Polyamines are essential molecules across all domains of life, but their role as signaling molecules in host-microbe interactions is increasingly recognized. However, because they are produced by both the host and the microbe, their dual origin makes their functional dissection challenging. The plant pathogen GMI1000 secretes large amounts of putrescine both and in the xylem sap of host plants. In this study, we investigated the genetic changes underlying its experimental evolution into a legume symbiont. We showed that the gene (RSc2277), which was repeatedly mutated during this process, encodes a putrescine exporter. Mutations in completely abolished putrescine excretion and enhanced bacterial proliferation within nodules during interaction with the legume . When these mutations occurred in symbionts already capable of intracellular infection, they further increased bacterial load in nodules and allowed the detection of nitrogenase activity. In addition, -mutated symbionts modulated host gene expression toward a more functional symbiotic state by repressing defense-related genes and inducing nodule development genes. These nodule development genes include genes encoding leghemoglobins and an arginine decarboxylase, a key enzyme in plant putrescine biosynthesis. These results indicate that bacterial and plant putrescine have distinct functions in legume symbiosis and highlight the complex role of polyamines in plant-microbe interactions.IMPORTANCERhizobia, the nitrogen-fixing symbionts of legumes, emerged through repeated and independent horizontal transfers of some essential symbiotic genes. However, these transfers alone are often insufficient to convert the recipient bacterium into a functional legume symbiont. In a laboratory experiment, we evolved the plant pathogen into a nodulating and intracellularly infecting symbiont of . This transition required genomic modifications in the recipient bacterium to activate its acquired symbiotic potential. Here, we demonstrated that one of these key adaptive modifications is the inactivation of bacterial putrescine export. This polyamine, when produced by the microsymbiont, appears to act as a negative signal for the plant. This study provides new insights into the distinct roles of bacterial- and plant-derived putrescine in plant-microbe interactions, highlighting their functional divergence despite being produced by both organisms.

Entry of herpesviruses into cells requires coordinated action of multiple viral glycoproteins, including gH/gL and gB, which comprise the core fusion machinery conserved in herpesviruses. The gH/gL heterodimer activates the gB fusion protein, triggering its refolding from a prefusion to a postfusion form to drive membrane merger. The cytoplasmic tail domain (CTD) of gB is proposed to act as an inhibitory clamp that stabilizes the prefusion state, with interactions between gH and gB CTDs destabilizing this clamp. We previously found that herpes simplex virus 1 (HSV-1) and saimiriine herpesvirus 1 (SaHV-1) gB homologs are functionally interchangeable but mediate reduced fusion when coexpressed with heterotypic gH/gL. To map the regions of gB responsible for species-specific interactions, we generated HSV-1/SaHV-1 gB chimeras by swapping the ectodomain, membrane-proximal region (MPR), transmembrane domain (TMD), and CTD segments. Our results show that homotypic CTD interactions alone are insufficient to trigger fusion, suggesting that gH/gL contacts the ectodomain of gB. We show that the HSV-1 gB CTD is hyperfusogenic relative to the SaHV-1 CTD, whereas the HSV-1 MPR is hypofusogenic relative to the SaHV-1 MPR. Together, these findings suggest that a functional interaction between the gH/gL-gB ectodomains contributes to fusion and that gB maintains a balance between promotion and restraint of fusion through coordinated contributions of its domains.

is a common sexually transmitted parasite that colonizes the human urogenital tract, causing infections that range from asymptomatic to highly inflammatory. As an extracellular pathogen, adherence to host epithelial cells is an important step to colonize the human host. Hence, understanding how the process of attachment to host cells is regulated remains an important goal in parasitology research and human health. -host interaction is regulated by changes in gene expression, but it is still largely unknown how these changes in transcriptional profiles are controlled, as very few transcriptional regulatory elements have been described. Our recent work highlighted the importance of epigenetics in the regulation of transcription, and a specific role for N6-methyladenine (6mA) in modulating three-dimensional chromatin structure has been suggested. Building on these findings, we analyzed here the role of 6mA and chromatin accessibility during the process of host-parasite interaction by integrating MeDIP-seq and assay for transposase-accessible chromatin sequencing data with RNA-seq in free vs host cell-attached parasites. Consistent with our previous results, we identified transcriptionally active and repressive regions flanked by 6mA modifications, observed both in the presence and absence of host cells. Importantly, we detected differentially accessible chromatin regions that influence the gene expression of key pathogenesis-related genes during host cell interaction. These findings highlight the importance of chromatin architecture in regulating gene expression during parasitic infection.

