mSphere最新文献

{"title":"Isolation of a highly virulent colibactin-positive tumor-promoting strain of <i>Escherichia coli</i> from the gut microbiota of an adult.","authors":"Allison M Weis, O'Connor J Matthews, Rickesha Bell, Nicole Lynn Pershing, Annika Dankwardt, Brittany A Fleming, Biljana Gigic, Martin Schneider, Sheetal Hardikar, Adetunji T Toriola, David Shibata, Christopher I Li, Doratha A Byrd, W Zac Stephens, Cornelia M Ulrich, Matthew A Mulvey, June L Round","doi":"10.1128/msphere.00219-26","DOIUrl":"https://doi.org/10.1128/msphere.00219-26","url":null,"abstract":"<p><p>Recent studies have pointed to critical roles for microbes in both exacerbation of and protection from the development of colon cancer. While much has been learned, the field remains understudied, with functional studies available for only a handful of bacteria. To identify novel microbes associated with colorectal cancer (CRC) development, we employed a preclinical chemical carcinogenesis CRC mouse model using germ-free mice that were colonized with human microbiotas. During the course of these studies, we identified a microbiota that exacerbated CRC, from which we isolated an <i>Escherichia coli</i> strain that had disseminated to the mouse kidneys. This strain, which we designated as AW001, was genetically similar to the reference adherent-invasive <i>E. coli</i> (AIEC) strain NC101 and encoded the DNA-damaging toxin colibactin. In relevant animal models, AW001 worsened both colitis and sepsis, making it a colitogenic AIEC-like strain with the capacity to cause invasive systemic infections similar to extraintestinal pathogenic <i>E. coli</i> (ExPEC). This strain will be a relevant tool to study human-associated intestinal <i>E. coli</i> strains capable of causing disease in mice.IMPORTANCEColorectal cancer (CRC) is a significant burden on human health. A growing body of work has pointed to critical roles for microbes in the exacerbation of and protection from the development of CRC. Specific <i>Escherichia coli</i> strains can produce colibactin, a genotoxin that has been implicated in exacerbating CRC. In this study, we tested human microbiotas in a mouse model of CRC and isolated a colibactin<i>-</i>positive <i>Escherichia</i> coli strain that led to tumorigenesis, disseminated from the gut to the mouse kidneys, caused death, and worsened both colitis and sepsis in murine models. Identification of this strain enhances our collective knowledge and adds an important tool for future studies on the role of microbes and CRC tumorigenesis.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0021926"},"PeriodicalIF":3.1,"publicationDate":"2026-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840399","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":"Identification of the Annexin A2-interacting domain of pneumococcal PsaA.","authors":"Prattay Dey, Yoonsung Hu, Faith Henson, Chaeyoung Kim, Nogi Park, Keun Seok Seo, Justin A Thornton","doi":"10.1128/msphere.00232-26","DOIUrl":"https://doi.org/10.1128/msphere.00232-26","url":null,"abstract":"<p><p><i>Streptococcus pneumoniae</i> is a gram-positive bacterium, colonizer of the human nasopharynx capable of causing severe invasive disease. Colonization of the nasopharynx is a prerequisite for the development of invasive disease and depends upon surface-bound bacterial proteins interacting with host cell receptors. Pneumococcal surface adhesin A (PsaA) is a highly conserved lipoprotein involved in the attachment to host airway cells via the host receptor Annexin A2 (ANXA2). However, the specific structural domains of PsaA responsible for ANXA2 recognition and binding remain unknown. Here, we employed a structure-based peptide approach to map the ANXA2-binding domain of PsaA. Five recombinant PsaA peptides were designed and generated based on the crystal structure of PsaA. Far-western blot analysis using ANXA2-overexpressing HEK293T/17 cell lysates revealed that the interaction with ANXA2 was specific to the C-terminal subdomain of PsaA. Cell-binding assays and flow cytometry further confirmed the interaction between the C-terminal subdomain of PsaA and cell surface-expressed ANXA2. Furthermore, polyclonal antibodies against the C-terminal subdomain significantly inhibited the binding of full-length PsaA to ANXA2-overexpressing cells, whereas antibodies against other subdomains did not. Consistently, polyclonal antibodies against the C-terminus of PsaA reduced the binding of <i>S. pneumoniae</i> to A549 lung epithelial cells to a greater extent than other antibodies. Together, these findings establish