mBio

We previously reported that the oxidative stress sensor Kelch-like ECH-associated protein 1 (Keap1) recognizes hepatitis B virus (HBV) X protein (HBx) to activate the NF-E2-related factor 2 (Nrf2) signaling pathway, thereby inhibiting HBV replication, and that HBx promotes K6-linked polyubiquitylation of Nrf2. However, the molecular mechanism remains unclear. Here, we investigated the role of HECT, UBA, and WWE domain-containing E3 ubiquitin ligase 1 (HUWE1) in HBx-mediated K6-linked polyubiquitylation of Nrf2 and its impact on HBV replication. Cell-based ubiquitylation assays demonstrated that HUWE1 knockdown reduced HBx-mediated K6-linked polyubiquitylation of Nrf2, while overexpression of wild-type HUWE1, but not the catalytically inactive HUWE1(C4341A) mutant, enhanced it, indicating that HUWE1 E3 ligase activity is required. Coimmunoprecipitation and proximity ligation assays demonstrated that HUWE1 interacts with HBx in the cytoplasm and binds Nrf2 only in the presence of HBx, suggesting that HBx bridges HUWE1 and Nrf2 into a ternary complex. Cycloheximide chase assays demonstrated that HUWE1 knockdown destabilized Nrf2 in HBx-expressing cells, supporting a role for HUWE1 in Nrf2 stabilization via K6-linked polyubiquitylation. Furthermore, HUWE1 knockdown or treatment with the HUWE1 inhibitor BI8626 significantly increased HBV RNA and pgRNA levels in HBV-infected cells. Collectively, these results demonstrate that HUWE1 promotes K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner to inhibit HBV replication. IMPORTANCEHepatitis B virus (HBV) chronically infects approximately 254 million people worldwide, yet host mechanisms that restrict viral replication remain incompletely understood. The Kelch-like ECH-associated protein 1 (Keap1)/ NF-E2-related factor 2 (Nrf2) signaling pathway is a central defense against oxidative stress. Under basal conditions, Nrf2 is degraded via Keap1/Cullin3-mediated K48-linked polyubiquitylation. We previously demonstrated HBV infection promotes Nrf2 stability through non-canonical K6-linked polyubiquitylation. Here, we identify the E3 ubiquitin ligase HUWE1 as the enzyme responsible for K6-linked polyubiquitylation of Nrf2. HBV X protein (HBx) recruits HUWE1 to Nrf2, forming a HUWE1/HBx/Nrf2 complex that switches Nrf2 ubiquitylation from K48 to K6, stabilizing Nrf2 and suppressing HBV replication. These findings reveal a novel antiviral mechanism exploiting a non-canonical ubiquitin code and highlight HUWE1 as a potential therapeutic target against chronic HBV infection.

Nitrogen fixation plays a central role in primary productivity and nitrogen cycling in aquatic ecosystems, yet its distribution among cyanobacterial lineages remains incompletely understood. Biological nitrogen fixation is energetically costly and highly oxygen-sensitive, imposing constraints in oxygenic phototrophs. The unicellular cyanobacterial genus Synechocystis has long been regarded as strictly non-diazotrophic. Here, we report that Synechocystis sp. LKSZ1 possesses a functional nitrogen fixation system. Comparative genomics revealed that LKSZ1 is distinct from other Synechocystis strains and uniquely harbors a complete nif gene. Phylogenetic and structural analyses indicate acquisition via horizontal gene transfer from filamentous cyanobacteria. Physiological assays demonstrated photoautotrophic growth under nitrogen-depleted conditions and nitrogenase activity under microoxic to anaerobic conditions. Disruption of nifK abolished both growth and activity. These findings show that ecological nitrogen limitation and host compatibility can enable functional integration of horizontally acquired nitrogen fixation.

