mBio最新文献

{"title":"Substrate identification of putative NCS1 and NCS2 nucleobase transporters in <i>Pseudomonas aeruginosa</i>.","authors":"Corey Kennelly, Arthur Prindle","doi":"10.1128/mbio.02434-24","DOIUrl":"https://doi.org/10.1128/mbio.02434-24","url":null,"abstract":"<p><p><i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that can salvage nucleobases from the environment to conserve nutrients that would otherwise be spent on <i>de novo</i> nucleotide biosynthesis. However, little is known regarding the substrate specificity of the 13 putative nucleobase transporters in <i>P. aeruginosa</i>. Here, using a combination of genetic and chemical approaches, we report substrate identifications for 10 putative nucleobase transporters in <i>P. aeruginosa</i>. Specifically, we individually expressed each transporter in a genetic background lacking all 13 putative nucleobase transporters and quantified growth on a panel of 10 nucleobases as sole nitrogen sources. We confirmed these expression-based substrate identifications using targeted genetic knockouts. In a complementary approach, we utilized four toxic nucleobase antimetabolites to characterize antimicrobial activity in these same strains. We identified the sole allantoin transporter as well as transporters for guanine, xanthine, uric acid, cytosine, thymine, uracil, and dihydrouracil. Furthermore, we associated at least five nucleobase transporters with hypoxanthine, which has been recently reported to be an antibiofilm cue in <i>P. aeruginosa</i>. These results provide an initial characterization of the putative nucleobase transporters in <i>P. aeruginosa</i>, significantly advancing our understanding of nucleobase transport in this clinically relevant organism.</p><p><strong>Importance: </strong><i>Pseudomonas aeruginosa</i> is a frequently multidrug-resistant opportunistic pathogen and one of the most common causes of healthcare-acquired infections. While nucleobases are known to support growth in nutrient-limited conditions, recent work showed that adenine and hypoxanthine can also decrease <i>P. aeruginosa</i> biofilm formation by disrupting c-di-GMP metabolism. Thus, nucleobase transport may be relevant to multiple aspects of <i>P. aeruginosa</i> biology and pathogenesis. However, there is currently little known about the transport of nucleobases in <i>P. aeruginosa</i>. Our work reports initial substrate identifications for 10 putative nucleobase transporters in <i>P. aeruginosa</i>, providing new tools to address previously difficult-to-test hypotheses relating to nucleobase transport in this organism.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546311","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Torque-speed relationship of the flagellar motor with dual-stator systems in <i>Pseudomonas aeruginosa</i>.","authors":"Haolin Wu, Zhengyu Wu, Maojin Tian, Rongjing Zhang, Junhua Yuan","doi":"10.1128/mbio.00745-24","DOIUrl":"https://doi.org/10.1128/mbio.00745-24","url":null,"abstract":"<p><p>The single polar flagellar motor in <i>Pseudomonas aeruginosa</i> is equipped with two stator systems, MotAB and MotCD, both driven by H⁺ ions. The torque-speed relationship for flagellar motors with two stator systems has not been explored previously. In this study, we developed a method that utilizes optical trapping and fluorescence labeling to measure the torque-speed relationships for the wild-type <i>P. aeruginosa</i> motor with dual stators and mutant strains with a single stator system, revealing surprising differences in them. Moreover, we found that the MotAB stators exhibit slip-bond behavior in load dependence, contrasting with the catch-bond behavior of the MotCD stators and <i>Escherichia coli</i> stators. Further examination of the solvent isotope and pH effects on the torque-speed relationships of these stator systems provided additional insights into their dynamics. Interestingly, we discovered that the torque of the wild-type motor is similar to the combined torque of motors with MotAB or MotCD stators, indicating an additive contribution from the two stator types in the wild-type motors. These findings underscore the enhanced adaptability of <i>P. aeruginosa</i> to a wide range of external environments with varying load conditions.IMPORTANCEWe developed a novel method to measure the flagellar motor torque-speed