Biology subjects

Young, V. B.

Publications and source records attributed to Young, V. B..

At least 19 recordsLinked to original sources

bioRxiv2026

High-fat diet rewires gut host-microbiota relationship to derive worse outcomes following Clostridioides difficile infection

AbstractClostridioides difficile is a leading cause of hospital-acquired enteric infections, with outcomes ranging from mild diarrhea to life-threatening pseudomembranous colitis and death. Dietary modulations can influence the course of antibiotic-induced C. difficile infection (CDI), and high-fat diets are associated with increased mortality. Here, we profiled host innate immune cells, cecal microbial composition, and the host transcriptome of normal diet (ND) and high-fat diet (HFD)-fed C57BL/6 male and female mice following experimental CDI during the acute and recovery phases. HFD-fed male mice exhibited delayed disease onset but increased mortality following CDI. During acute infection, HFD-fed males showed increased monocyte infiltration and expansion of CD103CD11b- and CD103-CD11b dendritic cell subsets; transcriptomic modules were enriched for antigen presentation and signatures consistent with T cell activation, coincident with enrichment of Escherichia and Lactococcus. In contrast, during recovery, HFD-fed males and females displayed sustained neutrophil and monocyte infiltration, depletion of eosinophils (prominent in males), tissue-resident Tim4/CD206 macrophage subsets and ILC3s, increased expression of Tlr4 and endothelial activation markers, downregulation of metabolic pathways, and failure to restore cecal microbial diversity. Collectively, these data indicate that high-fat feeding disrupts resolution programs after CDI and promotes progression to severe disease, with the most pronounced susceptibility in males.

microbiology↗

bioRxiv2025

Single-cell RNA sequencing identifies accumulation of Fcgr2b+ virtual memory like CD8 T cells with cytotoxic and inflammatory potential in aged mouse white adipose tissue

Aging and obesity are associated with pro-inflammatory changes in adipose tissue. Overlapping mechanisms, such as the infiltration of inflammatory macrophages and T cells into visceral adipose tissue, have been implicated in contributing inflammation. However, a comparative analysis from both states is needed to identify distinct regulatory targets. Here, we performed single-cell RNA sequencing of stromal vascular fractions (SVF) isolated from gonadal white adipose tissue (gWAT) of young mice fed either a normal or a high-fat diet, and aged mice fed a normal diet. Our analysis revealed that physiological aging, compared to high-fat diet induced obesity, was associated with accumulation of phenotypically distinct CD8 T cells resembling virtual memory (VM) CD8 T cells. These cells expressed high levels of Cd44, Sell, Il7r, Il2rb, lacked Itga4, and exhibited elevated Fcgr2b expression which was associated with pseudotime differentiation trajectories. Flow cytometry confirmed an age-associated increase in Fcgr2b+CD49d- VM like CD8 T cells in gWAT. Notably, these Fcgr2b-expressing cells exhibited a cytotoxic profile, and expressed granzyme M. Functional analysis using recombinant granzyme M revealed its potential in inducing inflammation in mouse fibroblasts and macrophages. Together, our study has identified Fcgr2b+CD49d- VM-like CD8 T cells in the adipose tissue of aged mice with regulatory, cytotoxic and inflammatory potential.

immunology↗

bioRxiv2025

Miniature bioreactor arrays for modeling functional and structural dysbiosis in inflammatory bowel disease

Alterations in the gut microbiota, known as gut dysbiosis, are associated with inflammatory bowel disease (IBD). There is a need for model systems that can recapitulate the IBD gut microbiome to better understand the mechanistic impact of differences in microbiota composition and its functional consequences in a controlled laboratory setting. To this end, we introduced fecal samples from patients with Crohns disease (CD) and ulcerative colitis (UC), as well as from healthy control subjects, to miniature bioreactor arrays (MBRAs) and analyzed the microbial communities over time. We then performed two functional assessments. First, we evaluated the colitogenic potential of the CD microbiotas in genetically susceptible germ-free IL-10-deficient mice and found that colitogenic capacity was preserved in a bioreactor-cultivated CD microbiota. Second, we tested impaired colonization resistance against Clostridioides difficile in UC microbiotas using the MBRA system and found that UC microbiotas were innately susceptible to C. difficile colonization while healthy microbiotas were resistant, consistent with what is seen clinically. Overall, our results demonstrate that IBD microbiotas perform comparably to healthy donor microbiotas in the MBRA system, successfully recapitulating microbial structure while preserving IBD-specific functional characteristics. These findings establish a foundation for further mechanistic research into the IBD microbiota using MBRAs.