Orientia tsutsugamushi (Ot) is an obligately intracellular bacterium that causes scrub typhus, a potentially severe infectious disease characterized by systemic inflammation and multiorgan dysfunction. We recently reported a protective role for IFN-{gamma} signaling in host defense against Ot infection; however, the underlying mechanisms remain obscure. Inducible nitric oxide synthase (iNOS, encoded by Nos2) is a key antimicrobial effector induced downstream of IFN-{gamma} signaling. Here, we used transgenic mouse models to further investigate the biological functions of iNOS. We first revealed the requirement of iNOS for the restriction of Ot growth in cultured bone marrow-derived macrophages. Using an intradermal mouse model, we found that while tissues of Nos2-/- and wild-type mice exhibited comparable bacterial burdens during acute infection phases, Nos2-/- mice developed eschar-like lesions similar to those observed in Ifngr1-/- mice, indicating a critical role for the IFN-{gamma}/iNOS axis in regulating skin pathology in scrub typhus. Notably, Nos2-/- mice displayed impaired bacterial clearance during the recovery phase (day 42), with persistent bacterial burdens in multiple organs accompanied by sustained immune activation and elevated inflammatory responses. Histopathological and biochemical analyses further revealed increased tissue damage and dysregulated physiological homeostasis in Nos2-/- mice during recovery. Mechanistically, iNOS deficiency resulted in heightened myeloid cell activation and prolonged expression of proinflammatory mediators, suggesting a dual contribution of iNOS in both antimicrobial defense and inflammation resolution. Collectively, these findings provide new insight into IFN-{gamma}-mediated defense mechanisms and imply the distinct roles of iNOS during different stages of scrub typhus. Author summaryScrub typhus is a potentially severe infectious disease caused by the bacterium Orientia tsutsugamushi (Ot), which is transmitted to humans through the bite of infected mites. Despite its global impact and expanding geographic distribution, the immune mechanisms that protect against this infection remain incompletely understood. In this study, we examined the role of inducible nitric oxide synthase (iNOS), an immune effector molecule that helps the host control infection. Using mouse models, we found that iNOS plays dual and stage-specific roles during Ot infection. Mice lacking iNOS developed dysregulated immune homeostasis during acute infection and exhibited skin lesions resembling the eschars observed in some patients with scrub typhus. In addition, these mice showed delayed bacterial clearance, prolonged inflammation, and increased tissue damage during the recovery phase. Our findings indicate that iNOS contributes not only to host antimicrobial defense but also to the control of excessive inflammation following infection. These results provide new insight into host defense mechanisms in scrub typhus and may help inform future therapeutic or preventive strategies.

Microbial degradation of suspended and sinking organic carbon regulates long-term oceanic carbon storage by controlling the efficiency of the biological pump. Yet microbial controls on carbon export and remineralization remain poorly constrained, limiting predictions of how ocean carbon cycling will respond to climate change. Here, we combined in situ sampling with ship-based incubations to quantify prokaryote-driven removal rates of suspended and sinking total organic carbon (TOC). Samples were collected below the mixed layer during three stages of a spring Phaeocystis pouchetii bloom in the Labrador Sea. Phaeocystis blooms can dominate regional phytoplankton biomass and are expected to increase under future climate. Removal rates were used as a proxy for carbon lability and combined with 16S rRNA metabarcoding and carbon composition analyses to link microbial community structure with substrate characteristics. Removal rates of sinking particles (0.02-0.06 d-1) were an order of magnitude higher than those of suspended TOC (0.002 d-1) during bloom-decline and non-bloom. In contrast, during late-bloom, suspended carbon exhibited rates of 0.01 d-1, comparable to sinking particles, and was enriched in exopolymer-rich colonies. Prokaryotic community composition varied primarily among bloom stages rather than carbon fractions, indicating that bloom stage-- and thus particle origin and composition--was the dominant control on bacterial degradation and assembly. Bacterial diversity peaked where carbon was refractory and originated from mixed phytoplankton. Together, these results demonstrate that suspended Phaeocystis-derived carbon can be rapidly remineralized when blooms produce exopolymer-rich colonies and highlight bloom stage as key regulator of microbial carbon processing and biological pump efficiency.