relationship by trapping a swimming bacterium using optical tweezers. Using the <i>P. aeruginosa</i> flagellar motor as a model system to investigate motor dynamics with dual stator types, we measured the torque-speed relationships for wild-type motors with dual stator types and mutants with a single type. We found drastic differences that stem from the varying load dependencies of stator stability. These variations enable bacteria to rapidly adjust their stator composition in response to external load conditions. Interestingly, we observed that the torque of the wild-type motor is akin to the cumulative torque of motors with either stator type, indicating an additive contribution from the two stator types in wild-type motors. The methodology we established here can be readily employed to study motor dynamics in other flagellated bacteria.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546315","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Aminoglycoside heteroresistance in <i>Enterobacter cloacae</i> is driven by the cell envelope stress response.","authors":"Ana J Choi, Daniel J Bennison, Esha Kulkarni, Hibah Azar, Haoyu Sun, Hanqi Li, Jonathan Bradshaw, Hui Wen Yeap, Nicholas Lim, Vishwas Mishra, Anna Crespo-Puig, Ewurabena A Mills, Frances Davies, Shiranee Sriskandan, Avinash R Shenoy","doi":"10.1128/mbio.01699-24","DOIUrl":"https://doi.org/10.1128/mbio.01699-24","url":null,"abstract":"&lt;p&gt;&lt;p&gt;&lt;i&gt;Enterobacter cloacae&lt;/i&gt; is a Gram-negative nosocomial pathogen of the ESKAPE (&lt;i&gt;Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas&lt;/i&gt;, and &lt;i&gt;Enterobacter&lt;/i&gt; spp.) priority group with increasing multi-drug resistance via the acquisition of resistance plasmids. However, &lt;i&gt;E. cloacae&lt;/i&gt; can also display forms of antibiotic refractoriness, such as heteroresistance and tolerance. Here, we report that &lt;i&gt;E. cloacae&lt;/i&gt; displays transient heteroresistance to aminoglycosides, which is accompanied with the formation of small colony variants (SCVs) with increased minimum inhibitor concentration (MIC) of gentamicin and other aminoglycosides used in the clinic, but not other antibiotic classes. To explore the underlying mechanisms, we performed RNA sequencing of heteroresistant bacteria, which revealed global gene expression changes and a signature of the CpxRA cell envelope stress response. Deletion of the &lt;i&gt;cpxRA&lt;/i&gt; two-component system abrogated aminoglycoside heteroresistance and SCV formation, pointing to its indispensable role in these processes. The introduction of a constitutively active allele of &lt;i&gt;cpxA&lt;/i&gt; led to high aminoglycoside MICs&lt;i&gt;,&lt;/i&gt; consistent with cell envelope stress response driving these behaviors in &lt;i&gt;E. cloacae&lt;/i&gt;. Cell envelope stress can be caused by environmental cues, including heavy metals. Indeed, bacterial exposure to copper increased gentamicin MIC in the wild-type but not in the Δ&lt;i&gt;cpxRA&lt;/i&gt; mutant. Moreover, copper exposure also elevated the gentamicin MICs of clinical isolates from bloodstream infections, suggesting that CpxRA- and copper-dependent aminoglycoside resistance is broadly conserved in &lt;i&gt;E. cloacae&lt;/i&gt; strains. Altogether, we establish that &lt;i&gt;E. cloacae&lt;/i&gt; relies on transcriptional reprogramming via the envelope stress response pathway for transient resistance to a major class of frontline antibiotic.IMPORTANCE&lt;i&gt;Enterobacter cloacae&lt;/i&gt; is a bacterium that belongs to the WHO high-priority group and an increasing threat worldwide due its multi-drug resistance. &lt;i&gt;E. cloacae&lt;/i&gt; can also display heteroresistance, which has been linked to treatment failure. We report that &lt;i&gt;E. cloacae&lt;/i&gt; shows heteroresistance to aminoglycoside antibiotics. These are important frontline microbicidal drugs used against Gram-negative bacterial infections; therefore, understanding how resistance develops among sensitive strains is important. We show that aminoglycoside resistance is driven by the activation of the cell envelope stress response and transcriptional reprogramming via the CpxRA two-component system. Furthermore, heterologous activation of envelope stress via copper, typically a heavy metal with antimicrobial actions, also increased