GalNAc) and mucin <i>O</i>-glycans increase susceptibility. Mixing fructose and GalNAc indicates the effects of GalNAc are dominant. Despite increasing ARB25 susceptibility, GalNAc did not reduce <i>BT1927</i> transcript or protein levels. Instead, GalNAc reduced the amount of BT1927 displayed on the cell surface and increased outer membrane vesiculation. Mutants in any of the three steps of the oxidative branch of the pentose phosphate pathway-grown in fructose-behaved similarly to wild-type cells grown in GalNAc, illuminating this pathway in the regulation of sugar-mediated phage resistance. Despite promoting strong resistance, cells grown in glucose/fructose sometimes displayed sub-populations that appeared to completely lack surface BT1927, suggesting another checkpoint exists to control whether this phage defense is deployed. Finally, we show the mucin sugar GalNAc increases susceptibility to several other phage, which has implications for <i>B. thetaiotaomicron</i> persistence in niches like the mucus layer.IMPORTANCEThe persistence of viruses that infect bacteria (bacteriophages or phages) in the human gut microbiome and their effects on bacterial physiology and host health are active areas of investigation. Our study investigates how various sugars and polysaccharides alter the susceptibility of the model gut symbiont <i>Bacteroides thetaiotaomicron</i> to lytic phages that are capable of infecting it. Our finding that the mucin sugar, <i>N</i>-acetylgalactosamine, and mucin <i>O</i>-glycans that contain this sugar reduce <i>B. thetaiotaomicron</i> resistance to multiple phages has implications for how this symbiont persists in different gut microhabitats, such as the mucus layer, and which defense mechanisms it can deploy to survive in these niches.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0017825"},"PeriodicalIF":3.0,"publicationDate":"2025-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145040239","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterization of two co-regulated response regulators in <i>Clostridioides difficile</i>.","authors":"Caitlin Lee Williams, Anchal Mehra, Emily K Harrison, Cynthia G Thomas, Jack Riley Palmer, D Kane Cooper, Elizabeth M Garrett, Rita Tamayo","doi":"10.1128/jb.00177-25","DOIUrl":"https://doi.org/10.1128/jb.00177-25","url":null,"abstract":"<p><p>As an obligate anaerobe, <i>Clostridioides difficile</i> grows exclusively in the host intestinal tract, necessitating mechanisms to sense and respond to the gut environment. The atypical signal transduction system encoded by <i>cmrRST</i> includes two OmpR-family response regulators, CmrR and CmrT. These regulators control multiple important phenotypes in <i>C. difficile</i>, including cell chaining, colony morphology, swimming motility, and biofilm formation. CmrR has a typical receiver domain with key conserved residues for phosphotransfer and conformational change, while CmrT is missing conserved residues and is likely a pseudoreceiver. Here, we used multiple methods to investigate dimer formation, including bacterial two-hybrid systems in both <i>Escherichia coli</i> and <i>C. difficile</i> and pull-down assays in <i>E. coli</i>. We detected CmrR homodimers and found that CmrR recognizes a specific DNA sequence found in multiple places in the genome, including upstream of <i>cmrRST</i>. CmrT formed homodimers in multiple assays, and mutation analysis of residues in its unusual active site suggests multiple mutations are needed to maximally reduce CmrT activity. Our data also suggest that CmrR and CmrT can form heterodimers, though the biological relevance of CmrR-CmrT heterodimers remains unclear. Our results suggest that CmrR and CmrT work together in transcriptional responses to stimuli.IMPORTANCETwo-component systems are nearly ubiquitous among bacteria and are one of the primary ways that bacteria respond to their environment. Atypical two-component proteins and systems are being identified in diverse bacteria, and studying these proteins helps us to understand the underlying mechanisms of these systems. CmrRST is an unusual two-component system that regulates many important phenotypes in <i>Clostridioides difficile</i>. This work begins to untangle the complex regulatory mechanisms by which CmrRST controls gene expression and furthers our understanding of the fundamental biology of <i>C. difficile</i>.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0017725"},"PeriodicalIF":3.0,"publicationDate":"2025-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145040308","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Functional analysis of the intracellular survival of <i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i> in THP-1 cells using CRISPR interference.","authors":"Jun Ho Lee, Eun-Seo Lee, Su Min Kyung, Xi-Rui Xiang, Hyun-Eui Park, Min-Kyoung Shin, Han Sang Yoo","doi":"10.1128/jb.00244-25","DOIUrl":"https://doi.org/10.1128/jb.00244-25","url":null,"abstract":"<p><p><i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i> (MAP) is a causative agent of Johne's disease in ruminants and a potential zoonotic agent linked with Crohn's disease in humans. Despite the possible risk to public health, few studies have focused on the virulence of MAP against human macrophages. Therefore, a functional analysis of mycobacterial genes associated with virulence, especially the intracellular survival of MAP, was performed after infection of MAP CRISPR interference (CRISPRi) mutants in the human THP-1 macrophages. MAP mutants were targeted to four genes (<i>mdh</i>, <i>pknG</i>, <i>MAP1981c</i>, and <i>icl</i>). The optimal concentration of anhydrotetracycline (ATc) was determined to be 5 µg/mL by measuring the survival of the cells and the downregulation of gene expression levels in the cells up to Day 3. The clump formation and intracellular survival of MAP were investigated using transmission electron microscopy and the colony-forming units, respectively. The clump formation of MAP mutants induced by CRISPRi was decreased in THP-1 macrophages at 24 and 72 h post-infection. The survival rates of the MAP mutants significantly decreased with increasing ATc concentration and time course of infection in MAP<i>-mdh</i>KD, <i>MAP1981c</i>KD, and MAP<i>-icl</i>KD. Conversely, the survival rate of THP-1 macrophages increased with increasing ATc concentration. Our results suggest that these genes might be closely related to MAP virulence along with intracellular survival in THP-1 macrophages. These data can provide novel insights into the utilization of CRISPRi in further research on MAP virulence by exploring intracellular survival using mycobacterial genes related to the virulence of MAP during host infection.</p><p><strong>Importance: </strong>Johne's disease, caused by <i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i> (MAP) is a worldwide issue in the dairy industry and has a possible connection to Crohn's disease (CD) in humans. Despite its potential contribution to the etiology of CD, there have been few studies focusing on the virulence of MAP against human macrophages. In the current study, we investigated MAP virulence along with intracellular survival in human THP-1 macrophages using functional analysis of MAP CRISPR interference (CRISPRi) mutants at the knockdown of genes associated with mycobacterial virulence. The identified potential genes represent novel candidate classes that could be necessary for MAP virulence by exploring intracellular survival during host infection and could provide novel insights for future studies on the utilization of CRISPRi.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0024425"},"PeriodicalIF":3.0,"publicationDate":"2025-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145040249","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}