that the C-terminus of PsaA is crucial for ANXA2 receptor recognition. Targeting this specific subdomain may be a promising strategy for developing next-generation protein-based pneumococcal vaccines that aim at blocking bacterial adherence.IMPORTANCE<i>Streptococcus pneumoniae</i> is a leading cause of millions of deaths worldwide each year due to its ability to transition from an asymptomatic colonizer to an invasive pathogen. Current pneumococcal conjugate and polysaccharide vaccines protect against pneumococcal disease, but overall colonization rates have remained stable. Since pneumococcus is an opportunistic pathogen, decreasing overall colonization rates is essential for preventing progression to disease. The significance of our research lies in mapping functional epitopes within key pneumococcal adhesins that play a critical role in bacterial adherence. Defining these adhesion epitopes is essential for the rational design of next-generation protein-based vaccines capable of blocking colonization and ultimately reducing the global burden of invasive pneumococcal diseases.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0023226"},"PeriodicalIF":3.1,"publicationDate":"2026-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147777006","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":"Hybrid capture RNA-seq defines temporal gene expression in <i>Rickettsia</i>.","authors":"Allison T Scott, Jon McGinn, Vincent L Butty, Stuart S Levine, Rebecca L Lamason","doi":"10.1128/msphere.00901-25","DOIUrl":"10.1128/msphere.00901-25","url":null,"abstract":"<p><p>Pathogenic <i>Rickettsia</i> species are obligate intracellular bacteria that must reside in a mammalian host or arthropod vector cell to survive. Although these bacteria transition between different intracellular environments during infection, they encode few putative transcription factors, and their gene regulatory networks are largely unknown. Because of their inextricable relationship with eukaryotic cells, transcriptional profiling of the pathogen is complicated by the abundance of contaminating host RNA, especially in infection conditions or stages where the bacterial burden is inherently low. Here, we employ a pathogen hybrid capture technique (PatH-Cap) to improve library preparation by enriching bacterial transcripts while depleting host and rRNA molecules. Using PatH-Cap, we explored transcriptional changes throughout the first 24 h of infection, including infection initiation-an infection stage that has been difficult to profile with standard library preparation methods. We then clustered genes based on their temporal trends, revealing cohorts of genes whose expression is up- or downregulated at different stages of infection. We also highlighted the diverse temporal expression trends of genes with known roles in growth and pathogenesis, including translation and cell division genes, secreted effectors, and secretion system components. Lastly, we identified 639 antisense RNA molecules, many of which also showed strong temporal trends. This work demonstrates that sensitive transcriptional profiling approaches like PatH-Cap hold great promise for dissecting gene expression networks driving infection in intracellular pathogens that have historically posed significant technical challenges.</p><p><strong>Importance: </strong>When investigating poorly annotated genomes, such as those in obligate intracellular bacteria, transcriptional analyses can reveal gene sets active under specific conditions and form the foundation for future targeted approaches. However, such systems-level analyses of dynamic gene expression changes during infection with Rickettsia species have been missing due to the limitations of standard RNA-seq library preparations. Here, we adapted the pathogen hybrid capture (PatH-Cap) method for the first time to any Rickettsia species. We leveraged this wealth of RNA-sequencing information to compare temporal trends between genes and investigate aspects of <i>Rickettsia parkeri</i> transcription regulation, such as predicting operon structure and identifying putative antisense RNA transcripts. This work establishes the most comprehensive analysis of temporal rickettsia gene expression to date, providing an important foundation for further analysis. Future work can apply the methods described here to investigate gene expression changes across different genetic or environmental perturbations, cellular contexts, or disease models.