Spa47 Oligomerization Plays a Key Role in ATPase Activation and Function in the Shigella Type III Secretion System

Abstract

Several Gram‐negative pathogens, including Shigella, utilize a conserved type three secretion system (T3SS) for infection. The type three secretion apparatus (T3SA) provides a unidirectional conduit for injection of effector proteins from the bacteria to the host cell cytoplasm. The conserved T3SS ATPase found at the base of the T3SA is believed to provide the energy for apparatus formation and effector secretion, however, the mechanisms of ATP hydrolysis, effector secretion, and apparatus formation by the T3SS ATPase remain unclear. We have recently shown that Spa47, the T3SS ATPase of Shigella, forms a unique activated oligomeric species during the course of purification. Using our recently published crystal structure of Spa47 as a guide, we modeled an activated Spa47 oligomer, finding that ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. To explore the dynamics of Spa47 protomer interaction in vitro and in vivo, we performed a series of ATP hydrolysis, fluorescence microscopy and Shigella invasion experiments. We observe a positive Hill coefficient with the monomeric Spa47 species that is not present with the oligomeric Spa47 species in vitro. Interestingly, we continue to see a positive Hill coefficient even with the addition of engineered ATPase inactive Spa47 point mutants to WT Spa47 monomer species. We have utilized live cell fluorescence microscopy to show that these ATPase inactive Spa47 point mutants localize to the T3SA though they do not support secretion. Furthermore, each of the ATPase inactive Spa47 point mutants as well as an oligomerization deficient Spa47 construct exhibits a dominant negative phenotype. Together, these results suggest that while ATP hydrolysis is not the major driving factor for Spa47 localization to the apparatus base, the Spa47 oligomer may undergo a series of concerted ATP hydrolysis events that are critical for apparatus function, similar to ATP synthase. As we better understand the nuances of T3SS ATPase mechanism and function, the results of this work will provide a strong platform for future work to perhaps target the T3SS ATPase for novel therapeutics.Support or Funding InformationNational Science Foundation Graduate Research Fellowship and National Institute of Health (1R15AI124108‐01A1)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.