Upon exposure to echinocandins, growing yeast cells begin to accumulate cell wall damage and eventually die, resulting in therapeutic effects. While resistance to echinocandins is well studied, tolerance and persistence mechanisms that may also contribute to clinical failures and relapses remain understudied. In time-kill assays with micafungin , the opportunistic pathogen exhibited biphasic kinetics of cell death. Modeling with exponential decay equations distinguished a fast-dying major population from a slow-dying minor population, indicative of persistence. A genome-wide forward-genetic screen revealed dozens of genes that appeared to regulate persistence and/or tolerance, but not resistance. Several of those genes encoded calcineurin and its upstream regulators. Using individual gene knockout mutants and FK506, we show that calcineurin signaling increases the lifespans of most cells through a process that is largely independent of Crz1, one of its downstream effectors. The formation of long-lived persister-like cells (i.e., persistence) was strongly dependent on calcineurin signaling, independent of Crz1. Pre-activation of calcineurin using genetic or chemical stressors, such as manogepix, strongly increased tolerance and persistence in , suggesting antagonism of echinocandin efficacy by this new antifungal. Calcineurin signaling was also necessary for the induction of tolerance and persistence in . The findings suggest that short-term administration of FK506 during the earliest stages of echinocandin treatment may improve clinical outcomes while possibly avoiding long-term immunosuppression.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a highly contagious disease that affects pigs, resulting in substantial economic losses in the global pig industry. A comprehensive understanding of viral-host protein interactions can facilitate the discovery of therapies for viral infection. In this study, we employ a 4D label-free quantitative proteomics approach to profile a comprehensive protein dynamics analysis in ASFV-infected pigs, identifying over 6,000 proteins across multiple organs. Our results reveal coordinated interorgan responses characterized by inflammatory activation and interferon signaling in defense against ASFV. The protein-protein interaction network analysis uncovers ASFV-induced functional modules, including the unfolded protein response (UPR), innate immune signaling, and inflammation, which are conserved across tissues. Notably, ASFV robustly activates all three branches of the UPR both and to promote viral replication. Furthermore, we identify that the virus-encoded protein D117L interacts with multiple UPR-related host proteins, thereby directly triggering UPR activation. Collectively, this study delineates the organ-specific proteomic landscape of ASFV infection, providing valuable insights into virus-host interactions and offering potential therapeutic targets for ASF.IMPORTANCEAfrican swine fever virus (ASFV) has caused severe consequences for the global pig industry. In this study, we conducted a multi-organ proteomic analysis using a 4D label-free quantitative proteomics approach and mapped the organ-specific proteomic landscape during ASFV infection. This work overcomes the limitations of most existing studies, which are primarily restricted to cell models and provide a more comprehensive understanding of ASFV infection and pathogenesis. Notably, the viral D117L protein is identified as a critical modulator of host cellular responses, directly subverting the unfolded protein response (UPR) pathway through specific interactions with host UPR-associated proteins. Collectively, our work lays the foundation for understanding the pathogenesis of ASFV, providing potential therapeutic strategies against African swine fever.

Bacterial flagella are assembled by a specialized type-III secretion system that exports structural subunits in a defined order. While the ATPase FliI is known to couple ATP hydrolysis to substrate translocation, its role in the transition between early and late secretion stages has remained unclear. Here, we systematically analyzed strains with mutations in the catalytic domain of FliI and found that early substrate export and hook-basal body formation can proceed with minimal FliI ATPase activity, whereas efficient triggering of the substrate specificity switch and late substrate export requires near-wild-type activity levels. Mutant strains showed delayed gene expression from class 3 promoters, prolonged early secretion, and impaired flagellar filament assembly, despite normal FliI localization and oligomerization. These findings support the involvement of FliI in controlling the temporal dynamics of flagellar assembly. We propose that FliI contributes to substrate switching, ensuring robust and orderly function of the flagellar type-III secretion system. This study highlights the multifaceted role of the type-III secretion system ATPase in optimizing the efficiency and robustness of flagellum assembly and establishes a mechanistic link between ATPase activity and substrate switching in type-III secretion systems.

