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Intestinal microbiota are essential for maintaining the host's immune homeostasis, but the mechanism is not fully understood. While microbial metabolite desaminotyrosine (DAT) is recognized for its protective role in viral immunity, its potential involvement in anti-parasitic defense remains unexplored. Here, we demonstrate that DAT orchestrates tuft cell hyperplasia and subsequent type 2 immunity, establishing critical defense against helminth infection. Mechanistically, DAT-mediated intestinal epithelial remodeling requires histone deacetylase 3 (HDAC3), as pharmacological inhibition of this epigenetic regulator abrogates both tuft cell expansion and impairs type 2 immune responses. Collectively, our findings not only explore DAT novel effects in anti-parasitic defense but also reveal a pathway whereby the small molecule metabolites calibrate intestinal type 2 immunity.IMPORTANCEA small molecule metabolite DAT drives tuft cell hyperplasia and type 2 immunity in the small intestine. DAT-mediated tuft cell hyperplasia depends on HDAC3 and an intact microbiota; our findings reveal how small molecule metabolites fine-tune intestinal type 2 defenses against parasites.

Integrative and conjugative elements (ICEs) are major mediators of horizontal gene transfer in bacteria. However, the role of recipient cells in their acquisition has received little attention. Using the ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis as minimal models, we combined genome-wide transposon mutagenesis with high-throughput mating assays to identify recipient factors required for ICE acquisition. The surface lipoprotein P48 emerged as the primary determinant of ICE uptake in both species. Structural and functional analyses revealed that P48 is the substrate-binding component of an ABC transporter with nucleoside-binding capacity. A single-point mutation that abolished nucleoside binding drastically reduced ICE acquisition, demonstrating that P48-mediated nucleoside recognition is essential for conjugative transfer. However, ICE uptake did not require nucleoside transport, as inactivation of the transporter permease blocked nucleoside analog toxicity but not ICE invasion. Loss of P48 also triggered transcriptional activation of vestigial ICE genes, suggesting that surface recognition affects the intracellular state of the recipient. Remarkably, ICE transfer from recipient-derived donors was unaffected by P48 loss, underscoring its acquisition-specific role. Together, these results reveal a previously unrecognized, surface-exposed recipient factor critical for efficient ICE transfer in mycoplasmas and identify nucleotide binding as a central function in conjugation. By demonstrating that recipient-encoded functions can directly control ICE dissemination, this work challenges the donor-centric paradigm of bacterial conjugation and suggests new strategies to restrict horizontal gene flow in pathogenic and synthetic mycoplasmas.IMPORTANCEIntegrative and conjugative elements (ICEs) are mobile DNA elements that drive bacterial conjugation, a major process by which bacteria exchange genes. Although conjugation has been studied for decades, the focus has been almost exclusively on donor cells and the ICE itself, leaving the role of recipient cells largely overlooked. Using the wall-less ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis as minimal models, we discovered that a single recipient lipoprotein is required for efficient ICE uptake. Our data show that nucleoside recognition by P48, but not transport, is critical for conjugation, revealing an unexpected mechanistic link between nutrient sensing and gene acquisition. These findings shift the paradigm of conjugation from a donor-driven process to one jointly determined by donor and recipient functions. By identifying a recipient-encoded determinant of ICE transfer, this work opens new avenues to control horizontal gene flow in both pathogenic and engineered bacteria.