The ability of the bacterial endosymbiont Wolbachia pipientis to block arboviruses in its mosquito host may be impinged by host genetic variation, leading to reduced efficacy in field releases. Across a large collection of Drosophila lines carrying natural genetic variation, we found that viral replication varied greatly in the absence of Wolbachia. However, the introduction of the symbiont reduced viral load in each background to similar levels, near the limit of detection. Therefore, Wolbachia-mediated viral blocking is seemingly robust against host genetic background. A genome-wide association study harnessing the variation in the viral loads across the Wolbachia-free set identified rhoGAP18B and betaCOP as host factors that contribute to SINV replication; furthermore, the gene products of which seemingly interact with each other in the context of cytoskeletal dynamics. These results shed light on host requirements for SINV replication and suggest possible avenues by which Wolbachia may encroach upon them during blocking.

Background and AimsThe rising incidence of Crohns disease (CD) in Westernized countries has been linked to changes in diet and increased consumption of food additives, yet the mechanisms by which these factors fuel intestinal inflammation remain unclear. Adherent-invasive Escherichia coli (AIEC), a pathobiont involved in CD pathogenesis, lacks a clear genetic hallmark but exhibits intestinal colonization and virulence traits, raising questions about the evolutionary forces promoting its emergence among select individuals. Here, we investigated how chronic exposure to two common dietary emulsifiers, carboxymethylcellulose (CMC) and polysorbate 80 (P80), along with host inflammation, drives AIEC genomic evolution and pathogenic potential. MethodsWild-type and Il10-deficient mice were monocolonized with AIEC and chronically exposed to CMC, P80, or water. Bacterial isolates were collected and analyzed for genomic diversification, mutations, and phenotype both in vitro and in vivo. ResultsEmulsifiers accelerated AIEC genomic diversification and selected for mutations linked to increased motility, invasion, and pro-inflammatory activity. Moreover, dietary emulsifier-evolved strains displayed a marked fitness advantage in vivo, outcompeting their counterparts in murine hosts, with the greatest advantage observed when evolution occurred under inflammatory conditions. Notably, evolutionary pathways and phenotypic outcomes were shaped by both emulsifier and the hosts inflammatory status, highlighting synergy between diet and host genetics in fostering pro-inflammatory pathobionts. ConclusionThese findings provide an evolutionary framework connecting modern dietary habits to the emergence of pathogenic AIEC strains, and underscore the importance of dietary interventions in individuals at risk for inflammatory bowel disease.

The type VI secretion system (T6SS) is a contractile nanoweapon widely employed by Gram-negative bacteria to gain competitive advantages by injecting effector proteins into recipient cells. Although the biochemical activities of T6SS effectors have been well characterized, how recipient factors modulate effector toxicity remains poorly understood. Using Agrobacterium C58 as a model, previous work identified the Escherichia coli ClpAP protease as a recipient susceptibility (RS) factor that enhances T6SS-mediated interbacterial competition. Agrobacterium C58 deploys two DNase effectors, Tde1 and Tde2, as the major antibacterial weapon. Here, we demonstrate that the recipient ClpAP protease and its adaptor ClpS enhanced C58-mediated interbacterial competition in a Tde2-dependent manner in both intra- and interspecies competition. Ectopic expression of Tde2 in E. coli caused growth inhibition and DNA cleavage in the presence of a functional ClpAPS protease complex, but not in any of the clpP, clpA or clpS mutants. Notably, Tde2 accumulated in these mutants but not in wild-type cells, whereas a catalytic variant accumulated regardless of ClpAPS status, suggesting that Tde2 is not directly degraded by ClpAPS. Instead, Tde2 depends on ClpAPS for full toxicity, likely through degradation of inhibitory N-degron substrate(s). Affinity purification of His-tagged Tde2 in a clpP mutant background, followed by mass spectrometry, identified eight N-degron substrate candidates. Tde2-mediated interbacterial competition was significantly reduced by overexpression of three candidates. Among them, the Tde2 DNase domain directly associated with guanosine 5-monophosphate reductase GuaC, supporting a model in which Tde2 toxicity is blocked by binding of GuaC. Collectively, our findings reveal an unanticipated layer of recipient-mediated regulation in T6SS competition and highlight proteolytic control of inhibitory substrates as a determinant of bacterial susceptibility during interbacterial conflict.

