mSphere最新文献

{"title":"Establishing an <i>ex vivo</i> porcine skin model to investigate the effects of broad-spectrum antiseptic on viable skin microbial communities.","authors":"E C Townsend, K Xu, K De La Cruz, L Huang, S Sandstrom, J J Meudt, D Shanmuganayagam, A Huttenlocher, A L F Gibson, L R Kalan","doi":"10.1128/msphere.00441-25","DOIUrl":"10.1128/msphere.00441-25","url":null,"abstract":"<p><p>Incomplete antiseptic efficacy against potentially pathogenic microbial taxa places some patients at disproportionate risk for developing a surgical site infection. Laboratory models capable of interrogating the effects of antiseptics on the skin and its complex microbial communities are desperately needed to improve and better tailor antiseptic formulations. This work aims to establish an <i>ex vivo</i> porcine skin model to explore the impact of topical antiseptics on complex skin microbial communities and superficial skin lipids. Microbiome samples were treated with propidium monoazide to selectively evaluate DNA from viable microorganisms. Bacterial abundances were assessed via viability-qPCR and quantitative culture. Viable community populations were evaluated with 16S rRNA gene sequencing. Epidermal biopsies were collected at multiple time points for lipidomic assessment via LC/MS. The <i>ex vivo</i> environment promoted shifts in porcine skin lipid composition and microbial communities over the experiment's duration. Compared to water-treated control skin, skin treated with the antiseptic chlorhexidine gluconate had significantly lower culturable counts and bioburden as determined by viability-qPCR. Compared to water-treated skin, viable microbial communities on CHG-treated skin displayed greater relative abundance of several gut-associated and Gram-negative bacterial taxa, including <i>SMB53</i>, <i>Turicibacter</i>, <i>Pseudomonas</i>, and <i>Proteus</i>. Collectively, these findings highlight the utility of an <i>ex vivo</i> porcine skin system for interrogating the impacts of antimicrobial disruption on complex microbial ecosystems, and ultimately, for the future testing and development of improved antiseptic formulations.</p><p><strong>Importance: </strong>Broad-spectrum antiseptics are widely used to prevent surgical site infection and as wound cleansing agents. The impacts of such agents on beneficial microbes in the skin microbiome are understudied. Here, we describe an <i>ex vivo</i> skin model to test the impacts of antiseptics or other topical agents on the healthy skin microbiome.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0044125"},"PeriodicalIF":3.1,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12482178/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144962209","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":"Fomite workshop recommendations addressing the role of surfaces in virus transmission in the built environment.","authors":"Sarah E Abney, Sally F Bloomfield, Stephanie A Boone, Todd Cutts, Charles P Gerba, M Khalid Ijaz, Amanda K Lyons, Jean-Yves Maillard, Kristina D Mena, Julie McKinney, Geun Woo Park, Elizabeth Scott, Kelly A Reynolds, William A Rutala, Syed A Sattar, David J Weber, Marc P Verhougstraete, Margaret M Williams, William A Furin, Amanda M Wilson, J Carrie Whitworth, Bahram Zargar","doi":"10.1128/msphere.00927-24","DOIUrl":"10.1128/msphere.00927-24","url":null,"abstract":"<p><p>The emergence of SARS-CoV-2 has led to a need to assess the role of fomites in viral transmission within the built environment. Assessing the role of fomites is necessary for developing intervention strategies for controlling emerging pathogens. A fomite workshop with experts was convened in November 2024 by academia, several government agencies, and public health officials to evaluate existing data and discuss how to mitigate risks. Fomite transmission is influenced by the nature of the built environment, population density and proximity, environmental factors (humidity, heat, etc.), virus survival, surface type, engineering controls (ventilation, physical barriers, etc.), and human behaviors. Based on our current data, direct contact with a contaminated surface/fomite, even for respiratory viruses, presents a risk of viral exposure and transmission by both contact with the fomite and resuspension in the air. Even respiratory viruses can be resuspended from fomites following human and pet movement, activities (e.g., vacuuming, toilet flushing, etc.), or changes in ventilation/indoor airflow. After resuspension from surfaces, microbes can be potentially inhaled (contributing to droplet and/or aerosol exposure) and/or re-deposited from primary to secondary fomites. Development of standard methods (molecular, chemical/physical, and infectivity assays) for detecting the presence of viruses on fomites and human behavior modeling would help to determine the most effective infection prevention strategies.