Claude Parsot

Structure and composition of the Shigella flexneri‘needle complex’, a part of its type III secreton

Type III secretion systems (TTSSs or secretons), essential virulence determinants of many Gram‐negative bacteria, serve to translocate proteins directly from the bacteria into the host cytoplasm. Electron microscopy (EM) indicates that the TTSSs of Shigella flexneri are composed of: (1) an external needle; (2) a transmembrane domain; and (3) a cytoplasmic bulb. EM analysis of purified and negatively stained parts 1, 2 and a portion of 3 of the TTSS, together termed the ‘needle complex’ (NC), produced an average image at 17 Å resolution in which a base, an outer ring and a needle, inserted through the ring into the base, could be discerned. This analysis and cryoEM images of NCs indicated that the needle and base contain a central 2–3 nm canal. Five major NC components, MxiD, MxiG, MxiJ, MxiH and MxiI, were identified by N‐terminal sequencing. MxiG and MxiJ are predicted to be inner membrane proteins and presumably form the base. MxiD is predicted to be an outer membrane protein and to form the outer ring. MxiH and MxiI are small hydrophilic proteins. Mutants lacking either of these proteins formed needleless secretons and were unable to secrete Ipa proteins. As MxiH was present in NCs in large molar excess, we propose that it is the major needle component. MxiI may cap at the external needle tip.

The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri

Bacteria of Shigella spp. are the causative agents of shigellosis. The virulence traits of these pathogens include their ability to enter into epithelial cells and induce apoptosis in macrophages. Expression of these functions requires the Mxi–Spa type III secretion apparatus and the secreted IpaA–D proteins, all of which are encoded by a virulence plasmid. In wild‐type strains, the activity of the secretion apparatus is tightly regulated and induced upon contact of bacteria with epithelial cells. To investigate the repertoire of proteins secreted by Shigella flexneri in conditions of active secretion, we determined the N‐terminal sequence of 14 proteins that are secreted by a mutant in which secretion was deregulated. Sequencing of the virulence plasmid pWR100 of the S. flexneri strain M90T (serotype 5) has allowed us to identify the genes encoding these secreted proteins and suggests that approximately 25 proteins are secreted by the type III secretion apparatus. Analysis of the G+C content and the relative positions of genes and open reading frames carried by the plasmid, together with information concerning the localization and function of encoded proteins, suggests that pWR100 contains blocks of genes of various origins, some of which were initially carried by four different plasmids.

THE SECRETION OF THE SHIGELLA FLEXNERI IPA INVASINS IS ACTIVATED BY EPITHELIAL CELLS AND CONTROLLED BY IPAB AND IPAD

Shigella species are enteropathogens that invade epithelial cells of the human colon. Entry into epithelial cells is triggered by the IpaB, IpaC and IpaD proteins which are translocated into the medium through the specific Mxi‐Spa machinery. In vitro, Shigella cells secrete only a small fraction of the Ipa proteins, the majority of which remains in the cytoplasm. We show here that upon interaction with cultured epithelial cells or in the presence of fetal bovine serum, S.flexneri release pre‐synthesized Ipa molecules from the cytoplasm into the environment. Evidence is presented that IpaB and IpaD are essential for both blocking secretion through the Mxi‐Spa translocon in the absence of a secretion‐inducing signal and controlling secretion of the Ipa proteins in the presence of a signal. Subcellular localization and analysis of the molecular interactions of the Ipa proteins indicate that IpaB and IpaD associate transiently in the bacterial envelope. We propose that IpaB and IpaD, by interacting in the secretion apparatus, modulate secretion.

Extracellular association and cytoplasmic partitioning of the IpaB and IpaC invasins of S. flexneri

Shigella species cause bacillary dysentery in humans by invading colonic epithelial cells. IpaB and IpaC, two major invasins of these pathogens, are secreted into the extracellular milieu. We show here that IpaB and IpaC form a complex in the extracellular medium and that each binds independently to a 17 kDa polypeptide, IpgC, in the bacterial cytoplasm. The IpgC polypeptide was found to be necessary for bacterial entry into epithelial cells, to stabilize the otherwise unstable IpaB protein, and to prevent the proteolytic degradation of IpaC that occurs through its association with unprotected IpaB. We propose that IpgC, which is not secreted and thus acts as a molecular chaperone, serves as a receptor that prevents premature oligomerization of IpaB and IpaC within the cytoplasm of Shigella cells.

The various and varying roles of specific chaperones in type III secretion systems

The type III secretion pathway is used by numerous Gram-negative pathogenic bacteria to deliver proteins within the membrane or the cytoplasm of eukaryotic cells with which these bacteria interact. Secretion is regulated by external signals. This requires that, before being secreted, proteins are stored in the cytoplasm where they need to be stabilised, separated from other interaction partners, and maintained in a secretion-competent state. Specialised, energy-independent chaperones play various roles in these functions by associating in the cytoplasm with proteins before their secretion. Some chaperones are also directly involved in modulating transcription in response to secretion.