microbiology↗

bioRxiv2024

Host origin of microbiota drives functional recovery and Clostridioides difficile clearance in mice

Colonization resistance provided by the gut microbiota is essential for resisting both initial Clostridioides difficile infection (CDI) and potential recurrent infection (rCDI). Although fecal microbiota transplantation (FMT) has been successful in treating rCDI by restoring microbial composition and function, mechanisms underlying efficacy of standardized stool-derived products remain poorly understood. Using a combination of 16S rRNA gene-based and metagenomic sequencing alongside metabolomics, we investigated microbiome recovery following FMT from human and murine donor sources in a mouse model of rCDI. We found that a human-derived microbiota was less effective in clearing C. difficile compared to a mouse-derived microbiota, despite successful microbial engraftment and recovery of bacterial functional potential. Metabolomic analysis revealed deficits in secondary metabolites, suggesting a functional remodeling between human microbes in their new host environment. Collectively, our data revealed additional environmental, ecological, or host factors involved in FMT-based recovery from rCDI. ImportanceClostridioides difficile is a significant healthcare-associated pathogen, with recurrent infections presenting a major treatment challenge due to further disruption of the microbiota after antibiotic administration. Despite the success of fecal microbiota transplantation (FMT) for the treatment of recurrent infection, the mechanisms mediating its efficacy remain largely underexplored. This study reveals that effectiveness of FMT may be compromised by a mismatch between donor microbes and the recipient environment, leading to deficits in key microbial metabolites. These findings highlight additional factors to consider when assessing the efficacy of microbial-based therapeutics for CDI and other conditions.

microbiology↗

bioRxiv2023

Flagellar switch inverted repeat sequence variation impacts Clostridioides difficile RT027/MLST1 virulence.

Clostridioides difficile RT027 strains cause infections that vary in severity from asymptomatic to lethal, but the molecular basis for this variability is poorly understood. Through comparative analyses of RT027 clinical isolates, we determined that isolates that exhibit greater variability in their flagellar gene expression exhibit greater virulence in vivo. C. difficile flagellar genes are phase-variably expressed due to the site-specific inversion of the flgB 5UTR region, which reversibly generates ON vs. OFF orientations for the flagellar switch. We found that longer inverted repeat (IR) sequences in this switch region correlate with greater disease severity, with RT027 strains carrying 6A/6T IR sequences exhibiting greater phenotypic heterogeneity in flagellar gene expression (60%-75% ON) and causing more severe disease than those with shorter IRs (> 99% ON or OFF). Taken together, our results reveal that phenotypic heterogeneity in flagellar gene expression may contribute to the variable disease severity observed in C. difficile patients.

microbiology↗

bioRxiv2023

Virulence and genomic diversity among clinical isolates of ST1 (BI/NAP1/027) Clostridioides difficile

Clostridioides difficile (C. difficile), a leading cause of nosocomial infection, produces toxins that damage the colonic epithelium and results in colitis that varies from mild to fulminant. Variation in disease severity is poorly understood and has been attributed to host factors (age, immune competence and intestinal microbiome composition) and/or virulence differences between C. difficile strains, with some, such as the epidemic BI/NAP1/027 (MLST1) strain, being associated with greater virulence. We tested 23 MLST1(ST1) C. difficile clinical isolates for virulence in antibiotic-treated C57BL/6 mice. All isolates encoded a complete Tcd pathogenicity locus and achieved similar colonization densities in mice. Disease severity varied, however, with 5 isolates causing lethal infections, 16 isolates causing a range of moderate infections and 2 isolates resulting in no detectable disease. The avirulent ST1 isolates did not cause disease in highly susceptible Myd88-/- or germ-free mice. Genomic analysis of the avirulent isolates revealed a 69 base-pair deletion in the N-terminus of the cdtR gene, which encodes a response regulator for binary toxin (CDT) expression. Genetic deletion of the 69 base-pair cdtR sequence in the highly virulent ST1 R20291 C. difficile strain rendered it avirulent and reduced toxin gene transcription in cecal contents. Our study demonstrates that a natural deletion within cdtR attenuates virulence in the epidemic ST1 C. difficile strain without reducing colonization and persistence in the gut. Distinguishing strains on the basis of cdtR may enhance the specificity of diagnostic tests for C. difficile colitis.