The pathogenesis of Cryptococcus neoformans is largely attributed to the impact of the polysaccharide capsule on the survival of the fungus in the harsh conditions of the host. These conditions include a robust immune response and nutrient limitation in different tissues. Little is known about the survival mechanisms of C. neoformans in nutrient-deprived conditions, and a key unanswered question is whether the fungus can use capsule material for nutrition during conditions of carbon limitation. We addressed this question by measuring alterations in capsule and cell wall in response to carbon limitation and found an influence on capsule porosity and density without a change in diameter. RNA-seq analysis of the response to carbon limitation identified transcripts for enzymes with potential relevance to polysaccharide changes, including carbohydrate-active enzymes. Subsequently, the impact of a selected set of enzymes was evaluated with capsule and cell wall-relevant mutants lacking Cas1 and Cas3 (O-acetylation of capsule polysaccharide), Chs1-8 (chitin synthases), Cps1 (hyaluronic acid synthase), and Kre64 (β-glucan specific glycosidase). Overall, our findings show that C. neoformans responds to carbon starvation by increasing capsular and cell wall permeability through interactions between cell wall components (α- and β-glucans and chitin) and capsules that alter their density and porosity. The results also suggest that C. neoformans does not substantially degrade the capsule polysaccharide under the conditions of carbon limitation employed in this study.IMPORTANCEThe World Health Organization recently placed Cryptococcus neoformans in the critical priority group of fungal pathogens that threaten human health. The elaboration of a polysaccharide capsule is a major contributor to the ability of C. neoformans to cause disease. However, the mechanisms of capsule formation are not well understood, and it is unknown whether the fungus can degrade the polysaccharide upon nutrient limitation. Here, we examined capsule degradation by starving the cells for glucose and monitoring changes in capsule permeability and binding of the dye calcofluor white to the cell wall. We found that permeability and dye binding increased with starvation. A parallel transcriptome analysis revealed candidate functions involved in the response to glucose availability, and subsequent tests with the corresponding mutants indicated an intricate connection between the cell wall and the capsule.

The filovirus, Ebola virus (EBOV), causes outbreaks of EBOV disease (EVD) throughout equatorial Africa. ERVEBO is a replication-competent recombinant vesicular stomatitis virus-vectored vaccine encoding the EBOV glycoprotein (recombinant vesicular stomatitis virus [rVSV]/EBOV), which is licensed to control EVD outbreaks. EVD outbreaks occur in regions endemic for Plasmodium-caused malaria. Plasmodium infections persist due in part to the parasite's ability to evade sterilizing immunity, which also dampens immune responses to heterologous vaccines. Acute murine Plasmodium infection at the time of rVSV/EBOV vaccination reduced vaccine-mediated protection against mouse-adapted EBOV (ma-EBOV) challenge. Decreased protection was associated with a Plasmodium-induced interferon gamma-mediated decrease of rVSV/EBOV replication in lymph node macrophages, resulting in reduced primary anti-EBOV glycoprotein antibody responses. Higher doses of rVSV/EBOV partially overcame the antibody deficits and elicited protective responses. Evidence of the negative impact of Plasmodium on the efficacy of low-dose rVSV/EBOV vaccine protocols supports the use of high antigen loads in the effective management of EVD outbreaks.

Importance: We show that a blood-stage murine Plasmodium infection negatively impacts the primary antibody response elicited by low-dose recombinant vesicular stomatitis virus (rVSV)/Ebola virus (EBOV) vaccination and results in reduced protection against a lethal dose of mouse-adapted EBOV. This defect occurs within the draining lymph node due to the elevation of interferon gamma elicited in Plasmodium yoelii (Py)-infected mice. The Py-imposed decrease in vaccine-mediated protection can be overcome with higher doses of rVSV/EBOV. While the strong protection conferred by rVSV/EBOV and significant side effects known to be associated with this vaccine have led to the suggestion that the vaccine dosage be reduced, our studies provide a rationale for maintaining the current higher dose.

Rapid and accurate diagnostics of bacterial infections are necessary for efficient treatment of antibiotic-resistant pathogens. Cultivation-based methods, such as antibiotic susceptibility testing (AST), are limited by bacterial growth rates and seldom yield results before treatment needs to start, increasing patient risk and contributing to antibiotic overprescription. Here, we present a deep-learning method that leverages patient data and available AST results to predict antibiotic susceptibilities that have not yet been measured. After training on three million AST results from 30 European countries, the method achieved an average accuracy of 93% across bacterial species and antibiotics. It predicted susceptibility with an average major error rate below 5% for quinolones, cephalosporins, and carbapenems, and below 8% and 14% for aminoglycosides and penicillins, respectively. Furthermore, the model predicted resistance with an average very major error rate below 10% for cephalosporins, carbapenems, and aminoglycosides, but with higher very major error rates for penicillins and quinolones. We combined the method with conformal prediction and demonstrated accurate estimation of the predictive uncertainty at the patient level. Our results suggest that artificial intelligence-based decision support may offer new means to meet the growing burden of antibiotic resistance.IMPORTANCEImproved diagnostic tools are vital for maintaining efficient treatment of antibiotic-resistant bacteria and for reducing antibiotic overconsumption. In our research, we introduce a new deep learning-based method capable of predicting untested antibiotic resistance phenotypes. The method uses transformers, a powerful artificial intelligence (AI) technique that efficiently leverages both antibiotic susceptibility tests (AST) and patient data simultaneously. The model produces predictions that can be used as time- and cost-efficient alternatives to results from cultivation-based diagnostic assays. Significantly, our study highlights the potential of AI technologies to address the increasing prevalence of antibiotic-resistant bacterial infections.

