mSphere最新文献

{"title":"Deletion of <i>RAP1</i> affects iron homeostasis, azole resistance, and virulence in <i>Candida albicans</i>.","authors":"Min-Chi Yang, Wei-Luen Huang, Hsuan-Yu Chen, Shin-Huey Lin, Yu-Shan Chang, Kuo-Yun Tseng, Hsiu-Jung Lo, I-Ching Wang, Chi-Jan Lin, Chung-Yu Lan","doi":"10.1128/msphere.00155-25","DOIUrl":"10.1128/msphere.00155-25","url":null,"abstract":"<p><p>Rap1 is a DNA-binding protein conserved from yeast to mammals for its role in telomeric maintenance. Here, to explore additional functions of <i>Candida albicans</i> Rap1, we performed RNA sequencing analysis. Experimental validations further showed that Rap1 plays a role in iron regulation, especially under low-iron conditions. Moreover, Rap1 was involved in iron acquisition and modulation of iron-related genes. Rap1 was found to be associated with fluconazole resistance in a low-iron condition. Finally, we demonstrated that the deletion of <i>RAP1</i> leads to reduced <i>C. albicans</i> virulence in a mouse model of infection. Together, this study reveals new functions of <i>C. albicans</i> Rap1, particularly in iron homeostasis, azole resistance, and virulence.</p><p><strong>Importance: </strong><i>Candida albicans</i> is an important pathogenic fungus that can cause superficial to life-threatening infections. Iron is essential for almost all organisms, yet it is highly restricted within the human host to defend against pathogens. To grow and survive in the iron-limited host environment, <i>C. albicans</i> has evolved multiple iron acquisition mechanisms. Understanding the regulation of iron homeostasis is, therefore, critical for elucidating <i>C. albicans</i> pathogenesis and virulence. This study explores the novel functions of <i>C. albicans</i> Rap1, with a focus on its contribution to iron acquisition and utilization. Our findings further highlight how iron availability impacts antifungal resistance and virulence through Rap1, providing insight into the complex iron regulatory machinery of <i>C. albicans</i>.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0015525"},"PeriodicalIF":3.7,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12108065/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144009034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Antibody-guided identification of <i>Achromobacter xylosoxidans</i> protein antigens in cystic fibrosis.","authors":"Cecilia Sahl, Sounak Chowdhury, Johan Malmström, Lisa I Påhlman","doi":"10.1128/msphere.00233-25","DOIUrl":"10.1128/msphere.00233-25","url":null,"abstract":"<p><p>Persistent bacterial airway infection is a hallmark feature of cystic fibrosis (CF). <i>Achromobacter</i> spp. are gram-negative rods that can cause persistent airway infection in people with CF (pwCF), but the knowledge of host immune responses to these bacteria is limited. The aim of this study was to investigate if patients develop antibodies against <i>Achromobacter xylosoxidans</i>, the most common <i>Achromobacter</i> species, and to identify the bacterial antigens that induce specific IgG responses. Seven serum samples from pwCF with <i>Achromobacter</i> infection were screened for antibodies against bacteria in an ELISA coated with <i>A. xylosoxidans</i>, <i>A. insuavis,</i> or <i>Pseudomonas aeruginosa</i>. Sera from pwCF with or without <i>P. aeruginosa</i> infection (<i>n</i> = 22 and 20, respectively) and healthy donors (<i>n</i> = 4) were included for comparison. Serum with high titers to <i>A. xylosoxidans</i> was selected for affinity purification of bacterial antigens using serum IgGs bound to protein G beads. The resulting IgG-antigen complexes were then analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Selected antigens of interest were produced in recombinant form and used in an ELISA to confirm the results. Four of the seven patients with <i>Achromobacter</i> infection had serum antibodies against <i>Achromobacter</i>. Using patient serum-IgG for affinity purification of <i>A. xylosoxidans</i> proteins, we identified eight antigens. Three of these, which were not targeted by anti-<i>P</i>. <i>aeruginosa</i> antibodies, were expressed recombinantly for further validation: dihydrolipoyl dehydrogenase (DLD), type I secretion C-terminal target domain-containing protein, and domain of uncharacterized function 336 (DUF336). While specific IgG against all three recombinant antigens was confirmed in the patient serum with high titers against <i>Achromobacter</i>, DLD and DUF336 showed the least binding to serum IgG from pwCF without <i>Achromobacter</i> spp. infection. Using serum IgG affinity purification in combination with LC-MS/MS and confirming the results using ELISA against recombinant proteins, we have identified bacterial antigens from <i>A. xylosoxidans</i>.IMPORTANCE<i>Achromobacter</i> species are opportunistic pathogens that can cause airway infections in people with cystic fibrosis. In this patient population, persistent <i>Achromobacter</i> infection is associated with low lung function, but the knowledge about bacterial interactions with the host is currently limited. In this study, we identify protein antigens that induce specific antibody responses in the host. The identified antigens may potentially be useful in serological assays, serving as a complement to culturing methods for the diagnosis and surveillance of <i>Achromobacter</i> infection.