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Intercellular spreading of shigellae is a prerequisite for shigellosis, although the molecular mechanisms underlying the phenomenon are still largely obscure. To elucidate some of these mechanisms, we performed random Tn10 insertion mutagenesis in Shigella flexneri YSH6000T and found a chromosomal locus in the NotI-J segment responsible for bacterial spreading. The locus affected in the mutant, designated vacJ, was neither involved in the invasion of epithelial cells nor in intracellular movement, but was required for intercellular spread. The vacJ mutant was capable of forming bacterium-containing membranous protrusions within the infected cell, but had diminished ability to move from the protrusions into the cytoplasm of the adjacent epithelial cells. Cloning and sequencing of the vacJ region indicated that the vacJ gene encoded a 28.0 kDa protein possessing a signal peptide at the N-terminus, which contained the motif characteristic of lipoproteins. The analysis of the vacJ product indicated that VacJ was exposed on the bacterial surface. The vacJ gene was distributed among shigellae and enteroinvasive Escherichia coli, and the constructed vacJ mutants failed to spread intercellularly, indicating that vacJ is a chromosomal gene essential for the pathogenicity of shigellae.
Mol Microbiol 1994 Jan
PMID:Identification and characterization of a chromosomal virulence gene, vacJ, required for intercellular spreading of Shigella flexneri. 814 44

Shigella flexneri kills macrophages through apoptosis, involving the induction of host cell DNA fragmentation and characteristic morphological changes. Shigella can only cause damage if it escapes from the phagolysosome into the cytoplasm. The S. flexneri cytotoxic genes have been localized to the ipa operon of shigella's virulence plasmid. ipaB, C and D deletion mutants are not invasive and therefore not cytotoxic. In order to distinguish genes involved in the escape from the phagolysosome as distinct from cytotoxicity, we constructed Shigella strains that secrete low amounts of Escherichia coli haemolysin (hly(low)). These strains can escape into the cytoplasm of the macrophage even in the absence of the invasion plasmid as verified by electron microscopy and resistance to chloroquine. Macrophages were infected with different ipa mutants expressing hly(low). Both delta ipaC hly(low) and delta ipaD hly(low) were cytotoxic whilst delta ipaB hly(low) and a hly(low) strain cured of shigella's pathogenicity plasmid were not. Furthermore, both delta ipaC hly(low) and delta ipaD hly(low) killed through apoptosis as shown by both changes in ultrastructural morphology and fragmentation of the host cell DNA. These results demonstrate that ipaB is essential for S. flexneri to induce apoptosis in macrophages.
Mol Microbiol 1994 Feb
PMID:IpaB mediates macrophage apoptosis induced by Shigella flexneri. 819 40

Our laboratories have independently identified a gene in Salmonella choleraesuis and Salmonella typhimurium that is necessary for efficient adherence and entry of these organisms into cultured epithelial cells. Introduction of a mutated gene into several Salmonella strains belonging to different serotypes rendered these organisms deficient for adherence and invasion of cultured cells. This effect was most pronounced in the host-adapted serotypes Salmonella gallinarum, S. choleraesuis, and Salmonella typhi. The nucleotide sequence of this gene, which we have termed invH, encodes a predicted 147-amino-acid polypeptide containing a signal sequence. The InvH predicted polypeptide is highly conserved in S. typhimurium and S. choleraesuis, differing at only three residues. The invH gene was expressed in Escherichia coli using a T7 RNA polymerase expression system and a polypeptide of approximately 16,000 molecular weight was observed, in agreement with the predicted size of its gene product. Upon fractionation, the expressed polypeptide was localized in the bacterial membrane fraction. Southern and colony hybridization analyses indicated that the invH gene is present in all Salmonella strains tested (91 strains belonging to 37 serotypes) with the exception of strains of Salmonella arizonae. No homologous sequences were detected in Yersinia, Shigella, Proteus, and several strains of enteroinvasive and enteropathogenic E. coli. Downstream from the S. choleraesuis (but not S. typhimurium) invH gene, a region with extensive homology to the insertion sequence IS3 was detected.
Mol Microbiol 1993 Jan
PMID:Cloning and molecular characterization of a gene involved in Salmonella adherence and invasion of cultured epithelial cells. 838 33

A large plasmid-encoded protein, VirG, on the bacterial surface is essential for the spreading of Shigella by eliciting polar deposition of filamentous actin in the cytoplasm of epithelial cells. VirG expression from the large plasmid is diminished greatly when it is introduced into Escherichia coli K-12 from Shigella. In an attempt to identify factors affecting VirG expression, we found that the absence of the ompT gene, encoding outer membrane protease OmpT, restored full production of VirG protein to E. coli K-12. Conversely, upon introduction of the ompT gene of E. coli K-12 into Shigella, spreading ability was completely abolished, probably because of the proteolytic degradation of VirG protein by OmpT. Analysis of the DNA sequence of the ompT region indicated that the absence of the ompT gene occurred in Shigella and enteroinvasive E. coli strains, and that the absent DNA segment corresponded to a remnant lambdoid phage structure found in E. coli K-12, which encompasses a 21 kb DNA segment spanning from argU through to the ompT genes. Since ompT is located near purE in E. coli K-12 and a virulence locus for provoking keratoconjunctivitis in the eyes of guinea-pigs, named kcpA, is located near purE in S. flexneri, and the two loci are involved in VirG expression, the KcpA- mutants of S. flexneri 2a constructed were examined for correlation between acquisition of ompT and VirG degradation. Our data suggest that the previous recognition of a kcpA locus in S. flexneri is the result of transfer of the ompT gene from E. coli K-12, giving rise to a KcpA- phenotype. These results indicate that the lack of OmpT protease confers upon Shigella the ability to spread into adjacent epithelial cells.
Mol Microbiol 1993 Aug
PMID:The absence of a surface protease, OmpT, determines the intercellular spreading ability of Shigella: the relationship between the ompT and kcpA loci. 841 95