Gasdermin B (GSDMB), a member of the spore-forming protein gasdermin (GSDM) family, is critical for inflammation and immunity and has been genetically linked to human diseases. Despite its prominent expression at mucosal surfaces, including the gastrointestinal and respiratory tracts, GSDMB's role in defending against viral pathogens at these barrier tissues remains poorly defined. Here, we reveal that porcine GSDMB (pGSDMB), which is highly expressed in the intestinal epithelium, is a potent innate restriction factor against porcine epidemic diarrhea virus (PEDV), a major enteric coronavirus. Mechanistically, PEDV infection activated caspase-3/6/7 to cleave pGSDMB at D237, generating an active N-terminal fragment (pGSDMB_1-237) that triggered pyroptotic cell death to limit viral propagation. Conversely, PEDV evolved a sophisticated countermeasure: the viral nonstructural proteins nsp1 and nsp15 cooperatively suppressed pGSDMB protein expression. This immune evasion required a critical region within nsp1 (86-110 amino acids) and the catalytic endoribonuclease residues (H226 and H241) of nsp15. Importantly, pGSDMB-mediated pyroptosis broadly inhibited replication of diverse swine enteric coronaviruses, including transmissible gastroenteritis virus and porcine deltacoronavirus. Our findings establish GSDMB as an executor of pyroptosis that guards the mucosal interface against coronavirus infection and unveils a novel viral strategy to circumvent this defense, highlighting new avenues for therapeutic intervention against coronaviruses.IMPORTANCEWhile gasdermin B (GSDMB) is genetically associated with mucosal inflammatory diseases like asthma, its function in host defense at mucosal barriers remains an open question. This study defines a critical role for GSDMB as a central innate immune executor against enteric coronaviruses. We demonstrate that porcine GSDMB (pGSDMB) is cleaved during infection to trigger pyroptotic cell death, thereby restricting the replication of porcine epidemic diarrhea virus (PEDV) and other swine enteric coronaviruses. Furthermore, we identify a novel immune evasion strategy whereby PEDV employs its nsp1 and nsp15 proteins to suppress pGSDMB expression, delineating the key viral domains required for this countermeasure. These findings bridge a significant knowledge gap by revealing GSDMB as a guardian of the mucosal interface and inform the development of potential broad-acting therapeutic strategies against coronaviruses.

Human metapneumovirus (HMPV) causes acute respiratory disease worldwide and is the second leading cause of lower respiratory infection and hospitalization in young children in the USA. There is no licensed vaccine or therapeutic. HMPV mutates rapidly; however, the specific genomic features that explain strain dominance remain undefined because there is limited routine genomic surveillance of HMPV. We analyzed prospectively collected nasal specimens and medical data from 8,000 pediatric acute respiratory infection cases and sequenced 219 HMPV whole genomes from Pittsburgh, PA, between 2016 and 2021. Only A2, B1, and B2 subgroups were detected. The dominant subgroup varied between seasons. Variants with an in-frame 111- or 180-nucleotide (nt) insertion that nearly duplicates the preceding flanking region in the 660-nt G gene (encodes the attachment protein) were the predominant A2 viruses detected by 2016-2017. Among B2 viruses, variants with smaller in-frame insertions in the same location of the G gene became dominant by 2017-2018. Each insertion length formed a distinct phylogenetic clade. The insertions are in the ectodomain and contain positively charged residues or predicted O-glycosylation sites. Epidemiological analysis revealed that HMPV infection was independently associated with age, insurance type, and comorbidities. Elevated disease severity was independently associated with age and comorbidities, although not with HMPV subgroup. To our knowledge, in the USA, this is the earliest detection of the A2 insertion variants and the first report of the B2 insertion variants. It is the largest population-based genomic HMPV study that provides a detailed phylodynamics and epidemiological analysis of prospectively collected clinical specimens.IMPORTANCEHuman metapneumovirus (HMPV) is a leading cause of lung infection and pediatric hospitalizations worldwide for which there is no licensed vaccine or therapeutic. Because HMPV mutates rapidly, understanding which mutations enhance its ability to multiply and spread is important for the development of interventions and treatments. We prospectively collected patient data and nasal specimens from children with symptoms of acute respiratory illness. The predominant A2 and B2 HMPV variants circulating in the population contained insertions in the attachment protein, which suggests that these insertions may be advantageous to the virus. Furthermore, our analysis suggests that age, insurance type, and underlying health conditions were associated with HMPV infection. Age and underlying health conditions were associated with elevated HMPV disease severity, whereas HMPV subgroup was not. This large HMPV genomic epidemiological study provides insight into patient factors associated with disease and the emergence of the dominant variants in the USA.