Formaldehyde is a highly toxic metabolite that can cause extensive damage to DNA and proteins, and strategies to mitigate formaldehyde toxicity are poorly understood. Methylotrophic bacteria, such as Methylobacterium extorquens, thrive on one-carbon compounds as sole sources of carbon and energy. These organisms are excellent models for discovering formaldehyde stress response systems because formaldehyde is an obligate intermediate in their central carbon metabolism. Here, we characterize an evolved def allele (defevo) that increases formaldehyde resistance in M. extorquens. The def gene encodes peptide deformylase (PDF, EC:3.5.1.88), an enzyme that contributes to protein processing by removing the formyl group from N-formylmethionine (fMet) on nascent peptides. The defevoallele has a single missense mutation that decreases PDF activity both in vitro and in vivo. Transcriptomic analysis of the defevo strain indicates there are pleiotropic effects of this mutation and a differential response to formaldehyde stress. We investigate possible mechanisms for the defevo mutants increased resistance to formaldehyde, including mitigation of formaldehyde-induced protein stress and altered membrane physiology. We find that the defevo allele selectively alleviates exogenous, but not endogenous, formaldehyde stress and identify a tradeoff in heat shock resistance. This study reports the first observation of lowered PDF activity benefiting a cellular physiological phenotype. Our work indicates that altered protein metabolism can mitigate the toxic effects of formaldehyde and furthers our understanding of the strategies that can protect cells from formaldehyde-induced damage. ImportanceFormaldehyde is a toxic chemical that can damage essential molecules inside of cells, yet all organisms inevitably produce it during normal metabolism. Despite its ubiquity, our understanding of strategies for how cells navigate formaldehyde toxicity is incomplete. This study focuses on Methylobacterium extorquens, which naturally generates high levels of formaldehyde as part of its growth on simple carbon compounds. We show herein that a single genetic change, which slows down how newly made proteins are processed during translation, can unexpectedly improve the bacteriums ability to resist formaldehyde stress. Further, we show that this single change has numerous effects on the cell, many of which may contribute to formaldehyde resistance.

Recent advances in high-throughput sequencing and novel culture techniques have revolutionized our understanding of the human microbiota. However, most studies primarily focused on bacterial communities, often overlooking the fungal component. Building upon our previous metagenomic analysis of the Inuit oropharyngeal microbiome 1, this study used culturomics to provide a more comprehensive view of both bacterial and fungal communities. We analyzed oropharyngeal swabs from the Qanuilirpitaa? 2017 Inuit Health Survey 2, demonstrating the complementarity of metagenomic and culturomic approaches. Our findings highlight the importance of culturomics in revealing low-abundance microorganisms, particularly fungi, which are often underrepresented in metagenomics data. Moreover, we designed an approach to isolate previously uncultivated species. We described two Pauljensenia sp., and provided insights into the phylogenetic relationship between Schaalia and Pauljensenia genera. This study underscores the necessity of a holistic approach to microbiome research, combining multiple techniques to fully elucidate microbial diversity in unique populations like the Inuit.

Bacteria use diverse mechanisms to interact with each other and with eukaryotic hosts, thereby shaping microbiome composition and influencing host health. One of these mechanisms is the production of outer membrane vesicles (OMVs), nanoscale structures that bud off from bacterial cells into the extracellular space. OMVs can deliver bioactive cargoes, including enzymes, RNA and DNA, enabling functions such as cell-to-cell communication, nutrient acquisition and immunomodulation. However, the role of OMVs in beneficial host-associated microbiomes remains unclear. Here, we investigated OMV production in the gut bacteria of the western honey bee (Apis mellifera), which forms a highly conserved and stable microbial community. Using electron microscopy, fluorescence labelling, and nanoparticle tracking analysis, we detected OMV production in every gram-negative species of the normal bee microbiota that we investigated. Vesicles were observed in gut contents of wild and laboratory-inoculated bees, but absent in bees lacking a microbiota. OMVs contained nucleic acids, with more RNA than DNA. Bacterial strains varied in OMV properties, including abundance, size, and zeta potential. These findings indicate that OMVs are likely significant mediators of interbacterial and host-microbe interactions in the bee gut.