microbiology↗

bioRxiv2022

Neutrophils prime unique transcriptional responses in intestinal organoids during infection with nontyphoidal Salmonella enterica serovars

Nontyphoidal strains of Salmonella enterica are a major cause of foodborne illnesses and infection with these bacteria result in inflammatory gastroenteritis. Neutrophils are a dominant immune cell type found at the site of infection in Salmonella-infected individuals, but how they regulate infection outcome is not well understood. Here we used a co-culture model of primary human neutrophils and human intestinal organoids to probe the role of neutrophils during infection with two of the most prevalent Salmonella serovars: Salmonella enterica serovar Enteritidis and Typhimurium. Using a transcriptomics approach, we identified a dominant role for neutrophils in mounting differential immune responses including production of pro-inflammatory cytokines, chemokines, and antimicrobial peptides. We also identified specific gene sets that are induced by neutrophils in response to Enteritidis or Typhimurium infection. By comparing host responses to these serovars, we uncovered differential regulation of host metabolic pathways particularly induction of cholesterol biosynthetic pathways during Typhimurium infection and suppression of RNA metabolism during Enteritidis infection. Together these findings provide insight into the role of human neutrophils in modulating different host responses to pathogens that cause similar disease in humans. ImportanceNontyphoidal serovars of Salmonella enterica are known to induce robust neutrophil recruitment in the gut during early stages of infection, but the specific role of neutrophils in regulating infection outcome of different serovars is poorly understood. Due to differences in human infection progression compared to small animal models, characterizing the role of neutrophils during infection has been challenging. Here we used a co-culture model of human intestinal organoids with human primary neutrophils to study the role of neutrophils during infection of human intestinal epithelium. Using a transcriptomics approach, we define neutrophil-dependent reprogramming of the host response to Salmonella, establishing a clear role in amplifying pro-inflammatory gene expression. Additionally, the host response driven by neutrophils differed between two similar nontyphoidal Salmonella serovars. These findings highlight the importance of building more physiological infection models to replicate human infection conditions to study host responses specific to individual pathogens.

immunology↗

bioRxiv2022

Differences in Gut Microbiota Assembly Alter Its Ability to Metabolize Dietary Polysaccharides and Resist Clostridioides difficile Colonization

The mammalian gut is home to a vibrant community of microbes. As the gut microbiota has evolved, its members have formed a complex yet stable relationships that prevent non-indigenous microorganisms, such as Clostridioides difficile, from establishing within the gut. Using a bioreactor model of the gut, we characterize how variation in microbial community assembly changes its ability to resist C. difficile. We established diluted microbial communities from healthy human stool in a bioreactor gut model and subsequently challenged them with vegetative C. difficile. 16S rRNA-gene sequencing and selective plating revealed that dilution progressively increases microbiota variability and decreases C. difficile colonization resistance. Using Dirichlet Multinomial Mixtures and linear discriminant analysis of effect size, we identified 19 bacterial taxa, including Bifidobacterium, Bacteroides and Lachnospiraceae, that associate with more resistant community types. Since these taxa are associated with butyrate production, which is tied to C. difficile colonization resistance, we performed another reactor experiment where we increased inulin concentrations prior to C. difficile challenge. Diluted communities concurrently lost their ability to produce additional butyrate in response to inulin, as measured by high performance liquid chromatography, and resist C. difficile colonization. These data demonstrate that a similar level of microbiota cohesiveness is required to prevent C. difficile colonization and metabolize inulin. It also suggests that metabolic activity of butyrate-producing microbes is tied to colonization resistance. Future work can leverage these findings to develop treatments that leverage knowledge of these ecological dynamics to improve efficacy. ImportanceThe microbes living in the human large intestine helps create an environment that is resistant to organisms that do not normally reside there, such as the pathogen Clostridioides difficile. Differences in ways in which microbial communities make an environment their home can change their ability to provide that resistance. To study those differences, we use a model of the intestine that incorporates only microbial variables (i.e. no host is involved). By diluting microbial communities to decrease their complexity, we show that communities lose their ability to resist C. difficile at a particular point and, at the same time, their ability to use inulin, a common dietary fiber, in ways that make the environment more toxic to C. difficile. These findings will help future researchers dissect the microbial components that create a resistant intestinal environment.