Undecaprenyl phosphate (C55-P) is a critical lipid carrier required for the transport of cell envelope precursors across the cytoplasmic membrane in bacteria. Recent studies have identified proteins of the DedA family and DUF368 domain family as C55-P flippases in both Gram-positive and Gram-negative organisms. However, their roles remain undefined in many clinically relevant pathogens. Here, we screened for DedA and DUF368 proteins in Pseudomonas aeruginosa and assessed their functional importance. We show that PA4029, a DedA family membrane protein, is involved in C55-P recycling. Deletion of PA4029 sensitizes cells to fosmidomycin and limits the emergence of spontaneous colistin-resistant mutants. Using native mass spectrometry, we demonstrate that PA4029 binds C55-P with high affinity and selectivity over membrane phospholipids, and that this interaction is disrupted by the C55-P targeting antibiotic amphomycin. We also show that a DUF368 protein, found in some Pseudomonas species lacking PA4029 orthologs, can functionally substitute for PA4029 in P. aeruginosa, suggesting divergent strategies for C55-P recycling in this genus. Together, these findings position PA4029 within the conserved DedA-mediated lipid carrier pathway and highlight its importance for cell envelope homeostasis and antibiotic resistance in P. aeruginosa.IMPORTANCEBacteria use lipid carrier undecaprenyl phosphate (C55-P) to build and maintain their cell envelope, which is necessary for survival and is the target of many antibiotics. Recent studies have implicated DedA family proteins in C55-P transport, but how these proteins function in important pathogens like Pseudomonas aeruginosa remains uncharacterized. In this work, we uncover a specific DedA protein, PA4029, and show its involvement in C55-P recycling and importance for bacteria's ability to develop resistance to the last-resort antibiotic colistin. These findings extend the relevance of DedA-mediated lipid transport to one of the most dreaded human pathogens.

Gut microbiomes can dramatically affect host health and fitness, but can shift rapidly under changing environmental conditions. Understanding the interplay between microbiota, environmental pressures, and host responses is critical for predicting species' resilience, particularly in populations transitioning from the wild to human care for conservation breeding. Although captivity can profoundly alter microbial communities and host physiology, the dynamics of these transitions across generations remain poorly understood. We evaluated gut microbiota and fitness metrics in the endangered Pacific pocket mouse (Perognathus longimembris pacificus) during the establishment of a conservation breeding and reintroduction program, spanning five generations. Microbiome composition shifted gradually, stabilizing into a distinct captivity-associated state after two to three generations. These transitions paralleled changes in host weight and reproductive performance, suggesting coordinated host-microbiome adaptation. In addition, we identified microbial taxa correlated with successful reproduction, highlighting potential microbial markers of fitness. Our findings provide the first characterization of gut microbiota in Pacific pocket mice and demonstrate how captivity shapes host-microbiome systems across generations. More broadly, they underscore the importance of considering microbiome dynamics in conservation management and suggest that microbial responses to environmental change may require multiple generations to reach a new stable state.IMPORTANCEIn human-altered landscapes, animals face numerous threats to their survival, yet little is known about how rapid environmental change affects host-microbiome dynamics across generations. Microbial communities play critical roles in host nutrition, immunity, and overall fitness, and shifts in composition may alter an organism's ability to adapt. We examined the gut microbiota of the endangered Pacific pocket mouse during the transition from wild to captive environments and across four descendant generations. We found that the microbiome did not immediately shift with captivity but instead stabilized into a distinct, captivity-associated state only after several generations. This study provides the first characterization of gut microbiota in pocket mice and is the first to show, at this resolution, how a wildlife species' microbiome adapts to environmental change while tracking health and fitness across generations. Our findings highlight the need to incorporate microbiome dynamics into conservation breeding and management strategies.