The inner membrane domain (IMD) is a metabolically active and laterally discrete membrane domain initially discovered in . The IMD correlates both temporally and spatially with the polar cell envelope elongation in . Whether or not a similar membrane domain exists in pathogenic species remains unknown. Here, we show that the IMD is a conserved membrane structure found in . We used two independent approaches, density gradient fractionation of membrane domains and visualization of IMD-associated proteins through fluorescence microscopy, to determine the characteristics of the plasma membrane compartmentalization in . Proteomic analysis revealed that the IMD is enriched in metabolic enzymes that are involved in the synthesis of conserved cell envelope components such as arabinogalactan and phosphatidylinositol mannosides. Using a fluorescent protein fusion of IMD-associated proteins, we demonstrated that this domain is concentrated in the subpolar region of the rod-shaped cells, where active cell envelope biosynthesis is taking place. Proteomic analysis further revealed the enrichment of enzymes involved in synthesis of phthiocerol dimycocerosates and phenolic glycolipids in the IMD. We validated the IMD association of two enzymes, α1,3-fucosyltransferase and fucosyl 4--methyltransferase, which are involved in the final maturation steps of phenolic glycolipid biosynthesis. Taken together, these data indicate that functional compartmentalization of membrane is an evolutionarily conserved feature found in both and , and utilizes this membrane location to enrich biosynthetic enzymes for its surface-exposed lipid virulence factors.IMPORTANCE remains an important public health threat, with more than one million deaths every year. The pathogen's ability to survive in the human host for decades highlights the importance of understanding how this bacterium regulates and coordinates its metabolism, cell envelope elongation, and growth. The IMD is a membrane structure that associates with the subpolar growth zone of actively growing mycobacterial cell, but its existence is only known in a non-pathogenic model, . Here, we demonstrated the presence of the IMD in , making the IMD an evolutionarily conserved plasma membrane compartment in mycobacteria. Furthermore, our study revealed that the IMD may function as the factory for synthesizing phenolic glycolipids, virulence factors produced by slow-growing pathogenic species.

Opsins are highly abundant retinal proteins in the membranes of photoheterotrophic bacteria. However, some microbial genomes encode an but lack the gene for the final enzyme in retinal synthesis. To account for this paradox, we hypothesized that bacterial opsins play a role in membrane structure and/or biogenesis independent of their potential for light-driven signaling or proton pumping. After purifying actinorhodopsin from a cell-free expression system and from membranes upon overexpression, we demonstrated both and that actinorhodopsin from . is a phospholipid scramblase, serving in its pentameric state as a retinal-independent phospholipid diffusion channel. Phospholipid headgroups move along a transbilayer path between actinorhodopsin protomers to equilibrate lipid content in the inner and outer leaflets. Two profound activities, membrane biosynthesis and capture of light energy, are thus facilitated by one ancient bacterial polypeptide. Light-dependent activity and light-independent phospholipid scrambling are shared functions of eukaryotic, archaeal, and bacterial rhodopsins.IMPORTANCECells are surrounded by membranes that concentrate metabolites and protect cellular contents. Most biomembranes are phospholipid bilayers, in which the phospholipids of each leaflet orient their greasy tails inward and polar groups outward. Bilayer biogenesis depends on phospholipids synthesized on the cytofacial side of the membrane reorienting to the extracellular membrane leaflet. This reorientation requires proteins, termed scramblases, and it was shown that rhodopsins-7-helix photoactive membrane proteins bound to the cofactor retinal-from organisms as widely divergent as mammals and archaea possess scramblase activity. Now we conclusively demonstrate, using purified proteins in laboratory membranes as well as computational approaches, that bacterial rhodopsins are also phospholipid scramblases. This work is important because it highlights a surprising commonality among bacteria, archaea, and eukaryotes and because it shows that rhodopsins-ancient proteins found in the last universal common ancestor-manifest two seemingly unrelated biochemical functions in one protein.

