The Mxi-Spa Type III Secretory Pathway ofShigella flexneri Requires an Outer Membrane Lipoprotein, MxiM, for Invasin Translocation

ABSTRACT Invasion of epithelial cells by Shigella flexneri is mediated by a set of translocated bacterial invasins, the Ipa proteins, and its dedicated type III secretion system, called Mxi-Spa. We show here that mxiM, part of the mxi-spa locus in the S. flexneri virulence plasmid, encodes an indispensable type III secretion apparatus component, required for both Ipa translocation and tissue culture cell invasion. We demonstrated that mature MxiM, first identified as a putative lipoprotein, is lipidated in vivo. Consistent with features of known lipoproteins, MxiM (i) can be labeled with [3H]palmitate and [2-3H]glycerol, (ii) is associated with the cell envelope, (iii) is secreted independently of the type III pathway, and (iv) requires an intact lipoprotein modification and processing site for full activity. The lipidated form of MxiM was detected primarily in the outer membrane, where it establishes a peripheral association with the inner leaflet. Through analysis of subcellular Ipa distribution in a mxiM null mutant background, MxiM was found to be required for the assembly and/or function of outer, but not inner, membrane regions of Mxi-Spa. This function probably requires interactions with other Mxi-Spa subunits within the periplasmic space. We discuss implications of these findings with respect to the function of MxiM and the structure of Mxi-Spa as a whole.

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MxiE Regulates Intracellular Expression of Factors Secreted by the Shigella flexneri 2a Type III Secretion System

Journal of Bacteriology ◽

10.1128/jb.184.16.4409-4419.2002 ◽

2002 ◽

Vol 184 (16) ◽

pp. 4409-4419 ◽

Cited By ~ 59

Author(s):

Colleen D. Kane ◽

Raymond Schuch ◽

William A. Day ◽

Anthony T. Maurelli

Keyword(s):

Type Iii Secretion ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Shigella Flexneri ◽

Type Iii Secretion System ◽

Growth Conditions ◽

Model Potential ◽

Secretion System ◽

Virulence Plasmid ◽

Type Iii

ABSTRACT The mxi-spa locus on the virulence plasmid of Shigella flexneri encodes components of the type III secretion system. mxiE, a gene within this locus, encodes a protein that is homologous to the AraC/XylS family of transcriptional regulators, but currently its role in pathogenesis remains undefined. We characterized the virulence phenotype of a nonpolar mxiE mutant and found that this mutant retained the ability to invade mammalian cells in tissue culture and secrete Ipas (type III effectors required for host cell invasion), although it was less efficient than wild-type Shigella at cell-to-cell spread. Despite its invasive properties in culture, the mxiE mutant was completely avirulent in an animal model. Potential targets for MxiE activation were identified by using promoter-green fluorescent protein fusions, and gene expression was examined under various growth conditions. Six MxiE-regulated genes were discovered: ospB, ospC1, ospE2, ospF, virA, and ipaH 9.8. Notably, activation of these genes only occurred within the intracellular environment of the host and not during growth at 37°C in liquid culture. Interestingly, all of the MxiE-regulated proteins previously have been shown to be secreted through the type III secretion system and are putative virulence factors. Our findings suggest that some of these Osp proteins may be involved in postinvasion events related to virulence. Since bacterial pathogens adapt to multiple environments during the course of infecting a host, we propose that Shigella evolved a mechanism to take advantage of a unique intracellular cue, which is mediated through MxiE, to express proteins when the organism reaches the eukaryotic cytosol.

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MxiM and MxiJ, Base Elements of the Mxi-Spa Type III Secretion System of Shigella, Interact with and Stabilize the MxiD Secretin in the Cell Envelope

Journal of Bacteriology ◽

10.1128/jb.183.24.6991-6998.2001 ◽

2001 ◽

Vol 183 (24) ◽

pp. 6991-6998 ◽

Cited By ~ 62

Author(s):

Raymond Schuch ◽

Anthony T. Maurelli

Keyword(s):