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0092724"},"PeriodicalIF":3.1,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12482177/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144962318","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":"Temporally segregated subpopulations of CDT and TcdA producer cells of <i>Clostridioides difficile</i>.","authors":"Sara Ramalhete, Isabel Roseiro, Carolina P Cassona, Carolina Alves Feliciano, Mónica Serrano, Adriano O Henriques","doi":"10.1128/msphere.00186-25","DOIUrl":"10.1128/msphere.00186-25","url":null,"abstract":"<p><p>Most strains of <i>Clostridioides difficile</i> produce two toxins, TcdA and TcdB, which are mainly responsible for the disease symptoms. TcdA and TcdB are coded for by genes in the pathogenicity locus (PaLoc). Some epidemic strains, however, such as R20291, of ribotype 027, additionally produce a binary toxin, CDT, coded for by genes in the CDT locus (CdtLoc). We analyzed the relationship between transcription of the TcdA and CDT encoding genes in strain R20291 using single-cell analysis with orthogonal fluorescence reporters. We found that most of the cells express <i>cdtA,</i> whereas a much smaller fraction expresses <i>tcdA,</i> and most of those also express <i>cdtA</i>. Expression of <i>cdtA</i> begins during exponential growth and persists during the stationary phase of growth, while the main period of <i>tcdA</i> transcription occurs as cells enter the stationary phase. This translates into early synthesis and release of CDT from the cells, whereas production of TcdA is detected mainly from the onset of the stationary phase. Both the PaLoc and CdtLoc also code for regulatory proteins, TcdR, a sigma factor, and CdtR, a response regulator of the LytR family, required for expression of the toxin-encoding genes, respectively. While CdtR contributes to the expression of <i>tcdA</i>, as shown before, we found reduced expression of <i>cdtA</i> in the absence of TcdR. Hence, TcdR and CdtR cross-regulate the PaLoc and the CdtLoc. Finally, and unlike the case for TcdA, we found no evidence for the association of CDT with mature spores.IMPORTANCEThe enteropathogen <i>Clostridioides difficile</i> causes a spectrum of intestinal diseases, ranging from mild diarrhea to severe conditions such as intestinal inflammation, perforation, and sepsis, that may lead to death, primarily through the production of two cytotoxins, TcdA and TcdB. Certain strains, however, such as those of ribotypes 027 and 078, additionally produce a binary toxin, CDT. Here, we employed single-cell analysis to investigate toxin gene expression in epidemic strain R20297 (RT027), commonly associated with severe infections. We found that CDT is synthesized early during growth, while TcdA is produced at the onset of the stationary phase, and that the two populations partially overlap. We also identify cross-regulation between two key regulatory proteins, TcdR and CdtR, which control TcdA/TcdB and CDT production. These insights into the mechanisms of toxin production at the population level may contribute to the development of targeted therapies for managing <i>C. difficile</i> infections and the resulting complications.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0018625"},"PeriodicalIF":3.1,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12482169/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144962402","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":"Erratum for Fattahi and Sadeghi Kalani, \"mRNA vaccine design using the proteome of <i>Theileria annulata</i> through immunoinformatics approaches\".","authors":"Roohollah Fattahi, Behrooz Sadeghi Kalani","doi":"10.1128/msphere.00665-25","DOIUrl":"https://doi.org/10.1128/msphere.00665-25","url":null,"abstract":"","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0066525"},"PeriodicalIF":3.1,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145192277","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fibroblasts enhance the growth and survival of adult feline small intestinal organoids.","authors":"Nicole D Hryckowian, Katelyn Studener, Waneska S Frizzarini, David Arranz-Solís, Roberto Sánchez-Sánchez, Laura J Knoll","doi":"10.1128/msphere.00290-25","DOIUrl":"10.1128/msphere.00290-25","url":null,"abstract":"<p><p>Intestinal