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0023325"},"PeriodicalIF":3.7,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12108089/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144036006","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Effect of urolithin A on intracellular survival of <i>Mycobacterium tuberculosis</i> by regulating AKT-FOXO1-mediated autophagy.","authors":"Jing Bi, Li Song, Qinglong Guo, Xi Chen, Yaqi Gong, Haojia Wu, Fan Zhang, Jingbin Wang, Guoliang Zhang","doi":"10.1128/msphere.00061-25","DOIUrl":"10.1128/msphere.00061-25","url":null,"abstract":"<p><p>Tuberculosis (TB), resulting from <i>Mycobacterium tuberculosis</i> (Mtb), is one of the leading causes of morbidity and mortality in humans worldwide. Host-directed therapy (HDT) is a novel approach for treating TB, particularly those with drug resistance. Urolithin A (UroA) produced through bioconversion of plant-derived ellagic acid by gut microbes has been proven to have multiple beneficial effects in a variety of diseases without showing undesired adverse reactions. However, whether UroA has antimycobacterial effect and the underlying mechanism has not yet been reported. Here, we found that UroA significantly inhibited Mtb growth within both macrophages and mice. Moreover, UroA promoted the activation of autophagy in Mtb-infected macrophages via the protein kinase B-Forkhead box protein O1 signaling pathway, which contributed to the antimycobacterial effect of UroA. Additionally, UroA suppressed the survival of clinically isoniazid (INH)-resistant Mtb (C2) within macrophages, and the combination of UroA and INH synergistically enhanced host elimination of Mtb H37Rv. Therefore, UroA may be utilized as a potential candidate for HDT and as an adjunctive therapy with first-line anti-TB drugs.IMPORTANCEHost-directed therapy (HDT) is a novel approach for treating tuberculosis (TB), particularly those with drug resistance. Urolithin A (UroA) produced through bioconversion of plant-derived ellagic acid by gut microbes has been proven to have multiple beneficial effects in a variety of diseases without showing undesired adverse reactions. We found that UroA significantly inhibited <i>Mycobacterium tuberculosis</i> (Mtb) growth within macrophages. Moreover, UroA suppressed the survival of clinically isoniazid (INH)-resistant Mtb (C2) within macrophages, and the combination of UroA and INH synergistically enhanced host elimination of Mtb H37Rv. Therefore, UroA may be utilized as a potential candidate for HDT and as an adjunctive therapy with first-line anti-TB drugs.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0006125"},"PeriodicalIF":3.7,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12108056/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144011800","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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 droplet digital PCR assay to measure pilin antigenic variation frequency in <i>Neisseria gonorrhoeae</i>.","authors":"Sarah J Quillin, Di Luo, Aoife Gavagan, Arthur Prindle, H Steven Seifert","doi":"10.1128/msphere.00094-25","DOIUrl":"10.1128/msphere.00094-25","url":null,"abstract":"<p><p>The strict human pathogen <i>Neisseria gonorrhoeae</i> (gonococcus [Gc]) infects an estimated 82 million individuals globally and is a World Health Organization-designated bacterial pathogen of public health importance due to escalating antimicrobial resistance. Gc vaccines have been hindered by Gc's ability to evade immune surveillance in part by varying its major surface antigens like the type IV pilus. We developed a quick and precise method for measuring pilin antigenic variation (Av) frequency using droplet digital PCR (ddPCR) technology. Two fluorescent probes were designed to detect either the hypervariable tail region of silent pilin locus <i>pilS</i>3-copy 1 (S3C1) or a sequence conserved in all <i>pilE</i> variants (CYS2). The appropriate frequency of pilin antigenic variation is measured by the proportion of <i>pilE</i> amplicons carrying the recombinant S3C1 copy relative to the total pilE amplicons measured by CYS2. The ddPCR assay is specific for RecA-dependent pilin antigenic variation. The reduced frequency of pilin Av in strains lacking RecA-modulating recombination protein RecX and the DNA helicase RecQ confirms the ability of the assay to measure changes in pilin Av frequency. We used the ddPCR assay to determine that pilin Av frequency is altered by the colony densities on a solid medium. The ddPCR assay is an accurate, efficient way to measure Gc pilin Av frequency.