ObjectivesAntimicrobial resistance (AMR) in Gram-negative pathogens is driven by complex and coordinated molecular mechanisms that remain incompletely characterized. This study integrated phenotypic, genomic, and quantitative proteomic analyses to characterize multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria circulating in an intensive care unit (ICU) in Northeastern Brazil. MethodsA total of 259 Gram-negative isolates collected between 2019 and 2021 underwent species identification, antimicrobial susceptibility testing, and targeted qPCR for resistance genes. Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa representing susceptible, MDR, and XDR phenotypes were selected for whole-genome sequencing and label-free quantitative proteomics. Differential protein abundance was assessed using Limma with |log2FC| > 1 and p < 0.05. ResultsK. pneumoniae (47%), A. baumannii (24%), and P. aeruginosa (21%) predominated. Carbapenem resistance reached 44%, 93%, and 61%, respectively, and MDR/XDR phenotypes occurred in >30% of isolates. Genomic analyses revealed dense resistomes with coexisting {beta}-lactamases (blaKPC, blaNDM, blaCTX-M, OXA) and widespread efflux systems. Proteomic profiling demonstrated phenotype-associated differences in outer membrane proteins, transport systems, regulatory proteins, and metabolic pathways. XDR isolates showed additional enrichment of envelope remodeling proteins, stress response mechanisms, and proteostasis-associated factors. ConclusionsMDR and XDR Gram-negative ICU pathogens exhibit coordinated resistance architecture characterized by accumulation of resistance genes and adaptive proteomic remodeling. Integrated multi-omics approaches provide mechanistic insight into antimicrobial resistance and support improved surveillance and therapeutic strategies. What is known?O_LIAntimicrobial resistance is a priority and a serious problem in global health, resulting in high rates of morbidity and mortality. C_LIO_LIKlebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa are on the World Health Organizations (WHO) priority list as major causes of morbidity and mortality worldwide. C_LIO_LIClassical characterization of susceptibility and resistance phenotypes does not capture the complexity of antimicrobial resistance and hampers effective control measures and actions to minimize the evolutionary dynamics of resistance in these bacteria. C_LI What is new?O_LIThe study characterizes the phenotypic pattern of antimicrobial susceptibility, the presence and sequencing of the resistome and virulome, and analyzes the label-free quantitative proteome of susceptible, MDR, and XDR phenotypes in strains of K. pneumoniae, A. baumannii, and P. aeruginosa circulating in hospital ICUs in Brazil. C_LIO_LIMDR and XDR gram-negative phenotypes are associated with a dense resistome, with widespread dissemination of beta-lactamase genes (bla_KPC, bla_NDM, bla_CTX-M, and OXA) and RND-type (MEXs) and acrAB-tolC efflux pumps, without changes in virulence genes. C_LIO_LIProteomic analysis demonstrated increased production of beta-lactamases, components of efflux pump systems, outer membrane protein synthesis, protection for oxidative stress mechanisms, proteins for iron acquisition, and systemic regulators. XDR strains additionally showed enhanced remodeling of the cell envelope, activation of proteostasis, and metabolic adaptation. C_LI

Salmonella Typhimurium is a versatile foodborne pathogen with a broad ecological range, making it an ideal model to better understand pathogen adaptations that allow them to infect multiple hosts and persist across diverse environments. We analyzed 745 genomes of S. Typhimurium isolated from three food animal sources (bovine, swine, and poultry) and two non-food animal sources (wild birds and the environment). We found that S. Typhimurium from food animal sources generally had a more open pangenome and harbored more antimicrobial resistance genes (ARGs) than non-food animal sources. Notably, swine isolates exhibited the most open pangenome and prevalent ARGs, likely as a result of horizontal gene transfer primarily mediated by plasmids. Despite similar core genome sizes, S. Typhimurium from different sources displayed distinct patterns of positive selection in the core genome that varied in both frequency and targeted functional categories. In contrast, although accessory genome sizes varied substantially across sources, the frequency of positive selection remained similar. Using machine learning, we further identified genetic variants (e.g., virulence factors) highly predictive of sources. These findings suggest that gain and loss of accessory genes and positive selection acting on core genes facilitate differential adaptation in S. Typhimurium, contributing to its broad ecological range.