microbiology↗

bioRxiv2022

Intestinal inflammation reversibly alters the microbiota to drive susceptibility to Clostridioides difficile colonization in a mouse model of colitis

Susceptibility to Clostridioides difficile infection (CDI) typically follows the administration of antibiotics. Patients with inflammatory bowel disease (IBD) have increased incidence of CDI, even in the absence of antibiotic treatment. However, the mechanisms underlying this susceptibility are not well understood. To explore the intersection between CDI and IBD, we recently described a mouse model where colitis triggered by the murine gut bacterium, Helicobacter hepaticus, in IL-10-/- mice led to susceptibility to C. difficile colonization without antibiotic administration. The current work disentangles the relative contributions of inflammation and gut microbiota in colonization resistance to C. difficile in this model. We show that inflammation drives changes in microbiota composition, which leads to CDI susceptibility. Decreasing inflammation with an anti-p40 monoclonal antibody promotes a shift of the microbiota back toward a colonization-resistant state. Transferring microbiota from susceptible and resistant mice to germ-free animals transfers the susceptibility phenotype, supporting the primacy of the microbiota in colonization resistance. These findings shine light on the complex interactions between the host, microbiota, and C. difficile in the context of intestinal inflammation, and may form a basis for the development of strategies to prevent or treat CDI in IBD patients. ImportancePatients with inflammatory bowel disease (IBD) have an increased risk of developing C. difficile infection (CDI), even in the absence of antibiotic treatment. Yet, mechanisms regulating C. difficile colonization in IBD patients remain unclear. Here, we use an antibiotic-independent mouse model to demonstrate that intestinal inflammation alters microbiota composition to permit C. difficile colonization in mice with colitis. Notably, treating inflammation with an anti-p40 monoclonal antibody, a clinically relevant IBD therapeutic, restores microbiota-mediated colonization resistance to the pathogen. Through microbiota transfer experiments in germ-free mice, we confirm that the microbiota shaped in the setting of IBD is the primary driver of susceptibility to C. diffiicile colonization. Collectively, our findings provide insight into CDI pathogenesis in the context of intestinal inflammation, which may inform methods to manage infection in IBD patients. More broadly, this work advances our understanding of mechanisms by which the host-microbiota interface modulates colonization resistance to C. difficile.

microbiology↗

bioRxiv2022

The gut bacterial community potentiates Clostridioides difficile infection severity

The severity of Clostridioides difficile infections (CDI) has increased over the last few decades. Patient age, white blood cell count, creatinine levels as well as C. difficile ribotype and toxin genes have been associated with disease severity. However, it is unclear whether there is an association between members of the gut microbiota and disease severity. The gut microbiota is known to interact with C. difficile during infection. Perturbations to the gut microbiota are necessary for C. difficile to colonize the gut. The gut microbiota can inhibit C. difficile colonization through bile acid metabolism, nutrient consumption and bacteriocin production. Here we sought to demonstrate that members of the gut bacterial communities can also contribute to disease severity. We derived diverse gut communities by colonizing germ-free mice with different human fecal communities. The mice were then infected with a single C. difficile ribotype 027 clinical isolate which resulted in moribundity and histopathologic differences. The variation in severity was associated with the human fecal community that the mice received. Generally, bacterial populations with pathogenic potential, such as Escherichia, Helicobacter, and Klebsiella, were associated with more severe outcomes. Bacterial groups associated with fiber degradation, bile acid metabolism and lantibiotic production, such as Anaerostipes and Coprobacillus, were associated with less severe outcomes. These data indicate that, in addition to the host and C. difficile, populations of gut bacteria can influence CDI disease severity. ImportanceClostridioides difficile colonization can be asymptomatic or develop into an infection, ranging in severity from mild diarrhea to toxic megacolon, sepsis, and death. Models that predict severity and guide treatment decisions are based on clinical factors and C. difficile characteristics. Although the gut microbiome plays a role in protecting against CDI, its effect on CDI disease severity is unclear and has not been incorporated into disease severity models. We demonstrated that variation in the microbiome of mice colonized with human feces yielded a range of disease outcomes. These results revealed groups of bacteria associated with both severe and mild C. difficile infection outcomes. Gut bacterial community data from patients with CDI could improve our ability to identify patients at risk of developing more severe disease and improve interventions which target C. difficile and the gut bacteria to reduce host damage.

