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Endosymbiosis in trypanosomatids involves a mutualistic association between a symbiotic bacterium and a host protozoan and represents an excellent model for studying metabolic coevolution and the origin of organelles. This work investigated the influence of the symbiont on the metabolism of Angomonas deanei by comparing wild-type and aposymbiotic strains under different nutritional conditions. The presence of the symbiont enhanced cell proliferation in the medium containing a single carbon source and increased O₂ consumption. Wild-type cells utilized oxidative phosphorylation to produce ATP, whereas aposymbiotic cells relied on substrate-level glycolysis, resulting in the excretion of greater amounts of fermentative products, such as acetate, succinate, and ethanol. Proteomic analysis revealed an increased expression of glycolytic and fermentative enzymes by the aposymbiotic strain and oxidative phosphorylation enzymes by symbiont-harboring cells. These findings highlight the role of the symbiotic bacterium in optimizing host metabolism and provide insights into the evolution of parasitism in trypanosomatids when A. deanei is compared with pathogenic species.

Importance: This work provides groundbreaking insights into the metabolic and evolutionary dynamics of endosymbiosis, a topic of central importance to cellular evolution. Angomonas deanei, a trypanosomatid species, has become a paradigm for investigating the evolution of eukaryotic cells and the origin of organelles through endosymbiosis. Harbored in the cytoplasm of this protozoan, the symbiont engages in intricate metabolic exchanges, offering a time window to analyze the processes and evolutionary history that underlie the establishment of permanent endosymbiotic relationships. By employing a multidisciplinary approach, we have uncovered how the symbiotic bacterium regulates the oxidative metabolism of the trypanosomatid, integrating glucose catabolism and optimizing energy production. Our discoveries have broad implications for understanding the metabolic integration of organelles, such as mitochondria and glycosomes, with the bacterial endosymbiont. Beyond unravelling the complexities of metabolic adaptations during symbiosis, our work may contribute to the general understanding of the evolutionary dynamics of parasitism within the Trypanosomatidae family.

Salmonella is a major foodborne pathogen causing substantial illness and economic burden globally. To address the lack of a centralized resource, we developed the Salmonella Serovar Wiki, a curated web portal providing comprehensive, serovar-specific information on more than 100 Salmonella serovars, including their geographical distribution, genomic characteristics, animal reservoirs, and outbreak history. Built on the Confluence platform, this Wiki consolidates data from sources such as the European Center for Disease Prevention and Control, Centers for Disease Control and Prevention, the U.S. Food and Drug Administration, and Rapid Alert System for Food and Feed, offering a valuable tool for academia, industry, and public health professionals.IMPORTANCEBy facilitating rapid access to serovar-specific information, the Salmonella Serovar Wiki aids both epidemiologists and environmental health professionals in hypothesis generation, outbreak investigations, and research into understudied serovars, ultimately enhancing food safety and public health outcomes.

Staphylococcus aureus is a common commensal of human skin and anterior nares but also a notable pathogen, causing a range of diseases from minor skin infections to serious, often fatal conditions. The global health community considers S. aureus a serious threat due to the rise of multidrug-resistant strains, including community-acquired methicillin-resistant S. aureus. Effective host tissue invasion by S. aureus requires precise spatiotemporal regulation of virulence factor expression, which can be controlled by small RNAs (sRNAs). Our lab has identified over 300 potential sRNAs in S. aureus USA300, highlighting their regulatory significance. Recently, our group characterized an sRNA named Teg41, which positively influences transcription from the PSMα promoter, enhancing toxin production and virulence. Teg41, located 203 bp downstream of the psmα transcription start site, exhibits relatively stable growth phase expression, a first for sRNAs in S. aureus. We demonstrate that the Teg41 and psmα promoters respond to different stimuli, and the Teg41 promoter has distinct regulatory regions. Single-cell analysis reveals non-homogeneous Teg41 expression within the bacterial population, with a subset of cells showing high expression. This study introduces new tools for studying divergent promoters and elucidates the Teg41-psmα expression relationship.IMPORTANCEStaphylococcus aureus is a leading cause of human infections and a major public health concern due to rising antibiotic resistance. Understanding how this pathogen regulates its virulence is critical to developing new therapeutic strategies. sRNAs are key regulators of bacterial gene expression, yet most remain uncharacterized. Here, we investigate Teg41, an sRNA that activates expression of the potent PSMα toxins. Using a novel dual-fluorescence reporter system and single-cell analysis, we uncover that Teg41 and its target promoter are regulated independently and heterogeneously across the bacterial population. These findings reveal new insights into sRNA-mediated regulation of virulence and provide innovative tools to dissect complex gene regulatory networks in S. aureus.