aminoglycoside MICs of the &lt;i&gt;E. cloacae&lt;/i&gt; type strain and clinical strains isolated from bloodstream infections. Our study suggests aminoglycoside recalcitrance in &lt;i&gt;E. cloacae&lt;/i&gt; could be broadly conserved and cautions against the undesir","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546295","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An orphan kinesin in <i>Trypanosoma brucei</i> regulates hook complex assembly and Golgi biogenesis.","authors":"Qing Zhou, Yasuhiro Kurasawa, Huiqing Hu, Thiago Souza Onofre, Ziyin Li","doi":"10.1128/mbio.02634-24","DOIUrl":"https://doi.org/10.1128/mbio.02634-24","url":null,"abstract":"<p><p>Kinesins are microtubule-based motor proteins that play diverse cellular functions by regulating microtubule dynamics and intracellular transport in eukaryotes. The early branching kinetoplastid protozoan <i>Trypanosoma brucei</i> has an expanded repertoire of kinetoplastid-specific kinesins and orphan kinesins, many of which have unknown functions. We report here the identification of an orphan kinesin named KIN-G that plays an essential role in maintaining hook complex integrity and promoting Golgi biogenesis in <i>T. brucei</i>. KIN-G localizes to the distal portion of the centrin arm of the flagellum-associated hook complex through association with the centrin arm protein TbCentrin4. Knockdown of KIN-G in <i>T. brucei</i> disrupts the integrity of the hook complex by reducing the length of the centrin arm and eliminating the shank part of the hook complex, thereby impairing flagellum attachment zone elongation and flagellum positioning, which leads to unequal cytokinesis. KIN-G associates with Golgi through a centrin arm-localized Golgi peripheral protein named CAAP1, which maintains Golgi-centrin arm association to facilitate Golgi biogenesis. Knockdown of KIN-G impairs Golgi biogenesis by disrupting CAAP1 at the centrin arm, thereby impairing the maturation of centrin arm-associated Golgi. <i>In vitro</i> microtubule gliding assays demonstrate that KIN-G is a plus end-directed motor protein, and its motor activity is required for hook complex assembly and Golgi biogenesis. Together, these results identify a kinesin motor protein for promoting hook complex assembly and uncover a control mechanism for Golgi biogenesis through KIN-G-mediated maintenance of Golgi-hook complex association.IMPORTANCE<i>Trypanosoma brucei</i> has a motile flagellum, which controls cell motility, cell morphogenesis, cell division, and cell-cell communication, and a set of cytoskeletal structures, including the hook complex and the centrin arm, associates with the flagellum. Despite the essentiality of these flagellum-associated cytoskeletal structures, their mechanistic roles and the function of their associated proteins remain poorly understood. Here, we demonstrate that the orphan kinesin KIN-G functions to promote the biogenesis of the hook complex and the Golgi apparatus. KIN-G exerts this function by mediating the association between centrin arm and Golgi through the centrin arm protein TbCentrin4 and a novel Golgi scaffold protein named CAAP1, thereby bridging the two structures and maintaining their close association to facilitate the assembly of the two structures. These findings uncover the essential involvement of a kinesin motor protein in regulating the biogenesis of the hook complex and the Golgi in trypanosomes.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546296","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"C→U transition biases in SARS-CoV-2: still rampant 4 years from the start of the COVID-19 pandemic.","authors":"Peter Simmonds","doi":"10.1128/mbio.02493-24","DOIUrl":"https://doi.org/10.1128/mbio.02493-24","url":null,"abstract":"<p><p>The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the pandemic and post-pandemic periods has been characterized by rapid adaptive changes that confer immune escape and enhanced human-to-human transmissibility. Sequence change is additionally marked by an excess number of C→U transitions suggested as being due to host-mediated genome editing. To investigate how these influence the evolutionary trajectory of SARS-CoV-2, 2,000 high-quality, coding complete genome sequences of SARS-CoV-2 variants collected pre-September 2020 and from each subsequently appearing