</p><p><strong>Importance: </strong>Gonorrhea is a sexually transmitted infectious disease of the human genital and nasopharyngeal mucosa caused by the host-restricted bacterium <i>Neisseria gonorrhoeae</i>. The rise of antibiotic-resistant gonorrhea is an urgent global threat to public health. Pilus antigenic variation is a gene conversion process that allows <i>N. gonorrhoeae</i> to evade host immune surveillance, and a mechanistic understanding of this process is crucial to understanding <i>N. gonorrhoeae</i> pathogenesis. This report shows that we can adopt a digital PCR methodology to quickly and accurately measure pilin antigenic variation.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0009425"},"PeriodicalIF":3.7,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12108084/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143973016","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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>Chlamydia trachomatis</i> TmeA promotes pedestal-like structure formation through N-WASP and TOCA-1 interactions.","authors":"Alix McCullough, C A Jabeena, Steve Huang, Brianna Steiert, Robert Faris, Mary M Weber","doi":"10.1128/msphere.00101-25","DOIUrl":"10.1128/msphere.00101-25","url":null,"abstract":"<p><p><i>Chlamydia trachomatis</i> (<i>C.t</i>.) is the causative agent of several human diseases, including the sexually transmitted infection chlamydia and the eye infection trachoma. As an obligate intracellular bacterial pathogen, invasion is critical for establishing infection and subsequent pathogenesis. During invasion, <i>C.t</i>. secretes effector proteins via its type III secretion system (T3SS), which manipulate host actin cytoskeletal regulation to promote bacterial entry. Previous studies identified the T3SS effector protein TmeA as a key factor in <i>C.t</i>. invasion, as it recruits and activates N-WASP. This interaction, in turn, activates the Arp2/3 complex, driving cytoskeletal rearrangements at the invasion site to drive <i>C.t</i>. uptake. In this study, we define the role of the N-WASP CRIB domain in mediating this interaction and demonstrate that TmeA functions as a mimic of Cdc42 as part of its established role in activating N-WASP. Additionally, we identified TOCA-1 as another host protein that directly interacts with TmeA. In other bacterial pathogens, notably an enterohemorrhagic <i>E. coli</i>, N-WASP and TOCA-1 are hijacked to mediate pedestal formation. Using siRNA-mediated knockdown of N-WASP and TOCA-1, followed by transmission electron microscopy, we found that both proteins are important for <i>C.t</i>.-mediated pedestal-like structure formation. Collectively, these findings expand our understanding of the intricacies of <i>C.t</i>. invasion, highlighting how TmeA-mediated interactions with N-WASP and TOCA-1 contribute to pedestal-like structure formation, which may represent an early step in <i>C.t</i>. infection.</p><p><strong>Importance: </strong><i>Chlamydia trachomatis</i> (<i>C.t.</i>) is an obligate intracellular bacterial pathogen that poses a significant threat to human health, being associated with various diseases, including chlamydia-the most prevalent bacterial sexually transmitted infection-and trachoma. Although often asymptomatic, chlamydia infections can lead to severe complications, such as infertility, ectopic pregnancy, and an increased risk of cervical and ovarian cancers. As an intracellular pathogen, host cell invasion is critical for <i>C.t.</i> survival and pathogenesis. In this study, we provide new insights into the interactions between the <i>C.t.</i> invasion effector protein TmeA and the host proteins N-WASP and TOCA-1, revealing that both host proteins are involved in pedestal-like structure formation during early stages of <i>C.t.</i> infection. These findings deepen our understanding of the mechanisms underlying TmeA-mediated host cell invasion and highlight a key pathway contributing to <i>C.t.</i>-mediated pathogenesis.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0010125"},"PeriodicalIF":3.7,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12108077/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144032126","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterizing antimicrobial activity of environmental <i>Streptomyces</i> spp. and oral bacterial and fungal isolates from <i>Canis familiaris</i> and <i>Felis catus</i>.","authors":"Audrey Cowen, Bonnie Yiu, Sara Fallah, Kirsten J Meyer, Emily Puumala, Yunjin Lee, Haley L Zubyk, Nicole Robbins, Justin R Nodwell, Jessie MacAlpine, Leah E Cowen","doi":"10.1128/msphere.00098-25","DOIUrl":"10.1128/msphere.00098-25","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Antimicrobials are a pillar of modern medicine, yet our limited arsenal of antibiotics and antifungals is currently threatened by widespread drug resistance. Ongoing efforts are focused on developing strategies to identify compounds that enhance the efficacy of current antimicrobials and develop novel, resistance-evasive therapeutic strategies. In this study, we characterized microbial isolates from two distinct environments to identify those that exhibit antimicrobial activity alone and in combination