Microbial communities are critical to the functioning of ecosystems and shape the ecology and evolution of host organisms. However, we have a limited understanding of how host-associated and free-living microbes differ in their structure and biogeography. Here, we test whether host-associated (fish gut) and free-living (lake bacterioplankton) microbes exhibit different metacommunity structure, spatial turnover, and consistency with neutral expectations using two independent lake systems. We characterized microbial communities in lake water (Vancouver Island and Sierra Nevada) and guts in two fish species (stickleback and brook trout) using 16S amplicon sequencing. We compared alpha and beta diversity within lakes, quantified spatial turnover (distance-decay), and tested for departure from neutral abundance-occurrence expectations between bacterioplankton and fish gut microbiomes. Fish microbiomes had lower alpha diversity compared to bacterioplankton, but higher beta diversity within lakes. Bacterioplankton were more similar across lakes yet showed stronger patterns of spatial turnover with distance than fish gut microbiomes. A neutral model explained a substantial proportion of abundance-occurrence relationships in bacterioplankton communities but performed poorly for fish-associated microbes. Our study indicates that host-associated and free-living microbes have disparate patterns of metacommunity structure and spatial turnover consistent with differences in the strength of neutral ecological processes. Fish microbiomes were less diverse at the local scale but more variable across space and time than bacterioplankton communities, suggestive of potentially strong local selection and/or reduced microbial exchange among hosts compared to environmental communities. Importantly, we observed highly consistent patterns across both lake systems despite differences in host species, sampling design, and region, demonstrating that differences in the distribution of host and environmental microbes are potentially widespread. This study demonstrates how host association fundamentally alters the diversity and spatial distribution of microbes, emphasizing the need to incorporate hosts into broader frameworks of microbial biogeography.

Bacterial ribosomal RNAs (rRNAs) are decorated with conserved nucleotide modifications, but the functionality of these modifications is often underexplored. MraW (RsmH) is a 16S rRNA methyltransferase that fine-tunes ribosomal function. We identified a loss-of-function allele in mraW that corrected a late-stage sporulation defect in Bacillus subtilis by bypassing a key sporulation checkpoint via altered translational regulation. Purified ribosomes isolated from {Delta}mraW cells displayed a [~]2-fold decrease in translation efficiency; in vivo, {Delta}mraW cells produced decreased levels of the sporulation checkpoint protein CmpA. This regulation was mediated by sequences from the 5 untranslated region and the coding sequence of cmpA, which form a step-loop structure that occlude early codons of the mRNA. Proteomic analysis revealed that MraW directly or indirectly regulates the production of multiple proteins, some of which form similar structural elements as the cmpA transcript. We propose that MraW modification of 16S rRNA enhances translation efficiency in general, and that specific transcripts, whose gene products are likely required in limiting quantities, have evolved structural features that act as a regulatory mechanism to govern protein levels. This type of regulation may be most apparent in bacteria which exhibit uncoupled transcription and translation. HIGHLIGHTSO_LIA conserved 16S rRNA modification enhances translation of structured mRNAs C_LIO_LIEarly mRNA stem-loops impose translational control dependent on ribosome modification C_LIO_LImRNA structure and rRNA modifications likely co-evolved to fine-tune protein dosage C_LI