Cell Surface ◽

Type Iii Secretion ◽

Cell Envelope ◽

General Structure ◽

Eukaryotic Cell ◽

Effector Proteins ◽

Complex Assembly ◽

Base Element ◽

Type Iii

ABSTRACT The type III secretion pathway is broadly distributed across many parasitic bacterial genera and serves as a mechanism for delivering effector proteins to eukaryotic cell surface and cytosolic targets. While the effectors, as well as the host responses elicited, differ among type III systems, they all utilize a conserved set of 9 to 11 proteins that together form a bacterial envelope-associated secretory organelle or needle complex. The general structure of the needle complex consists of a transenvelope base containing at least three ring-forming proteins (MxiD, MxiJ, and MxiG in Shigella) that is connected to a hollow needle-like extension that projects away from the cell surface. Several studies have shown that the initial steps in needle complex assembly require interactions among the base proteins, although specific details of this process remain unknown. Here we identify a role for another base element inShigella, MxiM, in interactions with the major outer-membrane-associated ring-forming protein, MxiD. MxiM affects several features of MxiD, including its stability, envelope association, and assembly into homomultimeric structures. Interestingly, many of the effects were also elicited by the inner-membrane-associated base element, MxiJ. We confirmed that MxiM-MxiD and MxiJ-MxiD interactions occur in vivo in the cell envelope, and we present evidence that together these base elements can form a transmembrane structure which is likely an important intermediary in the process of needle complex assembly.

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Induction of Necrosis in Human Neutrophils byShigella flexneri Requires Type III Secretion, IpaB and IpaC Invasins, and Actin Polymerization

Infection and Immunity ◽

10.1128/iai.68.3.1289-1296.2000 ◽

2000 ◽

Vol 68 (3) ◽

pp. 1289-1296 ◽

Cited By ~ 52

Author(s):

Mathias François ◽

Véronique Le Cabec ◽

Marie-Ange Dupont ◽

Philippe J. Sansonetti ◽

Isabelle Maridonneau-Parini

Keyword(s):

Type Iii Secretion ◽

Actin Polymerization ◽

Shigella Flexneri ◽

Cytochalasin D ◽

Host Cells ◽

Human Neutrophils ◽

Virulence Plasmid ◽

Type Iii ◽

Gene Coding ◽

Azurophil Granule

ABSTRACT Infection by Shigella flexneri is characterized by infiltration of neutrophils in the intestinal mucosa and by a strong inflammatory reaction. Although neutrophils are constitutively programmed to die by apoptosis, we show that isolated human neutrophils undergo necrosis 2 h after infection with virulent S. flexneri strain M90T but not with the virulence plasmid-cured strain BS176. This was demonstrated by the release of azurophil granule proteins concomitant with the release of lactate dehydrogenase (LDH), disruption of the plasma membrane, and absence of DNA fragmentation. Mutants with the mxiD1 gene, coding for an essential component of the secretion type III machinery, or the genes coding for IpaB or IpaC invasins deleted were not cytotoxic. Neutrophil necrosis occurred independently of the bacterial ability to leave phagosomes, and it involved actin polymerization, as the addition of cytochalasin D after phagocytosis of Shigella inhibited the release of LDH. In conclusion, Shigella kills neutrophils by necrosis, a process characterized by the release of tissue-injurious granular proteins. This probably contributes to disruption of the epithelial barrier, leading to the dysentery observed in shigellosis and allowingShigella to enter its host cells.

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Analysis of virulence plasmid gene expression defines three classes of effectors in the type III secretion system of Shigella flexneri

Microbiology ◽

10.1099/mic.0.27639-0 ◽

2005 ◽

Vol 151 (3) ◽

pp. 951-962 ◽

Cited By ~ 82

Author(s):

Tony Le Gall ◽

Maria Mavris ◽

Maria Celeste Martino ◽

Maria Lina Bernardini ◽

Erick Denamur ◽

...