(Mtb) arrests its growth at acidic pH when grown on specific single carbon sources, including propionate. However, Mtb grows well on propionate at pH 7.0, supporting that propionate can support growth as a sole carbon source. To understand the basis of the propionate-driven growth arrest at acidic pH, we performed a forward genetic selection for mutants that enable growth on propionate at pH 5.7. All the selected mutants had insertions in the two-component regulatory genes or . We hypothesized that growth arrest at acidic pH is caused by PhoPR diverting carbon from central carbon metabolism toward lipid anabolism and that when PhoPR is inactivated, growth is promoted through metabolizing propionate by the methylcitrate cycle (MCC) into pyruvate, a permissive carbon source for growth at acidic pH. Using chemical inhibition and mutants of the MCC pathway, we demonstrate that the enhanced growth is dependent on the MCC. Furthermore, stimulating lipid synthesis via the methylmalonyl-CoA pathway by adding vitamin B12 restricts growth in the mutant. Conversely, restricting lipid anabolism by inhibiting the triacylglycerol synthase enhances the growth of the mutant. Notably, CoA pools increased in the mutant grown on propionate, directly supporting our model. Given the role of CoA metabolism in pyrazinamide sensitivity, we examined Mtb sensitivity to pyrazinamide on propionate at acidic pH and, surprisingly, observed that pyrazinamide treatment of wild-type Mtb suppresses growth arrest on propionate at acidic pH. In contrast, the Δ mutant has enhanced sensitivity to pyrazinamide. Together, these findings support that propionate-driven growth arrest at acidic pH is caused by metabolic remodeling that is regulated by PhoPR and is associated with pyrazinamide sensitivity.IMPORTANCEWhen grown on certain single carbon sources, such as propionate, (Mtb) arrests its growth at acidic pH and establishes a state of non-replicating persistence. To understand the genetic basis of this growth restriction, a genetic selection was performed to identify mutants unable to arrest growth at acidic pH with propionate as a sole carbon source. The selection exclusively identified mutants in the PhoPR two-component regulatory system, which functions to modulate cell envelope lipids and redox homeostasis through the upregulation of lipid synthesis at acidic pH. Using genetic and chemical inhibition studies, we demonstrate that PhoPR arrests growth at acidic pH by diverting carbon away from the methyl citrate cycle toward lipid anabolism. Surprisingly, treatment of Mtb with pyrazinamide at acidic pH on propionate also enabled growth. Therefore, this study defines new mechanisms by which Mtb integrates environmental signaling to regulate growth, metabolism, and drug susceptibility. These findings are relevant to pathogenesis, as PhoPR is essential for growth in macrophages and animals, environments with varying pH and carbon source availability, depending on immune pressures. These data suggest that drug susceptibility may be impacted by enhanced growth and metabolic capacity of Mtb in acidic and propionate-rich environments, such as within the macrophage or the granuloma.

The role of commensal anaerobic bacteria in chronic respiratory infections is unclear, yet they can exist in abundances comparable to canonical pathogens . Their contributions to the metabolic landscape of the host environment may influence pathogen behavior by competing for nutrients and creating inhospitable conditions via toxic metabolites. Here, we show that the anaerobe-derived short-chain fatty acids (SCFAs) propionate and butyrate negatively affect physiology by disrupting branched-chain fatty acid (BCFA) metabolism. In turn, alterations to BCFA abundance impair growth, compromise membrane integrity, diminish expression of the accessory gene regulator quorum-sensing system, and increase sensitivity to membrane-targeting antimicrobials. Disrupted BCFA metabolism also reduced fitness in competition with , suggesting that airway microbiome composition and the metabolites they exchange can directly impact pathogen succession over time. The pleiotropic effects of SCFAs on fitness and their ubiquity as metabolites in the human host also suggest that they may be effective as adjuvants to traditional antimicrobial agents when used in combination.IMPORTANCE is a primary pathogen of chronic airway disease yet is also found in the upper airways of 30%-50% of the population to no obvious detriment. Thus, identifying the host and/or microbial factors that tip the balance between its commensal and pathogenic states may be key to its control. Here, we reveal that short-chain fatty acids produced by commensal microbiota promote a marked remodeling of the lipid membrane that, in turn, sensitizes the pathogen to antimicrobials, disrupts accessory gene regulator quorum signaling, and reduces its competitive fitness. Altogether, these data suggest that co-colonizing microbiota and the metabolites they exchange with may be key players in the microbial ecology of airway disease.

