Structural and functional studies of type three secretion virulence factors from gram-negative pathogenic bacteria

Abstract

Many pathogenic Gram-negative bacteria utilize type III secretion systems (TTSS) to alter the normal functions of target host epithelial cells. Of the 1.1 million deaths that are caused by Shigella each year, nearly a third are in children under five years of age. Salmonella enterica serovar Typhimurium is the leading cause of hospitalization and death due to food-borne gastroenteritis in the U.S. The pathogenesis of both of these species involves the invasion of epithelial cells of the gastrointestinal tract, which requires the use of a type III secretion system (TTSS). The Shigella type III secretion apparatus (TTSA) is composed of a basal body spanning both bacterial membranes and an exposed oligomeric needle. Host altering effectors are secreted through this energized conduit to promote bacterial invasion. The active needle-tip complex of S. flexneri is composed of a tip protein, IpaD, and two pore-forming translocators, IpaB and IpaC. Maturation of the needle-tip complex proceeds in a stepwise manner. IpaD is at the tip of the nascent TTSA needle where it controls the first step of TTS activation. The bile salt deoxycholate (DOC) binds to IpaD to induce recruitment of the first translocator, IpaB, into the maturing tip complex. The pore-forming translocators are bound by the class II chaperone, IpgC, within the bacterial cytoplasm in order to prevent premature association and degradation. Despite their importance in promoting Shigella virulence, few molecular level details are known regarding the interactions between IpgC and its targets, IpaB and IpaC. Additionally, the mechanism by which DOC serves to stabilize a conformational change within IpaD is poorly understood. Methods in structural biology, in particular X-ray crystallography, are extremely valuable in addressing such questions. We present here the crystal structures of IpgC (identifying an alternative quaternary state), DOC-bound IpaD and the N-terminal regions of both IpaB and SipB, the S. Typhimurium first translocator homolog. These structures have facilitated the functional analysis of crucial determinants of Gram-negative pathogens that would otherwise not have been possible. Additionally, these structural studies have revealed the critical α-helical nature of each protein subunit involved in mature needle-tip-translocon formation.