alpha, delta, BA.1, BA.2, BA.5, XBB, EG, HK, and JN.1 lineages were downloaded from NCBI Virus in April 2024. C→U transitions were the most common substitution during the diversification of SARS-CoV-2 lineages over the 4-year observation period. A net loss of C bases and accumulation of U's occurred at a constant rate of approximately 0.2%-0.25%/decade. C→U transitions occurred in over a quarter of all sites with a C (26.5%; range 20.0%-37.2%) around five times more than observed for the other transitions (5.3%-6.8%). In contrast to an approximately random distribution of other transitions across the genome, most C→U substitutions occurred at statistically preferred sites in each lineage. However, only the most C→U polymorphic sites showed evidence for a preferred 5'U context previously associated with APOBEC 3A editing. There was a similarly weak preference for unpaired bases suggesting much less stringent targeting of RNA than mediated by A3 deaminases in DNA editing. Future functional studies are required to determine editing preferences, impacts on replication fitness <i>in vivo</i> of SARS-CoV-2 and other RNA viruses, and impact on host tropism.</p><p><strong>Importance: </strong>Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the pandemic and post-pandemic periods has shown a remarkable capacity to adapt and evade human immune responses and increase its human-to-human transmissibility. The genome of SARS-CoV-2 is also increasingly scarred by the effects of multiple C→U mutations from host genome editing as a cellular defense mechanism akin to restriction factors for retroviruses. Through the analysis of large data sets of SARS-CoV-2 isolate sequences collected throughout the pandemic period and beyond, we show that C→U transitions have driven a base compositional change over time amounting to a net loss of C bases and accumulation of U's at a rate of approximately 0.2%-0.25%/decade. Most C→U substitutions occurred in the absence of the preferred upstream-base context or targeting of unpaired RNA bases previously associated with the host RNA editing protein, APOBEC 3A. The analyses provide a series of testable hypotheses that can be experimentally investigated in the future.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546297","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The vitamin D receptor is essential for the replication of pseudorabies virus.","authors":"Lei Zeng, Shu-Yi Wang, Meng-Hua Du, Bei-Bei Chu, Sheng-Li Ming","doi":"10.1128/mbio.02137-24","DOIUrl":"https://doi.org/10.1128/mbio.02137-24","url":null,"abstract":"<p><p>The vitamin D receptor (VDR) is a nuclear steroid receptor that regulates the expression of genes across various biological functions. However, the role of VDR in pseudorabies virus (PRV) infection has not yet been explored. We discovered that VDR positively influenced PRV proliferation because knockdown of VDR impaired PRV proliferation, whereas its overexpression promoted it. Additionally, we observed that PRV infection upregulated VDR transcription alongside 1,25-dihydroxyvitamin D3 (VD₃) synthesis, contingent on p53 activation. Furthermore, VDR knockdown hindered PRV-induced lipid synthesis, implicating VDR's involvement in this process. To decipher the mechanism behind VDR's stimulation of lipid synthesis during PRV infection, we conducted RNA sequencing (RNA-seq) and found significant enrichment of genes in the Ca²⁺ signaling pathway. Measurements of Ca²⁺ indicated that VDR facilitated Ca²⁺ absorption. Moreover, the PI3K/AKT/mTORC1 and AMPK/mTORC1 pathways were also enriched in our RNA-seq data. Interfering with VDR expression, or chelating Ca²⁺ using BAPTA-AM, markedly impacted the activation of PI3K/AKT/mTORC1 and AMPK/mTORC1 pathways, lipid synthesis, and PRV proliferation. In summary, our study demonstrates that PRV infection promotes VDR expression, thereby enhancing Ca²⁺ absorption and activating PI3K/AKT/mTORC1- and AMPK/mTORC1-mediated lipid synthesis. Our findings offer new insights into strategies for PRV prevention.IMPORTANCEVitamin D, beyond its well-known benefits for bone health and immune function, also plays a pivotal role in regulating gene expression through its receptor, the vitamin D receptor (VDR). Although VDR's influence spans multiple biological processes, its relationship with viral infections, particularly pseudorabies virus (PRV), remains underexplored. Our research illustrates a complex interplay where PRV infection boosts VDR expression, which in turn enhances Ca²⁺ absorption, leading to the activation of critical lipid synthesis pathways, PI3K/AKT/mTORC1 and AMPK/mTORC1. These findings not only deepen our understanding of the intricate dynamics between host molecular mechanisms and viral proliferation but also open avenues for exploring new strategies aimed at preventing PRV infection. By targeting components of the VDR-related signaling pathways, we can potentially develop novel therapeutic interventions against PRV and possibly other similar viral infections.