Acinetobacter baumannii is a high-priority multidrug-resistant pathogen that survives within host cells by hijacking intracellular defense pathways. Here, we identify a previously unrecognized signaling axis linking bacterial invasion to host lysosomal regulation. We show that A. baumannii activates calcium-independent phospholipase A2 (iPLA2), leading to increased lysophosphatidylcholine (LPC) production and calcium influx through the ORAI1 channel, which together drive activation and nuclear translocation of the lysosomal transcription factor EB (TFEB). Pharmacological inhibition or genetic silencing of iPLA2 or ORAI1 markedly impaired TFEB activation and lysosomal biogenesis. Mechanistically, we demonstrate that this pathway is initiated by the outer membrane protein A (OmpA), which promotes bacterial invasion and enhances iPLA2 activity, LPC production, and downstream TFEB signaling. Despite induction of lysosomal biogenesis, A. baumannii persists intracellularly by producing ammonia and alkalinizing the lysosomal environment, thereby counteracting host antibacterial activity. In vivo, infection induces activation of HLH-30, the TFEB ortholog, in Caenorhabditis elegans in an OmpA-dependent manner. Together, our finding define an OmpA-iPLA2-LPC-ORAI1-TFEB signaling axis that coordinates host lipid and calcium signaling with lysosomal responses, while revealing a bacterial counterstrategy that promotes intracellular survival.

AbstractMicrobial diversity is often assumed to be limited by the number of available resources, yet many communities persist well beyond that expectation. Understanding the mechanisms that enable such coexistence remains a central question in microbial ecology. Here, using a four-species bacterial consortium, we asked whether coexistence can emerge from interactions between species rather than from the external environment alone. Across 31 simple nutrient conditions, including 16 single-resource environments, all four species persisted and repeatedly reached stable coexistence. We then chose 27 additional conditions to further probe the boundaries of coexistence by varying resource concentrations, temporal dynamics, nutrient complexity and relief of auxotrophy-associated dependencies, and only observed the extinction of one species in one of these conditions. Although the community composition in each environment was largely shaped by species fitness on the supplied resources, experimental assays and consumer-resource modeling showed that the coexistence was not explained by resource supply, but rather by cross-feeding and niche partitioning of metabolic byproducts. These metabolic interactions were strong enough to sustain coexistence even for species unable to use the supplied resources directly. Furthermore, robust coexistence across environments appears to be an emergent property of microbial communities, ingrained in members metabolic byproduct profiles and niche differences. Our findings demonstrate how microbes can increase the chemical complexity of their environment sufficiently to maintain coexistence well beyond what is expected from external resource supply. SignificanceUnderstanding the drivers of microbial diversity is essential for managing natural ecosystems and designing synthetic microbiomes. This study challenges the conventional application of the competitive exclusion principle, demonstrating that a four-species consortium can coexist across 31 chemically and metabolically diverse one- and two-carbon source environments. By systematically testing and ruling out alternative stabilizing mechanisms, we show that co-existence is an emergent property of the consortium, sustained by metabolic cross-feeding and niche partitioning. Guided by computational models, we identify hallmarks of robust co-existence in simple environments, including high variance in resource affinities and growth on partner-derived metabolites. Our work demonstrates how microbes modify their environment to sustain high diversity and provides principles for designing synthetic microbiomes that persist across environments.

Kaposis Sarcoma Herpesvirus (KSHV), an enveloped double-stranded DNA virus, is the etiological agent of Kaposis sarcoma (KS), an endothelial cell-based tumor. KSHV is a leading cause of infection-related cancers in sub-Saharan Africa and immunocompromised individuals worldwide. Therefore, it is vital to identify the underlying mechanisms of viral infection and transmission to effectively identify specific therapeutic strategies and combat the disease. Here, we demonstrate that KSHV rewires the host cell lipidome during lytic infection. Bulk lipidomic analysis shows significant changes in the abundance of neutral lipids and phospholipids during lytic infection. We further investigated fatty acid-binding proteins (FABPs) to understand the underlying mechanisms that support KSHV pathogenesis. Using the doxycyclin-inducible iSLK.BAC16 cell line, we find that FABP genes are differentially regulated by lytic KSHV infection compared to latent infection. We report that FABP4 is significantly upregulated during lytic infection. Loss of FABP4 during lytic infection does not impact viral gene transcription however, lytic protein translation is reduced. Moreover, our intracellular and extracellular viral titers indicate that FABP4 affects maximal infectious virion production. This study highlights the role of FABP4 and its therapeutic potential as a target that facilitates KSHV infection and pathogenesis.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy characterized by local invasion, lymph node metastasis, and therapeutic resistance. Chronic periodontal disease has been linked to HNSCC progression, yet the responsible pathogens and underlying molecular mechanisms remain unclear. Here, we show that the keystone periodontal pathogen Porphyromonas gingivalis promotes HNSCC metastasis and chemoresistance through two internalin proteins that are secreted via the type IX secretion system (T9SS). These internalin proteins specifically bind the EC1 domain of E-cadherin through their curved solenoid-like leucine-rich repeats (LRRs), facilitating bacterial invasion and inducing epithelial-to-mesenchymal transition (EMT). Mechanistically, internalin-E-cadherin engagement drives {beta}-catenin nuclear translocation and activates p38 and JNK1/2 MAP kinase signaling pathways, enhancing tumor cell migration, metastatic dissemination, and resistance to cisplatin-induced apoptosis. Tissue microarrays detect internalin antigens in HNSCC specimens, supporting their in vivo relevance. Together, these findings establish a direct mechanistic link between an oral pathogen and HNSCC progression and extend the paradigm of internalin-E-cadherin interactions from microbial pathogenesis to cancer biology.