with current antimicrobials: (i) oral isolates from domesticated animals and (ii) environmental &lt;i&gt;Streptomyces&lt;/i&gt; spp. First, conditioned media prepared from bacterial and fungal oral isolates that were collected from &lt;i&gt;Canis familiaris&lt;/i&gt; and &lt;i&gt;Felis catus&lt;/i&gt; were screened for antibacterial and antifungal activity. Three supernatants from bacterial isolates exhibited antifungal activity against the human fungal pathogen &lt;i&gt;Candida albicans&lt;/i&gt; in the presence of subinhibitory concentrations of fluconazole, the most widely deployed antifungal. Additionally, two bacterial isolates displayed antibacterial activity against &lt;i&gt;Escherichia coli&lt;/i&gt; alone and in combination with the antibacterial ampicillin. Furthermore, 32 environmental isolates of confirmed and predicted &lt;i&gt;Streptomyces&lt;/i&gt; spp. were screened for activity against &lt;i&gt;C. albicans&lt;/i&gt; and &lt;i&gt;E. coli&lt;/i&gt;. Cell-free media harvested from isolates WAC5038 and WAC5287 exhibited antifungal activity against &lt;i&gt;Candida&lt;/i&gt; spp., while only the WAC5038-conditioned medium displayed antibacterial activity. Bioactivity-guided fractionation, coupled with UV/Vis absorbance spectra, suggested that the bioactive compound in WAC5287 has a similar absorbance spectrum to the antifungal class of polyenes, while the bioactive component of WAC5038 remains unknown. Overall, this work highlights a strategy to collect and screen environmental isolates for the identification of novel antimicrobials.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Importance: &lt;/strong&gt;The emergence and spread of antimicrobial resistance presents a global health challenge. As such, researchers are focused on developing pipelines to discover novel antimicrobials. In this study, we screened two distinct collections of microbes for antimicrobial activity. First, we collected bacterial and fungal isolates from the oral cavities of domesticated dogs and cats and identified these isolates using 16S (bacteria) and ITS (fungi) sequencing. Follow-up analyses confirmed that some conditioned media from bacterial isolates had antibacterial activity against &lt;i&gt;Escherichia coli&lt;/i&gt; and antifungal activity against &lt;i&gt;Candida albicans&lt;/i&gt; both alone and in combination with the current antimicrobial drugs. Additionally, screening 32 predicted or confirmed Streptomyces environmental isolates for antifungal and antibacterial activity identified two isolates with antifungal activity (WAC5038 and WAC5287), with only one isolate demonstrating antibacterial activity (WAC5038).","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0009825"},"PeriodicalIF":3.7,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12108073/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143983285","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Role of the multiple telomeric repeat arrays in integration, persistence, and efficacy of the commercial CVI988 vaccine.","authors":"Luca D Bertzbach, Yu You, Tereza Vychodil, Ahmed Kheimar, Lisa Kossak, Mohammad A Sabsabi, Andelé M Conradie, Benedikt B Kaufer","doi":"10.1128/msphere.00142-25","DOIUrl":"10.1128/msphere.00142-25","url":null,"abstract":"<p><p>Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes fatal T cell lymphomas in chickens. Oncogenic MDV strains can integrate their genome into the host telomeres of latently infected and tumor cells. This integration process is facilitated by telomeric repeat arrays (TMR) present at the ends of the MDV genome, which consist of the hexanucleotide (TTAGGG)_<i>n</i> that is identical to host telomere sequences. In addition, integration of the virus genome is crucial for the development of lymphomas. Live-attenuated vaccines play a vital role in protecting chickens against this deadly disease, yet our understanding of their biology remains limited. Intriguingly, the commercial gold standard MDV vaccine, the live-attenuated MDV strain CVI988, also possesses TMR at the ends of its genome. In this study, we investigated the role of the multiple TMR arrays (mTMR) in vaccine virus integration, latency, reactivation, and protection against very virulent MDV. Our data revealed that the mTMR present in CVI988 are important for virus genome integration and maintenance in latently infected cells <i>in vitro</i>. In addition, virus latency, reactivation, and vaccine efficacy were reduced in an mTMR deleted mutant compared to the wild-type vaccine. These results provide valuable insights into the biology of this important vaccine virus and shed light on the roles of the mTMR in vaccine integration, latency, and protection against very virulent MDV.IMPORTANCEMarek's disease virus (MDV) is an oncogenic herpesvirus and causes lethal lymphomas in chickens. The gold standard vaccine is the live-attenuated MDV strain CVI988 (a.k.a. Rispens). CVI988 is extensively used in chickens worldwide due to its high efficacy in preventing disease and lymphomas. The CVI988 vaccine harbors telomere arrays (TMR) at the ends of its genome. TMR facilitate genome integration of oncogenic MDV strains into the host telomeres. This study provides critical insights into the biology of the widely used MDV vaccine strain CVI988, demonstrating the crucial role of mTMR in viral genome integration, latency, and protection against very virulent MDV. Furthermore, our findings enhance the understanding of MDV vaccine biology and may guide future strategies to improve Marek's disease control.