mSphere

Respiratory viruses pose a major threat to human health. Environmental virus persistence in respiratory secretions is essential for successful spread, yet studies have focused largely on a select few viruses in non-physiological matrices. Here, we compared the stability of structurally distinct human respiratory viruses, adenovirus, influenza virus, and rhinovirus, in deposited particles of pooled human saliva under indoor-relevant relative humidity (RH) conditions. Adenovirus exhibited <2.2-log10 decay over 6 h regardless of RH, while rhinovirus exhibited significantly greater degradation (>3.8-log10, at 50% and 80% RH). Influenza virus showed minimal decay at 20% and 50% RH but degraded significantly more (3-log10) at 80% RH. To distinguish aspects of human saliva that are key determinants in driving virus decay, we recapitulated saliva composition using the inorganic solute and protein concentrations determined via ion-coupled plasma mass spectrometry and proteomics, respectively. Rhinovirus was significantly more stable in simulated saliva than in human saliva at 50% RH, while influenza virus and adenovirus behaved similarly in both matrices. These findings emphasize virus-specific environmental persistence and highlight limitations of relying on a single virus and particle matrix for defining public health strategies. Ultimately, these insights will contribute to approaches for mitigating environmental transmission of distinct respiratory pathogens.

Protein-protein interactions (PPIs) influence critical biological processes in pathogenic microorganisms, such as the human fungal pathogen, Cryptococcus neoformans. Fungal thermotolerance and stress response pathways are key virulence determinants that directly impact pathogen adaptation and survival and the infection process. To establish a comprehensive baseline of PPIs in C. neoformans and explore these interactions to infer functional roles for uncharacterized proteins, we applied size exclusion chromatography coupled with mass spectrometry to the secreted and cellular proteomes of the fungi. As a result, 216 and 1699 unique proteins were identified across 24 secretome and proteome fractions, respectively. The predicted secretome networks included expected proteins associated with vesicles and virulence, indicating a role in extracellular defense. Whereas the cryptococcal proteome highlighted interactions among proteins with defined roles in fungal virulence for protein stability and thermotolerance, including two previously uncharacterized proteins, CNAG_00287 and CNAG_05199, putatively involved in complex formation with heat-shock proteins (HSP). Based on sequence and structure homology, we propose that CNAG_00287 is a tetratricopeptide repeat-containing co-chaperone that modulates Hsp 70 activity and CNAG_05199 functions as a Hsp70. We validated the thermotolerance role of CNAG_00287 in heat-related stress, as its absence significantly impaired fungal growth in nutrient-limited media at 37 {degrees}C. Together, this work resolves virulence-associated PPIs within C. neoformans and reveals new molecular regulators of thermotolerance that underpin fungal pathogenicity.

The opportunistic pathogen Pseudomonas aeruginosa can store carbon by polymerizing (R)-3-hydroxy fatty acids into polyhydroxyalkanoates (PHA) and forming large intracellular carbonosomes. These structures primarily function as stores of excess carbon that can be employed during nutrient imbalances, though they have been increasingly reported to have secondary functions in stress responses. A range of Proteobacteria can form carbonosomes, and they have been well characterised in strains used in bioplastic production and wastewater treatment. However, the occurrence, regulation and functions of carbonosomes in pathogenic bacteria remain poorly understood. Using flow cytometry and fluorescence microscopy following staining of carbonosomes with BODIPY 493/503, we characterised carbonosome abundance and dynamics in growth-arrested P. aeruginosa starved of nitrogen but provided with excess carbon. We found that carbonosomes substantially impact the energetic and biosynthetic output of growth-arrested P. aeruginosa, affecting protein biosynthesis, flagellar motility, and ATP/NADPH pools. The production and use of carbonosomes was found to confer a strong fitness advantage to growth-arrested P. aeruginosa during fluctuating nutrient conditions. Small carbonosomes were observed during the lag phase of growth in rich media and the ability to produce these structures was conserved across several different laboratory and clinical strains, although with different dynamics and maximal amounts. For example, the Liverpool epidemic strain LESB58 showed very little BODIPY staining while the closely related LESB65 strain showed high levels of staining. Crucially, we found that growth-arrested cells depolymerize carbonosomes upon tobramycin exposure and that carbonosome depolymerization significantly increases P. aeruginosa tobramycin sensitivity. Together, our observations suggest that carbonosomes significantly impact the physiology, metabolism, and antibiotic susceptibility of P. aeruginosa.

