Biology subjects

Fletcher, J. R.

Publications and source records attributed to Fletcher, J. R..

8 recordsLinked to original sources

bioRxiv2026

Butyrate synergizes with glucose to promote anaerobic growth of Staphylococcus aureus via anaplerotic metabolism and stress response pathways

Short-chain fatty acids (SCFAs) like butyrate and propionate are abundant microbiota-derived metabolites that influence bacterial physiology in host-associated niches like the gastrointestinal tract. However, their effects on Staphylococcus aureus under varying nutritional conditions remain incompletely understood. Here we investigated how SCFAs interact with glucose or galactose to regulate anaerobic growth, biofilm formation, and global transcription in S. aureus. Both SCFAs inhibit anaerobic growth in a dose-dependent manner but biofilm was differentially affected, with butyrate promoting and propionate suppressing biofilm formation. Glucose and galactose alleviated SCFA-mediated growth inhibition, with glucose exerting the strongest effect. Notably, glucose enhanced butyrate-associated growth and biofilm formation beyond glucose alone, whereas galactose produced more modest effects. Enzymatic and genetic analyses indicated that SCFA-sugar biofilms contain proteins and extracellular DNA and involve VraSR-dependent regulation. Transcriptomic profiling revealed broad metabolic reprogramming, including induction of urease genes, amino acid biosynthesis, and stress response pathways. Synergistic effects between butyrate and glucose were partially dependent on anaplerotic metabolism via pyruvate carboxylase, linking oxaloacetate regeneration from pyruvate to S. aureus SCFA adaptation. Together these findings demonstrate that the nutritional environment dictates whether SCFAs impair S. aureus growth or reprogram its physiology, promoting metabolic adaptation and biofilm formation under sugar-replete conditions.

microbiology↗

bioRxiv2025

Cross-feeding interactions between Fusobacterium nucleatum and the glycan forager Segatella oris

Fusobacterium nucleatum is a common member of the oral microbiota frequently associated with extra-oral infections and diverse polymicrobial environments, including chronic airway diseases and colorectal tumors. Yet, its interactions with co-colonizing microbiota remain poorly defined. Here, we investigate cross-feeding interspecies dynamics between F. nucleatum and Segatella oris, a glycan-foraging anaerobe enriched in airways and gastrointestinal tumors. Using broth cultures, cell-free supernatants, and co-culture on primary human airway epithelial cells, we identify bidirectional interactions that shape nutrient acquisition, biofilm formation, gene expression, and host responses. While mucin or S. oris supernatants modestly enhanced F. nucleatum growth, both conditions triggered transcriptional remodeling, including induction of the nan operon for sialic acid catabolism, suggesting reliance on glycan degradation by S. oris. Conversely, S. oris exhibited differential expression of multiple polysaccharide utilization loci (PULs) when exposed to F. nucleatum or its metabolites. Biofilm formation by F. nucleatum was strongly inhibited by S. oris, indicative of antagonistic interactions. Dual and triple RNA-seq revealed that epithelial responses were predominately shaped by F. nucleatum, with enrichment of inflammatory and cancer-associated pathways; however, co-colonization with S. oris modulated the magnitude and specificity of host gene expression. These findings demonstrate that glycan-mediated cross-feeding and microbial interactions shape the physiology and pathogenic potential of F. nucleatum in mucosal environments. This work underscores the importance of modeling polymicrobial communities under host-relevant conditions to better understand pathobiont behavior at the epithelial interface. ImportanceFusobacterium nucleatum is increasingly recognized as a pathobiont in mucosal diseases, including colorectal cancers and chronic airway infections, yet its functional interactions with co-colonizing microbiota remain poorly understood. Here, we demonstrate that F. nucleatum engages in bidirectional interactions with Segatella oris, a glycan-foraging anaerobe also enriched in mucin-rich environments. Through nutrient cross-feeding, antagonism, and transcriptional modulation, these interactions shape bacterial behavior and the host epithelial response. Notably, glycan degradation by S. oris enables F. nucleatum access to sialic acids, while F. nucleatum suppresses expression of multiple polysaccharide utilization loci in S. oris, revealing a reciprocal ecological influence. Co-colonization of the airway epithelial surface also modulates gene expression linked to inflammation and cancer. These findings advance our understanding of polymicrobial dynamics at mucosal interfaces and highlight the importance of incorporating microbe-microbe-host interactions into reductionist models of infection and disease.