microbiology↗

bioRxiv2021

The Food Additive Xanthan Gum Drives Adaptation of the Human Gut Microbiota

The diets of industrialized countries reflect the increasing use of processed foods, often with the introduction of novel food additives. Xanthan gum is a complex polysaccharide with unique rheological properties that have established its use as a widespread stabilizer and thickening agent1. However, little is known about its direct interaction with the gut microbiota, which plays a central role in digestion of other, chemically-distinct dietary fiber polysaccharides. Here, we show that the ability to digest xanthan gum is surprisingly common in industrialized human gut microbiomes and appears to be contingent on the activity of a single bacterium that is a member of an uncultured bacterial genus in the family Ruminococcaceae. We used a combination of enrichment culture, multi-omics, and recombinant enzyme studies to identify and characterize a complete pathway in this uncultured bacterium for the degradation of xanthan gum. Our data reveal that this keystone degrader cleaves the xanthan gum backbone with a novel glycoside hydrolase family 5 (GH5) enzyme before processing the released oligosaccharides using additional enzymes. Surprisingly, some individuals harbor a Bacteroides species that is capable of consuming oligosaccharide products generated by the keystone Ruminococcaceae or a purified form of the GH5 enzyme. This Bacteroides symbiont is equipped with its own distinct enzymatic pathway to cross-feed on xanthan gum breakdown products, which still harbor the native linkage complexity in xanthan gum, but it cannot directly degrade the high molecular weight polymer. Thus, the introduction of a common food additive into the human diet in the past 50 years has promoted the establishment of a food chain involving at least two members of different phyla of gut bacteria.

microbiology↗

bioRxiv2021

Protection from lethal Clostridioides difficile infection via intraspecies competition for co-germinant

Clostridioides difficile, a Gram-positive, spore-forming bacterium, is the primary cause of infectious nosocomial diarrhea. Antibiotics are a major risk factor for C. difficile infection (CDI) as they disrupt the gut microbial community, enabling increased germination of spores and growth of vegetative C. difficile. To date the only single-species bacterial preparation that has demonstrated efficacy in reducing recurrent CDI in humans is non-toxigenic C. difficile. Using multiple infection models we determined that pre-colonization with a less virulent strain is sufficient to protect from challenge with a lethal strain of C. difficile, surprisingly even in the absence of adaptive immunity. Additionally, we showed that protection is dependent on high levels of colonization by the less virulent strain and that it is mediated by exclusion of the invading strain. Our results suggest that reduction of amino acids, specifically glycine following colonization by the first strain of C. difficile is sufficient to decrease germination of the second strain thereby limiting colonization by the lethal strain. ImportanceAntibiotic-associated colitis is often caused by infection with the bacterium Clostridioides difficile. In this study we found that reduction of the amino acid glycine by pre-colonization with a less virulent strain of C. difficile is sufficient to decrease germination of a second strain. This finding demonstrates that the axis of competition for nutrients can include multiple life stages. This work is important, as it is the first to identify a possible mechanism through which pre-colonization with C. difficile, a current clinical therapy, provides protection from reinfection. Furthermore, our work suggests that targeting nutrients utilized by all life stages could be an improved strategy for bacterial therapeutics that aim to restore colonization resistance in the gut.

microbiology↗

bioRxiv2020

Comparative transcriptional profiling of the early host response to infection by typhoidal and non-typhoidal Salmonella serovars in human intestinal organoids