A chromosomal fragment from Salmonella typhimurium, when cloned in Escherichia coli, generates a haemolytic phenotype. This fragment carries two genes, termed slyA and slyB. The expression of slyA is sufficient for the haemolytic phenotype. The haemolytic activity of E. coli carrying multiple copies of slyA is found mainly in the cytoplasm, with some in the periplasm of cells grown to stationary phase, but overexpression of SlyB, a 15 kDa lipoprotein probably located in the outer membrane, may lead to enhanced, albeit unspecific, release of the haemolytic activity into the medium. Polyclonal antibodies raised against a purified SlyA-HlyA fusion protein identified the overexpressed monomeric 17 kDa SlyA protein mainly in the cytoplasm of E. coli grown to stationary phase, although smaller amounts were also found in the periplasm and even in the culture supernatant. However, the anti-SlyA antibodies reacted with the SlyA protein in a periplasmic fraction that did not contain the haemolytic activity. Conversely, the periplasmic fraction exhibiting haemolytic activity did not contain the 17 kDa SlyA protein. Furthermore, S. typhimurium transformed with multiple copies of the slyA gene did not show a haemolytic phenotype when grown in rich culture media, although the SlyA protein was expressed in amounts similar to those in the recombinant E. coli strain. These results indicate that SlyA is not itself a cytolysin but rather induces in E. coli (but not in S. typhimurium) the synthesis of an uncharacterised, haemolytically active protein which forms pores with a diameter of about 2.6 nm in an artificial lipid bilayer. The SlyA protein thus seems to represent a regulation factor in Salmonella, as is also suggested by the similarity of the SlyA protein to some other bacterial regulatory proteins. slyA- and slyB-related genes were also obtained by PCR from E. coli, Shigella sp. and Citrobacter diversus but not from several other gram-negative bacteria tested.
Mol Gen Genet 1995 Dec 15
PMID:SlyA, a regulatory protein from Salmonella typhimurium, induces a haemolytic and pore-forming protein in Escherichia coli. 854 13

Entry of Shigella flexneri into epithelial cells involves secretory proteins, the Ipa proteins, and their dedicated secretion apparatus, the Mxi-Spa translocon, which is encoded by the mxi and spa operons. We have characterized the mxiG gene that is located at the proximal part of the mxi operon. Inactivation of mxiG abolished lpa secretion, which indicates that MxiG is an essential component of the Mxi-Spa translocon. Immunoblotting analysis of membrane fractions suggests that the 42 kDa MxiG protein is associated with both the inner and outer membranes. Taking advantage of the complementation of the mxiG mutant by a plasmid carrying a wild-type copy of mxiG (which restored Ipa secretion, entry into HeLa cells, and cell-to-cell spread) we mutagenized the mxiG gene carried by the complementing plasmid to replace the RGD motif of MxiG by RAD. This mutation (mxiG*), which had no effect on the stability of the protein, did not affect Ipa secretion in vitro or entry into HeLa cells, but impaired intercellular dissemination. Therefore, MxiG and possibly proteins secreted by the Mxi-Spa translocation are involved not only in entry but also in spread of Shigella between epithelial cells.
Mol Microbiol 1995 Aug
PMID:MxiG, a membrane protein required for secretion of Shigella spp. Ipa invasins: involvement in entry into epithelial cells and in intercellular dissemination. 855 65

Swarming by Proteus mirabilis is characterized by cycles of rapid population migration across surfaces, following differentiation of typical vegetative rods into long, hyperflagellated, virulent swarm cells. A swarm-defective TnphoA insertion mutant was isolated that was not defective in cell motility, differentiation or control of the migration cycle, but was specifically impaired in the ability to undergo surface translocation as a multicellular mass. The mutation, previously shown to compromise urinary tract virulence, was located within a 1112 bp gene that restored normal swarming of the mutant when expressed in trans. The gene encoded a 40.6 kDa protein that is related to putative sugar transferases required for lipopolysaccharide (LPS) core modification in Shigella and Salmonella. The immediately distal open reading frame encoded a protein that is related to dehydrogenases involved in the synthesis of LPS O-side-chains, enterobacterial common antigen and extracellular polysaccharide (PS). Gel electrophoresis and electron microscopy showed that the mutant still made LPS but it had lost the ability to assemble a surface (capsular) PS, which gas-liquid chromatography and mass spectrometry indicated to be an acidic type II molecule rich in galacturonic acid and galactosamine. We suggest that this surface PS facilitates translocation of differentiated cell populations by reducing surface friction.
Mol Microbiol 1995 Sep
PMID:A cell-surface polysaccharide that facilitates rapid population migration by differentiated swarm cells of Proteus mirabilis. 859 35

