Biology subjects

Payne, S. M.

Publications and source records attributed to Payne, S. M..

6 recordsLinked to original sources

bioRxiv2025

The VapBC Toxin Antitoxin System Enhances Shigella flexneri Fitness Through Coordination of Metabolic Stress Adaptation and Virulence

Shigella flexneri is a facultative intracellular pathogen that causes bacillary dysentery by invading and replicating within intestinal epithelial cells. Successful intracellular survival requires the bacterium to balance metabolic adaptation with the sustained expression of virulence programs by the type III secretion system (T3SS). Toxin- antitoxin (TA) systems, including the highly conserved type II VapBC module, are classically associated with plasmid maintenance via post-segregational killing, but their broader roles during infection remain poorly understood. Here, we investigate the function of the VapBC system during S. flexneri infection. We show that the vapBC operon is activated in response to intracellular stress and that VapC-dependent cleavage of initiator tRNAfMet occurs specifically during infection. The stringent response, primarily mediated by SpoT, appears to influence operon responsiveness by maintaining vapB expression levels, while iron limitation emerges as a strong activator of vapC transcription and activity. Deletion of vapBC impairs T3SS activation and bacterial dissemination, despite normal invasion, and induces a strong host interferon response, including the upregulation of guanylate-binding proteins (GBPs), known to restrict bacterial spread. Transcriptomic profiling of the{Delta} vapBC mutant reveals downregulation of core metabolic genes and upregulation of envelope stress and other TA modules, indicating a loss of intracellular homeostasis. These findings uncover a novel role for VapBC in promoting Shigella fitness by coordinating stress adaptation, virulence expression, and immune evasion, thereby sustaining the bacteriums intracellular lifestyle.

microbiology↗

bioRxiv2024

Vibrio cholerae CsrA controls ToxR levels by increasing the stability and translation of toxR mRNA

Regulation of colonization and virulence factor production in response to environmental cues is mediated through several regulatory factors in Vibrio cholerae, including the highly conserved RNA-binding global regulatory protein CsrA. We have shown previously that CsrA increases synthesis of the virulence-associated transcription factor ToxR in response to specific amino acids (NRES) and is required for the virulence of V. cholerae in the infant mouse model of cholera. In this study, we mapped the 5 untranslated region (5 UTR) of toxR and showed that CsrA can bind directly to an RNA sequence encompassing the 5 UTR, indicating that the regulation of ToxR levels by CsrA is direct. Consistent with this observation, the 5 UTR of toxR contains multiple putative CsrA binding sequences (GGA motifs), and mutating these motifs disrupted the CsrA-mediated increase in ToxR. Optimal binding of CsrA to a defined RNA oligonucleotide required the bridging of two GGA motifs within a single RNA strand. To determine the mechanism of CsrA regulation, we assayed toxR transcript levels, stability, and efficiency of translation. Both the amount of toxR mRNA in NRES and the stability of the toxR transcript were increased by CsrA. Using an in vitro translation assay, we further showed that synthesis of ToxR was greatly enhanced in the presence of purified CsrA, suggesting a direct role for CsrA in the translation of toxR mRNA. We propose a model in which CsrA binding to the 5 UTR of the toxR transcript promotes ribosomal access while precluding interactions with RNA-degrading enzymes. IMPORTANCEVibrio cholerae is uniquely adapted to life in marine environments as well as in the human intestinal tract. Global regulators such as CsrA, which help translate environmental cues into an appropriate cellular response, are critical for switching between these distinct environments. Understanding the pathways involved in relaying environmental signals is essential for understanding both the environmental persistence and the intestinal pathogenesis of this devastating human pathogen. In this study, we demonstrate that CsrA directly regulates synthesis of ToxR, a key virulence factor of V. cholerae. Under conditions favoring high levels of active CsrA in the cell, such as in the presence of particular amino acids, CsrA increases ToxR protein levels by binding to the toxR transcript and enhancing both its stability and translation. By responding to nutrient availability, CsrA is perfectly poised to activate the virulence gene regulatory cascade at the preferred site of colonization, the nutrient-rich small intestinal mucosa.

microbiology↗

bioRxiv2024

Structural Determinants of Vibrio cholerae FeoB Nucleotide Promiscuity

Ferrous iron (Fe2+) is required for the growth and virulence of many pathogenic bacteria, including Vibrio cholerae (Vc), the causative agent of the disease cholera. For this bacterium, Feo is the primary system that transports Fe2+ into the cytosol. FeoB, the main component of this system, is regulated by a soluble cytosolic domain termed NFeoB. Recent reanalysis has shown that NFeoBs can be classified as either GTP-specific or NTP-promiscuous, but the structural and mechanistic bases for these differences were not known. To explore this intriguing property of FeoB, we solved the X-ray crystal structures of VcNFeoB in both the apo and GDP-bound forms. Surprisingly, this promiscuous NTPase displayed a canonical NFeoB G-protein fold like GTP-specific NFeoBs. Using structural bioinformatics, we hypothesized that residues surrounding the nucleobase could be important for both nucleotide affinity and specificity. We then solved the X-ray crystal structures of N150T VcNFeoB in the apo and GDP-bound forms to reveal H-bonding differences surround the guanine nucleobase. Interestingly, isothermal titration calorimetry revealed similar binding thermodynamics of the WT and N150T proteins to guanine nucleotides, while the behavior in the presence of adenine nucleotides was dramatically different. AlphaFold models of VcNFeoB in the presence of ADP and ATP showed important conformational changes that contribute to nucleotide specificity among FeoBs. Combined, these results provide a structural framework for understanding FeoB nucleotide promiscuity, which could be an adaptive measure utilized by pathogens to ensure adequate levels of intracellular iron across multiple metabolic landscapes.