organoids are important cell culture models that complement live animal studies of many intestinal pathogens. Adult feline small intestinal organoids are needed for infectious disease research but are difficult to work with due to slow growth and premature senescence. We introduce a method of co-culturing adult feline small intestinal organoids with growth-inhibited human foreskin fibroblast feeder cells to enhance organoid proliferation and survival. With feeder cells, feline jejunal and ileal organoids survived at least 9 months in culture until cryopreservation. Fibroblast supplementation increased the maximum size of cat and mouse intestinal organoids as well. The increased longevity and size of these organoids are a significant improvement on current methods. These organoids also supported pre-sexual development of the medically important parasite <i>Toxoplasma gondii</i>, as evidenced by expression of the merozoite-specific marker GRA11b. This GRA11b positivity was higher in mature cat organoid-derived monolayers grown for 21 days prior to infection, compared with monolayers grown for 10 days. These methods have high potential to reduce the number of cats used for infectious disease research and may be applicable for intestinal cells from other animals that are difficult to culture.IMPORTANCEMany microbial pathogens are acquired orally through contaminated food or water. Being able to model these infections in cell culture has been greatly enhanced by the development of intestinal organoid technology. One of the species that hosts several infections is cats, but cat intestinal organoids have been notoriously difficult to grow. Here, we describe a co-culture method with fibroblast cells that dramatically improves the longevity of adult cat intestinal organoids. These cat organoid cells can support the pre-sexual development stages of the intestinal pathogen <i>Toxoplasma gondii</i>, a parasite whose sexual cycle is restricted to cats and is the reason that pregnant women are told not to change the litter box. These culture conditions will be a resource to study other cat intestinal pathogens and intestinal organoids from other animals that are difficult to culture.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0029025"},"PeriodicalIF":3.1,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12482142/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144799734","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":"Prevalence, diversity, and parasitism of tailed prophages in <i>Vibrio harveyi</i>.","authors":"Ruijie Ma, Huiying Zhong, Rui Zhang","doi":"10.1128/msphere.00228-25","DOIUrl":"10.1128/msphere.00228-25","url":null,"abstract":"<p><p><i>Vibrio harveyi</i>, a pathogenic bacterium, contains prophages that significantly influence its pathogenesis and evolutionary traits. Investigating the prevalence, evolution, and ecological roles of these prophages is of great importance as <i>V. harveyi</i> is responsible for luminous bacteriosis in aquatic organisms. In this study, 13 tailed prophages were identified from 55 globally sourced <i>V. harveyi</i> genomes, with prophage-bacterium junctions precisely annotated. These prophages exhibited distinct parasitic mechanisms, including Mu-type transposition, site-specific recombination, and a plasmid-like non-integrated state, reflecting their adaptive plasticity. Proteome-based phylogenetic analysis classified these prophages into eight subfamilies and nine genera, with half representing novel taxonomic singletons. Network analysis of <i>V. harveyi</i> prophages and a large set of prophages across <i>Vibrio</i> species revealed distinct prophage distribution patterns, including broad cross-species dissemination and clade-specific or strain-specific colonization. Further genomic analysis identified homologs of experimentally validated virulence factors associated with motility and biofilm formation, suggesting a potential role of these prophages in enhancing bacterial pathogenicity and adaptive fitness. CRISPR spacer matching provided the intra-species lytic history for 7 out of 13 identified prophages, underscoring their involvement in horizontal transfer of virulence traits. In summary, this study established a comprehensive genomic database of <i>V. harveyi</i> prophages, shedding light on their diversity, prevalence, and parasitic strategies.IMPORTANCEUnderstanding how prophages parasitize <i>Vibrio harveyi</i> holds significant commercial implications, given the pathogen's notoriety for inducing vibriosis across