Pre-prolipoprotein phosphatidylglycerol diacylglyceryl transferase (Lgt) catalyzes the first step in the lipoprotein modification pathway and is essential for growth and viability of Helicobacter pylori. We explored the fate of H. pylori cells under Lgt depletion conditions to obtain insight into Lgt function on morphology and physiology. Downregulation of lgt expression led to growth arrest and a heterogenous population of small rod-shaped cells and small round cells with spiky protrusions, a loose outer membrane and disintegrated flagella. H. pylori overcomes a defect in lipoprotein modification through alteration of its gene expression profile resulting in so-called revertants. Outer membrane protein synthesis, carbon metabolism and nutrient uptake was reduced, and protein translation and flagellar biogenesis increased. Lgt specific regulation was observed in phospholipid biosynthesis resulting in an initial increase in cardiolipin followed by an increase in lysophospholipids at the time of adaptation. Suppressors of {Delta}lgt that were selected on solid media under restrictive conditions either lacked chromosomal mutations or had mutations in the cagUT-tetR regulon controlling the expression of lgt. A suppressor without mutations had an altered expression profile that led to downregulation of a DNA methyltransferase and upregulation of HP_0014, a motility associated glycosyltransferase family protein. Transmission electron microscopy and single cell video microscopy showed that a subpopulation of cells is motile in the revertant while the suppressor regained full motility with higher average speed compared to the wild type strain. Overall, our data show that lipoprotein modification by Lgt is important for membrane and flagellar integrity and that the heterogenous character of H. pylori cultures allows individual cells to overcome this cell envelope stress and survive. Our findings are important for antibacterial development strategies targeting essential integral membrane enzymes involved in lipoprotein modification and stimulate exploration of synergetic activity between known antibiotics and novel antibacterial agents targeting novel cellular pathways.

Objective: To evaluate the specimen-level performance of a multichannel electronic nose (e-nose) sensor array in classifying stool specimens into relevant Clostridioides difficile categories. Design: Retrospective single-center validation study with reference to polymerase chain reaction (PCR) and C. difficile toxin testing. Participants: A convenience sample of 31 discarded stool specimens with PCR and confirmatory toxin testing. Categorized into PCR-positive (+)/toxin+, PCR+/toxin-negative (-), and PCR-. Methods: Specimens were sampled in controlled headspace by two e-nose sensor arrays in 10-minute sessions with 5-second sampling intervals. The primary endpoint was one-versus rest classification performance (sensitivity, specificity, positive predictive value, and accuracy) into three classes: PCR+/toxin+, PCR+/toxin-, and PCR-. Data were normalized and the region of interest was extracted for training and testing. Random forests were utilized for classification of normalized data with a test-train split at the specimen-level. Results: There were 457 unique 10-minute sampling events (147 PCR+/toxin+, 183 PCR+/toxin-, 127 PCR-). Sensors 1 and 2 captured 20,331 and 23,538 unique 5-second exposures within the region of interest, respectively. Testing data classification accuracy was 0.79 [0.78, 0.81] and 0.84 [0.83, 0.85] for Sensors 1 and 2. Sensitivity for PCR+/toxin+ exposures was 0.82 [0.79, 0.84] and 0.94 [0.92, 0.95], and specificity was 0.91[0.90, 0.93] and 0.93 [0.92, 0.94] for Sensors 1 and 2. Conclusion: An e-nose sensor array partially differentiated PCR-classified C. difficile specimen classes at the exposure level under headspace-controlled conditions. Performance was strongest for PCR+/toxin+ exposures. Findings support further prospective validation in the hospital room setting.