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546314","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unraveling the role of UilS, a urea-induced acyl-homoserine lactonase that enhances <i>Serratia marcescens</i> fitness, interbacterial competition, and urinary tract infection.","authors":"Marisel R Tuttobene, Brayan S Arango Gil, Gisela Di Venanzio, Javier F Mariscotti, Rodrigo Sieira, Mario F Feldman, María Soledad Ramirez, Eleonora García Véscovi","doi":"10.1128/mbio.02505-24","DOIUrl":"https://doi.org/10.1128/mbio.02505-24","url":null,"abstract":"<p><p><i>Serratia marcescens</i>, a member of the Enterobacteriaceae family, is an opportunistic human pathogen and a frequent cause of urinary tract infections. Clinical isolates often exhibit resistance to multiple antibiotics, posing challenges for successful treatment. Understanding its pathogenic mechanisms is crucial for elucidating new potential targets to develop effective therapeutic interventions and manage <i>S. marcescens</i> infections. This work identifies <u>u</u>rea-<u>i</u>nduced <u>l</u>actonase of <i><u>S</u>erratia</i> (UilS), a lactonase encoded in the <i>S. marcescens</i> RM66262 strain isolated from a patient with a urinary tract infection. The study explores the bacterium's response to urea, a major component of urine, and its impact on <i>uilS</i> expression. We found that UilS degrades acyl-homoserine lactones (AHL) autoinducers traditionally associated with quorum sensing mechanisms. Surprisingly, UilS is able to degrade self and non-self AHL, exhibiting quorum-quenching activity toward <i>Pseudomonas aeruginosa</i>. We found that LuxR regulates <i>uilS</i> expression that is enhanced in the presence of AHL. In addition, urea-dependent induction of UilS expression is controlled by the transcriptional response regulator CpxR. UilS confers fitness advantage to <i>S. marcescens</i>, especially in the presence of urea, emphasizing the adaptive plasticity of strains to modulate gene expression based on environmental signals and population density. We also discovered a novel bacterial killing capacity of <i>S. marcescens</i> that involves UilS, indicating its importance in the interspecies interaction of <i>Serratia</i>. Finally, we found that a <i>uilS</i> mutant strain displays attenuated colonization in a mouse model of catheter-associated urinary tract infection. <i>uilS</i> is present in clinical but absent in environmental isolates, suggesting an evolutionary adaptation to host-specific selective pressures.</p><p><strong>Importance: </strong>This work reveals the acyl-homoserine lactonase <u>u</u>rea-<u>i</u>nduced <u>l</u>actonase of <i><u>S</u>erratia</i> as a novel virulence factor of <i>Serratia marcescens</i>, unraveling a potential target to develop antimicrobial strategies and shedding light on the complex regulatory network governing pathogenicity and adaptation to host environments.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-throughput phenotype-to-genotype testing of meningococcal carriage and disease isolates detects genetic determinants of disease-relevant phenotypic traits.","authors":"Robeena Farzand, Mercy W Kimani, Evangelos Mourkas, Abdullahi Jama, Jack L Clark, Megan De Ste Croix, William M Monteith, Jay Lucidarme, Neil J Oldfield, David P J Turner, Ray Borrow, Luisa Martinez-Pomares, Samuel K Sheppard, Christopher D Bayliss","doi":"10.1128/mbio.03059-24","DOIUrl":"https://doi.org/10.1128/mbio.03059-24","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Genome-wide association studies (GWAS) with binary or single phenotype data have successfully identified disease-associated genotypes and determinants of antimicrobial resistance. We