diverse aquatic species and causing substantial economic losses in the global aquaculture industry. We report here 13 well-curated prophage genomes identified from 55 globally collected <i>V. harveyi</i> genomes. Notably, these prophages exhibited previously unrecognized genomic diversity, along with distinct parasitic strategies and hierarchical distribution patterns. In-depth analysis of their genetic profiles identified multiple homologs of experimentally validated virulence determinants involved in regulating bacterial motility and biofilm formation. Lytic history was detected for over half of these prophages, suggesting their role in driving the dissemination of virulence traits within the species.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0022825"},"PeriodicalIF":3.1,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12482185/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961690","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":"GacA regulates symbiosis and mediates lifestyle transitions in <i>Pseudomonas</i>.","authors":"Youqing Luo, Apsara Srinivas, Casey Guidry, Carolee Bull, Cara H Haney, Corri Hamilton","doi":"10.1128/msphere.00277-25","DOIUrl":"10.1128/msphere.00277-25","url":null,"abstract":"<p><p>Through horizontal gene transfer, closely related bacterial strains assimilate distinct sets of genes, resulting in significantly varied lifestyles. However, it remains unclear how strains properly regulate horizontally transferred virulence genes. We hypothesized that strains may use components of the core genome to regulate diverse horizontally acquired genes. To investigate how closely related bacteria assimilate and activate horizontally acquired DNA, we used a model consisting of strains in the <i>brassicacearum</i>/<i>corrugata</i>/<i>mediterranea</i> (BCM) subclade <i>of Pseudomonas fluorescens</i>, including <i>Pseudomonas</i> species N2E2 and N2C3, which exhibit contrasting lifestyles on the model plant <i>Arabidopsis. Pseudomonas</i> sp. N2E2 is a plant commensal and contains genes encoding biosynthetic enzymes for the antifungal compound 2,4-diacetylphloroglucinol (DAPG). In contrast, <i>Pseudomonas</i> sp. N2C3 lacks DAPG biosynthesis and has gained a pathogenic island encoding syringomycin (SYR)- and syringopeptin (SYP)-like toxins from the plant pathogen <i>Pseudomonas syringae</i>. This causes a transition in lifestyle from plant-protective N2E2 to plant-pathogenic N2C3. We found that N2E2 and N2C3 share a highly conserved two-component system GacA/S, a known regulator of DAPG and SYR/SYP. Using knockout mutations, we found that a <i>ΔgacA</i> mutation resulted in loss of expression of SYR/SYP virulence genes and returned pathogenic N2C3 to a plant commensal lifestyle. Our study further explored the conservation of regulatory control across strains by demonstrating that GacA genes from both distant and closely related <i>Pseudomonas</i> strains could functionally complement one another across the genus.IMPORTANCEEmerging pathogens represent a significant threat to humans, agriculture, and natural ecosystems. Bacterial horizontal gene transfer (HGT) aids in the acquisition of novel genes that facilitate adaptation to new environments. Our work shows a novel role for GacA in orchestrating the regulatory changes necessary for virulence and lifestyle transitions facilitated by HGT. These findings suggest that the GacA/S system plays a key role in mediating transitions across diverse <i>Pseudomonas</i> symbiotic lifestyles. This work provides insights into the mechanisms that drive the emergence of pathogenic strains and highlights potential targets for managing bacterial threats to plant health.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0027725"},"PeriodicalIF":3.1,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12482163/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145015822","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":"Comparative single-cell genomics of two uncultivated <i>Naegleria</i> species harboring <i>Legionella</i> cobionts.","authors":"Jamie McGowan, Estelle S Kilias, James Lipscombe, Elisabet Alacid, Tom Barker, Leah Catchpole, Seanna McTaggart, Sally D Warring, Karim Gharbi, Thomas A Richards, Neil Hall, David Swarbreck","doi":"10.1128/msphere.00352-25","DOIUrl":"10.1128/msphere.00352-25","url":null,"abstract":"<p><p>Amoeboflagellates of the genus <i>Naegleria</i> are free-living protists ubiquitously found in soil and freshwater habitats worldwide. They include the \"brain-eating amoeba\" <i>Naegleria fowleri</i>, an opportunistic