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0090125"},"PeriodicalIF":3.1,"publicationDate":"2026-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147776989","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":"Tubulin polyglutamylases TTLL4C and TTLL6B are essential for maintaining cytoskeletal integrity in <i>Trypanosoma brucei</i>.","authors":"Lucas Brehm, Moritz Röder, Stephanie Lamer, Marinus Thein, Andreas Schlosser, Carlo Unverzagt, Klaus Ersfeld","doi":"10.1128/msphere.00909-25","DOIUrl":"https://doi.org/10.1128/msphere.00909-25","url":null,"abstract":"<p><p>Tubulin polyglutamylation is a post-translational modification that modulates microtubule interactions with associated proteins and motor proteins, thereby contributing to the regulation of microtubule dynamics. Although its roles are well established in higher eukaryotes, the functional significance of this modification in protozoan parasites remains poorly understood. In the present study, we characterize two polyglutamylases, TTLL4C and TTLL6B, in the parasite <i>Trypanosoma brucei</i>, a protozoan organism that possesses a subpellicular, nematic array of highly stable microtubules. Using gene knockout and overexpression approaches combined with immunofluorescence, western blotting, and mass spectrometry, we show that TTLL4C functions as an initiator for α-tubulin polyglutamylation, specifically catalyzing monoglutamylation at residue E445. Loss of TTLL4C perturbs posterior cytoskeletal architecture, resulting in blunt cell ends and reduced cell length. By contrast, TTLL6B functions as an elongase with preferential activity on β-tubulin, extending polyglutamate chains after initiation. TTLL6B depletion results in an elongated cell morphology, organelle mispositioning, and delayed cytokinesis. Together, these findings delineate complementary roles of TTLL4C and TTLL6B in maintaining cytoskeletal integrity and cell shape in <i>T. brucei</i>, underscoring the importance of balanced polyglutamylation for morphogenesis and cell cycle progression in kinetoplastids.</p><p><strong>Importance: </strong>Post-translational modifications of microtubules, collectively known as the tubulin code, are increasingly recognized as key determinants in the modulation of microtubule properties. Notably, these modifications have been implicated in the pathogenesis of several diseases, including specific forms of neurodegeneration and ciliopathies. A comprehensive understanding of this regulatory layer is therefore of considerable biological and medical significance. Moreover, the conservation of tubulin post-translational modifications across eukaryotic evolution underscores their fundamental cellular importance. In this study, the protozoan parasite <i>Trypanosoma brucei</i> is used as a model system to examine the functional roles of two microtubule polyglutamylases, TTLL4C and TTLL6B. The findings reveal that these enzymes are essential for maintaining cytoskeletal integrity, cell morphology, organelle positioning, and normal cell growth. Collectively, this work advances our understanding of microtubule regulation and highlights the broader cellular functions governed by tubulin polyglutamylation.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0090925"},"PeriodicalIF":3.1,"publicationDate":"2026-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147777025","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":"Associations of antimicrobial and multidrug resistance in <i>Escherichia coli</i> from the fecal flora and housing environment of calves on dairy farms with atmospheric variables.","authors":"Véronique Bernier Gosselin, Martin Wegmann, Romane Zufferey, Vincent Perreten, Mireille Meylan","doi":"10.1128/msphere.00847-25","DOIUrl":"https://doi.org/10.1128/msphere.00847-25","url":null,"abstract":"<p><p>The objective of this study was to describe the non-linear relationships between antimicrobial resistance to at least one tested antimicrobial (AMR) or multidrug resistance (MDR; resistance to at least three antimicrobial drug classes) of <i>Escherichia coli</i> isolates from calf feces and from the calf environment on dairy farms, and farm-level atmospheric variables, as well as selected calf- and herd-level factors. Data sets of 601 fecal isolates and 777 environmental isolates, collected from 57 dairy farms on 204 and 212 sampling dates, respectively, were combined with atmospheric data closest to each farm (monthly average of values prior to each sampling date). By employing random forest models, we found that the maximum age of the sampled calves in the pooled fecal sample showed the highest importance for the outcome MDR in the calf fecal data set. For the outcome AMR