describe a novel phenotype-to-genotype approach for a major bacterial pathogen that involves simultaneously testing for associations among multiple disease-related phenotypes and linkages between phenotypic variation and genetic determinants. High-throughput assays quantified variation among 163 &lt;i&gt;Neisseria meningitidis&lt;/i&gt; serogroup W ST-11 clonal complex isolates for 11 phenotypic traits. A comparison of carriage and two disease subgroups detected significant differences between groups for eight phenotypic traits. Candidate genotypic testing indicated that indels in &lt;i&gt;csw&lt;/i&gt;, a capsular biosynthesis gene, were associated with reduced survival in antibody-depleted heat-inactivated serum. GWAS testing detected 341 significant genetic variants (3 single-nucleotide polymorphisms and 338 unitigs) across all traits except serum bactericidal antibody-depleted assays. Growth traits were associated with variants of capsular biosynthesis genes, carbonic anhydrase, and an iron-uptake system while adhesion-linked variation was in &lt;i&gt;pilC2&lt;/i&gt;, &lt;i&gt;marR,&lt;/i&gt; and &lt;i&gt;mutS&lt;/i&gt;. Multiple phase variation states or combinatorial phasotypes were associated with significant differences in multiple phenotypes. Controlling for group effects through regression and recursive random forest approaches detected group-independent effects for &lt;i&gt;nalP&lt;/i&gt; with biofilm formation and &lt;i&gt;fetA&lt;/i&gt; with a growth trait. Through random forest testing, nine phenotypes were weakly predictive of MenW:cc11 sub-lineage, original or 2013, for disease isolates while three characteristics separated carriage and disease isolates with &gt;80% accuracy. This study demonstrates the power of combining high-throughput phenotypic testing of pathogenically relevant isolate collections with genomics for identifying genetic determinants of specific disease-relevant phenotypes and the pathobiology of microbial pathogens.IMPORTANCENext-generation sequencing technologies have led to the creation of extensive microbial genome sequence databases for several bacterial pathogens. Mining of these databases is now imperative for unlocking the maximum benefits of these resources. We describe a high-throughput methodology for detecting associations between phenotypic variation in multiple disease-relevant traits and a range of genetic determinants for &lt;i&gt;Neisseria meningitidis&lt;/i&gt;, a major causative agent of meningitis and septicemia. Phenotypic variation in 11 disease-related traits was determined for 163 isolates of the hypervirulent ST-11 lineage and linked to specific single-nucleotide polymorphisms, short sequence variants, and phase variation states. Application of machine learning algorithms to our data outputs identified combinatorial phenotypic traits and genetic variants predictive of a disease association. This app","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546304","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Trehalose polyphleates participate in <i>Mycobacterium abscessus</i> fitness and pathogenesis.","authors":"Silke Malmsheimer, Wassim Daher, Yara Tasrini, Claire Hamela, John Jairo Aguilera-Correa, Christian Chalut, Graham F Hatfull, Laurent Kremer","doi":"10.1128/mbio.02970-24","DOIUrl":"https://doi.org/10.1128/mbio.02970-24","url":null,"abstract":"<p><p>Mycobacteria produce a large repertoire of surface-exposed lipids with major biological functions. Among these lipids, trehalose polyphleates (TPPs) are instrumental in the infection of <i>Mycobacterium abscessus</i> by the therapeutic phage BPs. However, while the biosynthesis and transport of TPPs across the membrane by MmpL10 have been reported, the role of TPPs in host infection remains enigmatic. Here, we addressed whether the loss of TPPs influences interactions with macrophages and the virulence of <i>M. abscessus</i>. As anticipated, the deletion of <i>mmpL10</i> in smooth (S) and rough (R) variants of <i>M. abscessus</i> abrogated TPP production, which was rescued upon gene complementation. Importantly, infection of human THP-1 cells with the <i>mmpL10</i> mutants was associated with decreased intramacrophage survival and a reduced proportion of infected cells. The rough <i>mmpL10</i> mutant showed an impaired capacity to block phagosomal acidification and was unable