virG, a plasmid-coded virulence gene of Shigella flexneri: identification of the virG protein and determination of the complete coding sequence

Lett, Marie‐Claire; Sasakawa, Chihiro; Okada, Nobuhiko; Sakai, Takaaki; Makino, Sou‐ichi; Yamada, Masatoshi; Komatsu, Keiko; Yoshikawa, Masanosuke

doi:10.1128/jb.171.1.353-359.1989

Cited by 235 publications

(204 citation statements)

References 26 publications

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“…Forty-five mutants that were invasive but were defective in plaque formation, indicating mutations affecting intracellular multiplication or spread to adjacent cells, were selected for further study. The DNA sequences adjacent to the TnphoA inserts were analysed in the mutants and among these were icsA (virG ), a gene previously shown to be required for plaque formation (Bernardini et al, 1989;Lett et al, 1989), and a number of genes associated with LPS synthesis (Table 1). The S. flexneri LPS O-antigen-synthesis genes identified in this screen included rfbA, -B, -C, -F, and -G. These genes are involved in different steps in synthesis of the O-antigen repeating unit and are encoded on the S. flexneri chromosome (Macpherson et al, 1994;Morona et al, 1994) (Fig.…”

Section: Isolation Of Strains With Chromosomal Lps-synthesis Gene Mutmentioning

confidence: 99%

Effect of mutations in Shigella flexneri chromosomal and plasmid‐encoded lipopolysaccharide genes on invasion and serum resistance

Hong

Payne

1997

Molecular Microbiology

117

120

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SummaryThis study shows that both length and distribution of lipopolysaccharide (LPS) are important for Shigella flexneri invasion and virulence. Mutants were generated in the chromosomal LPS synthesis genes rfa, rfb, and rol, and in a plasmid-encoded O-antigen chain-length regulator, cld pHS-2 . LPS analysis showed that mutations in rfb genes and in a candidate rfaL gene either eliminated the entire O-antigen side chains or produced chains of greatly reduced length. Mutation in a previously unidentified gene, rfaX, affected the LPS core region and resulted in reduced amounts of O-antigen. Mutants defective in cld pHS-2 or rol had different distributions of O-antigen chain lengths. The results of tissue-culture cell invasion and plaque assays, the Seré ny test, and serum-sensitivity assay suggested roles for the different LPS synthesis genes in bacterial survival and virulence; rfaL, rfaX and rfb loci are required for serum resistance and intercellular spread, but not for invasion; cld pHS-2 is required for resistance to serum killing and for full inflammation in the Seré ny test, but not for invasion or intercellular spread, while rol is required for normal invasiveness and plaque formation, but not for serum resistance. Thus, O-antigen synthesis and chain-length regulation genes encoded on both the chromosome and the small plasmid pHS-2 play important roles in S. flexneri invasion and virulence.

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Section: Isolation Of Strains With Chromosomal Lps-synthesis Gene Mutmentioning

confidence: 99%

Effect of mutations in Shigella flexneri chromosomal and plasmid‐encoded lipopolysaccharide genes on invasion and serum resistance

Hong

Payne

1997

Molecular Microbiology

117

120

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“…VirG is a surface-exposed outer membrane protein of bacterium, composed of 1102 amino acids, which contains three distinctive domains: the Nterminal signal sequence (residues 1-52), the surfaceexposed 706-amino-acid a-domain (residues 53-758) and the membrane-embedded 344-amino-acid C-terminal b-core (residues 759-1102) (Lett et al, 1989;Goldberg et al, 1993;Suzuki et al, 1995). The N-terminal two-thirds of the VirG a-domain, which contains six glycine-rich repeats, is essential for mediating actin assembly of Shigella in host cells, as the a-domain is involved in interacting with host proteins such as vinculin and neural Wiskott-Aldrich syndrome protein (N-WASP) (Suzuki et al, 1996;Egile et al, 1999;Suzuki and Sasakawa, 2001).…”

Section: Introductionmentioning

confidence: 99%

Neural Wiskott-Aldrich syndrome protein (N-WASP) is the specific ligand for Shigella VirG among the WASP family and determines the host cell type allowing actin-based spreading