Objective Heme-dependent metronidazole (MTZ) resistance has emerged in Clostridioides difficile and is primarily mediated by a T-to-G single-nucleotide polymorphism (SNP) in the promoter of nimB (PnimBG). We aimed to develop a CRISPR-Cas12a-based assay for rapid detection of the PnimBG mutation associated with MTZ resistance. Methods A series of primers and CRISPR RNAs (crRNAs) were designed and optimized to identify the most effective combination for discriminating between PnimBG (MTZ-resistant) and PnimBT (MTZ-susceptible) alleles. Detection was evaluated by visual fluorescence using blue-light and UV imaging systems, as well as by quantitative fluorescence measurements. Results The CRISPR-Cas12a assay accurately differentiated PnimBG and PnimBT alleles in C. difficile. In a collection of clinical isolates, the assay correctly identified all 11 MTZ-susceptible isolates carrying the PnimBT allele and all 10 MTZ-resistant isolates carrying the PnimBG allele. Fluorescence signals were readily detected both visually and quantitatively. Furthermore, colony PCR using crude colony lysates yielded results comparable to those obtained with purified genomic DNA, enabling rapid colony-based detection without conventional DNA extraction. Conclusions The CRISPR-Cas12a assay enables rapid molecular detection of the PnimBG mutation associated with heme-dependent MTZ resistance in C. difficile. This approach could complement conventional susceptibility testing for MTZ resistance detection.

Candida auris is an emerging multidrug-resistant fungal pathogen. However, little is known about the role of long noncoding RNAs (lncRNAs) in the human immune response to this fungus. We reanalyzed RNA-sequencing data from peripheral blood mononuclear cells obtained from three healthy donors. The cells were exposed to live C. auris, purified mannan, purified {beta} glucan, or RPMI control for 4 h and 24 h. We identified 607 putative novel polyadenylated lncRNA loci that were absent from GENCODE Release 48. Of the 449 novel loci tested for differential expression, 102 responded to at least one stimulus. Novel-lncRNA responses were limited at 4 h but increased markedly at 24 h. At 24 h, 78 novel lncRNAs responded to live C. auris, 34 to mannan, and 11 to {beta}-glucan. Many of these responses were specific to a particular stimulus. Among the 102 responsive lncRNAs, 94 were significantly co-expressed with protein coding genes. The coding-gene partners of the most highly connected lncRNAs were associated with innate immunity, defense responses, lysosomal functions, and signaling pathways. Temporal analysis identified 19 novel lncRNAs whose responses changed between 4 and 24 h. Fourteen were also differentially expressed and were selected for further analysis. Combining temporal response, co-expression, and genomic proximity identified 4 candidate cis-associated pairs: MSTRG.32011-TLR2, MSTRG.5763-DUSP5, MSTRG.25627-HCK, and MSTRG.18159 ITGB3.These findings reveal a predominantly late and stimulus-specific novel-lncRNA response to C. auris and provide prioritized candidates for experimental validation.

In 2024, Chicago experienced a measles virus outbreak. Air samples collected as part of routine respiratory virus surveillance from three hospitals that received measles patients were retrospectively analyzed. Measles virus was detected in air samples from one emergency department across five weeks when measles patients were present and another when no known cases were present. Two of three viral sequences derived from air samples were identical to measles patients. The source of one diverging sequence could not be identified and may represent a case not identified by traditional surveillance methods. Air sampling may supplement measles investigation, surveillance and response.

Co-administration of phages and antibiotics has been proposed as a therapeutic approach against antibiotic-resistant bacteria. The relationship, however, between antibiotic resistance and phage resistance in clinical isolates is unclear. Here, we examine associations between phage and antibiotic resistance profiles across a panel of Pseudomonas aeruginosa clinical isolates from the Centers for Disease Control (CDC) and Food and Drug Administration (FDA) Antimicrobial Resistance Isolate (ARI) Bank comprising 55 clinical strains with full genome sequences and antibiotic susceptibility testing (AST) data for 11 clinically relevant antibiotics. As phages in this study, we use three well-characterized, morphologically distinct phages, OMKO1, Luz19, and PAML31-1. We screen for phage resistance using a growth suppression assay, then conduct statistical analysis against antibiotic MIC (Minimum Inhibitory Concentration) data provided by the CDC to define association patterns across this dataset. We find multiple significant susceptibility correlations between pairs of antibiotics and phages, and a positive overall association between average phage resistance and antibiotic resistance across the 55 strains, even controlling for phylogenetic associations (=0.358, p<0.005). We conclude that phage and antibiotic resistance are positively associated across this clinical isolate collection, suggesting that the two resistance phenotypes are not independent in P. aeruginosa. These findings have implications for the development of phage-antibiotic cocktails.