to co-localize with Galectin-3, a marker of phagosomal membrane damage. This suggests that TPPs participate, directly or indirectly, in phagolysosomal fusion and in phagosomal membrane damage to establish cytosolic communication. The TPP defect that affects the fitness and virulence of <i>M. abscessus</i> was further demonstrated in zebrafish embryos using a rough clinical strain resistant to phage BPs and harboring a frameshift mutation in <i>mmpL10</i>. Infection with this strain was correlated with a slight decrease in embryo survival and a reduced bacterial burden as compared to the corresponding parental and complemented derivatives. Together, these results indicate that TPPs are important surface lipids contributing to the pathogenicity of <i>M. abscessus</i>.IMPORTANCETrehalose polyphleates (TPPs) are complex lipids associated with the mycobacterial cell surface and were identified 50 years ago. While the TPP biosynthetic pathway has been described recently, the role of these lipids in the biology of mycobacteria remains yet to be established. The wide distribution of TPPs across mycobacterial species suggests that they may exhibit important functions in these actinobacteria. Here, we demonstrate that <i>Mycobacterium abscessus,</i> an emerging multidrug-resistant pathogen that causes severe lung diseases in cystic fibrosis patients, requires TPPs for survival in macrophages and virulence in a zebrafish model of infection. These findings support the importance of this underexplored family of lipids in mycobacterial pathogenesis.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546316","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Suppression of ZBP1-mediated NLRP3 inflammasome by the tegument protein VP22 facilitates pseudorabies virus infection.","authors":"Zicheng Ma, Depeng Liu, Wandi Cao, Lei Guo, Kesen Liu, Juan Bai, Xingyi Li, Ping Jiang, Xing Liu","doi":"10.1128/mbio.01945-24","DOIUrl":"https://doi.org/10.1128/mbio.01945-24","url":null,"abstract":"<p><p>The interaction between Z-DNA binding protein 1 (ZBP1) and the NLR family pyrin domain-containing 3 (NLRP3) inflammasome has been uncovered in several viral infections. However, the role of this molecular pathway during infection with the alpha-herpesvirus pseudorabies virus (PRV) remains largely elusive. Here, we report that during PRV infection, ZBP1-mediated NLRP3 inflammasome activation is inhibited by the viral tegument protein VP22, thereby facilitating viral infection. Through a combination of RNA sequencing and genetic studies, we demonstrate that PRV VP22 functions as a virus-encoded virulence factor by evading the inhibitory effects of ZBP1 on virus infection. Importantly, the replication and pathogenicity of a recombinant PRV lacking VP22 are significantly increased in ZBP1-deficient cells and mice. Mechanistically, PRV VP22 interacts with ZBP1, impeding the recruitment of receptor-interacting protein kinase 3 and Caspase-8, thereby inhibiting NLRP3 activation. Furthermore, we show that the N-terminal 1-50 amino acid domain of VP22 dominantly destabilizes ZBP1-mediated function. Taken together, these findings identify a functional link between PRV infection and ZBP1-mediated NLRP3 inflammatory response, providing novel insights into the pathogenesis of PRV and other herpesviruses.</p><p><strong>Importance: </strong>Z-DNA binding protein 1 (ZBP1) functions as a pivotal innate immune sensor that regulates inflammatory cell death during viral infections. However, its role in pseudorabies virus (PRV) infection remains unknown. Here, we demonstrate that ZBP1 serves as a restrictive factor by triggering the activation of the NLR family pyrin domain-containing 3 inflammasome, a process counteracted by PRV-encoded protein VP22. Furthermore, VP22 interferes with the interaction between ZBP1 and receptor-interacting protein kinase 3/Caspase-8, particularly through its N-terminal 1-50 amino acids. Importantly, deficiency in ZBP1 enhances the replication and virulence of recombinant viruses lacking VP22 or its N-terminal 1-50 amino acids. These findings reveal how PRV escapes ZBP1-mediated inflammatory responses during infection, potentially informing the rational design of therapeutic interventions.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546312","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}