IpaD Localizes to the Tip of the Type III Secretion System Needle of Shigella flexneri

Abstract

Shigella flexneri, the causative agent of shigellosis, is a gram-negative bacterial pathogen that initiates infection by invading cells within the colonic epithelium. Contact with host cell surfaces induces a rapid burst of protein secretion via the Shigella type III secretion system (TTSS). The first proteins secreted are IpaD, IpaB, and IpaC, with IpaB and IpaC being inserted into the host cell membrane to form a pore for translocating late effectors into the target cell cytoplasm. The resulting pathogen-host cross talk results in localized actin polymerization, membrane ruffling, and, ultimately, pathogen entry. IpaD is essential for host cell invasion, but its role in this process is just now coming to light. IpaD is a multifunctional protein that controls the secretion and presentation of IpaB and IpaC at the pathogen-host interface. We show here that antibodies recognizing the surface-exposed N terminus of IpaD neutralize Shigella's ability to promote pore formation in erythrocyte membranes. We further show that MxiH and IpaD colocalize on the bacterial surface. When TTSS needles were sheared from the Shigella surface, IpaD was found at only the needle tips. Consistent with this, IpaD localized to the exposed tips of needles that were still attached to the bacterium. Molecular analyses then showed that the IpaD C terminus is required for this surface localization and function. Furthermore, mutations that prevent IpaD surface localization also eliminate all IpaD-related functions. Thus, this study demonstrates that IpaD localizes to the TTSA needle tip, where it functions to control the secretion and proper insertion of translocators into host cell membranes.

Bioinformatic and Biochemical Evidence for the Identification of the Type III Secretion System Needle Protein ofChlamydia trachomatis

“…In this case, the alteration in cross-linking pattern could correspond to states in which CdsF is interacting with different proteins. For example, CdsF likely interacts with translocator proteins embedded in the inclusion membrane in the context of a native infection, whereas in EBs it could interact with capping proteins similar to those described for Shigella or Yersinia needles (13,41). Regardless of the mechanism, it is therefore probable that the different dominant bands seen here in the cross-linking ladders during active T3S (RBs) and inactive T3S (EBs) represent an alteration in the functional state CdsF.…”

Identification of the MxiH Needle Protein Residues Responsible for Anchoring Invasion Plasmid Antigen D to the Type III Secretion Needle Tip

“…The needle is ϳ50 nm in length, has an outer diameter of 7.0 nm, and possesses an inner channel that is ϳ2.0 -3.0 nm in diameter (10,11). We recently demonstrated that invasion plasmid antigen D (IpaD) localizes to the tip of the TTSA needle (12), where it controls secretion of the translocator proteins IpaB and IpaC and directs their insertion into host cell membranes (13). From its position at the TTSA needle tip, IpaD is responsible for recruiting and stably maintaining IpaB to this site in the presence of bile salts (14).…”

“…From its position at the TTSA needle tip, IpaD is responsible for recruiting and stably maintaining IpaB to this site in the presence of bile salts (14). This is an essential step for Shigella invasiveness, and the interactions between IpaD and MxiH that are responsible for tip complex stabilization are thus critical for Shigella pathogenesis (12,14).…”

Intracellular type III secretion by cytoplasmic Shigella flexneri promotes caspase-1-dependent macrophage cell death

“…Purified IpaB and IpaC integrate into host-cell plasma membranes (De Geyter et al, 1997, and IpaB induces liposome fusions in vitro (Hume et al, 2003). IpaD localizes to the tip of the needle prior to secretion and likely serves as a scaffold for the assembly of the hydrophobic IpaBC translocon (Espina et al, 2006). IpaB and IpaC also have effector functions: while microinjected IpaC triggers actin polymerization and filopodia formation in epithelial cells (Tran Van Nhieu et al, 1999), microinjected IpaB triggers cell death in macrophages (Chen et al, 1996).…”