Biology subjects

Hryckowian, A. J.

Publications and source records attributed to Hryckowian, A. J..

12 recordsLinked to original sources

bioRxiv2026

EndD mediates butyrate-dependent toxin release in Clostridioides difficile

Clostridioides difficile is an urgent threat to human health. Current treatments for C. difficile infections (CDIs) are antibiotics and microbiome restoration therapy (MRT) for recurrent cases. However, antibiotics contribute to antibiotic resistance and recurrent CDIs and the long-term sustainability and accessibility of MRTs remains to be determined. Since a dysbiotic gut microbiome is the primary risk factor for CDI, a better understanding of the interactions between C. difficile, the microbiome, and the host will aid development of treatments with improved precision. Emerging evidence supports that butyrate, a prominent end product of gut microbiome metabolism, is a key determinant of C. difficile pathogenesis. Notably, C. difficile releases more of its toxins TcdA and TcdB in butyrate-rich environments. Here, we demonstrate that butyrate-dependent toxin release is not driven by two previously characterized modes of toxin release (e.g., TcdE-dependent secretion or Cwp19-dependent autolysis). Instead, butyrate enhances the expression of a broadly conserved endolysin (EndD), which is responsible for butyrate-dependent toxin release. We additionally demonstrate that endD-dependent toxin release does not universally occur under all growth conditions and that its expression is dependent on the late-stage sporulation sigma factor SigK. Overall, our findings provide deeper insight into butyrate-dependent effects on C. difficile pathogenesis and set the stage for future work to better understand the molecular and genetic underpinnings of endD regulation.

microbiology↗

bioRxiv2026

Phage-based microbiome manipulation reveals ecological interactions within gut communities

Mechanistic understanding of gut ecology is limited by the availability of tools for precise manipulation of microbiome composition. Here, we isolate lytic phages to enable targeted removal of gut commensal Escherichia fergusonii (Ef) from complex, undefined stool-derived in vitro communities. A single phage drove resistance without fitness cost in monoculture, but resistant Ef exhibited reduced fitness in communities, enabling expansion of closely related Proteobacteria. Resistance arose via reversible promoter inversion linked to outer-membrane function. A phage cocktail overcame resistance to achieve Ef knockout across communities with minimal collateral effects. Using knockout communities, we show that Ef is necessary and sufficient for preventing Salmonella invasion. Replacement with an Ef transposon-mutant library revealed that community-specific fitness defects are enriched in genes involved in outer-membrane assembly. Disruption of these genes sensitized Ef to antagonistic community members, highlighting interspecies warfare as a key driver of microbiome ecology. These results establish phage-mediated perturbation as a framework for linking species to community-level function and for enabling precision microbiome engineering.

microbiology↗

bioRxiv2024

Impacts of perR on oxygen sensitivity, gene expression, and murine infection in Clostridioides difficile 630Δerm

Clostridioides difficile infection (CDI), characterized by colitis and diarrhea, afflicts approximately half a million people in the United States every year, burdening both individuals and the healthcare system. C. difficile 630{Delta}erm is an erythromycin-sensitive variant of the clinical isolate C. difficile 630 and is commonly used in the C. difficile research community due to its genetic tractability. 630{Delta}erm possesses a point mutation in perR, an autoregulated transcriptional repressor that regulates oxidative stress resistance genes. This point mutation results in a constitutively de-repressed PerR operon in 630{Delta}erm. To address the impacts of perR on phenotypes relevant for oxygen tolerance and relevant to a murine model of CDI, we corrected the point mutant to restore PerR function in 630{Delta}erm (herein, 630{Delta}erm perRWT). We demonstrate that there is no difference in growth between 630{Delta}erm and a 630{Delta}erm perRWT under anaerobic conditions or when exposed to concentrations of O2 that mimic those found near the surface of the colonic epithelium. However, 630{Delta}erm perRWT is more sensitive to ambient oxygen than 630{Delta}erm, which coincides with alterations in expression of a variety of perR-dependent and perR-independent genes. Finally, we show that 630{Delta}erm and 630{Delta}erm perRWT do not differ in their ability to infect and cause disease in a well-established murine model of CDI. Together, these data support the hypothesis that the perR mutation in 630{Delta}erm arose as a result of exposure to ambient oxygen and that the perR mutation in 630{Delta}erm is unlikely to impact CDI-relevant phenotypes in laboratory studies. ImportanceClostridioides difficile is a diarrheal pathogen and a major public health concern. To improve humans understanding of C. difficile, a variety of C. difficile isolates are used in research, including C. difficile 630{Delta}erm. 630{Delta}erm is a derivative of the clinical isolate 630 and is commonly studied because it is genetically manipulable. Previous work showed that a mutation in perR in 630{Delta}erm results in a dysregulated oxidative stress response, but no work has been done to characterize perR-dependent effects on the transcriptome or to determine impacts of perR during infection. Here, we identify transcriptomic differences between 630{Delta}erm and 630{Delta}erm perRWT exposed to ambient oxygen and demonstrate that there is no strain-based difference in burdens in murine C. difficile infection.