et al. 2002

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SummaryShigella, the causative agent of bacillary dysentery, is capable of directing its movement within host cells by forming an actin comet tail. The VirG (IcsA) protein expressed at one pole of the bacterium recruits neural Wiskott-Aldrich syndrome protein (N-WASP), a member of the WASP family, which in turn stimulates actin-related protein (Arp) 2/3 complex-mediated actin polymerization. As all the WASP family proteins induce actin polymerization by recruiting Arp2/3 complex, we investigated their involvement in Shigella motility. Here, we show that VirG binds to N-WASP but not to the other WASP family proteins. Using a series of chimeras obtained by swapping N-WASP and WASP domains, we demonstrated that the specificity of VirG to interact with N-WASP lies in the N-terminal region containing the pleckstrin homology (PH) domain and calmodulin-binding IQ motif of N-WASP. A conformational change in N-WASP was important for the VirG-N-WASP interaction, as elimination of the C-terminal acidic region, which is responsible for the intramolecular interaction with the central basic region of N-WASP, affected the specific binding to VirG. We observed that, in haematopoietic cells such as macrophages, polymorphonuclear leucocytes (PMNs) and platelets, WASP was predominantly expressed, whereas the expression of N-WASP was greatly suppressed. Indeed, unlike Listeria, Shigella was unable to move in macrophages at all, (V) domain, a domain (C) with homology to the actindepolymerizing protein cofilin and, finally, a C-terminal acidic (A) segment (Miki et al., 1996). The C-terminal VCA domain interacts with the actin-related protein (Arp) 2/3 complex after the stimulation of actin polymerization by the complex (Rohatgi et al., 1999). N-WASP exists in two conformations: an inactive form in which the VCA domain is masked, and an active form in which it is exposed to promote actin polymerization by interaction with the Arp2/3 complex. The activation of N-WASP requires Cdc42 or PtdIns(4,5)P2 bound to N-WASP (Miki et al., 1998b;Rohatgi et al., 1999;2000;Prehoda et al., 2000). It has been reported that N-WASP is required for generating the actin tail from motile S. flexneri (Suzuki et al., 1998;Egile et al., 1999). Arp2/3 complex is also required for actin-based motility of Shigella, in which N-WASP stimulates the actin nucleation activity of the Arp2/3 complex in vitro (Egile et al., 1999).In mammalian cells, five WASP family members, N-WASP, WASP and WAVE1-3, have been identified and characterized to date (Suetsugu et al., 1999); however, none of them except N-WASP has been elucidated for involvement in the actin-based motility of Shigella in infected cells. It has been shown that human platelet extracts supported Shigella actin-based motility as added N-WASP in vitro (Egile et al., 1999). Recent reports using N-WASP-deficient mouse embryonic fibroblasts (Lommel et al., 2001;Snapper et al., 2001) confirmed our previous work indicating that N-WASP is required for Shigella motility in mammalian cells (Suzuki et al., 1998). However...

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“…The virulence plasmid-encoded protein product of the icsA (virG) gene is necessary and sufficient for actinbased motility (Makino et al, 1986;Bernardini et al, 1989;Lett et al, 1989;Goldberg and Theriot, 1995;Kocks et al, 1995). IcsA, a 120 kDa outer membrane protein, is located at a single pole on the surface of the bacterium, at the junction with the actin tail (Goldberg et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

Actin-based motility is sufficient for bacterial membrane protrusion formation and host cell uptake

Monack

Theriot

2001

Cellular Microbiology

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SummaryShigella flexneri replicates in the cytoplasm of host cells, where it nucleates host cell actin filaments at one pole of the bacterial cell to form a`comet tail' that propels the bacterium through the host's cytoplasm. To determine whether the ability to move by actinbased motility is sufficient for subsequent formation of membrane-bound protrusions and intercellular spread, we conferred the ability to nucleate actin on a heterologous bacterium, Escherichia coli. Previous work has shown that IcsA (VirG), the molecule that is necessary and sufficient for actin nucleation and actin-based motility, is distributed in a unipolar fashion on the surface of S. flexneri. Maintenance of the unipolar distribution of IcsA depends on both the S. flexneri outer membrane protease IcsP (SopA) and the structure of the lipopolysaccharide (LPS) in the outer membrane. We co-expressed IcsA and IcsP in two strains of E. coli that differed in their LPS structures. The E. coli were engineered to invade host cells by expression of invasin from Yersinia pseudotuberculosis and to escape the phagosome by incubation in purified listeriolysin O (LLO) from Listeria monocytogenes. All E. coli strains expressing IcsA replicated in host cell cytoplasm and moved by actin-based motility. Actin-based motility alone was sufficient for the formation of membrane protrusions and uptake by recipient host cells. The presence of IcsP and an elaborate LPS structure combined to enhance the ability of E. coli to form protrusions at the same frequency as S. flexneri, quantitatively reconstituting this step in pathogen intercellular spread in a heterologous organism. The frequency of membrane protrusion formation across all strains tested correlates with the efficiency of unidirectional actin-based movement, but not with bacterial speed.

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virG, a plasmid-coded virulence gene of Shigella flexneri: identification of the virG protein and determination of the complete coding sequence

Cited by 235 publications

References 26 publications

Effect of mutations in Shigella flexneri chromosomal and plasmid‐encoded lipopolysaccharide genes on invasion and serum resistance

Effect of mutations in Shigella flexneri chromosomal and plasmid‐encoded lipopolysaccharide genes on invasion and serum resistance

Neural Wiskott-Aldrich syndrome protein (N-WASP) is the specific ligand for Shigella VirG among the WASP family and determines the host cell type allowing actin-based spreading

Actin-based motility is sufficient for bacterial membrane protrusion formation and host cell uptake

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