Urbanization is accelerating at an unprecedented pace, with 70% of the global population projected to live in cities by 2050. This shift presents significant challenges and opportunities for fostering sustainable urban ecosystems aligned with the United Nations Sustainable Development Goals. Microbiomes-the diverse communities of microorganisms that underpin ecosystem function-are increasingly recognized for their vital role in nutrient cycling, climate regulation, biodiversity support, and human well-being. However, their consideration and integration in urban design remain underexplored, often limited to disease mitigation. The emerging field of microbiome-integrated urban design seeks to leverage microbial activity to enhance urban health and resilience through a multispecies framework. To address critical gaps, the Probiotic Cities Working Group convened a global interdisciplinary workshop, engaging experts from ecology, architecture, urban planning, immunology, and social sciences. Using reverse brainstorming and thematic analysis, participants identified eight core themes and 40 priority research questions (via a modified Delphi technique). These themes span communication and policy, pollution prevention, interdisciplinary collaboration, experimental design, ethics, and public perception of microbiomes. A binomial concordance analysis revealed strong consensus on the top-ranked questions, which address urgent needs such as improving science communication, defining success metrics, and promoting evidence-based microbiome interventions. This paper discusses the top-ranked priority research questions and their broader implications for microbiome science, urban health, and sustainable development. By focusing on these priorities, researchers, policymakers, and practitioners can foster a transformative agenda to integrate microbiomes into urban design, advancing resilient and equitable cities for the future.

Actin proteins are common to all domains of life and exhibit ATP-dependent polymerization to form filaments. In bacteria, four families of bacterial actin-like proteins (BALPs) have been identified and characterized. These BALPs are involved in plasmid partitioning (ParM), cell division (FtsA), magnetosome positioning (MamK), and cell morphology (MreB). Here, we report the identification of a fifth family of BALP, FcmB. Using the model filamentous cyanobacterium Nostoc punctiforme, we demonstrate that FcmB is a BALP that regulates cell morphology in filamentous cyanobacteria. Deletion of fcmB, or fcmC, which encodes an FcmB-interacting protein, resulted in the loss of rod morphology, similar to the phenotype reported for mreB mutants in other bacteria, including cyanobacteria. However, despite the apparent functional similarity, fcmB is not a paralog of mreB, but rather was acquired by horizontal gene transfer of a plasmid partitioning system and subsequent integration into the chromosome. Fluorescent protein fusions and immunofluorescence demonstrate that FcmB forms membrane-bound filaments which wrap around the circumference of the cell, while FcmC is localized to discrete membrane-associated foci and is essential for proper membrane localization of FcmB. Protein-protein interactions were detected between FcmB and FcmC, but not MreB, indicating that FcmB and MreB do not form heterofilaments. It is currently unclear how FcmBC exerts its effect on cell morphology, but both mreB and fcmB are ubiquitous in the developmentally complex heterocyst-forming filamentous cyanobacteria, and the presence of two discrete systems modulating cell morphology may be critical for the remarkable degree of phenotypic plasticity observed in these organisms.IMPORTANCEFilament-forming actin proteins are found in nearly all living organisms. In bacteria, four families of actin proteins have been defined, with biological functions in plasmid partitioning, cell division, magnetosome positioning, and cell morphology. Here, we identify and characterize FcmB, a fifth family of bacterial actin proteins found in filamentous cyanobacteria, and demonstrate that this family evolved from plasmid partitioning actins but influences cell morphology rather than DNA segregation. Filamentous cyanobacteria exhibit substantial phenotypic plasticity and typically contain both FcmB and MreB, the other actin family known to regulate cell morphology. The presence of two distinct families of actin proteins influencing cell morphology may play a critical role in the ability of these organisms to rapidly alter their cell shape.