Microbial communities typically comprise a few dominant taxa alongside numerous rare members. While dominant formation is generally attributed to species-level traits that confer adaptation to specific environments, it remains unclear how the rare biosphere influences which taxa ultimately become dominant. We constructed thousands of sub-communities from a soil microbiome using appropriate dilution to preserve the dominants but allow stochastic variation in rare assemblages. Following cultivation under an identical growth condition, dominant taxa varied substantially across replicates, which were determined to be driven primarily by species generalist competitive capacities rather than their initial relative abundances. Notably, the identity of the most abundant taxon in each sub-community was modulated by interactions with rare community members. Co-occurrence and metabolic resource overlap analyses revealed predominantly negative interactions among dominant taxa, while rare taxa exhibited variable and asymmetric interaction patterns. A refined consumer-resource model supported these observations, showing that species are first "nominated" as dominant candidates based on generalist competitive capacities, and then "voted" into the most abundant taxon in a community through collective feedback from rare taxa. The findings imply that dominant emergence in microbial communities is not solely determined by intrinsic species traits but is also critically shaped by interactions with rare taxa. The proposed "nomination-voting" model highlights a collective role of the rare biosphere in shaping community structure and offers a new framework for understanding microbial assembly.IMPORTANCEMicrobial ecosystems are almost always dominated by only a few species, but their diversity resides in the rare biosphere. These rare members are usually considered passive passengers with little influence, yet our work reveals that they can collectively determine which species to become the most abundant taxon. We describe this process as a "nomination-voting" system: competitive traits nominate potential dominants, while rare taxa vote for the ultimate winner through their complex interactions. Recognizing this hidden but decisive role of rare microbes provides a new perspective on community assembly and underscores how subtle ecological interactions shape community outcomes. This assembly framework offers new opportunities for the prediction, manipulation, and stabilization of agriculture, health, and environmental microbiomes.

is among the most abundant bacterial genera in the healthy human colon, comprising approximately 10-15% of the total gut microbiota. Species within this genus ferment complex carbohydrates, including pectin, to produce butyrate, a short-chain fatty acid with anti-inflammatory and anti-carcinogenic properties. Butyrate is the primary energy source for colonocytes and in is synthesized via the butyryl-CoA:acetate CoA transferase pathway. Reduced levels of are often associated with increased abundance of and may be linked to early-onset colorectal cancer. Here, genomic analysis of strains revealed that several lack antibiotic resistance genes, suggesting a favorable safety profile. Additional genome mining revealed multiple biosynthetic gene clusters (BGCs) involved in the synthesis of secondary metabolites, including ranthipeptides, which may exhibit antimicrobial activity. Understanding the functional roles of these BGCs, particularly their potential to inhibit , is critical for advancing microbiome-based therapies. Moreover, developing effective delivery strategies to maintain populations in the colon is essential for promoting gut health and preventing disease.

Exported polysaccharides play crucial functions in bacteria. Polysaccharide biosynthesis in the ubiquitous Wzx/Wzy- and ABC-transporter-dependent pathways starts with the transfer of a sugar-1-phosphate from a nucleotide-sugar donor to undecaprenyl phosphate, a reaction catalyzed by a phosphoglycosyl transferase (PGT). Both reaction substrates are limited and shared among multiple glycoconjugate pathways, raising the question of how bacteria regulate these pathways. In s, EpsZ, which belongs to the family of large monotopic PGTs (monoPGTs), starts the Wzx/Wzy-dependent exopolysaccharide (EPS) biosynthesis. The Dif chemosensory system regulates EPS biosynthesis by an unknown mechanism via the phosphorylated single-domain response regulator EpsW (EpsW~P). Here, we show that EpsW~P stimulates EPS biosynthesis at the post-translational level. Moreover, MiniTurbo-based proximity labeling experiments suggest that EpsW~P interacts directly with EpsZ. Additionally, heterologous expression of these two proteins in demonstrates that EpsW stimulates EpsZ enzymatic activity. WbaP, the prototype large monoPGT, forms a functional homodimer, with dimerization involving a distinct cytoplasmic β-hairpin. However, AlphaFold-based structural modeling shows that EpsZ lacks this β-hairpin, suggesting an alternative mechanism for dimerization. Structural modeling of an EpsZ-EpsW heterocomplex suggests that EpsW~P, by direct interaction, promotes the formation of the stable EpsZ dimer. These findings suggest a new model for the regulation of polysaccharide biosynthesis in which EpsW~P allosterically facilitates the formation of the active, dimeric conformation of EpsZ, thereby activating EPS biosynthesis at its initial step. Genomics and structural modeling suggest that the regulation of large monoPGTs by a single-domain response regulator is widespread in myxobacteria and potentially beyond.IMPORTANCEBacteria produce various polysaccharides with important biological functions and biotechnological applications. Polysaccharide synthesis is energy-costly and requires substrates that are in limited supply, raising the question of how bacteria regulate these pathways. Here, we explored the regulation of exopolysaccharide biosynthesis in . We demonstrate that the phosphorylated single-domain response regulator EpsW activates exopolysaccharide biosynthesis at the post-translational level by stimulating the activity of the phosphoglycosyl transferase EpsZ. By interacting with EpsZ, phosphorylated EpsW facilitates the formation of the active, dimeric conformation of EpsZ, thereby activating exopolysaccharide biosynthesis at its initial step. We propose that this previously unrecognized regulatory mechanism is broadly conserved, not only in myxobacteria but also beyond.