Schistosoma mansoni infection is associated with alterations of the host gut microbial community structure, yet its impact on microbial genome dynamics and horizontal gene transfer remains poorly understood. In this study, we investigated the activity of Mobile Genetic Elements (MGEs), and of Insertion Sequences (IS) in particular, in the gut microbiome of wild-type (WT) and human-microbiota-associated (HMA) mice. Taxonomic profiling revealed significant differences in bacterial diversity between WT and HMA mice, with reduced richness and alpha diversity observed in HMA gut microbiomes regardless of the infection status. Principal Coordinates Analysis demonstrated clear separation between microbial communities, reflecting differences in host background and bacterial composition. In contrast, differences were observed between plasmid and viral community profiles and between these and bacterial diversity, suggesting that different MGEs may follow distinct ecological dynamics within the gut microbiome. IS elements within bacterial genomes were widespread across all microbiome communities analysed in this study, with higher insertion counts observed not only in HMA compared to WT mice, but also in infected compared to uninfected mice. IS elements were predominantly associated with dominant gut bacterial taxa, particularly members of Bacteroidia and Clostridia. At the genomic level, insertion events occurred more frequently in intergenic regions than in coding sequences, suggesting a preferential selective pressure towards alteration of gene expression rather than gene disruption. Furthermore, IS activity was dominated by a subset of IS families, with consistent patterns observed across experimental groups. Notably, infection was associated with increased insertion frequencies across multiple taxa and IS families, suggesting enhanced mobilisation under conditions of host-associated stress (the physiological and molecular pressure experienced by microorganisms, pathogens, or the host itself during their interactions within a symbiotic or infected environment) and microbiome perturbation. Together, these findings demonstrate that S. mansoni infection may promote IS mobilisation in the mouse gut microbiome, potentially contributing to microbial genome plasticity and facilitating horizontal gene transfer within host-associated microbial communities.

Disruption of the intestinal microbiota increases susceptibility to Clostridioides difficile infection (CDI), creating an opportunity for prophylactic interventions before symptomatic disease develops. We investigated whether Escherichia coli Nissle 1917 (EcN) engineered to express the bacteriophage-derived endolysin CD27L could enhance pathogen inhibition and attenuate CDI. Using a plasmid-cured EcN chassis and pMUT2-derived vectors, we generated strains expressing CD27L under constitutive or anaerobically regulated promoter control. The constitutive strain, BioEcN1, reduced viable C. difficile recovery relative to parental EcN and was selected for animal studies. BioEcN1 remained recoverable from monoassociated mice without in vivo antibiotic selection, with similar recovery on selective and nonselective media supporting construct retention. Prophylactic administration reduced clinical disease, body-weight loss, and intestinal histopathological injury in germ-free mice. Observed survival was 100% in BioEcN1-treated challenged mice versus 50% in untreated challenged mice during the monitored period. Protection was accompanied by lower fecal and cecal pathogen burdens. In mice harboring a human-derived dysbiotic microbiota, prophylactic BioEcN1 also reduced clinical and stool-consistency scores, with lower early fecal pathogen recovery and less colon shortening. These findings establish preclinical proof of concept for prophylactic administration of an endolysin-expressing probiotic to attenuate CDI.