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0014225"},"PeriodicalIF":3.7,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12108085/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144030085","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"TEAL-Seq: targeted expression analysis sequencing.","authors":"Georgia Doing, Priya Shanbhag, Isaac Bell, Sara Cassidy, Efthymios Motakis, Elizabeth Aiken, Julia Oh, Mark D Adams","doi":"10.1128/msphere.00984-24","DOIUrl":"10.1128/msphere.00984-24","url":null,"abstract":"<p><p>Metagenome sequencing enables the genetic characterization of complex microbial communities. However, determining the activity of isolates within a community presents several challenges, including the wide range of organismal and gene expression abundances, the presence of host RNA, and low microbial biomass at many sites. To address these limitations, we developed \"targeted expression analysis sequencing\" or TEAL-seq, enabling sensitive species-specific analyses of gene expression using highly multiplexed custom probe pools. For proof of concept, we targeted about 1,700 core and accessory genes of <i>Staphylococcus aureus</i> and <i>S. epidermidis</i>, two key species of the skin microbiome. Two targeting methods were applied to laboratory cultures and human nasal swab specimens. Both methods showed a high degree of specificity, with >90% reads on target, even in the presence of complex microbial or human background DNA/RNA. Targeting using molecular inversion probes demonstrated excellent correlation in inferred expression levels with bulk RNA-seq. Furthermore, we show that a linear pre-amplification step to increase the number of nucleic acids for analysis yielded consistent and predictable results when applied to complex samples and enabled profiling of expression from as little as 1 ng of total RNA. TEAL-seq is much less expensive than bulk metatranscriptomic profiling, enables detection across a greater dynamic range, and uses a strategy that is readily configurable for determining the transcriptional status of organisms in any microbial community.IMPORTANCEThe gene expression patterns of bacteria in microbial communities reflect their activity and interactions with other community members. Measuring gene expression in complex microbiome contexts is challenging, however, due to the large dynamic range of microbial abundances and transcript levels. Here we describe an approach to assessing gene expression for specific species of interest using highly multiplexed pools of targeting probes. We show that an isothermal amplification step enables the profiling of low biomass samples. TEAL-seq should be widely adaptable to the study of microbial activity in natural environments.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0098424"},"PeriodicalIF":3.7,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12108068/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144021195","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Meningococcal vaccine 4CMenB elicits a robust cellular immune response that targets but is not consistently protective against <i>Neisseria gonorrhoeae</i> during murine vaginal infection.","authors":"Joseph J Zeppa, Jamie E Fegan, Pauline Maiello, Epshita A Islam, Isaac S Lee, Christine Pham, Laura-Lee Caruso, Scott D Gray-Owen","doi":"10.1128/msphere.00940-24","DOIUrl":"10.1128/msphere.00940-24","url":null,"abstract":"<p><p>Retrospective epidemiological studies suggest that the licensed serogroup B meningococcal vaccine 4CMenB (Bexsero) provides some protection against the closely related pathogen <i>Neisseria gonorrhoeae</i> in humans. This result has been replicated in murine models of gonococcal colonization, with a gonococci-reactive humoral response and more rapid clearance of vaginal infection. However, immunization with 4CMenB consistently elicits a robust humoral response but does not protect all individuals; hence, the correlates of protection remain undefined. Herein, we exploit the fact that 4CMenB promotes gonococcal clearance in only a subset of immunized mice to perform a broad analysis of the adaptive response in animals that are or are not protected. We observe that 4CMenB vaccination induces high levels of anti-neisserial antibodies in both serum and the vaginal lumen, and a robust cellular response highlighted by an increase in both conventional naïve and memory populations as well as unconventional lymphocyte subsets. Multiplex and flow cytometry results show that 4CMenB vaccination generates a robust, multi-faceted cytokine response that