Functional insights into the Shigella type III needle tip IpaD in secretion control and cell contact

Abstract

SummaryType III secretion apparatus (T3SA) are complex nanomachines that insert a translocation pore into the host cell membrane through which effector proteins are injected into the cytosol. In Shigella, the pore is inserted by a needle tip complex that also controls secretion. IpaD is the key protein that rules the composition of the tip complex before and upon cell contact or Congo red (CR) induction. However, how IpaD is involved in secretion control and translocon insertion remains not fully understood. Here, we report the phenotypic analysis of 20 10-amino acids deletion variants all along the coiled-coil and the central domains of IpaD (residues 131-332). Our results highlight three classes of T3S phenotype; (i) wild-type secretion, (ii) constitutive secretion of all classes of effectors, and (iii) constitutive secretion of translocators and early effectors, but not of late effectors. Our data also suggest that the composition of the tip complex defines both the T3SA inducibility state and late effectors secretion. Finally, we shed light on a new aspect regarding the contact of the needle tip with cell membrane by uncoupling the Shigella abilities to escape macrophage vacuole, and to insert the translocation pore or to invade nonphagocytic cells.

Control of type III secretion activity and substrate specificity by the cytoplasmic regulator PcrG

“…Slow-secreting, "constitutive on" needle-subunit mutants in Shigella similarly turn on effector secretion without the upregulation of secretion rate seen in the presence of Congo red or in strains lacking the needle tip-associated translocator proteins IpaB and IpaD (47,48). Conversely, small deletions in the needle tip protein IpaD can selectively up-regulate secretion of early export substrates, including the pore-forming translocator proteins IpaB and IpaC, but not late effectors (7). Because IpaB and IpaC proteins are secreted at low levels before triggering of effector secretion in a process termed leakage, these ipaD mutants modulate the secretion activity.…”

“…In most systems, the needle tip protein prevents premature effector secretion, most likely by allosterically constraining the T3SS in an effector secretion "off" conformation (6)(7)(8)(9)(10). PcrG, the needle tip protein chaperone, as well as PopN, a member of the YopN/MxiC family of proteins, control effector secretion from the bacterial cytoplasm in Pseudomonas aeruginosa.…”

Deoxycholate-Enhanced Shigella Virulence Is Regulated by a Rare π-Helix in the Type Three Secretion System Tip Protein IpaD

“…14,33 To determine the effect of the engineered IpaD mutations on translocator expression and secretion profiles in S. flexneri , the ipaD null S. flexneri strain (ΔIpaD) was complemented with each of the engineered IpaD mutants and grown overnight in tryptic soy broth (TSB) containing 0.1 mg/mL ampicillin and 0.05 mg/mL kanamycin. The bacteria were gently separated from the culture supernatant via centrifugation and the soluble, secreted proteins were isolated by precipitation in the presence of 5% trichloroacetic acid (TCA) followed by centrifugation at 20,000 x g. The resulting protein pellet was washed with acetone, resuspended in SDS sample buffer (210 mM Tris pH 6.8, 100 mM NaCl, 3 mM EDTA, 200 mM SDS, 3 mM NaN 3 , 0.03% Bromophenol Blue, 30% Glycerol), separated by SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes.…”

The Inside Story of Shigella Invasion of Intestinal Epithelial Cells

“…Host-cell sensing has been proposed to involve interaction between IpaB and cellsurface receptors, such as hyaluronic acid receptor CD44. Interestingly, IpaD thought to connect IpaB and the MxiH-needle, also appears to control the various stages of secretion between early and late effectors on cell contact (Schiavolin et al 2013). The precise mechanism controlling the switch from nonactive to active secretion following tip contact is not known.…”