microbiology↗

bioRxiv2024

Commensal-derived short-chain fatty acids disrupt lipid membrane homeostasis in Staphylococcus aureus

The role of commensal anaerobic bacteria in chronic respiratory infections is unclear, yet they can exist in abundances comparable to canonical pathogens in vivo. Their contributions to the metabolic landscape of the host environment may influence pathogen behavior by competing for nutrients and creating inhospitable conditions via toxic metabolites. Here, we reveal a mechanism by which the anaerobe-derived short chain fatty acids (SCFAs) propionate and butyrate negatively affect Staphylococcus aureus physiology by disrupting branched chain fatty acid (BCFA) metabolism. In turn, BCFA impairment results in impaired growth, diminished expression of the agr quorum sensing system, as well as increased sensitivity to membrane-targeting antimicrobials. Altered BCFA metabolism also reduces S. aureus fitness in competition with Pseudomonas aeruginosa, suggesting that airway microbiome composition and the metabolites they produce and exchange directly impact pathogen succession over time. The pleiotropic effects of these SCFAs on S. aureus fitness and their ubiquity as metabolites in animals also suggests that they may be effective as sensitizers to traditional antimicrobial agents when used in combination.

microbiology↗

bioRxiv2023

Distilling Mechanistic Models From Multi-Omics Data

High-dimensional multi-omics data sets are increasingly accessible and now routinely being generated as part of medical and biological experiments. However, the ability to infer mechanisms of these data remains low due to the abundance of confounding data. The gap between data generation and interpretation highlights the need for strategies to harmonize and distill complex multi-omics data sets into concise, mechanistic descriptions. To this end, a four-step analysis approach for multiomics data is herein demonstrated, comprising: filling missing data and harmonizing data sources, inducing sparsity, developing mechanistic models, and interpretation. This strategy is employed to generate a parsimonious mechanistic model from high-dimensional transcriptomics and metabolomics data collected from a murine model of Clostridioides difficile infection. This approach highlighted the role of the Stickland reactor in the production of toxins during infection, in agreement with recent literature. The methodology present here is demonstrated to be feasible for interpreting multi-omics data sets and it, to the authors knowledge, one of the first reports of a successful implementation of such a strategy.

systems biology↗

bioRxiv2022

Staphylococcus aureus overcomes anaerobe-derived short-chain fatty acid stress via FadX and the CodY regulon

Chronic rhinosinusitis (CRS) is characterized by immune dysfunction, mucus hypersecretion, and persistent infection of the paranasal sinuses. While Staphylococcus aureus is a primary CRS pathogen, recent sequence-based surveys have found increased relative abundances of anaerobic bacteria, suggesting that S. aureus may experience altered metabolic landscapes in CRS relative to healthy airways. To test this possibility, we characterized the growth kinetics and transcriptome of S. aureus in supernatants of the abundant CRS anaerobe Fusobacterium nucleatum. While growth was initially delayed, S. aureus ultimately grew to similar levels as in the control medium. The transcriptome was significantly affected by F. nucleatum metabolites, with the agr quorum sensing system notably repressed. Conversely, expression of fadX, encoding a putative propionate coA-transferase, was significantly increased, leading to our hypothesis that short chain fatty acids (SCFAs) produced by F. nucleatum could mediate S. aureus growth behavior and gene expression. Supplementation with propionate and butyrate, but not acetate, recapitulated delayed growth phenotypes observed in F. nucleatum supernatants. A fadX mutant was found to be more sensitive than wild type to propionate, suggesting a role for FadX in the S. aureus SCFA stress response. Interestingly, spontaneous resistance to butyrate, but not propionate, was frequently observed. Whole genome sequencing and targeted mutagenesis identified codY mutants as resistant to butyrate inhibition. Together, these data show that S. aureus physiology is dependent on its co-colonizing microbiota and metabolites they exchange, and indicate that propionate and butyrate may act on different targets in S. aureus to suppress its growth. ImportanceS. aureus is an important CRS pathogen, yet is found in the upper airways of 30-50% of people without complications. The presence of strict and facultative anaerobic bacteria in CRS sinuses has recently spurred research into bacterial interactions and how they influence S. aureus physiology and pathogenesis. We show here that propionate and butyrate produced by one such CRS anaerobe, F. nucleatum, alter the growth and gene expression of S. aureus. We show that fadX is important for S. aureus to resist propionate stress, and that the CodY regulon mediates growth in inhibitory concentrations of butyrate. This work highlights the possible complexity of S. aureus-anaerobe interactions, and implicates membrane stress as a possible mechanism influencing S. aureus behavior in CRS sinuses.