spans numerous T cell subsets (T_H1, T_H2, T_reg, and T_H17) and that non-T non-B lymphocytes play an important role in this response, as indicated by an unbiased principal component analysis. Together, this work provides the first comprehensive analysis of the robust humoral and complex cellular response to 4CMenB so as to reveal the effector mechanisms that may contribute to immunity against vaginal gonococcal infection.IMPORTANCEGonorrhea, a sexually transmitted infection caused by the human-specific pathogen <i>Neisseria gonorrhoeae</i> (Ngo), is a growing public health concern due to its rise in prevalence and increasing antibiotic resistance against first-line agents. There is currently no vaccine available for this important bacterium due, in part, to our lack of understanding of immune correlates of protection. Interestingly, a human-approved vaccine (4CMenB; Bexsero) against a related pathogen (<i>N. meningitidis</i>; a cause of meningitis) has demonstrated some protection against gonorrhea in epidemiologic studies. Herein, we provide the first detailed analysis of cellular and antibody-mediated immune responses to this vaccine in animals protected against gonococcal colonization. These findings provide new understanding regarding immune correlates of protection against <i>N. gonorrhoeae</i>, providing new insight into immune protection and helping guide the development of a much-needed vaccine.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0094024"},"PeriodicalIF":3.7,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12108064/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144019829","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Signal sequences target enzymes and structural proteins to bacterial microcompartments and are critical for microcompartment formation.","authors":"Elizabeth R Johnson, Nolan W Kennedy, Carolyn E Mills, Shiqi Liang, Swetha Chandrasekar, Taylor M Nichols, Grant A Rybnicky, Danielle Tullman-Ercek","doi":"10.1128/msphere.00962-24","DOIUrl":"10.1128/msphere.00962-24","url":null,"abstract":"<p><p>Spatial organization of pathway enzymes has emerged as a promising tool to address several challenges in metabolic engineering, such as flux imbalances and off-target product formation. Bacterial microcompartments (MCPs) are a spatial organization strategy used natively by many bacteria to encapsulate metabolic pathways that produce toxic, volatile intermediates. Several recent studies have focused on engineering MCPs to encapsulate heterologous pathways of interest, but how this engineering affects MCP assembly and function is poorly understood. In this study, we investigated the role of signal sequences, short domains that target proteins to the MCP core, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterized two novel Pdu signal sequences on the structural proteins PduM and PduB, which constitute the first report of metabolosome signal sequences on structural proteins rather than enzymes. We then explored the role of enzymatic and structural Pdu signal sequences on MCP assembly by deleting their encoding sequences from the genome alone and in combination. Deleting enzymatic signal sequences decreased the MCP formation, but this defect could be recovered in some cases by overexpressing genes encoding the knocked-out signal sequence fused to a heterologous protein. By contrast, deleting structural signal sequences caused similar defects to knocking out the genes encoding the full-length PduM and PduB proteins. Our results contribute to a growing understanding of how MCPs form and function in bacteria and provide strategies to mitigate assembly disruption when encapsulating heterologous pathways in MCPs.IMPORTANCESpatially organizing biosynthetic pathway enzymes is a promising strategy to increase pathway throughput and yield. Bacterial microcompartments (MCPs) are proteinaceous organelles that many bacteria natively use as a spatial organization strategy to encapsulate niche metabolic pathways, providing significant metabolic benefits. Encapsulating heterologous pathways of interest in MCPs could confer these benefits to industrially relevant pathways. Here, we investigate the role of signal sequences, short domains that target proteins for encapsulation in MCPs, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterize two novel signal sequences on structural proteins, constituting the first Pdu signal sequences found on structural proteins rather than enzymes, and perform knockout studies to compare the impacts of enzymatic and structural signal sequences on MCP assembly. Our results demonstrate that enzymatic and structural signal sequences play critical but distinct roles in Pdu MCP assembly and provide design rules for engineering MCPs while minimizing disruption to MCP assembly.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0096224"},"PeriodicalIF":3.7,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12108088/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144030088","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}