Since the discovery of Shigella as the aetiologic agent of acute dysentery almost 100 years ago, this organism has been described as a non-motile and nonflagellated organism that invades the human colonic mucosa. In this study, the production of flagella by prototypic strains of all four Shigella species and, moreover, by fresh clinical isolates was demonstrated by electron microscopy. The flagellum of Shigella (flash) is approximately 10 microns long and 12-14 nm in diameter and is typically seen emanating from one pole of the bacterium. Flash is composed of a putative structural polypeptide subunit of 33-38 kDa that shares immunological similarities with Escherichia coli, Salmonella spp., and Proteus mirabilis flagellins, and with the recently described recombinant Shigella flagellins (FliCSS and FliCSF) expressed in E. coli K-12. A fliCSS-specific oligo probe hybridized with all four Shigella species, while a fliCSF probe hybridized with all Shigella flexneri and Shigella dysenteriae strains, but not with all Shigella sonnei or Shigella boydii strains, indicating genetic divergence among their flagellin genes. Shigella exhibits motility in low-concentration motility agar under physiological growth conditions. The expression of flash and motility appears to be strictly regulated by unidentified genetic and environmental factors. These heretofore undescribed features may allow the bacteria to circumvent the natural intestinal mucosal defences leading to bacterial colonization and disease. The motility of shigellae may represent an evolutionary adaptation important for bacterial survival.
Mol Microbiol 1995 Oct
PMID:Expression of flagella and motility by Shigella. 859 61

The gram negative rod Shigella flexneri uses it surface protein IcsA to induce host cell actin assembly and to achieve intracellular motility. Yet, the IcsA protein lacks the oligoproline sequences found in ActA, the surface protein required for locomotion of the gram positive rod Listeria monocytogenes. Microinjection of a peptide matching the second ActA oligoproline repeat (FEFPPPPTDE) stops Listeria locomotion (Southwick, F.S., and D.L. Purich. 1994a. Proc. Natl. Acad. Sci. USA. 91:5168-5172), and submicromolar concentrations (intracellular concentration 80-800 nM) similarly arrest Shigella rocket-tail assembly and intracellular motility. Coinjection of a binary solution containing profilin and the ActA analogue increased the observed rates of intracellular motility by a factor of three (mean velocity 0.90 +/- 0.07 mu m/s, SD n=16 before injection vs 0.3 +/- 0.1 mu m/s, n=33 postinjection, intracellular concentration = 80 nM profilin plus 80 nM ActA analogue). Recent evidence suggests the ActA analogue may act by displacing the profilin-binding protein VASP (Pistor, S.C., T. Chakaborty, V. Walter, and J. Wehland. 1995. Curr. Biol. 5:517-525). At considerably higher intracellular concentrations (10 muM), the VASP oligoproline sequence (GPPPPP)3 thought to represent the profilin-binding site (Reinhard, M., K. Giehl, K. Abel, C. Haffner, T. Jarchau, V. Hoppe, B.M. Jockusch, and U. Walter. 1995. EMBO (Eur. Mol. Biol. Organ.) J. 14:1583-1589) also inhibited Shigella movement. A binary mixture of the VASP analogue and profilin (each 10 muM intracellular concentration) led to a doubling of Shigella intracellular migration velocity (0.09 +/- 0.06 mu m/s, n = 25 preinjection vs 0.18 +/- 0.10 mu m/s, n = 61 postinjection). Thus, the two structurally divergent bacteria, Listeria and Shigella, have adopted convergent mechanisms involving profilin recognition of VASP oligoproline sequences and VASP recognition of oligoproline sequences in ActA or an ActA-like host protein to induce host cell actin assembly and to provide the force for intracellular locomotion and cell-cell spread.
 ...
PMID:Recognition of two classes of oligoproline sequences in profilin-mediated acceleration of actin-based Shigella motility. 860 12

The adsorption specificity of T4 is determined by the tip of the gene 37 tail fibers which bind to receptors on the bacterial surface. T4 infects only Escherichia coli and closely related Shigella species, but rare host range mutants can be isolated that infect Yersinia pseudotuberculosis I, an evolutionally distant bacterium. Some of these mutations result in amino acid residue substitutions in the C-terminal portion of gene 37, but others involve unequal exchanges between a series of sequence motifs (His boxes) in the same region. The duplication or mutational alteration of this segment apparently suffices for phage adsorption to a Yersinia receptor. It is suggested that recombination between the His box sequences can generate diversity in phage host range by shuffling receptor recognition domains.
J Mol Biol 1996 May 24
PMID:Bacteriophage T4 host range is expanded by duplications of a small domain of the tail fiber adhesin. 863 4


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