in the calf data set and for both outcomes in the environment data set, all the atmospheric variables had the highest importance. In <i>E. coli</i> from the calf environment, AMR was negatively associated with the average, maximum, and minimum temperature in the month prior to sampling. In both the calf and environment data sets, both AMR and MDR were positively associated with increasing deviation from historical temperature. These findings warrant confirmation in larger data sets.IMPORTANCEThe dynamics of expansion and transmission of resistant bacterial populations and of antimicrobial resistance (AMR) genes within as well as between human, animal, and environmental reservoirs are complex. An association between average minimum ambient temperature and AMR rates in selected human pathogenic bacteria has recently been reported. The present study highlights the relative importance of atmospheric variables for the outcomes AMR and multidrug resistance (MDR), as well as associations between atmospheric variables and the AMR outcomes in two <i>Escherichia coli</i> data sets from calves' fecal and environmental samples from Swiss dairy farms. In both the calf and environment data sets, both AMR and MDR were positively associated with increasing deviation from historical temperature. In the face of rapid anthropogenic global warming, these findings warrant further research on the association between AMR in livestock species and atmospheric variables.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0084725"},"PeriodicalIF":3.1,"publicationDate":"2026-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147776999","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":"Trehalose improves cold tolerance of <i>Pediococcus pentosaceus</i> OL77 and enhances low-temperature oat silage fermentation.","authors":"Jikuan Chai, Chaosheng Liao, Zeliang Ju, Xin Liu, Xinyi Qu, Jie Bai, Guiqin Zhao","doi":"10.1128/msphere.00169-26","DOIUrl":"https://doi.org/10.1128/msphere.00169-26","url":null,"abstract":"<p><p>Low temperature remains a major bottleneck in silage fermentation, especially in cold regions and high-altitude areas. This study investigated the regulatory role of compatible solutes in enhancing the cold adaptation of <i>Pediococcus pentosaceus</i> OL77 and their synergistic application in oat silage under suboptimal temperatures (5°C-15°C). RT-qPCR analysis showed that expression of the cold shock protein gene <i>CspP</i> was sharply induced at 5°C, indicating its central role in cold-stress response. Exogenous trehalose and betaine significantly downregulated <i>CspP</i> expression, promoted bacterial growth, accelerated acid production, and enhanced metabolic stability. In silage trials, the OL77 + trehalose treatment resulted in improved lactic acid production, reduced ammonia nitrogen accumulation, and suppression of yeasts and molds across all tested temperatures. A random forest model identified 11 core variables critical for silage quality, highlighting the combined treatment as the most effective across all temperature conditions. These findings offer a robust theoretical and practical framework for enhancing low-temperature silage through synergistic application of compatible solutes and psychrophilic lactic acid bacteria and demonstrate the potential of machine learning in fermentation process optimization.IMPORTANCELow temperature is a major constraint on silage fermentation in cold and high-altitude regions. This study shows that trehalose improves the cold adaptation and fermentation performance of <i>Pediococcus pentosaceus</i> OL77, highlighting a practical strategy for improving oat silage quality under suboptimal temperatures.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0016926"},"PeriodicalIF":3.1,"publicationDate":"2026-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147776973","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":"Wavyleaf basketgrass (<i>Oplismenus undulatifolius</i>) invasion is associated with changes in soil microbial communities.","authors":"Michael R Fulcher, Anthony Tritz, Vanessa Beauchamp, Carrie A Wu","doi":"10.1128/msphere.00895-25","DOIUrl":"https://doi.org/10.1128/msphere.00895-25","url":null,"abstract":"<p><p>Introduced invasive plants can alter the composition of resident soil microbial communities, which may disrupt ecosystem function and facilitate continued invasion success. Wavyleaf basketgrass (<i>Oplismenus undulatifolius</i>) is a high-risk, non-native invasive plant currently confined to the Mid-Atlantic United States but with the potential to colonize forest