IpaD of Shigella flexneri Is Independently Required for Regulation of Ipa Protein Secretion and Efficient Insertion of IpaB and IpaC into Host Membranes

Abstract

Shigella flexneri causes human dysentery after invading the cells of the colonic epithelium. The best-studied effectors of Shigella entry into colonocytes are the invasion plasmid antigens IpaC and IpaB. These proteins are exported via a type III secretion system (TTSS) to form a pore in the host membrane that may allow the translocation of other effectors into the host cytoplasm. TTSS-mediated secretion of IpaD is also required for translocation pore formation, bacterial invasion, and virulence, but the mechanistic role of this protein is unclear. IpaD is also known to be involved in controlling Ipa protein secretion, but here it is shown that this activity can be separated from its requirement for cellular invasion. Amino acids 40 to 120 of IpaD are not essential for IpaD-dependent invasion; however, deletions in this region still lead to constitutive IpaB/IpaC secretion. Meanwhile, a central deletion causes only a partial loss of control of Ipa secretion but completely eliminates IpaD's invasion function, indicating that IpaD's role in invasion is not a direct outcome of its ability to control Ipa secretion. As shigellae expressing ipaD N-terminal deletion mutations have reduced contactmediated hemolysis activity and are less efficient at introducing IpaB and IpaC into erythrocyte membranes, it is possible that IpaD is responsible for insertion of IpaB/IpaC pores into target cell membranes. While efficient insertion of IpaB/IpaC pores is needed for optimal invasion efficiency, it may be especially important for Ipa-dependent membrane disruption and thus for efficient vacuolar escape and intercellular spread.

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).…”

“…This mutation also prevents polymerization of needles in vivo, resulting in secretion of MxiH C⌬5 monomers but a phenotype that is otherwise like a mxiH null mutant (19). To obtain uniformly 15 N, 13 Clabeled MxiH C⌬5 , mxiH⌬5/pET22b in E. coli Tuner(DE3) was grown in minimal medium supplemented with 15 N NH 4 Cl and 13 C-glucose as the sole nitrogen and carbon sources, respectively (21). After reaching mid log phase, bacteria were induced to produce protein with 1 mM isopropyl-1-thio-␤-D-galactopyranoside and grown at 15°C overnight.…”

“…NMR Spectroscopy-Uniformly 15 N-or 15 N, 13 C-labeled MxiH C⌬5 and unlabeled IpaD were dissolved in NMR buffer (10 mM sodium phosphate, pH 6.0, 10 mM NaCl, 10% D 2 O, and 90% H 2 O). NMR data were acquired at 25°C on a Bruker Avance 800 MHz spectrometer fitted with a cryogenic triple-resonance probe and a Z-axis pulse field gradient.…”

“…NMR data were processed using NMRPipe (23) and analyzed using NMRView (24). Backbone resonances of free MxiH C⌬5 were assigned using 1 H-15 N HSQC, HNCA, CBCA(CO)NH, and HNCACB acquired using a 0.8 mM 15 N, 13 C-MxiH C⌬5 sample as described for BsaL C⌬5 (21). Chemical shift mapping was done by acquiring two-dimensional 1 H-15 N HSQC at MxiH C⌬5 :IpaD molar ratios of 0.5, 1, 2, 4, and 8.…”

“…First, invasion of Henle 407 cells and contact-mediated hemolysis were used to detect the proper introduction of IpaB/IpaC translocons into targeted cells, which is dependent upon IpaD localization to the TTSA needle tip (10,(12)(13)(14). Second, overnight secretion of Ipa proteins provided a measure of functional needle formation.…”