Keyword(s):

Type Iii Secretion ◽

Expression Profiles ◽

Shigella Flexneri ◽

Transcriptional Regulators ◽

Host Cells ◽

Virulence Plasmid ◽

Dependent Manner ◽

Regulation Of Expression ◽

Type Iii ◽

Plasmid Genes

Proteins directly involved in entry and dissemination of Shigella flexneri into epithelial cells are encoded by a virulence plasmid of 200 kb. A 30-kb region (designated the entry region) of this plasmid encodes components of a type III secretion (TTS) apparatus, substrates of this apparatus and their dedicated chaperones. During growth of bacteria in broth, expression of these genes is induced at 37 °C and the TTS apparatus is assembled in the bacterial envelope but is not active. Secretion is activated upon contact of bacteria with host cells and is deregulated in an ipaB mutant. The plasmid encodes four transcriptional regulators, VirF, VirB, MxiE and Orf81. VirF controls transcription of virB, whose product is required for transcription of entry region genes. MxiE, with the chaperone IpgC acting as a co-activator, controls expression of several effectors that are induced under conditions of secretion. Genes under the control of Orf81 are not known. The aim of this study was to define further the repertoires of virulence plasmid genes that are under the control of (i) the growth temperature, (ii) each of the known virulence plasmid-encoded transcriptional regulators (VirF, VirB, MxiE and Orf81) and (iii) the activity of the TTS apparatus. Using a macroarray analysis, the expression profiles of 71 plasmid genes were compared in the wild-type strain grown at 37 and 30 °C and in virF, virB, mxiE, ipaB, ipaB mxiE and orf81 mutants grown at 37 °C. Many genes were found to be under the control of VirB and indirectly of VirF. No alteration of expression of any gene was detected in the orf81 mutant. Expression of 13 genes was increased in the secretion-deregulated ipaB mutant in an MxiE-dependent manner. On the basis of their expression profile, substrates of the TTS apparatus can be classified into three categories: (i) those that are controlled by VirB, (ii) those that are controlled by MxiE and (iii) those that are controlled by both VirB and MxiE. The differential regulation of expression of TTS effectors in response to the TTS apparatus activity suggests that different effectors might be required at different times following contact of bacteria with host cells.

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The outer membrane phospholipase A is essential for membrane integrity and type III secretion in Shigella flexneri

Open Biology ◽

10.1098/rsob.160073 ◽

2016 ◽

Vol 6 (9) ◽

pp. 160073 ◽

Cited By ~ 6

Author(s):

Xia Wang ◽

Feng Jiang ◽

Jianhua Zheng ◽

Lihong Chen ◽

Jie Dong ◽

...

Keyword(s):

Bacterial Infection ◽

Outer Membrane ◽

Mutant Strain ◽

Type Iii Secretion ◽

Shigella Flexneri ◽

Phospholipase A ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Type Iii ◽

Outer Membrane Phospholipase A

Outer membrane phospholipase A (OMPLA) is an enzyme located in the outer membrane of Gram-negative bacteria. OMPLA exhibits broad substrate specificity, and some of its substrates are located in the cellular envelope. Generally, the enzymatic activity can only be induced by perturbation of the cell envelope integrity through diverse methods. Although OMPLA has been thoroughly studied as a membrane protein in Escherichia coli and is constitutively expressed in many other bacterial pathogens, little is known regarding the functions of OMPLA during the process of bacterial infection. In this study, the proteomic and transcriptomic data indicated that OMPLA in Shigella flexneri , termed PldA, both stabilizes the bacterial membrane and is involved in bacterial infection under ordinary culture conditions. A series of physiological assays substantiated the disorganization of the bacterial outer membrane and the periplasmic space in the ΔpldA mutant strain. Furthermore, the ΔpldA mutant strain showed decreased levels of type III secretion system expression, contributing to the reduced internalization efficiency in host cells. The results of this study support that PldA, which is widespread across Gram-negative bacteria, is an important factor for the bacterial life cycle, particularly in human pathogens.

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

Molecular Microbiology ◽

10.1046/j.1365-2958.2000.02179.x ◽

2000 ◽

Vol 38 (4) ◽

pp. 760-771 ◽

Cited By ~ 277

Author(s):

Carmen Buchrieser ◽

Philippe Glaser ◽

Christophe Rusniok ◽

Hafed Nedjari ◽

Helene d'Hauteville ◽

...