understory across the eastern United States. In this study, we characterized soil microbial communities from locations spanning the invaded range using amplicon sequencing to understand the impacts of wavyleaf basketgrass establishment on resident soil microbiomes. We compared the diversity and structure of microbial communities from invaded and uninvaded forest soils, as well from wavyleaf basketgrass rhizospheres. Invasion by wavyleaf basketgrass was associated with an increase in fungal diversity within sampling locations but a decrease in diversity across sampling locations. Changes in the relative abundance of specific sequence variants indicated a small number of resident microbes may be amplified in wavyleaf basketgrass rhizospheres. Finally, fungal alpha diversity was correlated with soil chemistry variables in uninvaded plots but not in invaded plots, and increased plant ground cover attributed to wavyleaf basketgrass invasion was positively correlated with fungal diversity. Together, these patterns suggest that wavyleaf basketgrass recruits diverse microbial associates from the environment, homogenizes soil microbiomes across invaded locations, and overrides existing environmental selection pressures exerted by soil chemistry profiles. Ongoing expansion of the species' invaded range may produce similar impacts in new environments.</p><p><strong>Importance: </strong>Understanding whether and how microbial communities are altered by plant invasion provides important information about the impact of introduced species on natural resources, nutrient cycling, and biodiversity that influence subsequent land management and ecosystem restoration decisions. We document biotic homogenization of resident soil microbes across geographically disparate locations following a relatively recent plant invasion. We further provide evidence suggesting microbial community changes are linked to the enrichment of specific taxa from the invasive plant's rhizosphere and possible buffering of these communities against other environmental selective pressures.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0089525"},"PeriodicalIF":3.1,"publicationDate":"2026-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147776964","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":"Small is big: growing impact of small molecule mass spectrometry in infectious disease drug development.","authors":"Laura-Isobel McCall","doi":"10.1128/msphere.00860-25","DOIUrl":"https://doi.org/10.1128/msphere.00860-25","url":null,"abstract":"<p><p>Small molecule analysis by mass spectrometry (MS) in drug discovery and development has historically focused on confirmation of synthesis products and on drug quantification in pharmacokinetic and pharmacodynamic studies. However, advances in sensitivity, throughput, and cost of mass spectrometers, alongside improvements in data analysis pipelines, have led to increasing contributions of small molecule MS across drug discovery and development stages. In this minireview, I will discuss these recent technical advances in the context of infectious disease drug discovery and development, highlighting applications to high throughput screening and hit identification, the discovery of novel mechanisms of action, personalized treatment and diagnostics, and fighting treatment failure.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0086025"},"PeriodicalIF":3.1,"publicationDate":"2026-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147776967","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":"pH-responsive substrate switching in mycobacterial type VII ESX secretion.","authors":"Owen A Collars, Richard L Hernandez, Simon D Weaver, Rebecca J Prest, Caleb Manu, Gopinath Viswanathan, Rachel M Cronin, Bradley S Jones, David M Tobin, Matthew M Champion, Patricia A Champion","doi":"10.1128/msphere.00056-26","DOIUrl":"10.1128/msphere.00056-26","url":null,"abstract":"<p><p>During infection, pathogenic mycobacteria reside within phagosomes of varying acidity based on the macrophage activation state. The ESX-1 secretion system (early secreted antigen 6 kilodaltons [ESAT-6] system 1) delivers protein virulence factors essential for phagosome lysis, facilitating infection. The mechanisms underlying ESX-1 lytic activity in heterogeneous environments remain unknown. Here, we show that the canonical Type VII secretion system, ESX-1, orchestrates substrate switching in response to different environments. Growing <i>Mycobacterium marinum</i> at acidic pH resulted in substrate switching <i>in vitro</i>. Substrate switching was accompanied by significant changes to the levels of ESX-1 substrate transcripts and to the levels of both ESX-1 substrates and chaperones at the protein level. We showed that specific ESX-1 transcripts were significantly upregulated <i>in vivo</i> and that distinct substrate sets are required in an acidic infection model.