Site‐selective alterations within the hemolysin A non‐polar core

Abstract

The secretion of virulence factors often aids bacterial pathogenesis. 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. In fact, whooping cough, meningitis, and certain food‐borne illnesses have been attributed to TPS pathway containing gram‐negative bacterial species. Systematically, TpsA members are activated concomitant with Tps‐dependent secretion across the outer membrane. Upon secretion, TpsA members elicit a variety of functions including cytolysis, adhesion, contact‐growth inhibition, and iron sequestration. Collectively, these TpsA functions provide advantageous invasion and proliferation strategies within host cells. Structurally, TpsA members can be divided into a two‐partner secretion (TPS) domain and a functional domain. All TPS domains are constructed from a 300‐residue right‐handed, parallel β‐helix structure, and recognize their cognate TpsB partner. In order to further understand the relationship between TPS domains and TpsA structure and function, we have implemented a truncated form of hemolysin A (HpmA265), a TpsA member from Proteus mirabilis. In previous studies, HpmA265 was structurally separated into three sequential folding subdomains: polar core, non‐polar core, and carboxy‐terminus. Specifically, these research investigations targeted valine 158 (V158) and phenylalanine 215 (F215) located within the first and last parallel β‐strands of the non‐polar core subdomain. A series of site‐selective variations were established at both V158 and F215. These variant forms of HpmA265 were characterized structurally via protease sensitivity and protein folding techniques, while functionality was ascertained within a template‐assisted hemolysis assay. Structurally, the V158 variants have destabilized the unfolding transitions associated with both the polar and non‐polar core subdomains, while leaving functionality unaffected. Site‐selective variants at F215 have selectively destabilized the non‐polar core subdomain, while leaving the unfolding transition attributed to the polar core subdomain unaffected. Additionally, the F215 variants do not affect template‐assisted hemolysis. Therefore, our results have been able to dissect the structural stability within the non‐polar core subdomain from template‐assisted function. These results have expanded the understanding for the implementation of TPS domains within gram‐negative bacteria.Support or Funding InformationFunding for this research was provided by: University Wisconsin – La Crosse Faculty Research Grant Program (TMW) and University Wisconsin – La Crosse Undergraduate Research and Creativity Grant Program (JDG).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.