Tuesday, April 1, 2025
4/1/2025
Human gut Bacteroidales defense mechanisms against emerging antibacterial toxins - PROJECT SUMMARY The human colon harbors a rich microbial community engaged in fierce competition for nutrients and territory. In healthy individuals, the Gram-negative bacterial order Bacteroidales comprise a large component of the colonic bacteria and secrete many antibacterial molecules to antagonize closely related members. Recently, our laboratory identified a large family of antibacterial, pore-forming toxins produced by various members of Bacteroidales that are structurally similar to the pore-forming cholesterol-dependent cytolysins (CDC) termed CDC-like (CDCL) toxins. Unlike the CDCs, the CDCLs assemble a pore using two proteins where one large monomer functions as a membrane platform to recruit ~30 smaller monomers, which lack a receptor binding domain. Metagenomic analysis revealed these 2-component CDCLs are produced by various members of Bacteroidales that have been associated with health and disease states and are widespread in the gut microbiota of diverse human populations. We determined the CDCLs are an antibacterial toxin that targets closely related members of Bacteroidales. Therefore, the production of CDCLs could impact the composition of the gut microbiota thereby affecting human health. The CDCLs require proteolytic activation, which occurs on the cell surface of sensitive species and is mediated by a cysteine protease, DpnB. Interestingly, we have discovered sensitive species can resist the effects of the CDCLs by two different methods. The first is mediated by slipped strand mutation within a conserved 7-adenine nucleotide region within the gene encoding DpnB. This region is found within many Bacteroidales cysteine proteases which suggest sensitive species have evolved a common resistance mechanism against CDCL-mediated killing. However, the loss of DpnB unexpectedly results in an altered secretome where some proteins are upregulated and others lost, potentially shifting its metabolic profile and influencing its ability to compete within the gut microbiome. This suggests that the regulation of DpnB may respond to other environmental factors that could affect its metabolism. The second resistance mechanism is linked to a stress response controlled by a sigma/anti-sigma factor pair. This stress response upregulates and down regulates multiple genes but does not affect DpnB expression. How or whether any of the genes regulated by this stress response protects from CDCL-mediated killing remains unexplored. Herein studies are proposed to answer three specific questions: i) how does the loss of DpnB alter the bacterial fitness within the gut microbiome?, ii) if sensitive species use slipped strand mutagenesis to regulate the expression of cysteine proteases, does DpnB revert to a functional state?, and iii) what component(s) of the stress response are necessary to protect the cell from the CDCLs and how do these components disrupt the assembly of the pore complex? The results from these experiments will provide insight into how diverse members of Bacteroidales protect against a novel family of antibacterial toxins and how these resistance mechanisms alter bacterial fitness within the gut microbiome.
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