is a deadly fungal pathogen. Upon entering a mammalian host, it deploys a voluminous polysaccharide capsule that is necessary for it to survive host defenses and maintain an infection. Capsule expansion is regulated transcriptionally, as deletion of many transcription factors (TFs) alters the capsule. Thus, we set out to map the transcriptional regulatory network of that is, to identify the TFs that directly regulate each gene in the genome. First, we carried out RNA-seq of 120 single-TF-deletion strains, together with wild-type controls. We then applied NetProphet3, a TF network mapping algorithm, to predict the direct functional targets of each TF. Unexpectedly, analysis of this network indicated that there are no TFs that primarily regulate genes involved in capsule formation. Rather, the TFs that play a role in deploying the capsule also regulate many other genes and processes. Comparison to a TF network map we built for , a distantly related model yeast, identified pairs of TFs that are functionally orthologous-that is, their targets are enriched for orthologous genes. In many cases, these pairs are different from the ones identified by sequence homology alone. We suggest that network analyses should be used to complement sequence comparison when searching for functionally orthologous TFs. Our network map can be searched and visualized at https://cryptococcus.net/ .

is one of the most common fungal pathogens, yet much remains unknown about how its virulence factors cooperate to promote pathogenicity. To investigate this, CRISPR-Cas9 technology was used to create a panel of 19 single, double, triple, and quadruple deletion mutant strains targeting four established virulence factors: (adhesin/invasin), (candidalysin toxin), (hypha formation regulator), and (protease). , the deletion of each gene had differing impacts across multiple characterization assays. The ∆/∆ mutant was unable to form hyphae under inducing conditions, leading to downstream impairment of epithelial invasion. The ∆/∆ mutant exhibited significantly reduced adhesion and invasion into epithelial cells, resulting in attenuated cellular damage. The ∆/∆ mutant displayed significantly reduced epithelial damage, cell signaling, and immune activation. The phenotype of the ∆/∆ mutant resembled that of wild type but was unable to degrade protein. In an immunocompromised murine model of oropharyngeal infection, hyphal growth and candidalysin production were the dominant drivers of elevated fungal burden, innate immune responses, and mortality. Following a 5-day infection with ∆/∆ and ∆/∆ single gene deletion strains, mice had survival rates of 100% and 80%, respectively, compared to 15% in wild-type infected mice. Notably, 100% survival was also observed following challenge with all ∆/∆ and ∆/∆ combination mutants. This study demonstrates that specific virulence attributes act in combination to promote mucosal infection, with hyphal growth and candidalysin production being a critical driver of oropharyngeal infection.IMPORTANCE has been classified by the WHO as a "critical priority" pathogen, highlighting the urgent need for a greater understanding of the mechanisms that enable it to cause disease. possesses numerous virulence attributes, but how they synergize during infection is not well understood. Here, using reverse genetics, we dissect the individual and combinatorial roles of four virulence factors (Als3p, candidalysin, hyphal growth, and Sap2p) and in an murine model of oropharyngeal candidiasis. Increasing the number of gene deletions correlated with reduced oral fungal burden, with hyphal growth and candidalysin together being critical for infection, inflammation, and mortality during oropharyngeal infection. These findings demonstrate that virulence attributes act cooperatively as a collective network to promote pathogenicity, a finding also observed in plant fungal pathogens. Our approach has identified specific fungal virulence factors that can be targeted for new treatment strategies against infections.