pathogen that causes primary amoebic meningoencephalitis, a rare but fatal infection of humans. Beyond their direct pathogenicity, protists can also act as environmental reservoirs for intracellular bacterial pathogens, such as <i>Legionella</i> spp., to persist and multiply in the environment. In this study, we carried out single-cell genome sequencing of two uncultivated <i>Naegleria</i> species isolated from the River Leam in England. From single cells, we generated two highly complete <i>Naegleria</i> genomes. Phylogenetic analyses placed these species as close relatives of <i>Naegleria fultoni</i> and <i>Naegleria pagei</i>. Exploring <i>Naegleria</i> evolutionary genomics, we identified gene families encoding antistasin-like domains, which have been characterized as factors that inhibit coagulation in blood-feeding leeches. Antistasin-like domains were identified in all sequenced <i>Naegleria</i> species and their close relative <i>Willaertia magna,</i> yet are otherwise largely restricted to animal genomes. Significantly, we recovered highly complete bacterial genomes from each <i>Naegleria</i> single-cell sample. Phylogenomic analysis revealed that both bacteria belong to the Legionellaceae family. Both bacterial genomes encode comprehensive sets of secretion systems and effector arsenals. We identified putative <i>Legionella</i> effectors that resemble TAL (Transcription activator-like) effectors from plant pathogenic <i>Xanthomonas</i> spp. in terms of protein sequence and predicted structure, representing a potentially novel class of <i>Legionella</i> effectors. Our study highlights the advantages of single-cell environmental genomics approaches, which enable direct association of intracellular pathogens with their hosts to better understand the evolution of host-pathogen interactions.IMPORTANCEBeyond their direct pathogenic potential, amoebae and other protists found in the environment can indirectly threaten human health by serving as reservoirs for intracellular bacterial pathogens to persist, evolve, and multiply in the environment. Despite their importance, protist-bacterial interactions remain poorly understood. In this study, we employed single-cell genomics to sequence the genomes of two uncultivated <i>Naegleria</i> amoebae, both harboring novel <i>Legionella</i> bacteria. From individual cells, we recovered highly complete eukaryotic and bacterial cobiont genome assemblies. Our work demonstrates the power of single-cell sequencing approaches in directly linking intracellular pathogens to their hosts to better understand the evolution of protist-bacterial interactions and the role that protists play in facilitating bacterial pathogens to persist long term in the environment.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0035225"},"PeriodicalIF":3.1,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12482156/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144962267","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":"Analysis of <i>Treponema pallidum</i> subsp. <i>pallidum</i> predicted outer membrane proteins (OMPeomes) in 21 clinical samples: variant sequences are predominantly surface-exposed.","authors":"Petra Pospíšilová, Pavla Fedrová, Eliška Vrbová, Christopher M Hennelly, Farhang Aghakhanian, Kelly L Hawley, Everton B Bettin, Timothy C Davenport, Sylvia M Bruisten, Hélène C A Zondag, Philippe A Grange, Nicolas Dupin, Natasha Arora, Angel A Noda, Arlene C Seña, Melissa J Caimano, Juan C Salazar, Jonathan J Juliano, M Anthony Moody, Justin D Radolf, Jonathan B Parr, David Šmajs","doi":"10.1128/msphere.00213-25","DOIUrl":"10.1128/msphere.00213-25","url":null,"abstract":"<p><p>The incidence of syphilis, a sexually transmitted disease caused by the <i>Treponema pallidum</i> subsp. <i>pallidum</i> (TPA), has been surging globally despite effective antibiotic therapy. A new strategy for syphilis control is the development of a multi-component syphilis vaccine with global efficacy, which requires the identification of surface-exposed candidate vaccinogens and the determination of their antigenic diversity within circulating TPA strains. To improve the quality of sequences from repetitive and paralogous regions of the TPA genome, we have developed a sequencing scheme that allows amplification and long-read sequencing of 25 targets encoding TPA proteins including 15 outer membrane proteins. We tested this approach on a set of 21 clinical TPA strains, mostly of European origin preselected by MLST typing. A total of 462 (88%) of 525 amplicons were sequenced. Of 58 new alleles identified