Respiratory viruses, including coronaviruses, generally initiate infection in the upper respiratory tract, where mechanistic studies of host-virus interactions require models amenable to genetic manipulation. Human nasal air-liquid interface (ALI) cultures are realistic models of the nasal epithelium, but robust CRISPR/Cas9 engineering workflows for this primary cell system have been lacking. Here, we established an integration-free CRISPR/Cas9 workflow for genetic engineering of human nasal basal cells, which are subsequently differentiated into gene-edited ALI cultures. GFP-based sorting of RNP-transfected nasal basal cells substantially increases editing, achieving knockout efficiencies of 70-90% across gene targets (B2M, SLC35A1, ACE2, and TMPRSS2). Genetic disruption of ACE2 markedly reduces infection by SARS-CoV-2 and hCoV-NL63, confirming its important role for both viruses in the human nasal epithelium. TMPRSS2 knockout substantially reduces replication of both viruses, demonstrating SARS-CoV-2 depending on TMPRSS2 in the human nasal epithelium and providing genetic evidence in primary human cell cultures that hCoV-NL63 relies on TMPRSS2. This platform enables systematic genetic perturbation in a physiologically relevant model of the primary site of respiratory virus infection, validates ACE2 and TMPRSS2 as targets for medical countermeasures against SARS-CoV-2 and hCoV-NL63, and provides a versatile approach to identify host dependency factors for both circulating and emerging respiratory viruses.

Protein kinase R (PKR) is an antiviral sensor that induces translational shutdown and stress granule (SG) formation through the phosphorylation of eIF2. Although previous studies have proposed that hepatitis C virus (HCV) non-structural protein 5A (NS5A) inhibits PKR dimerization, other studies consistently observe PKR activation and SG formation during HCV infection. To address this discrepancy, we interrogated the ability of NS5A from different genotypes and strains to regulate canonical PKR-eIF2 activation in a variety of cell culture models. Regardless of the genotype or strain tested, HCV NS5A failed to inhibit PKR dimerization or dsRNA-induced PKR activation, nor did it rescue downstream steps in the PKR-eIF2 pathway, such as translation halt and SG formation, in contrast to known viral PKR antagonists. Similarly, NS5A did not antagonise PKR activation when expressed in the context of other HCV non-structural proteins. We also tested the role of the host factor cyclophilin A (CypA), which interacts with both NS5A and PKR. However, the absence of CypA did not affect downstream PKR signaling or the activity of viral PKR antagonists or NS5A. Collectively, these findings indicate that NS5A does not effectively antagonise the PKR-eIF2 pathway and further support previous findings that HCV replication occurs in the presence of an active PKR response.

Human noroviruses (HuNoVs) are the most common cause of non-bacterial foodborne illnesses worldwide. HuNoVs are notoriously difficult to culture, and current cell culture systems, such as human intestinal enteroids have limitations in efficiency and scalability, which has limited their applications in food safety studies. Recently, it has been shown that transformed salivary gland cell lines (including NS-SV-TT-DC) allow for replication and passaging of norovirus GII.4. Herein, we examined whether we can independently observe viral replication in salivary gland cells and if other strains of human norovirus can replicate in these cells. For this purpose, we first evaluated the replication of several norovirus strains in NS-SV-TT-DC cells by either infecting them with vesicle-cloaked viruses or total viruses in stool filtrates. Low levels of viral replication was observed and vesicle isolation did not improve viral replication. Next, we screened for replication of 50 norovirus samples from 9 genotypes, and 14 isolates demonstrated over a two-fold viral replication. We observed that the initial viral load in the samples does not have a significant effect on replication efficiency. We were also able to passage several isolates. Subsequently, we examined whether interferon inhibitors such as Ruxolitinib, TPCA-1, and BX795 would improve viral replication and observed negligible effects. Finally we tested multiple supplements including MgCl2, bile salt, ceramide, and TAK-779. Most supplements had little or no effect on HuNoV replication in NS-SV-TT-DC cells. Bile salt and TAK-779 showed limited, genotype-specific benefits. Altogether these results indicate that salivary gland cells allow limited replication of certain HuNoV isolates.