Salmonella enterica represents over 2500 serovars associated with a wide-ranging spectrum of disease; from self-limiting gastroenteritis to invasive infection caused by non-typhoidal serovars (NTS) and typhoidal serovars, respectively. Host factors strongly influence infection outcome as malnourished or immunocompromised individuals can develop invasive infections from NTS, however, comparative host responses to individual serovars have been difficult to perform due to reliance on poorly representative model systems. Here we used human intestinal organoids (HIOs), a three-dimensional "gut-like" in vitro system derived from human embryonic stem cells, to elucidate similarities and differences in host responses to NTS and typhoidal serovars. HIOs discriminated between the two most prevalent NTS, Salmonella enterica serovar Typhimurium (STM) and Salmonella enterica serovar Enteritidis (SE), and typhoidal serovar Salmonella enterica serovar Typhi (ST) in epithelial cell invasion, replication and transcriptional responses. Pro-inflammatory signaling and cytokine output was reduced in ST-infected HIOs compared to NTS infections, reflecting early stages of NTS and typhoidal diseases. While we predicted that ST would induce a distinct transcriptional profile from the NTS strains, more nuanced expression profiles emerged. Notably, pathways involved in cell cycle, metabolism and mitochondrial functions were downregulated in STM-infected HIOs and upregulated in SE-infected HIOs. These results correlated with elevated levels of reactive oxygen species production in SE-infected HIOs compared to mock-infected HIOs. Collectively, these results suggest that the HIO model is well suited to reveal host transcriptional programming specific to individual Salmonella serovars, and that individual NTS may provoke unique host epithelial responses during intestinal stages of infection. Author SummarySalmonella enterica is the major causative agent of bacterial infections associated with contaminated food and water. Salmonella enterica consists of over 2500 serovars of which Typhimurium (STM), Enteritidis (SE) and Typhi (ST) are the three major serovars with medical relevance to humans. These serovars elicit distinctive immune responses and cause different diseases in humans, including self-limiting diarrhea, gastroenteritis and typhoid fever. Differences in the human host response to these serovars are likely to be a major contributing factor to distinct disease outcomes but are not well characterized, possibly due to the limitations of human-derived physiological infection models. Unlike immortalized epithelial cell culture models, human intestinal organoids (HIOs) are three-dimensional structures derived from embryonic stem cells that differentiate into intestinal mesenchymal and epithelial cells, mirroring key organizational aspects of the intestine. In this study, we used HIOs to monitor transcriptional changes during early stages of STM, SE and ST infection. Our comparative analysis showed that HIO inflammatory responses are the dominant response in all infections, but ST infection induces the weakest upregulation of inflammatory mediators relative to the other serovars. In addition, we identified several cellular processes, including cell cycle and mitochondrial functions, that were inversely regulated between STM and SE infection despite these serovars causing similar localized intestinal infection in humans. Our findings reinforce HIOs as an emerging model system to study Salmonella serovar infection, and provide global host transcriptional response profiles as a foundation for understanding human infection outcomes.

microbiology↗

bioRxiv2020

Salmonella enterica serovar Typhimurium SPI-1 and SPI-2 shape the transcriptional landscape of epithelial cells in a human intestinal organoid model system

The intestinal epithelium is a primary interface for engagement of the host response by foodborne pathogens, like Salmonella enterica serovar Typhimurium (STm). While interaction of STm with the mammalian host has been well studied in vitro in transformed epithelial cell lines or in the complex intestinal environment in vivo, few tractable models recapitulate key features of the intestinal epithelium. Human intestinal organoids (HIOs) contain a polarized epithelium with functionally differentiated cell subtypes, including enterocytes and goblet cells. HIOs contain luminal space that supports bacterial replication and are more amenable to experimental manipulation than animals while more reflective of physiological epithelial responses. Here we use the HIO model to define transcriptional responses of the host epithelium to STm infection, also determining host pathways dependent on Salmonella Pathogenicity Island-1 (SPI-1) and -2 (SPI-2) encoded Type 3 secretion systems (T3SS). Consistent with prior findings, we find that STm strongly stimulates pro-inflammatory gene expression. Infection-induced cytokine gene expression was rapid, transient and largely independent of SPI-1 T3SS-mediated invasion, likely due to continued luminal stimulation. Notably, STm infection led to significant down-regulation of host genes associated with cell cycle and DNA repair, an effect that required SPI-1 and SPI-2 T3SS. The transcriptional profile of cell cycle-associated target genes implicates multiple miRNAs as likely mediators of STm-dependent cell cycle suppression. These findings from Salmonella-infected HIOs delineate common and distinct contributions of SPI-1 and SPI-2 T3SSs in inducing early host responses during enteric infection and reveal host cell cycle as a potential target during STm intracellular infection. ImportanceSalmonella enterica serovar Typhimurium (STm) causes a significant health burden worldwide, yet host responses to initial stages of intestinal infection remain poorly understood. Due to differences in infection outcome between mice and humans, evaluating physiological host responses driven by major virulence determinants of Salmonella have been difficult to date. Here we use the 3D human intestinal organoid model to define early responses to infection with wildtype STm and mutants defective in the SPI-1 or SPI-2 Type 3 secretion systems. Both secretion system mutants show defects in a mouse model of oral Salmonella infection but the specific contributions of each secretion system are less well understood. We show that STm upregulates pro-inflammatory pathways independently of either secretion system while downregulation of host cell cycle pathways is dependent on both SPI-1 and SPI-2. These findings lay the groundwork for future studies investigating how SPI-1- and SPI-2-driven host responses affect infection outcome and show the potential of this model to study host-pathogen interactions with other serovars to understand how initial interactions with the intestinal epithelium may affect pathogenesis.

