Investigation of the structure and function of type III secretion needle protein MxiH from Shigella flexneri

Shigella flexneri causes a severe though self-limiting form of dysentery in humans that is clinically known as shigellosis. Shigellosis is characterized by fever, intense abdominal pain and frequent scanty stools containing blood and mucus. The organism is typically transmitted through contaminated water. Pathogenesis of Shigella requires the action of a type III secretion system (T3SS) that the organism uses to promote bacterial invasion of colonic epithelial cells. T3SSs are composed of three major structures: a cytoplasmic ATPase "bulb", a trans-periplasmic "basal body" and an extracellular "needle". The external needle is a hollow rod-like structure that extends ∼60 nm from the bacterial surface and is a helical assembly of MxiH monomers.; Following ingestion, S. flexneri travels to the colon where it uses its T3SS to induce cytoskeletal changes in epithelial cells that allow pathogen entry. After the T3S needle tip contacts a target cell, a signal is transmitted to the base to activate type III secretion. Following activation, the T3SS inserts "translocator" proteins (IpaB and IpaC) into the target cell membrane to provide the final portion of a conduit linking the bacterial and host cell cytoplasms. "Effector" proteins are then released into the host cytoplasm to subvert normal cellular processes and promote bacterial uptake. Exactly how signals are transmitted from the tip of the needle to the base (a distance of ∼800 A) is not clear. Here we show that mutations in MxiH, the T3SS needle monomer, affect the secretion status and activation of the T3SS suggesting that the host cell contact signal is transmitted via the needle itself. We then describe the solution properties of purified MxiH and key MxiH mutants using circular dichroism (CD) spectroscopy. CD spectroscopy was also used to compare solution properties of needle proteins from the Gram-negative pathogens Salmonella typhimurium and Burkholderia pseudomallei. Lastly, we used electron microscopy to visualize T3SS needles to show that IpaD, a type III secreted translocator protein, resides at the tip of the T3SS needle under non-inducing conditions. With a better understanding of T3S function, we plan to devise new strategies for combating shigellosis.