Pseudomonas aeruginosa is one of the most common pathogenic bacteria in the clinic. Its large genome and strong genetic plasticity enable it to survive in various environments, posing a significant threat to patient health. We previously identified a key effector protein, TesG, secreted by the type I secretion system, which plays a crucial role in the chronic infection process of P. aeruginosa. However, the underlying mechanism remains incompletely understood. In this study, we newly discovered that TesG can induce alternative activation of macrophages and explored its mechanisms through a series of in vivo and in vitro experiments. We found that TesG promotes the expression of the NLRC5 protein, thereby inducing the polarization of macrophages toward the M2 phenotype. The activation of macrophages induced by TesG primarily occurs through the currently known mechanisms of NLRC5. These findings suggest that P. aeruginosa can regulate the alternative activation of macrophages through the TesG/NLRC5 signaling pathway during chronic infection, significantly aiding its evasion of the host immune system killing. In summary, our data highlight the complex infection strategies developed by pathogenic bacteria to achieve chronic infection.IMPORTANCEDuring the transition from acute to chronic Pseudomonas aeruginosa infection, bacteria modulate the host's immune microenvironment to evade immune responses, ensuring long-term survival. Clinical studies have confirmed that the effector protein TesG (secreted by a type I secretion system) can serve as a potential clinical biomarker for chronic P. aeruginosa lung infections. Our findings indicate that TesG promotes the alternative activation of macrophages through the regulation of NLRC5, thereby suppressing inflammatory responses and facilitating the progression of chronic pulmonary infections. These discoveries enhance our understanding of the complex interplay between P. aeruginosa and the host, laying the groundwork for developing precise diagnostic and therapeutic strategies targeting chronic pulmonary infections.

Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by inflammation and progressive fibrosis of the biliary tree. PSC pathogenesis remains poorly understood, and there are no effective therapies. Previous studies have observed associations between colonic and biliary microbiome alterations and PSC. We aimed to determine whether bacterial isolates cultured from PSC patient bile induce disease-associated phenotypes in cells, specifically cell death, epithelial permeability, inflammation, and changes in host-protective pathways. Bile was collected from PSC patients by endoscopic retrograde cholangiography and from non-PSC controls undergoing cholecystectomies. Biliary bacteria were cultured anaerobically, and 50 colonies per sample were identified by 16S sequencing. No bacteria were isolated from non-PSC controls, while bacteria were cultured from most PSC patients. The PSC bile microbiomes exhibited reduced diversity compared to the gut or oral cavity, with one or two species predominating. The effects of supernatants from seven PSC-associated bacterial isolates on cellular phenotypes were characterized using human colonic (Caco-2), hepatic (HepG2), and biliary (EGI-1) cells. Overall, PSC-associated bacteria produced factors cytotoxic to hepatic and biliary cells. An Enterococcus faecalis isolate, and to a lesser extent a Veillonella parvula isolate, induced epithelial permeability, while Escherichia coli, Fusobacterium necrophorum, and Klebsiella pneumoniae isolates induced inflammatory cytokines in biliary cells. Our data suggest that bacteria cultured from PSC bile induce cellular changes characteristic of PSC pathogenesis, with different isolates inducing distinct cellular responses. Our work provides a starting point for future research into bacterial contributions to PSC with the eventual goal of developing therapies for this disease.IMPORTANCEPrimary sclerosing cholangitis (PSC) is a chronic liver disease in which inflammation and scarring of the bile ducts cause bile to build up in the liver, leading to liver damage and eventually liver failure. The causes of this disease are poorly understood, and the only current treatment is a liver transplant. To develop new treatments, we must first better understand what leads to this disease. We examined whether bacteria isolated from PSC patient bile can cause disease-related responses in human biliary, liver, and intestinal cells. We observed that different PSC-associated bacteria can induce distinct disease-related cellular changes, including inflammation and cell death. These data suggest that the microbial community in PSC patients may indeed be linked to disease development. Our findings provide new starting points for further exploration into the poorly understood origins of PSC.

