Contribution of beta circuit completion towards virulence factor stability and function
Authors: Grosskopf Julian , Weaver Todd , Grilley Daniel 
Published: June 2021

As bacterial pathogens gain antibacterial resistance, few mechanisms remain available for halting pathogenicity, giving a frightening glimpse into a post‐antibacterial era. However, the secretion of virulence factors is often imperative to bacterial pathogenesis within the human body and is becoming a larger target for antibiotic treatment. The two‐partner secretion (TPS) pathway, harboring both A (TpsA) and B‐components (TpsB), is the most commonly used gram‐negative virulence factor secretion system currently known. In fact, whooping cough, meningitis, UTIs, and certain food‐borne illnesses have been attributed to gram‐negative bacterial species containing the TPS system. Systematically, TpsA members are activated concomitant with TpsB‐dependent secretion across the outer membrane. Upon secretion, TpsA members elicit a variety of functions including cytolysis, adhesion, contact‐growth inhibition, and iron sequestration thus allowing the pathogen to invade and proliferate within the host, advantageously. Structurally, TpsA members can be divided into a TPS domain and a functional (virulent) domain. All TPS domains are constructed from a ~300‐residue right‐handed, parallel, β‐helix, and recognize their cognate TpsB membrane‐bound partner during secretion. Fundamentally, TpsA β‐helix structures are built from consecutive β‐circuits, where each β‐circuit is constructed from three parallel β‐strands. In order to further understand the relationship between complete TpsA β‐circuit establishment, structural stability and function we have implemented a truncated form of hemolysin A (HpmA265) from Proteus mirabilis. In previous studies, HpmA265 was structurally separated into three sequential folding subdomains: polar core, non‐polar core, and carboxy‐terminus. Structurally, the carboxy‐terminal subdomain harbors a partial, two‐stranded β‐circuit. Previous investigations replaced valine 158 (V158) and phenylalanine 215 (F215), as located within the first and last parallel β‐strands of the non‐polar core subdomain, with polar residues. The site‐selective alteration of V158 demonstrated increased function, while merging the unfolding transitions associated with the non‐polar and carboxy‐terminal subdomains. Alternatively, site‐selective alteration of F215 demonstrated decreased function, while destabilizing the transitions associated with both the non‐polar and carboxy‐terminal subdomains. Ultimately, template‐assisted activity has been interrelated to extent of β‐circuit structural destabilization within the carboxy‐terminal subdomain. Specifically, this research expands upon the previous V158 and F215 results by probing the structural and functional relationship via progressive amino‐acid extension to the HpmA265 carboxy‐terminal subdomain. Ultimately, the structural and functional effects of final β‐circuit completion will be ascertained using protein unfolding and hemolytic functional measurements.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.