Conversion of PtdIns(4,5)P2 into PtdIns(5)P by the S.flexneri effector IpgD reorganizes host cell morphology

Phosphoinositides play a central role in the control of several cellular events including actin cytoskeleton organization. Here we show that, upon infection of epithelial cells with the Gram‐negative pathogen Shigella flexneri, the virulence factor IpgD is translocated directly into eukaryotic cells and acts as a potent inositol 4‐phosphatase that specifically dephosphorylates phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2] into phosphatidylinositol 5‐monophosphate [PtdIns(5)P] that then accumulates. Transfection experiments indicate that the transformation of PtdIns(4,5)P2 into PtdIns(5)P by IpgD is responsible for dramatic morphological changes of the host cell, leading to a decrease in membrane tether force associated with membrane blebbing and actin filament remodelling. These data provide the molecular basis for a new mechanism employed by a pathogenic bacterium to promote membrane ruffling at the entry site.

MxiD, an outer membrane protein necessary for the secretion of the Shigella flexneri Ipa invasins

The invasive phenotype of Shigella flexneri is conferred by a 220 kb virulence plasmid, pWR100, that encodes both the Ipa proteins, which are involved in the entry process, and factors which are required for the export and correct localization of the Ipa proteins. We have characterized the mxiD gene, whose expression, like that of the ipa operon, is regulated by temperature. After inactivation of mxiD, the mutant strain was unable to invade HeLa cells and to provoke keratoconjunctivitis in guinea‐pigs. Analysis of culture supernatants indicated that wild‐type S. flexneri secretes about nine polypeptides and that secretion of several of these, including IpaA, IpaB, and IpaC, is abolished in the mxiD mutant. Examination of the membrane proteins of the wild‐type and mxiD strains suggested that MxiD is an outer membrane protein. Amino acid sequence comparison revealed that MxiD is homologous to the YscC protein of Yersinia enterocolitica and to the C‐terminal region of the PulD protein of Klebsiella pneumoniae. Both YscC and PulD are involved in extracellular protein secretion. These results indicate that MxiD is an essential component of the Ipa secretion apparatus.

Enhanced secretion through the Shigella flexneri Mxi-Spa translocon leads to assembly of extracellular proteins into macromolecular structures.

Genes required for entry of Shigella flexneri into epithelial cells in vitro are clustered in two adjacent loci, one of which encodes secretory proteins, the IpaA–D proteins, and the other their dedicated secretion apparatus, the Mxi–Spa translocon. Ipa secretion, which is induced upon contact of bacteria with epithelial cells, is prevented during growth in vitro. Here, we show that ipaB and ipaD mutations lead to enhanced secretion of a set of about 15 proteins. These extracellular proteins and some Ipas associate in organized structures consisting of extended sheets. Growth of the wild‐type strain in the presence of Congo red is shown to induce protein secretion through the Mxi–Spa translocon. Cultures grown to stationary phase in the presence of Congo red contain extracellular filaments whose composition and morphology are similar to those produced by the hyper‐secreting ipaB and ipaD mutants.

SepA, the major extracellular protein of Shigella flexneri: autonomous secretion and involvement in tissue invasion

In addition to lpa proteins and lcsA, which are involved in entry into epithelial cells and intercellular spread, respectively, Shigella secretes a 110 kDa protein, designated SepA. We report the identification, cloning, and nucleotide sequence determination of the sepA gene, analysis of SepA secretion, and construction and characterization of a sepA mutant. The sepA gene is carried by the virulence plasmid and codes for a 150 kDa precursor. Upon secretion, which does not involve accessory proteins encoded by the virulence plasmid, the precursor is converted to a mature protein of 110 kDa by two cleavages removing an N‐terminal signal sequence and a C‐terminal fragment. Extensive similarities were detected between the sequence of the first 500 residues of mature SepA and the N‐terminal region of lgA1 proteases from Neisseria gonorrhoeae and Haemophilus influenzae, the Tsh haemagglutinin of an avian pathogenic Escherichia coli, and the Hap protein involved in adhesion and penetration of H. influenzae. The C‐terminal domain of the SepA precursor, which is not present in the secreted protein, exhibits sequence similarity with pertactin of Bordetella pertussis and the ring‐forming protein of Helicobacter mustelae. Construction and phenotypic characterization of a sepA mutant indicated that SepA is required neither for entry into cultured epithelial cells nor for intercellular dissemination. However, in the rabbit ligated ileal loop model, the sepA mutant exhibited an attenuated virulence, which suggests that SepA might play a role in tissue invasion.

Secretion of predicted Inc proteins of Chlamydia pneumoniae by a heterologous type III machinery.

Chlamydia spp. are strictly intracellular pathogens that grow inside a vacuole, called an inclusion. They possess genes encoding proteins homologous to components of type III secretion machineries, which, in other bacterial pathogens, are involved in delivery of bacterial proteins within or through the membrane of eukaryotic host cells. Inc proteins are chlamydial proteins that are associated with the inclusion membrane and are characterized by the presence of a large hydrophobic domain in their amino acid sequence. To investigate whether Inc proteins and other proteins exhibiting a similar hydropathic profile might be secreted by a type III system, we used a heterologous secretion system. Chimeras were constructed by fusing the N‐terminal part of these proteins with a reporter, the Cya protein of Bordetella pertussis, and these were expressed in various strains of Shigella flexneri. We demonstrate that these hybrid proteins are secreted by the type III secretion system of S. flexneri, thereby providing evidence that IncA, IncB and IncC are secreted by a type III mechanism in chlamydiae. Moreover, we show that three other proteins from Chlamydia pneumoniae, all of which have in common the presence of a large hydrophobic domain, are also secreted by S. flexneri type III secretion machinery.