microbiology↗

bioRxiv2023

Insertion sequence driven evolution of the microbiota revealed by a novel metagenomics pipeline

Insertion sequence (IS) elements are mobile genetic elements in bacterial genomes that support adaptation. We developed a database of IS elements coupled to a computational pipeline that identifies IS element insertions in the microbiota. We discovered that diverse IS elements insert into the genomes of intestinal bacteria regardless of human host lifestyle. These insertions target bacterial accessory genes that aid in their adaptation to unique environmental conditions. Using IS expansion in Bacteroides, we show that IS activity leads to insertion "hot spots" in accessory genes. We show that IS insertions are stable and can be transferred between humans. Extreme environmental perturbations force IS elements to fall out of the microbiota and many fail to rebound following homeostasis. Our work shows that IS elements drive bacterial genome diversification within the microbiota and establishes a framework for understanding how strain level variation within the microbiota impacts human health.

genomics↗

bioRxiv2023

Exogenous butyrate inhibits butyrogenic metabolism and alters expression of virulence genes in Clostridioides difficile

The gut microbiome engenders colonization resistance against the diarrheal pathogen Clostridioides difficile but the molecular basis of this colonization resistance is incompletely understood. A prominent class of gut microbiome-produced metabolites important for colonization resistance against C. difficile is short chain fatty acids (SCFAs). In particular, one SCFA (butyrate) decreases the fitness of C. difficile in vitro and is correlated with C. difficile- inhospitable gut environments, both in mice and in humans. Here, we demonstrate that butyrate-dependent growth inhibition in C. difficile occurs under conditions where C. difficile also produces butyrate as a metabolic end product. Furthermore, we show that exogenous butyrate is internalized into C. difficile cells, is incorporated into intracellular CoA pools where it is metabolized in a reverse (energetically unfavorable) direction to crotonyl-CoA and (S)-3-hydroxybutyryl-CoA and/or 4-hydroxybutyryl-CoA. This internalization of butyrate and reverse metabolic flow of butyrogenic pathway(s) in C. difficile coincides with alterations in toxin production and sporulation. Together, this work highlights butyrate as a signal of a C. difficile inhospitable environment to which C. difficile responds by producing its diarrheagenic toxins and producing environmentally-resistant spores necessary for transmission between hosts. These findings provide foundational data for understanding the molecular and genetic basis of how C. difficile growth is inhibited by butyrate and how butyrate serves as a signal to alter C. difficile virulence in the face of a highly competitive and dynamic gut environment. IMPORTANCEThe gut microbiome engenders colonization resistance against the diarrheal pathogen Clostridioides difficile but the molecular basis of this colonization resistance is incompletely understood, which hinders the development of novel therapeutic interventions for C. difficile infection (CDI). We investigated how C. difficile responds to butyrate, an end-product of gut microbiome community metabolism which inhibits C. difficile growth. We show that exogenously-produced butyrate is internalized into C. difficile, which inhibits C. difficile growth by interfering with its own butyrate production. This growth inhibition coincides with the expression of virulence-related genes. Future work to disentangle the molecular mechanisms underlying these growth and virulence phenotypes will likely lead to new strategies to restrict C. difficile growth in the gut and minimize its pathogenesis during CDI.

microbiology↗

bioRxiv2022

Butyrate differentiates permissiveness to Clostridioides difficile infection and influences growth of diverse C. difficile isolates

