The Tripartite Type III Secreton of Shigella flexneri Inserts Ipab and Ipac into Host Membranes

Abstract

Bacterial type III secretion systems serve to translocate proteins into eukaryotic cells, requiring a secreton and a translocator for proteins to pass the bacterial and host membranes. We used the contact hemolytic activity of Shigella flexneri to investigate its putative translocator. Hemolysis was caused by formation of a 25-Å pore within the red blood cell (RBC) membrane. Of the five proteins secreted by Shigella upon activation of its type III secretion system, only the hydrophobic IpaB and IpaC were tightly associated with RBC membranes isolated after hemolysis. Ipa protein secretion and hemolysis were kinetically coupled processes. However, Ipa protein secretion in the immediate vicinity of RBCs was not sufficient to cause hemolysis in the absence of centrifugation. Centrifugation reduced the distance between bacterial and RBC membranes beyond a critical threshold. Electron microscopy analysis indicated that secretons were constitutively assembled at 37°C before any host contact. They were composed of three parts: (a) an external needle, (b) a neck domain, and (c) a large proximal bulb. Secreton morphology did not change upon activation of secretion. In mutants of some genes encoding the secretion machinery the organelle was absent, whereas ipaB and ipaC mutants displayed normal secretons.

Polymerization of a single protein of the pathogen Yersinia enterocolitica into needles punctures eukaryotic cells

“…Compared with wild type, no differences were found in the morphology of the needles of the yopD mutant (data not shown). Overall, the needles of Y. enterocolitica resembled those described for S. typhimurium and S. flexneri (14)(15)(16)(17).…”

“…To investigate the function of the YscF needles, we used the contact hemolytic activity of the bacteria as a simple model system. As with S. flexneri (15), contact hemolysis by Yersinia did not occur without centrifugation ( Fig. 2A).…”

“…In E. coli, the surface structures containing the protein EspA are required for translocation of the protein EspB (13). Apart from these filamentous appendages, needle-like surface structures have been described for S. typhimurium and Shigella flexneri (14)(15)(16)(17). These needles contain the homologous proteins PrgI and MxiH, respectively, and are part of a cylindrical organelle that consists of at least three more proteins of the type III secretion system (14,(16)(17)(18).…”

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

“…7,[10][11][12] IpaB then interacts with cholesterol and sphingolipid rich host cell membranes, which can be mimicked using defined liposomes. 8 The interaction of IpaB with host cells or cholesterol/sphingolipid rich liposomes recruits IpaC, the second translocator, 8,13 to assemble the final T3SA needle tip complex, which results in formation of the translocon pore. 14 This event completes the unidirectional conduit between Shigella and host cell cytoplasm and the concomitant full induction of effector protein translocation although whether this occurs as a single step or stepwise process that requires the T3SA is not entirely clear.…”

Molecular model of a type III secretion system needle: Implications for host-cell sensing

“…Many of the 25 proteins from which the T3SS is constructed are similar in either sequence or function to proteins of the bacterial flagellum (5), and export of the axial flagellar components is highly analogous to export through a virulence T3SS (6). However, T3SSs (and not flagellar systems) undergo host-contact-mediated activation and translocation of effector proteins into host cells, apparently through direct contact of the external distal tip of the apparatus with the host cell (7)(8)(9). The role of the needle has been probed by using mutagenic studies of the needle-subunit proteins from Yersinia and Shigella (10,11).…”