Abstract The upper respiratory tract (URT) microbiome plays a vital role in respiratory health, with older children (5-14 years old) serving as key reservoirs for the transmission of bacteria to younger individuals (< 5 years old). However, the URT microbiome in this age group remains poorly characterised, particularly in relation to colonisation by the pathobiont Streptococcus pneumoniae. To address this gap, we analysed URT samples collected over eight visits from 134 healthy children aged 5-14 years in The Gambia, using culture-dependent methods to detect S. pneumoniae and culture-independent 16S rRNA sequencing to characterise the microbiome in parallel. 16S rRNA sequencing identified Streptococcus and Prevotella as the dominant microbiome constituents. When integrating longitudinal sampling data, we observed temporal instability with frequent switches in microbiome profiles over time. URT samples colonised with S. pneumoniae were associated with increased abundance of Moraxella, Streptococcus and Haemophilus, whereas non-colonised URT samples were enriched with Granulicatella and Filifactor. S. pneumoniae colonisation was also associated with lower Shannon diversity compared with non-colonised samples. Age and season were the strongest determinants of microbiome composition; increasing age was associated with decreasing prevalence and abundance of taxa including Haemophilus and Moraxella, while gram-positive taxa were predominantly enriched in the dry season. These findings provide new insight into the URT microbiome of older children in The Gambia and highlight Granulicatella and Filifactor as potential contributors to colonisation resistance against S. pneumoniae.

Background Group B Streptococcus (GBS) is a leading cause of bacterial sepsis globally. Late-onset (LO) GBS disease is preceded by colonization within the gastrointestinal tract in vulnerable neonates. Previous studies demonstrate that Candida albicans (C. albicans) promotes GBS colonization of host tissues including the bladder and vagina. However, its impact on GBS colonization of the intestinal tract is unknown. Methods We established a new animal model to study GBS and C. albicans co-colonization model in the context of the newborn host to examine C. albicans impact on GBS and further evaluated fluconazole prophylactic treatment to these same outcomes. Physical interactions between organisms in vivo were imaged and GBS gene expression was evaluated. Results C. albicans increased GBS burden in the neonatal gut and GBS was physically colocalized with C. albicans hyphae as evidenced by the colocalization analysis. Fluconazole treatment of co-colonized mice decreased extraintestinal GBS dissemination. C. albicans presence upregulated genes encoding proteins responsible for GBS adhesion and invasion of intestinal epithelial cells. Conclusion C. albicans enhances GBS burden and pathogenicity in the neonatal gut. These findings highlight the need for further investigation into C. albicans as a potential risk factor for GBS colonization and LO disease in human infants.

Microsporidia are divergent fungal pathogens that infect a wide range of hosts. Microsporidia have evolved highly reduced genomes, resulting in the loss of many metabolic pathways. Encephalitozoon intestinalis is a human-infecting microsporidian species with the smallest known eukaryotic genome (2.3 Mbp). As an obligate intracellular parasite, E. intestinalis is dependent on its host for replication. Our current understanding of E. intestinalis cell biology remains limited. This is largely due to genetic intractability, which has made live-cell imaging challenging, and its small size (~2 m), which is within the diffraction limit of conventional microscopy. Here we develop a live-cell imaging technique, "silhouette microscopy", that does not rely on genetic manipulation of microsporidia, and enables us to study the live cell dynamics of E. intestinalis replication in host cells, from entry to exit. We report the timing of cell division, kinetics of the parasite life cycle, and dynamics of the parasitophorous vacuole within which E. intestinalis resides. The parasitophorous vacuole undergoes frequent fission and fusion, and ultimately ruptures to facilitate parasite release from the host cell. To gain a higher resolution understanding into the cell biology of E. intestinalis development, we performed ultrastructure expansion microscopy (U-ExM) on infected cells. U-ExM reveals the organization of the E. intestinalis cytoskeleton and development of the specialized invasion organelle, the polar tube. The combination of live-cell imaging and U-ExM provide a platform to study the E. intestinalis life cycle, and sheds light on the dynamics and cell biology of E. intestinalis in host cells.