A disrupted "dysbiotic" gut microbiome engenders susceptibility to the diarrheal pathogen Clostridioides difficile by impacting the metabolic milieu of the gut. Diet, in particular the microbiota accessible carbohydrates (MACs) found in dietary fiber, is one of the most powerful ways to affect the composition and metabolic output of the gut microbiome. As such, diet is a powerful tool for understanding the biology of C. difficile and for developing alternative approaches for coping with this pathogen. One prominent class of metabolites produced by the gut microbiome are short chain fatty acids (SCFAs), the major metabolic end products of MAC metabolism. SCFAs are known decrease the fitness of C. difficile in vitro and that high intestinal SCFA concentrations are associated with reduced fitness of C. difficile in animal models of C. difficile infection (CDI). Here, we use controlled dietary conditions (8 diets that differ only by MAC composition) to show that C. difficile fitness is most consistently impacted by butyrate, rather than the other two prominent SCFAs (acetate and propionate), during murine model CDI. We similarly show that butyrate concentrations are lower in fecal samples from humans with CDI relative to healthy controls. Finally, we demonstrate that butyrate impacts growth in diverse C. difficile isolates. These findings provide a foundation for future work which will dissect how butyrate directly impacts C. difficile fitness and will lead to the development of diverse approaches distinct from antibiotics or fecal transplant, such as dietary interventions, for mitigating CDI in at-risk human populations. IMPORTANCEClostridioides difficile is a leading cause of infectious diarrhea in humans and it imposes a tremendous burden on the healthcare system. Current treatments for C. difficile infection (CDI) include antibiotics and fecal microbiota transplant, which contribute to recurrent CDIs and face major regulatory hurdles, respectively. Therefore, there is an ongoing need to develop new ways to cope with CDI. Notably, a disrupted "dysbiotic" gut microbiota is the primary risk factor for CDI but we incompletely understand how a healthy microbiota resists CDI. Here, we show that a specific molecule produced by the gut microbiota, butyrate, is negatively associated with C. difficile burdens in humans and in a mouse model of CDI and that butyrate impedes the growth of diverse C. difficile strains in pure culture. These findings help to build a foundation for designing alternative, possibly diet-based, strategies for mitigating CDI in humans.

microbiology↗

bioRxiv2021

Independent host- and bacterium-based determinants protect a model symbiosis from phage predation

Summary/AbstractBacteriophages (phages) are diverse and abundant constituents of microbial communities worldwide, and are capable of modulating bacterial populations in diverse ways. Here we describe a novel phage, {phi}HNL01, which infects the marine bacterium Vibrio fischeri. We use culture-based approaches to demonstrate that mutations in the exopolysaccharide locus of V. fischeri render this bacterium resistant to infection by {phi}HNL01, highlighting the extracellular matrix as a key determinant of phage tropism in this interaction. Additionally, using the natural symbiosis between V. fischeri and the squid Euprymna scolopes, we show that during colonization, V. fischeri is protected from phage present in the ambient seawater. Taken together, these findings shed light on independent yet synergistic host- and bacterium-based strategies for resisting symbiosis-disrupting phage predation, and present important implications for understanding these strategies in the context of host-associated microbial ecosystems.

microbiology↗

bioRxiv2020

Bacteroides thetaiotaomicron-infecting bacteriophage isolates inform sequence-based host range prediction

Our emerging view of the gut microbiome largely focuses on bacteria and less is known about other microbial components such as of bacteriophages (phages). Though phages are abundant in the gut, very few phages have been isolated from this ecosystem. Here, we report the genomes of 27 phages from the United States and Bangladesh that infect the prevalent human gut bacterium Bacteroides thetaiotaomicron. These phages are mostly distinct from previously sequenced phages with the exception of two, which are crAss-like phages. We compare these isolates to existing human gut metagenomes, revealing similarities to previously inferred phages and additional unexplored phage diversity. Finally, we use host tropisms of these phages to identify alleles of phage structural genes associated with infectivity. This work provides a detailed view of the guts "viral dark matter" and a framework for future efforts to further integrate isolation- and sequencing-focused efforts to understand gut-resident phages.

microbiology↗

bioRxiv2020

Gold-standard diagnostics are tarnished by lytic bacteriophage

BackgroundA fundamental clinical and scientific concern is how lytic bacteriophage, as well as antibiotics, impact diagnostic positivity. MethodsCholera was chosen as a model disease to investigate this important question. Patients with diarrheal disease were enrolled at two remote hospitals in Bangladesh. Diagnostic performance was assessed as a function of lytic bacteriophage detection, as well as exposure to the first-line antibiotic azithromycin detected by mass spectrometry. ResultsAmong diarrheal samples positive by nanoliter quantitative PCR for Vibrio cholerae (n=78/849), the odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by 89% (OR 0.108; 95%CI 0.002-0.872) and 87% (OR 0.130; 95%CI 0.022-0.649) when lytic bacteriophage were detected, respectively. The odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by more than 99% (OR 0.00; 95% CI: 0.00-0.28) and 89% (OR 0.11; 95% CI: 0.03-0.44) when azithromycin was detected, respectively. ConclusionsEstimations of cholera burden may improve by accommodating for the negative effect of antimicrobial exposure on diagnostic positivity. Furthermore, the findings herein challenge our current approach to interpreting and developing bacterial diagnostics given variable rates of lytic bacteriophage and antibiotic exposure.