microbiology↗

bioRxiv2021

The Stickland fermentation precursor trans-4-hydroxyproline differentially impacts the metabolism of Clostridioides difficile and commensal Clostridia

An intact gut microbiota confers colonization resistance against Clostridioides difficile through a variety of mechanisms, likely including competition for nutrients. Recently, proline was identified as an important environmental amino acid that C. difficile uses to support growth and cause significant disease. A post-translationally modified form, trans-4-hydroxyproline, is highly abundant in collagen, which is degraded by host proteases in response to C. difficile toxin activity. The ability to dehydrate trans-4-hydroxyproline via the HypD glycyl radical enzyme is wide-spread amongst gut microbiota, including C. difficile and members of the commensal Clostridia, suggesting that this amino acid is an important nutrient in the host environment. Therefore, we constructed a C. difficile {Delta}hypD mutant and found that it was modestly impaired in fitness in a mouse model of infection, and was associated with an altered microbiota when compared to mice challenged with the wild type strain. Changes in the microbiota between the two groups were largely driven by members of the Lachnospiraceae family and the Clostridium genus. We found that C. difficile and type strains of three commensal Clostridia had significant alterations to their metabolic gene expression in the presence of trans-4-hydroxyproline in vitro. The proline reductase (prd) genes were elevated in C. difficile, consistent with the hypothesis that trans-4-hydroxyproline is used by C. difficile to supply proline for fermentation. Similar transcripts were also elevated in some commensal Clostridia tested, although each strain responded differently. This suggests that the uptake and utilization of other nutrients by the commensal Clostridia may be affected by trans-4-hydroxyproline metabolism, highlighting how a common nutrient may be a signal to each organism to adapt to a unique niche. Further elucidation of the differences between them in the presence of hydroxyproline and other key nutrients will be important to determining their role in nutrient competition against C. difficile.

microbiology↗

bioRxiv2020

Age-related reductions in the number of serial sarcomeres contribute to shorter fascicle lengths but not elevated passive tension

We investigated age-related changes to fascicle length (FL), sarcomere length (SL), and serial sarcomere number (SSN), and how this affects passive force. Following mechanical testing to determine passive force, the medial gastrocnemius muscle of young (n=9) and old (n=8) Fisher 344BN hybrid rats was chemically fixed at the optimal muscle length for force production; individual fascicles were dissected for length measurement, and laser diffraction was used to assess SL. Old rats had [~]14% shorter FL than young, which was driven by a [~]10% reduction in SSN, with no difference in SL ([~]4%). Passive force was greater in the old compared to young rats at long muscle lengths. Shorter FL and reduced SSN in the old rats could not entirely explain increased passive forces for absolute length changes, owing to a slight reduction in SL in old, resulting in similar SL at long muscle lengths. Summary StatementThis study sought to explain the increased passive tension observed for muscles of older individuals owing to age-related changes to muscle architecture.

physiology↗

bioRxiv2020

Clostridioides difficile exploits toxin-mediated inflammation to alter the host nutritional landscape and exclude competitors from the gut microbiota

Introductory paragraphClostridioides difficile is a bacterial pathogen that causes a range of clinical disease from mild to moderate diarrhea, pseudomembranous colitis, and toxic megacolon. Typically, C. difficile infections (CDIs) occur after antibiotic treatment, which alters the gut microbiota, decreasing colonization resistance against C. difficile. Disease is mediated by two large toxins and the expression of their genes is induced upon nutrient depletion via the alternative sigma factor TcdR. Using tcdR mutants in two strains of C. difficile, we defined how toxin-induced inflammation alters C. difficile metabolism, tissue gene expression, and the gut microbiota to determine how inflammation by the host may be beneficial to C. difficile. Here we show that C. difficile metabolism is significantly different in the face of inflammation, with changes in many carbohydrate and amino acid uptake and utilization pathways. Host gene expression signatures suggest that degradation of collagen and other components of the extracellular matrix by matrix metalloproteinases is a major source of peptides and amino acids that supports C. difficile growth in vivo. Lastly, the inflammation induced by C. difficile toxin activity alters the gut microbiota, excluding members from the genus Bacteroides that are able to compete against C. difficile for the same essential nutrients released from collagen degradation.

microbiology↗