Phosphomimetic Tyrosine Mutations in Spa47 Inhibit Type Three Secretion ATPase Activity and Shigella Virulence Phenotype.

Koleton D Hardy,Nicholas E Dickenson

doi:10.3390/pathogens11020202

Abstract

Shigella is a highly infectious human pathogen responsible for 269 million infections and 200,000 deaths per year. Shigella virulence is absolutely reliant on the injection of effector proteins into the host cell cytoplasm via its type three secretion system (T3SS). The protein Spa47 is a T3SS ATPase whose activity is essential for the proper function of the Shigella T3SS needle-like apparatus through which effectors are secreted. A phosphoproteomics study recently found several Shigella T3SS proteins, including Spa47, to be tyrosine phosphorylated, suggesting a means of regulating Spa47 enzymatic activity, T3SS function, and overall Shigella virulence. The work presented here employs phosphomimetic mutations in Spa47 to probe the effects of phosphorylation at these targeted tyrosines through in vitro radiometric ATPase assays and circular dichroism as well as in vivo characterization of T3SS secretion activity, erythrocyte hemolysis, and cellular invasion. Results presented here demonstrate a direct correlation between Spa47 tyrosine phosphorylation state, Spa47 ATPase activity, T3SS function, and Shigella virulence. Together, these findings provide a strong foundation that leads the way to uncovering the specific pathway(s) that Shigella employ to mitigate wasteful ATP hydrolysis and effector protein secretion when not required as well as T3SS activation in preparation for host infection and immune evasion.

Highlights

Shigella are Gram-negative, rod-shaped, facultative anaerobic bacteria that are closely related to Escherichia coli and belong to the large family Enterobacteriaceae
While shigellosis infections disproportionately burden developing regions of the world where there is limited access to clean drinking water and antibiotic treatment, there is an urgent concern surrounding the rapid emergence of antibiotic-resistant Shigella strains in both developing and industrialized nations throughout the world [4,5]
The findings presented here clearly demonstrate the importance of the identified Spa47 tyrosine residues in both Spa47 function and Shigella virulence, providing a strong foundation for follow-up studies uncovering the specific regulatory pathway(s) responsible for Spa47 regulation as well as identifying a priority target for the development of much needed non-antibiotic T3SS ATPase inhibitors

Summary

Introduction

Shigella are Gram-negative, rod-shaped, facultative anaerobic bacteria that are closely related to Escherichia coli and belong to the large family Enterobacteriaceae. While shigellosis infections disproportionately burden developing regions of the world where there is limited access to clean drinking water and antibiotic treatment, there is an urgent concern surrounding the rapid emergence of antibiotic-resistant Shigella strains in both developing and industrialized nations throughout the world [4,5]. These recent developments underscore the need to better understand the mechanism(s) that Shigella uses to infect human hosts and identify potential targets for the formulation of efficient non-antibiotic countermeasures against shigellosis

Methods

Results

Conclusion