microbiology↗

bioRxiv2020

Loss of RpoS results in attenuated Escherichia coli colonization of human intestinal organoids and a competitive disadvantage within the germ-free mouse intestine

Pluripotent stem-cell-derived human intestinal organoids (HIOs) are three-dimensional, multicellular structures that model a previously uncolonized, naive intestinal epithelium in an in vitro system. We recently demonstrated that microinjection of the non-pathogenic Escherichia coli strain, ECOR2, into HIOs induced morphological and functional maturation of the HIO epithelium, including increased secretion of mucins and cationic antimicrobial peptides. In the current work, we use ECOR2 as a biological probe to investigate the bacterial response to colonization of the HIO lumen. In E. coli and other Gram-negative bacteria, adaptation to environmental stress is regulated by the general stress response sigma factor, RpoS. We generated an isogenic {triangleup}rpoS ECOR2 mutant to compare challenges faced by a bacterium during colonization of the HIO lumen relative to the germ-free mouse intestine, which is currently the best available system for studying the initial establishment of bacterial populations within the gut. We demonstrate that loss of RpoS significantly decreases the ability of ECOR2 to colonize HIOs, though it does not prevent colonization of germ-free mice. Rather, the {triangleup}rpoS ECOR2 exhibits a fitness defect in the germ-free mouse intestine only in the context of microbial competition. These results indicate that HIOs pose a differentially restrictive luminal environment to E. coli during colonization, thus increasing our understanding of the HIO model system as it pertains to studying the establishment of intestinal host-microbe symbioses. ImportanceTechnological advancements have and will continue to drive the adoption of organoid-based systems for investigating host-microbe interactions within the human intestinal ecosystem. Using E. coli deficient in the RpoS-mediated general stress response, we demonstrate that the type or severity of microbial stressors within the HIO lumen differ from those of the in vivo environment of the germ-free mouse gut. This study provides important insight into the nature of the HIO microenvironment from a microbiological standpoint.

microbiology↗

bioRxiv2020

Intestinal inflammation and altered gut microbiota associated with inflammatory bowel disease renders mice susceptible to Clostridioides difficile colonization and infection