biochemistry↗

bioRxiv2023

(p)ppGpp is required for Virulence of Shigella flexneri

Infection by the enteric pathogen Shigella flexneri requires transit through the gastrointestinal tract and invasion of and replication within the cells of the host colonic epithelium. This process exposes the pathogen to a range of diverse microenvironments. Further, the unique composition and physical environment of the eukaryotic cell cytosol represents a stressful environment for S. flexneri, and extensive physiological adaptations are needed for the bacterium to thrive. In this work, we show that disrupting synthesis of the stringent response alarmone (p)ppGpp in S. flexneri diminished expression of key virulence genes, including ipaA, ipaB, ipaC and icsA, and it reduced bacterial invasion and intercellular spread. Deletion of the (p)ppGpp synthase gene relA alone had no effect on S. flexneri virulence, but disruption of both relA and the (p)ppGpp synthase/hydrolase gene spoT resulted in loss of (p)ppGpp synthesis and virulence. While the relA spoT deletion mutant was able to invade a cultured human epithelial cell monolayer, albeit at reduced levels, it was unable to maintain the infection and spread to adjacent cells, as indicated by loss of plaque formation. Complementation with spoT on a plasmid vector restored plaque formation. Thus, SpoT alone is sufficient to provide the necessary level of (p)ppGpp for virulence. These results indicate that (p)ppGpp is required for S. flexneri virulence and adaptation to the intracellular environment, adding to the repertoire of signaling pathways that affect Shigella pathogenesis.

microbiology↗

bioRxiv2022

Bacteroides thetaiotaomicron outer membrane vesicles modulate virulence of Shigella flexneri

The role of the gut microbiota in the pathogenesis of Shigella flexneri remains largely unknown. To understand the impact of the gut microbiota on S. flexneri virulence, we examined the effect of interspecies interactions with Bacteroides thetaiotaomicron (Bt), a prominent member of the gut microbiota, on S. flexneri invasion. When grown in Bt conditioned medium, S. flexneri showed reduced invasion of human epithelial cells. This decrease in invasiveness of S. flexneri resulted from a reduction in the level of S. flexneris master virulence regulator VirF. Reduction of VirF corresponded with a decrease in expression of a secondary virulence regulator virB, as well as expression of S. flexneri virulence genes required for invasion, intracellular motility, and spread. Repression of S. flexneri virulence factors by Bt conditioned medium was not caused by either a secreted metabolite or protein, but rather, was due to the presence of Bt outer membrane vesicles (OMVs) in the conditioned medium. The addition of purified Bt OMVs to S. flexneri growth medium recapitulated the inhibitory effects of Bt conditioned medium on invasion, virulence gene expression, and virulence protein levels. Total lipids extracted from either Bt cells or Bt OMVs also recapitulated the effects of Bt condition medium on expression of the S. flexneri virulence factor IpaC, indicating that Bt OMV lipids, rather than a cargo contained in the vesicles, are the active factor responsible for the inhibition of S. flexneri virulence. ImportanceShigella flexneri is the causative agent of bacillary dysentery in humans. Shigella spp. are one of the leading causes of diarrheal morbidity and mortality, especially among children in low and middle-income countries. The rise of antimicrobial resistance combined with the lack of an effective vaccine for Shigella heightens the importance of studies aimed at better understanding previously uncharacterized aspects of Shigella pathogenesis. Here, we show that conditioned growth medium from the commensal bacteria Bacteroides thetaiotaomicron represses the invasion of S. flexneri. This repression is due to the presence of B. thetaiotaomicron outer membrane vesicles. These findings establish a role for interspecies interactions with a prominent member of the gut microbiota in modulating the virulence of S. flexneri and identify a novel function of outer membrane vesicles in interbacterial signaling between members of the gut microbiota and an enteric pathogen.

microbiology↗

bioRxiv2022

Vibrio cholerae alkalizes its environment via citrate metabolism to inhibit enteric growth

Vibrio cholerae is a Gram-negative pathogen, living in constant competition with other bacteria in both marine environments and during human infection. One competitive advantage of V. cholerae is the ability to metabolize diverse carbon sources such as chitin and citrate. We observed that when V. cholerae strains were grown on a medium with citrate, the mediums chemical composition turned into a hostile alkaline environment for Gram-negative bacteria such as Escherichia coli and Shigella flexneri. We found that although the ability to exclude competing bacteria was not contingent on exogenous citrate, V. cholerae citrate metabolism mutants {Delta}oadA-1, {Delta}citE, and {Delta}citF mutants were not able to inhibit S. flexneri or E. coli growth. Lastly, we demonstrated that while the V. cholerae mediated increased medium pH was necessary for the enteric exclusion phenotype, secondary metabolites such as bicarbonate (protonated to carbonate in the raised pH) from the metabolism of citrate enhanced the ability to inhibit the growth of E. coli. These data provide a novel example of how V. cholerae outcompetes other Gram-negative bacteria.

microbiology↗