Advanced in vitro models that better recapitulate human intestinal physiology are needed to improve translational relevance and reduce reliance on animal experimentation. Microfluidic gut-on-chip systems provide controlled architectural and mechanical microenvironments that more closely resemble native tissue than conventional static cultures. However, how these features influence epithelial responses to microbial stimuli remains incompletely understood. In this study, we compared conventional two-dimensional (2D) Caco-2 monolayers with a perfused three-dimensional (3D) gut-on-chip model exhibiting crypt-like epithelial organization. Microbial challenge was employed as a biologically relevant perturbation to investigate barrier-associated responses under distinct culture conditions. Following exposure to the probiotic Escherichia coli Nissle 1917 (EcN), epithelial morphology, barrier-associated gene expression, inflammatory signaling, and temporal response patterns were assessed over time. In a complementary approach, susceptibility to bacterial stress was examined using increasing doses of Citrobacter rodentium (C. rodentium). Both culture systems responded dynamically to microbial exposure; however, differences in response patterns were observed between static and perfused cultures. EcN induced transient alterations in epithelial organization followed by recovery-associated responses, while C. rodentium caused dose-dependent structural and transcriptional changes. Notably, epithelial damage became apparent at lower bacterial loads in conventional 2D cultures, whereas the chip-based model maintained tissue integrity under comparable conditions. Together, these findings indicate that epithelial architecture and the surrounding culture environment influence barrier-associated responses to microbial challenge. The study highlights the utility of gut-on-chip systems for investigating host-microbe interactions and provides a framework for functional comparison of advanced intestinal in vitro models.

Microsporidia are a group of parasites that exhibit vast morphological, ecological, and genetic diversity. To catalog this diversity, we previously generated a database of attributes for microsporidia species. Here we expand this database to include species described since 2021, previously missed descriptions, and provisional species defined by clustering GenBank 18S rRNA sequences, resulting in 1,795 species. Using these data, we describe when and where microsporidia have been reported, their hosts and host habitats, the tissues they infect, and their spore morphology. A phylogeny of 18S sequence clusters shows that closely related microsporidia tend to infect closely related hosts, such that sequence identity is predictive of host taxonomy. Clustering also reveals previously unappreciated genetic diversity within some named species, in particular the human pathogen Vittaforma corneae, suggesting that these may comprise multiple species. We have also generated a web portal through which new species information can be submitted, which will facilitate regular updates. This database provides a resource for systematic analysis of microsporidia properties and a reference for taxonomic descriptions.

Cytolysin is a toxin-bacteriocin commonly encoded by Enterococcus faecalis that can target erythrocytes, neutrophils, and macrophages as well as other Gram-positive bacteria. Cytolysin can lyse human, horse, rabbit, and mouse erythrocytes, but not sheep erythrocytes (SEs). It is hypothesized that SEs are resistant to cytolysin because they lack phosphatidylcholine (PC) in their membranes, while susceptible erythrocytes have PC-rich membranes, suggesting PC as a cytolysin binding target. Here, we isolated a urinary E. faecalis strain C33 that can efficiently lyse SEs and found that this activity was conserved among several E. faecalis clinical isolates. We demonstrated that although C33 cytolysin was necessary for SE lysis, it was not sufficient, suggesting a secondary factor enhances cytolysin activity against SEs. Through liposome assays we found that while PC was required for the activity of DS16 cytolysin, PC was dispensable for C33 cytolysin activity. To assess the biological significance of C33 cytolysin in the bladder microenvironment, we assessed C33 cytolysin activity against bladder epithelial cells and the urinary microbiota. We found that C33 cytolysin was more cytotoxic to human bladder epithelial cells than the canonical DS16 cytolysin. We also found greater inhibition of co-isolated urinary microbiota species Streptococcus parasanguinis, Enterococcus raffinosus, Staphylococcus capitis, and Corynebacterium coyleae by C33 cytolysin. Interestingly, E. faecalis C33 also inhibited the protective urogenital taxa Lactobacillus crispatus and Bifidobacterium longum. Together, these results reveal a new PC-independent cytolysin activity among clinical E. faecalis isolates that may support survival and competition in the urinary microenvironment.