Clostridioides difficile has emerged as a noteworthy pathogen in patients with inflammatory bowel disease (IBD). Concurrent IBD and CDI is associated with increased morbidity and mortality compared to CDI alone. IBD is associated with alterations of the gut microbiota, an important mediator of colonization resistance to C. difficile. Here, we describe and utilize a mouse model to explore the role of intestinal inflammation in susceptibility to C. difficile colonization and subsequent disease severity in animals with underlying IBD. Helicobacter hepaticus, a normal member of the mouse gut microbiota, was used to trigger inflammation in the distal intestine akin to human IBD in mice that lack intact IL-10 signaling. Development of IBD resulted in a distinct intestinal microbiota community compared to non-IBD controls. We demonstrate that in this murine model, IBD was sufficient to render mice susceptible to C. difficile colonization. Mice with IBD were persistently colonized by C. difficile, while genetically identical non-IBD controls were resistant to C. difficile colonization. Concomitant IBD and CDI was associated with significantly worse disease than unaccompanied IBD. IL-10-deficient mice maintained gut microbial diversity and colonization resistance to C. difficile in experiments utilizing an isogenic mutant of H. hepaticus that does not trigger intestinal inflammation. These studies in mice demonstrate that the IBD-induced microbiota is sufficient for C. difficile colonization and that this mouse model requires intestinal inflammation for inducing susceptibility to CDI in the absence of other perturbations, such as antibiotic treatment. IMPORTANCEThe incidence of CDI continues to increase significantly among patients with IBD, independent of antibiotic use, yet the relationship between IBD and increased risk for CDI remains to be understood. However, antibiotic-induced perturbations of the gut microbiota may mask mechanisms specific to IBD-induced C. difficile susceptibility and infection. Our study sought to describe and utilize a mouse model to specifically explore the relationship between the IBD-induced gut microbial community and susceptibility to C. difficile colonization and CDI development. We demonstrate that IBD is sufficient for C. difficile colonization and infection in mice and results in significantly worse disease than IBD alone, representing a murine model that recapitulates human IBD and CDI comorbidity. Furthermore, this model requires IBD-induced inflammation to sculpt a microbiota permissible to C. difficile colonization. Use of this model will aid in developing new clinical approaches to predict, diagnose, and treat C. difficile infection in the IBD population.

microbiology↗

bioRxiv2020

A plasmid locus associated with Klebsiella clinical infections encodes a microbiome-dependent gut fitness factor

Introductory paragraphGut colonization by the pathogen Klebsiella pneumoniae (Kp) is consistently associated with subsequent Kp disease1-5, and patients are predominantly infected with their colonizing strain1,2. However, colonizing strains likely vary in their potential to cause infection. We previously identified the plasmid-encoded tellurium resistance (ter) operon as highly associated with infection when compared to asymptomatic colonization in hospitalized patients1. The ter operon bestows resistance to the toxic compound tellurite oxide (TeO3-2), but this is unlikely to be its physiological function, as tellurium and TeO3-2 are exceedingly rare. Here we show that terC is necessary and terZABCDEF is sufficient for phenotypic TeO3-2 resistance. Next, we demonstrate that ter is encoded on a diverse group of plasmids without known plasmid-encoded virulence genes, suggesting an independent role in infection. Finally, our studies indicate that ter is a gut fitness factor, and its fitness advantage is conferred only when specific gut microbiota constituents are present. Collectively, these data reveal the Kp ter operon that is highly associated with human infection likely acts early in pathogenesis as a horizontally-transferrable fitness factor promoting robust gut colonization in the presence of the indigenous microbiota.

microbiology↗

bioRxiv2020

Aging dampens the intestinal innate immune response during Clostridioides difficile infection and is associated with altered intestinal eosinophil mobilization

Clostridioides (formerly Clostridium) difficile is the most common cause of hospital-acquired infection, and advanced age is a risk factor for C. difficile infection. Disruption of the intestinal microbiota and immune responses contribute to host susceptibility and severity of C. difficile infection. However, the impact of aging on the cellular immune response associated with C. difficile infection in the setting of advanced age remains to be well described. This study explores the effect of age on cellular immune responses in C. difficile infection as well as disease severity. Young adult mice (2-3 months old) and aged mice (22-28 months old) were rendered susceptible to C. difficile infection with cefoperazone and then infected with C. difficile strains of varying disease-causing potential. Aged mice infected with C. difficile develop more severe clinical disease, compared to young mice. Tissue-specific CD45+ immune cell responses occurred at the time of peak disease severity in the cecum and colon of all mice infected with a high-virulence strain of C. difficile; however, significant deficits in intestinal neutrophils and eosinophils were detected in aged mice. Interestingly, while C. difficile infection in young mice was associated with a robust increase in cecal and colonic eosinophils, there was a complete lack of an intestinal eosinophil response in aged counterparts accompanied by a simultaneous increase in blood eosinophils with severe disease. These findings demonstrate that age-related alterations in immune responses are associated with significantly worse C. difficile infection and support a key role for intestinal eosinophils in mitigating C. difficile-mediated disease severity.

immunology↗