in comparison to the SS14 and Nichols TPA reference strains, the majority encoded new protein sequences (<i>n</i> = 55; 94.8%). The 55 variant protein sequences were encoded by 99 individual TPA loci, where single amino acid replacements occurred most frequently (<i>n</i> = 50), followed by replacements of two to three amino acids (<i>n</i> = 35) and differences comprising four or more residues (<i>n</i> = 14); the latter included six intra-strain recombination events. Most differences were localized to predicted surface-exposed regions, consistent with adaptive evolution of bacterial determinants that function at the host-pathogen interface. Clinical strains having the same allelic profiles from different localities differed in several loci, suggesting that geographical origin significantly contributes to genetic diversity of circulating strains.IMPORTANCEOur findings underscore the importance of analyzing TPA clinical samples isolated from diverse geographical regions in order to understand TPA OMP variability.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0021325"},"PeriodicalIF":3.1,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12482175/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144962295","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":"TgAP2X-7 is a novel cell cycle-regulated transcription factor that plays an essential role in <i>Toxoplasma</i> tachyzoite propagation.","authors":"Padmaja Mandadi, Ramu Anandakrishnan, Rajshekhar Y Gaji","doi":"10.1128/msphere.00438-25","DOIUrl":"10.1128/msphere.00438-25","url":null,"abstract":"<p><p>Apicomplexan AP2 (ApiAP2) family proteins are a family of transcription factors that are known to regulate gene expression in apicomplexan pathogens, including <i>Toxoplasma</i>. In this study, we focused on TgAP2X-7, a member of the APiAP2 family that is predicted to be essential for <i>Toxoplasma</i> fitness. Endogenous tagging of TgAP2X-7 followed by immunofluorescence analysis revealed that it's a cell cycle-regulated nuclear protein with peak expression in the G1 phase. Since TgAP2X-7 is predicted to be essential for parasite growth, we adopted an auxin-inducible degron (AID) based conditional knockdown approach to understand its function. Phenotypic analysis of the conditional knockdown mutant of TgAP2X-7 showed that the protein is indeed essential for <i>Toxoplasma</i> propagation <i>in vitro,</i> and loss of this transcription factor results in a major defect in invasion and a minor defect in replication. Examination with cell division markers indicated that the absence of TgAP2X-7 results in defects in endodyogeny. Transcriptomic analysis indicated that loss of TgAP2X-7 leads to dysregulation of global gene expression in the parasite, including genes required for host-cell invasion, metabolism, and gene expression. Additionally, Cleavage Under Targets and Tagmentation (CUT&TAG) analysis suggests that TgAP2X-7 likely binds to an 11 bp motif [C/T/G]GCATGCA[G/C/A][C/T/G][G/A] in the parasite genes' promoter region. Together, these findings suggest that TgAP2X-7 is a novel transcriptional regulator in <i>Toxoplasma</i> that governs the expression of genes required for parasite propagation.IMPORTANCE<i>Toxoplasma gondii</i> is a protozoan parasite that can cause life-threatening disease in mammals; hence, identifying key factors required for parasite growth and pathogenesis is important to develop novel therapeutics. In this study, we identify and characterize a member of the Apicomplexan AP2 (ApiAP2) family, TgAP2X-7, a developmentally regulated transcription factor. By generating conditional mutant TgAP2X-7, we show that this protein is required for <i>Toxoplasma</i> propagation <i>in vitro,</i> and the absence of this protein results in parasites with significantly reduced competency in invasion, moderate deficiency in replication, and defects in cell division. Importantly, TgAP2X-7-deficient parasites show global changes in gene expression profile, including decreased expression of genes important for <i>Toxoplasma</i> entry into the host cell. Additionally, we identified an 11 bp DNA motif likely recognized by this transcription factor. Hence, this study provides an initial insight into the function of a novel cell cycle-regulated transcription factor essential for <i>Toxoplasma</i> growth.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0043825"},"PeriodicalIF":3.1,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12482157/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145015899","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}