Biology subjects

Shull, L. M.

Publications and source records attributed to Shull, L. M..

4 recordsLinked to original sources

bioRxiv2025

BetaH proteolysis unleashes an electrostatic-homing antibacterial polymorphic toxin

Contact-dependent or diffusible proteinaceous polymorphic toxin systems (PTSs) mediate widespread bacterial competition. While bioinformatic analyses have identified diverse PTSs across bacterial phyla, experimental validation in Gram-positive species remains limited. Here, we characterize a diffusible PTS encoded by the Staphylococcus aureus S8-Ntox35 locus. We demonstrate that this system mediates inter-genus antagonism against Listeria monocytogenes via a bactericidal, heat-labile protein, and that toxicity depends on extracellular cleavage of the BetaH domain by an S8 peptidase. This processed peptide resembles a cationic antimicrobial peptide (CAMP) and facilitates intoxication of target cells by the Ntox35 RNase domain. Target cell resistance is impacted by known CAMP defense pathways, including DltABCD and MprF, and experimental evolution identified the ABC transporter AnrAB as essential for intoxication. Unexpectedly, disruption of AnrAB abolished Ntox35 susceptibility, while simultaneously sensitizing cells to the proposed CAMP like activity of the processed BetaH domain. These findings reveal a novel mechanism of inter-genus antagonism among Firmicutes and establish a functional role for extracellular processing and ABC transporter-mediated susceptibility in PTS activity. Our work expands the known repertoire of diffusible toxins in Gram-positive bacteria and sets the foundation for broader ecological and mechanistic investigation of S8-PTS systems. SignificancePolymorphic toxin systems (PTSs) are widely used by bacteria to inhibit competitors, but diffusible proteinaceous toxins have been largely characterized in Gram-negative species. Here, we identify and mechanistically characterize a diffusible PTS in Staphylococcus aureus that mediates inter-genus antagonism in the Firmicutes phylum. This system employs a secreted S8 peptidase to cleave a BetaH-toxin fusion, releasing a previously caged cationic amphipathic -helix that facilitates membrane targeting and intoxication of susceptible cells. We further show that toxin activity requires the target cell ABC transporter AnrAB, revealing a novel route of entry and an evolutionary tradeoff between toxin susceptibility and antimicrobial resistance. Together, these findings uncover a new mode of bacterial competition and highlight a broadly distributed toxin system with ecological relevance across Firmicutes.

microbiology↗

bioRxiv2024

Analysis of genetic requirements and nutrient availability for Staphylococcus aureus growth in cystic fibrosis sputum

Staphylococcus aureus is one of the most common pathogens isolated from the lungs of people with cystic fibrosis (CF), but little is known about its ability to colonize this niche. We performed a Tn-seq screen to identify genes necessary for S. aureus growth in media prepared from ex vivo CF sputum. We identified 19 genes that were required for growth in all sputum media tested and dozens more that were required for growth in at least one sputum medium. Depleted mutants of interest included insertions in many genes important for surviving metal starvation as well as the primary regulator of cysteine metabolism cymR. To investigate the mechanisms by which these genes contribute to S. aureus growth in sputum, we quantified low-molecular-weight thiols, nutrient transition metals, and the host metal-sequestration protein calprotectin in sputum from 11 individuals with CF. In all samples, the abundance of calprotectin exceeded nutrient metal concentration, explaining the S. aureus requirement for metal-starvation genes. Further, all samples contain potentially toxic quantities of cysteine and sufficient glutathione to satisfy the organic sulfur requirements of S. aureus. Deletion of the cysteine importer genes tcyA and tcyP in the {Delta}cymR background restored growth to wild-type levels in CF sputum, suggesting that the mechanism by which cymR is required for growth in sputum is to prevent uncontrolled import of cysteine or cystine from this environment. Overall, this work demonstrates that calprotectin and cysteine limit S. aureus growth in CF sputum. IMPORTANCEStaphylococcus aureus is a major cause of lung infections in people with cystic fibrosis (CF). This work identifies genes required for S. aureus growth in this niche, which represent potential targets for anti-Staphylococcal treatments. We show that genes involved in surviving metal starvation are required for growth in CF sputum. We also found that the primary regulator of cysteine metabolism, CymR, plays a critical role in preventing cysteine intoxication during growth in CF sputum. To support these models, we analyzed sputum from 11 individuals with CF to determine concentrations of calprotectin, nutrient metals, and low-molecular-weight thiols, which have not previously been quantified together in the same samples.

microbiology↗

bioRxiv2022

Bile Salts and N-Acetyl-D-Glucosamine induce transcription of the vieSAB Operon in Vibrio cholerae El Tor

The vieSAB operon regulates motility, biofilm formation and cholera toxin production in the classical biotype of V. cholerae. Classical biotype mutants lacking the cyclic-di-GMP phosphodiesterase and response regulator vieA or its cognate sensor kinase vieS fail to effectively colonize the infant mouse small intestine, are less motile than the wild-type strain, and are hyper-biofilm formers. However, deletion of vieA or vieS in the currently-circulating El Tor biotype does not have a demonstrable effect on these phenotypes. To begin to understand the role of vieSAB in the El Tor biotype, we studied its regulation and the effect of vieA expression on bacterial physiology, separating the functional roles of VieAs enzymatic and DNA-binding activities. We identify bile salts and N-acetylglucosamine, which can be found at high concentration in the small intestine, as stimulating expression of vieSAB during in vitro growth. We show that, in contrast to many response regulators, VieA does not regulate its own expression. Together, these data indicate that V. cholerae upregulates vieSAB in response to small-intestinal signals, allowing VieA to alter the physiology of the cell through its transcriptional regulation and cyclic-di-GMP signaling activities in order to adapt to the human host.

microbiology↗

bioRxiv2020

Topologically correct synthetic reconstruction of pathogen social behavior found in deep tissue sites

Within deep tissue sites, extracellular bacterial pathogens often replicate in clusters that are surrounded by immune cells. Disease is modulated by interbacterial interactions as well as bacterial-host cell interactions resulting in microbial growth, phagocytic attack and secretion of host antimicrobial factors. To overcome the limited ability to manipulate these infection sites, we established a system for Yersinia pseudotuberculosis (Yptb) growth in microfluidics-driven microdroplets that regenerates microbial social behavior in tissues. Chemical generation of nitric oxide (NO) in the absence of immune cells was sufficient to reconstruct microbial social behavior, as witnessed by expression of the NO-inactivating protein Hmp on the extreme periphery of microcolonies, mimicking spatial regulation in tissues. Similarly, activated macrophages that expressed inducible NO synthase (iNOS) drove peripheral expression of Hmp, allowing regeneration of social behavior observed in tissues. These results argue that topologically correct microbial tissue growth and associated social behavior can be reconstructed in culture.

microbiology↗