Gingival inflammation is associated with dysbiotic oral biofilms characterized by reduced nitrate-reducing capacity and diminished nitric oxide (NO) bioavailability. While dietary nitrate has been shown to influence oral microbial activity, the effects of sustained, localized nitrate delivery on oral biofilm ecology and gingival inflammation remain incompletely defined. In this randomized, double-blind, placebo-controlled trial, 30 adults with gingival bleeding were assigned to receive localized prebiotic nitrate (~0.989 mmol per dose) or placebo for 21 days. The primary outcome was mean bleeding on probing (mBOP). Secondary outcomes included modified Gingival Index (mGI), Quigley-Hein plaque index (QHPI), salivary nitrite (as a proxy for NO bioavailability), oral pH, and microbiome composition assessed by 16S rRNA gene sequencing. Prebiotic nitrate supplementation formulation delivered in a slow-release chewing gum significantly reduced mBOP (25.7% to 15.3%; p = 0.0002) compared to placebo chewing gum. Salivary nitrite levels and oral pH increased, indicating enhanced nitrate metabolism. Microbiome analysis demonstrated enrichment of nitrate-reducing taxa, including Rothia mucilaginosa and Neisseria spp., and a relative reduction in inflammation-associated genera such as Prevotella and Porphyromonas. Localized prebiotic nitrate formula delivered in a functional chewing gum was associated with reduced gingival inflammation and shifts in oral microbiome composition consistent with enhanced nitrate-reducing capacity critical in nitric oxide formation. These findings support a role for biofilm-directed nutritional modulation as a non-antimicrobial approach for managing gingival inflammation and improving nitric oxide bioavailability.

Gut microbial biotransformation of poorly absorbable polyphenols into bioactive, bioavailable metabolites is increasingly recognized as a key mechanism underlying their health benefits of polyphenols. Microbial ellagic acid (EA)-to-urolithin conversion represents a typical example, but the environmental factors that facilitate such metabolism remain underexplored. We discovered that urolithin production by a gut commensal bacterium, Gordonibacter urolithinfaciens (G. uro), is metabolically repressed by arginine. To overcome such limitations, we developed PhenolBoost Medium (PBM) that induces a metabolic shift by suppressing the arginine deiminase pathway while activating pyruvate metabolism and hydrogen production in G. uro, thereby driving urolithin dehydroxylation. Transcriptomic profiling and 13C-isotopic tracing analysis revealed that pyruvate metabolism in PBM upregulates hydrogenase expression, facilitating the dehydroxylation of EA. PBM also promoted the complete conversion of EA to urolithin A in G. uro-Enterocloster bolteae co-culture, and other polyphenol biotransformations. In addition, co-culturing G. uro with hydrogen-producing Bacteroides species significantly increased urolithin production. Furthermore, an arginine-limited, pyruvate-enriched dietary regimen proved effective in vivo, resulting in significantly higher urolithin production and bioavailability in a mouse model. Our findings reveal the critical role of hydrogen in facilitating polyphenol dehydroxylation, and offer a viable nutritional strategy for boosting microbial production of beneficial metabolites from polyphenols.