Dietary components influence microbial composition in the digestive tract. Although often viewed as energy sources, dietary components are likely to shape microbial determinants of intestinal colonization beyond metabolism. Here, we report that a dietary long-chain fatty acid enhances the yeast colonization of the murine gut partly by eliciting modifications to the fungal cell surface. Mice fed an oleic acid-rich diet were readily colonized by and exhibited higher fungal load in feces compared with rodents fed an isocaloric control diet. Surprisingly, β-oxidation, a catabolic process to break down fatty acids for energy production, was dispensable for to colonize the high oleic acid diet-fed mice. 16S rRNA analysis detected rather modest differences in the bacterial communities between control and oleic acid-rich diets. We identified as an oleic acid-induced kinase that dictates cell wall mannan exposure and binding to intestinal mucin under anaerobic conditions. Furthermore, oleic acid induced the expression of several transcription factors that positively regulate intestinal colonization via remodeling of the fungal cell surface. We posit that in environments largely devoid of oxygen like the large intestine, dietary oleic acid favors a cell surface configuration that enhances gut occupation.IMPORTANCE is a fungal pathobiont that inhabits the digestive tract of most human adults. The fungus has roles in health and disease because it modulates prominent immune-inflammatory host responses from the gut, and in individuals with debilitated defenses, it can disseminate from the gastrointestinal tract, producing life-threatening infections. Here, we investigate how a dietary component shapes physiology and ultimately its ability to inhabit the mammalian gut.

Mucosal immunity plays a crucial role in protection against respiratory viruses. However, the mechanisms underlying mucosal responses and their impact on COVID-19 outcomes are not well understood, as mucosal immunity is compartmentalized and not always reflected in the bloodstream. This study examined primary immune responses in 584 mucosal and blood specimens collected over a month from previously naïve adults and children with COVID-19. Various laboratory techniques were utilized to quantify and characterize viral RNA, antigens, antibodies, and cytokines in the samples, including PCR, sequencing, ELISA, and Luminex. Comprehensive system analysis uncovered distinctive characteristics associated with mild COVID-19 disease progression, including markedly early and elevated induction of mucosal IFN-α and IP-10, followed by increased levels of IL-1RA and IgG. Individuals experiencing mild COVID-19 demonstrated synchronized mucosal and systemic immune responses, with a gradual increase in antibody production that resulted in enhanced neutralization potency, potentially conferring greater protection against future infection. In contrast, individuals with moderate and severe COVID-19 exhibited diminished IFN-α and IP-10 responses and dysregulated mucosal and systemic immune responses marked by rapid and robust yet less effective humoral immunity, potentially driven by high antigen and cytokine levels in both compartments. Collectively, these findings underscore that early mucosal immune responses may play a pivotal role in attenuating COVID-19 disease severity. Additionally, they suggest that primary mucosal immune responses to novel viruses influence clinical outcomes, providing critical insights necessary for developing prognostic indicators, treatments, and mucosal vaccines that confer protection against SARS-CoV-2 and emerging respiratory pathogens.

Microbial methane generation (methanogenesis) is an important metabolic process in the terrestrial deep biosphere and is an analog to early Earth as it is proposed to be one of the most ancient metabolisms on Earth. Signs of methanogenesis in meteorite impact craters are of particular interest in this respect as these settings are proposed hot spots for deep microbial colonization of the upper crust. Yet, reports of active deep rock-hosted methanogenesis are scarce, particularly for methylotrophic methanogenesis, while reports from terrestrial meteorite impact craters are completely lacking. Here, we used indigenous communities in cultures enriched from 400-m deep fluids to confirm and characterize active methane production from several carbon donors, including indigenous oil, in a terrestrial impact crater at Siljan, Sweden. Metagenomic and metatranscriptomic data of the methane-producing cultures revealed a consortium dominated by sp. KB-1 and Methanogranum gryphiswaldense, mediating methanogenesis solely via the methyl-reduction pathway, and resulting in a δC isotope enrichment of up to 98.6‰. These results provide insights into methylotrophic methanogenesis in deep subsurface environments in general, and in particular in fractured meteorite impact structures.IMPORTANCEThis study revealed that microbes enriched from groundwater in a 380-m deep borehole within the Siljan meteorite impact crater in Sweden were capable of producing methane, a key greenhouse gas. This is especially significant because it is the first proof of active methanogens in an impact crater and showing a specific pathway of methane production-methylotrophic methanogenesis-is present in the deep terrestrial subsurface, an environment that is typically hard to study. These findings shed light on life in extreme conditions on Earth and show that meteorite craters can be biological hotspots, rich with ancient life processes.

