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Phosphomimetic Tyrosine Mutations in Spa47 Inhibit Type Three Secretion ATPase Activity and Shigella Virulence
Koleton D Hardy1, Nicholas E Dickenson1
1Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322, USA.
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.
Insights
Shigella virulence relies on its type three secretion system (T3SS). This study shows Spa47 tyrosine phosphorylation regulates T3SS activity and bacterial infection, offering new therapeutic targets.
Area of Science:
- Microbiology and Immunology
- Molecular Biology
- Pathogen Virulence Mechanisms
Background:
- Shigella is a major human pathogen causing millions of infections and deaths annually.
- Shigella virulence depends on effectors delivered by the type three secretion system (T3SS).
- Spa47, a T3SS ATPase, is crucial for T3SS apparatus function and effector secretion.
Purpose of the Study:
- To investigate the role of Spa47 tyrosine phosphorylation in regulating Shigella T3SS function and virulence.
- To understand how phosphorylation affects Spa47 ATPase activity and its impact on bacterial pathogenesis.
Main Methods:
- Utilized phosphomimetic mutations in Spa47 to mimic phosphorylation.
- Performed in vitro radiometric ATPase assays and circular dichroism.
- Conducted in vivo characterization of T3SS secretion, erythrocyte hemolysis, and cellular invasion.
Main Results:
- Demonstrated a direct correlation between Spa47 tyrosine phosphorylation state and its ATPase activity.
- Showed that Spa47 phosphorylation directly impacts T3SS function and overall Shigella virulence.
- Linked phosphorylation to modulation of effector protein secretion and bacterial invasion.
Conclusions:
- Spa47 tyrosine phosphorylation is a key regulatory mechanism for Shigella T3SS.
- Findings provide insights into pathways controlling T3SS activation and immune evasion.
- This research opens avenues for developing strategies against Shigella infections.