</p><p><strong>Importance: </strong>Pathogenic mycobacteria cause chronic and acute disease. Mycobacterial pathogens promote infection by transporting bacterial proteins into the host using ESX/Type VII secretion systems. The ESX-1 system secretes proteins into the phagosome that release the bacteria into the cytoplasm and promote bacterial survival in the macrophage. We show that <i>Mycobacterium marinum</i>, an animal pathogen and model for studying ESX-1 and tuberculosis, switches which ESX-1 proteins are secreted in response to acidic pH, an infection relevant signal. We demonstrate that protein secretion reflects changes in substrate transcripts and in substrate and chaperone protein levels. Finally, we leveraged two infection models to support that ESX-1 substrate switching likely occurs during infection. Our findings support a model in which mycobacterial pathogens use different proteins to lyse macrophage phagosomes of different pH.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0005626"},"PeriodicalIF":3.1,"publicationDate":"2026-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147777047","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":"Cryopreservation method for <i>Entamoeba histolytica</i> trophozoites.","authors":"Makoto Kazama, Fumika Mi-Ichi","doi":"10.1128/msphere.00889-25","DOIUrl":"10.1128/msphere.00889-25","url":null,"abstract":"<p><p><i>Entamoeba histolytica</i>, a protozoan parasite, causes amebiasis, which is a global public health problem. Clinical manifestation and pathogenesis of amebiasis are closely associated with the proliferation and tissue invasion abilities of <i>E. histolytica</i> trophozoites. At the same time, <i>E. histolytica</i> trophozoites are studied in a broad range of biology research fields. As such, the development of <i>in vitro</i> culture of <i>E. histolytica</i> trophozoites greatly facilitated amebic research. Now, a standard method for cryopreservation of <i>E. histolytica</i> trophozoites is required because available methods either do not give a high enough revival rate, which can significantly delay projects, or they are not widely adopted. Here, we attempted to optimize the conditions for <i>E. histolytica</i> trophozoite cryopreservation, including cell density, cooling rate, cell freezing reagent, and freezing profile. We found an optimized condition that reproducibly yielded >30% revival, regardless of storage period in liquid nitrogen (up to 365 days). This optimized condition is that <i>E. histolytica</i> trophozoites are suspended in 0.5 mL CELLBANKER 2 in a 1 mL cryotube at 2 × 10⁶ cells/mL and frozen from 4 to -40°C at a rate of -0.2°C/min using VIA Freeze Uno. This cryopreservation method can minimize the risk of losing <i>E. histolytica</i> trophozoite lines required to continue projects, facilitating amebic research.</p><p><strong>Importance: </strong>Amebiasis, which is caused by <i>Entamoeba histolytica</i> infection, is the third deadliest parasitic disease globally. Proliferation of <i>E. histolytica</i> trophozoites and their invasion into the host tissues cause amebiasis symptoms and pathogenesis. <i>E. histolytica</i> trophozoites are also important in multiple biology research topics. Therefore, <i>E. histolytica</i> trophozoites are a common subject in academic as well as clinical fields. A standard method for <i>in vitro</i> culture of <i>E. histolytica</i> trophozoites is well established. By contrast, a widely adopted practical method for cryopreservation of <i>E. histolytica</i> trophozoites is not yet available. This hampers the advancement of amebic research, as the required <i>E. histolytica</i> trophozoite lines sometimes cannot be revived from cryopreservation. In this study, we varied parameters critical to the revival rate, namely, cell density, cooling rate, freezing reagent, and freezing profile, and present an optimized cryopreservation method for <i>E. histolytica</i> trophozoites, which gives reproducibly high revival rates.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0088925"},"PeriodicalIF":3.1,"publicationDate":"2026-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13123703/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147574874","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}