microbiology↗

bioRxiv2019

The clinical drug candidate ebselen attenuates inflammation and promotes microbiome recovery after antibiotic treatment for Clostridium difficile infection

Clostridium difficile infection (CDI) is an enteric bacterial disease that is increasing in prevalence worldwide. C. difficile capitalizes on gut inflammation and microbiome dysbiosis to establish infection, with symptoms ranging from watery diarrhea to toxic megacolon. We recently reported that the safe in human clinical drug candidate ebselen (NCT03013400, NCT01452607, NCT00762671, NCT02603081) has biochemical, cell-based and in vivo efficacy against the bacterial toxins of C. difficile. Here, we show that ebselen treatment reduces recurrence rates and decreases colitis in a hamster relapse model of CDI. Furthermore, ebselen treatment does not alter microbiome diversity but promotes its recovery back to that of healthy controls after antibiotic-induced dysbiosis in both healthy and C. difficile-infected mice. This increased microbiome recovery upon ebselen treatment correlates with a decrease in host-derived inflammatory markers suggesting that the anti-inflammatory properties of ebselen, combined with its anti-toxin function, help to mitigate the major clinical challenges of CDI, including recurrence, microbial dysbiosis, and colitis.

microbiology↗

bioRxiv2019

Multiple phase-variable mechanisms, including capsular polysaccharides, modify bacteriophage susceptibility in Bacteroides thetaiotaomicron

A variety of cell surface structures, including capsular polysaccharides (CPS), dictate interactions between bacteria and their environment including their viruses (bacteriophages). Members of the prominent human gut Bacteroidetes characteristically produce several phase-variable CPS, but their contributions to bacteriophage interactions are unknown. We used engineered strains of the human symbiont Bacteroides thetaiotaomicron, which differ only in the CPS they express, to isolate bacteriophages from two locations in the United States. Testing each of 71 bacteriophages against a panel of strains that express wild-type phase-variable CPS, one of eight different single CPS, or no CPS at all, revealed that each phage infects only a subset of otherwise isogenic strains. Deletion of infection-permissive CPS from B. thetaiotaomicron was sufficient to abolish infection for several individual bacteriophages, while infection of wild-type B. thetaiotaomicron with either of two different bacteriophages rapidly selected for expression of non-permissive CPS. Surprisingly, acapsular B. thetaiotaomicron also escapes complete killing by these bacteriophages, but surviving bacteria exhibit increased expression of 8 distinct phase-variable lipoproteins. When constitutively expressed, one of these lipoproteins promotes resistance to multiple bacteriophages. Finally, both wild-type and acapsular B. thetaiotaomicron were able to separately co-exist with one bacteriophage for over two months in the mouse gut, suggesting that phase-variation promotes resistance but also generates sufficient numbers of susceptible revertants to allow bacteriophage persistence. Our results reveal important roles for Bacteroides CPS and other cell surface structures that allow these bacteria to persist despite bacteriophage predation and hold important implications for using bacteriophages therapeutically to target gut symbionts.

microbiology↗

bioRxiv2018

Microbiota Accessible Carbohydrates Facilitate Clearance of Clostridium difficile

Clostridium difficile (Cd) is an opportunistic diarrheal pathogen and Cd infection (CDI) represents a major healthcare concern, causing an estimated 15,000 deaths per year in the United States alone1. Several enteric pathogens, including Cd, leverage inflammation and the accompanying microbial dysbiosis to thrive in the distal gut2. Although diet is among the most powerful available tools for affecting the health of humans and their relationship with their microbiota, investigation into the effects of diet on CDI has been limited. Here, we show in mice that the consumption of microbiota accessible carbohydrates (MACs) found in dietary plant polysaccharides has a significant impact on CDI. Specifically, using a model of antibiotic-induced CDI that typically resolves within 12 days of infection, we demonstrate that MAC-deficient diets perpetuate CDI. We show that Cd burdens are suppressed through ...

microbiology↗