Keyword(s):

Type Iii Secretion ◽

Shigella Flexneri ◽

Virulence Plasmid ◽

Type Iii

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Influence of the Salmonella typhimuriumPathogenicity Island 2 Type III Secretion System on Bacterial Growth in the Mouse

Infection and Immunity ◽

10.1128/iai.67.1.213-219.1999 ◽

1999 ◽

Vol 67 (1) ◽

pp. 213-219 ◽

Cited By ~ 149

Author(s):

Jacqueline E. Shea ◽

Carmen R. Beuzon ◽

Colin Gleeson ◽

Rosanna Mundy ◽

David W. Holden

Keyword(s):

Bacterial Growth ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Virulence Plasmid ◽

Temperature Sensitive ◽

Wild Type ◽

Type Iii ◽

Virulence Attenuation

ABSTRACT We have investigated the in vivo growth kinetics of aSalmonella typhimurium strain (P11D10) carrying a mutation in ssaJ, a Salmonella pathogenicity island 2 (SPI2) gene encoding a component of a type III secretion system required for systemic growth in mice. Similar numbers of mutant and wild-type cells were recovered from the spleens and livers of BALB/c mice up to 8 h after inoculation by the intraperitoneal route. Thereafter, the numbers of wild-type cells continued to increase logarithmically in these organs, whereas those of P11D10 remained relatively static for several days before being cleared. Gentamicin protection experiments on spleen cell suspensions recovered from infected mice showed that viable intracellular wild-type bacteria accumulated over time but that intracellular P11D10 cells did not. Infection experiments were also performed with wild-type and P11D10 cells carrying the temperature-sensitive plasmid pHSG422 to distinguish between bacterial growth rates and killing in vivo. At 16 h postinoculation there were 10-fold more wild-type cells than mutant cells in the spleens of infected mice, but the numbers of cells of both strains carrying the nonreplicating plasmid were very similar, showing that there was little difference in the degree of killing sustained by the two strains and that the SPI2 secretion system must be required for bacterial replication, rather than survival, in vivo. The SPI2 mutant phenotype in mice is similar to that of strains carrying mutations in the Salmonella virulence plasmid spv genes. To determine if these two sets of genes interact together, a double mutant strain carrying SPI2 and spv mutations was constructed and compared with strains carrying single mutations in terms of virulence attenuation. These experiments failed to provide any evidence showing that the SPI2 and spv gene products interact together as part of the same virulence mechanism.

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Identification of the cis-Acting Site Involved in Activation of Promoters Regulated by Activity of the Type III Secretion Apparatus in Shigella flexneri

Journal of Bacteriology ◽

10.1128/jb.184.24.6751-6759.2002 ◽

2002 ◽

Vol 184 (24) ◽

pp. 6751-6759 ◽

Cited By ~ 46

Author(s):

Maria Mavris ◽

Philippe J. Sansonetti ◽

Claude Parsot

Keyword(s):

Type Iii Secretion ◽

Wild Type Strain ◽

Shigella Flexneri ◽

Virulence Plasmid ◽

Cis Acting ◽

Type Iii ◽

Genes Encoding ◽

In The Wild ◽

Necessary And Sufficient ◽

Transcriptional Fusions

ABSTRACT Bacteria of Shigella spp. use a virulence plasmid-encoded type III secretion (TTS) system to invade the colonic epithelium in humans. The activity of the TTS apparatus is tightly regulated in the wild-type strain and is induced upon contact of bacteria with epithelial cells, whereas it is deregulated, i.e., constitutively active, in some mutants. Under conditions of deregulated secretion, approximately 20 proteins are secreted, including VirA, OspB to OspG, and at least three members of the IpaH family, all of which are encoded by the virulence plasmid. Conditions inducing or deregulating the activity of secretion also induce the transcription of virA and four ipaH genes. The transcription of virA and ipaH9.8 requires both MxiE, a transcriptional activator of the AraC family, and IpgC, the chaperone of IpaB and IpaC, acting as a coactivator. Using reporter plasmids containing lacZ transcriptional fusions, we showed that the ipaH7.8. ipa4.5. ospC1, and ospF promoters are activated under conditions of deregulated secretion and that both MxiE and IpgC are necessary and sufficient for their activation in both Shigella flexneri and Escherichia coli. Promoter mapping and deletion analysis of the ipaH9.8. virA, and ospC1 promoters identified a 17-bp motif, the MxiE box, which overlaps the −35 region and is essential for the activation of these promoters. The presence of eight MxiE boxes on the virulence plasmid suggests that 11 genes encoding secreted proteins may be regulated by the activity of secretion. We also present evidence that at least one ipaH gene that is carried by the chromosome is controlled by MxiE and IpgC.

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