() causes both pulmonary and extrapulmonary infections in humans, and its virulence is largely attributed to the community-acquired respiratory distress syndrome (CARDS) toxin. CARDS toxin is transiently expressed during growth in the serum-rich SP4 medium, detected predominantly in the cytoplasm, with none secreted into the medium. Here, we show that CARDS toxin synthesis has increased at least fivefold during growth in serum-free minimal media, with approximately 40% of the toxin being released into the extracellular environment. In infected human alveolar epithelial cells, the toxin is visualized both in association with and in free form around the perinuclear region, confirming its secretion. Using density gradient centrifugation, transmission electron microscopy, and immunoblotting, we demonstrate that secretes extracellular vesicles enriched with CARDS toxin. Approximately 35% of the secreted toxin is associated with vesicles, while the remainder exists in a free-soluble form. The vesicle-bound toxin shows increased resistance to proteolytic degradation compared to the free-soluble toxin. Both forms of the purified toxin are biologically active and induce varying degrees of cytotoxicity in human epithelial cells based on their distinct trafficking patterns, which highlights their distinct roles in host-cell interactions. Together, these findings reveal a dual mode of CARDS toxin secretion as a key feature of pathogenicity, providing a significant benefit in toxin delivery and thereby enhancing virulence, expanding the scope of infection.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in antigenically distinct variants that challenge vaccine-induced immunity. The KP.2 monovalent mRNA vaccine was deployed in 2024 to address immune escape by emerging SARS-CoV-2 subvariants. We assessed neutralizing antibody responses in 56 adults with varied exposure histories following KP.2 vaccination against emerging variants including LP.8.1, LF.7.1, NB.1.8.1, XFG, and BA.3.2. While KP.2 vaccination enhanced neutralization against homologous variants, substantial reductions in neutralizing activity were observed against emerging Omicron variants across all exposure groups. Exposure history showed some influence on neutralization breadth, with self-reported vaccination-only participants exhibiting better cross-neutralization compared to individuals with hybrid immunity. Antigenic cartography revealed substantial antigenic distances between KP.2 and emerging variants, highlighting significant immune escape potential that threatens vaccine protection.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, producing variants that escape vaccine-induced immunity. The current work shows that KP.2 monovalent vaccination provides limited protection against antigenically distant Omicron variants (LP.8.1, LF.7.1, NB.1.8.1, XFG and BA.3.2). These findings highlight the ongoing challenge of maintaining vaccine effectiveness against evolving SARS-CoV-2 variants and argue for continuous updating of vaccines.

Infections caused by multidrug-resistant enterococci, particularly vancomycin-resistant (VRE), present significant therapeutic challenges. Daptomycin, a last-line treatment for VRE, often loses efficacy due to the emergence of resistance. In this study, we revealed the critical role of the gene as a key determinant of daptomycin susceptibility and resistance in . We showed that in the absence of a functional , daptomycin-resistant mutants did not emerge , and derivatives of clinical daptomycin-resistant strains engineered to lack functional were rendered even more sensitive to daptomycin than wild-type daptomycin-susceptible strains. These findings indicated that functional is critical for key known mechanisms of daptomycin resistance, and mutations in have phenotypic dominance to those that otherwise confer resistance. Therefore, inhibiting the activity of the gene product is predicted to prevent or reverse resistance, offering a promising new strategy for extending the efficacy of daptomycin for treating enterococcal infections.IMPORTANCEDaptomycin is one of the few remaining effective antibiotics for treating vancomycin-resistant enterococcal infections but is limited by the emergence of resistance during protracted therapy. Here, we show that without a functional gene, daptomycin-resistant mutants do not arise under conditions where wild-type strains readily generate daptomycin-resistant mutants. Furthermore, we show that loss of function mutation of the gene in daptomycin-resistant clinical isolates renders them more susceptible to daptomycin than wild-type . This indicates that an effective small molecule inhibitor of LafB activity or gene expression would be a useful adjunctive for extending and restoring the therapeutic utility of daptomycin.