The APOE4 allele is the greatest known genetic factor for sporadic or late-onset Alzheimer's Disease (LOAD). Gut microbiome (GMB) dysbiosis can lead to poorer outcomes in disease. The intersection of sex, APOE genotype, inflammation, and gut microbiota is incompletely understood. Previous studies in humans and humanized APOE mice have demonstrated APOE-genotype-specific differences in the GMB. However, most of these studies were unable to resolve bacteria to the species level. It remains unclear how GMB changes with age and sex in the context of APOE genotype. In this study, humanized male mice with either APOE 2, 3, or 4 genotype were bred with the same two C57BL/6J sisters to standardize microbiomes across lines and monitor divergence based on APOE allele. Stool samples were collected at breeder set up and from the heterozygous (F1) and homozygous (F2) generations at wean and 6 months old. Stool was assessed via shallow shotgun sequencing to enable species and strain-level taxonomic resolution. The heterozygous pups' microbiome resembled each other at wean across all genotypes. However, the heterozygous pups and their homozygous offspring continued to diverge, particularly the APOE2 females. In homozygous mice, the GMB demonstrated significant divergence at 6 months of age based on sex and APOE genotype. In comparison to their APOE3 and APOE4 counterparts, APOE2 females and males demonstrated an increased quantity of bacteria associated with anti-inflammatory profiles, including in the Lachnospiraceae family (Lachnospiraceae bacterium UBA3401) and decreased quantities in the Turicibacteraceae family (higher levels are associated with LOAD).IMPORTANCEThe APOE4 allele is implicated as a significant risk factor for many diseases, including cardiovascular disease (responsible for more deaths than any other disease) and sporadic or late-onset Alzheimer's Disease (accounts for an estimated 60%-80% of all dementia cases). It is known that the gut microbiome (GMB) is affected by different genotypes and disease states. Mouse model studies have environmental and genetic controls, allowing a specific gene to be studied. This study aims at discovering key GMB species differences allowing for future therapeutic targets. The GMB of the experimental mice was standardized, and genotype and sex-specific divergence was observed with species and even strain level taxonomic resolution. Reported here are the first data demonstrating GMB divergence over time driven by APOE genotype from an inherited source and the first data to identify APOE genotype-specific bacteria species that may serve as therapeutic targets in APOE-driven disease.

The human microbiome varies extensively between individuals. While there are numerous studies investigating the effects of inter-individual differences on microbiome composition, there are few studies investigating inter-individual effects on microbial modulation of the host or host-specific effects. To address this knowledge gap, we colonized human bronchial epithelial air-liquid interface tissue cultures generated from six different adults with one of three phylogenetically diverse bacteria and compared how each microbe differentially modulated host gene expression in each of the six donors. Microbial treatment had the strongest effect on transcription, followed by donor-specific effects. Gene pathways differed markedly in their donor and microbe specificity; interferon expression was highly donor-dependent, while transcription of epithelial barrier and antibacterial innate immunity genes was predominantly microbially driven. Moreover, we evaluated whether microbial regulation of alternative splicing was modulated by the donor. Strikingly, we found significant nonredundant, donor-specific regulation of alternative splicing exclusively in the gram-positive commensal microbes. These findings highlight that microbial effects on the human airway epithelium are not only species-specific but also deeply individualized, underscoring the importance of the host context in shaping microbe-induced transcriptional and splicing responses.IMPORTANCEMicrobiota are integral regulators of host gene expression, utilizing diverse mechanisms that are shaped by the interplay between microbiome composition and inter-individual differences, i.e., host-specific factors. While previous studies have characterized inter-individual variation in microbiome composition and the effects of variable microbiome composition on the host, the extent to which host-specificity itself regulates host-microbe interactions remains poorly understood. In this study, we address this gap by characterizing changes in epithelial gene expression from six different human donors following colonization with one of three phylogenetically diverse bacteria. By systematically comparing donor-specific responses, we demonstrate that host specificity is a key determinant of the host transcriptional response to microbial colonization. Importantly, we demonstrate that the effects of host specificity are not uniform, but instead are dependent on the colonizing microbe. Our findings underscore the complexity of host-microbe relationships and establish host specificity as a significant factor shaping host-microbe interactions.