Serotype 1a O-antigen modification: molecular characterization of the genes involved and their novel organization in the Shigella flexneri chromosome.
(17/922)
The factors responsible for serotype 1a O-antigen modification in Shigella flexneri were localized to a 5.8-kb chromosomal HindIII fragment of serotype 1a strain Y53. The entire 5.8-kb fragment and regions up- and downstream of it (10.6-kb total) were sequenced. A putative three-gene operon, which showed homology with other serotype conversion genes, was identified and shown to confer serotype 1a O-antigen modification. The serotype conversion genes were flanked on either side by phage DNA. Multiple insertion sequence (IS) elements were located within and upstream of the phage DNA in a composite transposon-like structure. Host DNA homologous to the dsdC and the thrW proA genes was located upstream of the IS elements and downstream of the phage DNA, respectively. The sequence analysis indicates that the organization of the 10.6-kb region of the Y53 chromosome is unique and suggests that the serotype conversion genes were originally brought into the host by a bacteriophage. Several features of this region are also characteristic of pathogenicity islands. (+info)
The Shigella flexneri bacteriophage Sf6 tailspike protein (TSP)/endorhamnosidase is related to the bacteriophage P22 TSP and has a motif common to exo- and endoglycanases, and C-5 epimerases.
(18/922)
The temperate bacteriophage Sf6 infects Shigella flexneri strains of serotype X or Y, converting them into serotypes 3a or 3b, respectively. The tailspike protein (TSP) of Sf6 possesses endo-1,3-alpha-L-rhamnosidase (endorhamnosidase) activity which results in cleavage of the lipopolysaccharide O-antigen receptor during the adsorption of the phage to the cell surface. When used in Southern hybridization, a P22 gene 9 (encoding P22 TSP) DNA probe hybridized with restriction fragment Pstl-7 of Sf6. DNA sequencing and analysis of Pstl-7 and the adjacent Pstl-8 fragment revealed an open reading frame (ORF1) of 1872 bp (624 amino acids) bearing amino acid sequence homology to the bacteriophage P22 TSP N-terminal head-binding domain. High conservation of key residues was suggestive of similar secondary and tertiary N-terminal protein structure and a similar function of the Sf6 TSP in this region. In addition, an amino acid sequence motif (DFGX3DGX6AX3A) was identified between residues 164 and 184 which was also found to exist in various prokaryotic and eukaryotic exo-/endoglycanases, C-5 epimerases and bacteriophage proteins. Expression of ORF1 from a T7 promoter produced a 67 kDa protein (detected by L-[35S]methionine labelling and SDS-PAGE). Assay of heat-treated cytoplasmic extracts containing the ORF1-encoded protein by incubation with whole Sh. flexneri Y cells demonstrated that O-antigen hydrolysis activity was present; ORF1 therefore encodes Sf6 TSP. Sf6 TSP exhibited specific and preferential activity for long-chain Sh. flexneri serotype X or Y O-antigen, cleavage of which resulted in the release of oligosaccharide fragments, consistent with octasaccharides in size, as detected by fluorophore-assisted carbohydrate electrophoresis (FACE). (+info)
Shigellosis and Escherichia coli diarrhea: relative importance of invasive and toxigenic mechanisms.
(19/922)
Shigellae and dysentery-like Escherichia coli must invade the epithelium of the colon to cause disease which can present as dysentery, diarrhea, or both. This paper addresses the possible role of a Shigella dysenteriae-like (Shiga-like) toxin in the pathogenesis of shigellosis and E. coli diarrheal diseases. The possibility for such a role is suggested by the following observations: 1) diarrhea, considered to be a result of secretion of water by the small bowel, is frequently observed in shigellosis, a large bowel disease. 2) Even though shigellae do not invade the jejunum of monkeys fed Shigella flexneri, jejunal secretion is seen in animals with diarrhea. 3) The Shiga toxin of S. dysenteriae has enterotoxic activity and other serotypes of shigellae produce Shiga-like toxins. 4) E. coli 015 RDEC-1 causes a diarrheal disease and frequently death in young rabbits. This organism neither produces E. coli enterotoxins nor is it invasive, but it may produce low levels of a Shiga-like toxin. (+info)
Membrane vesicles derived from Pseudomonas aeruginosa and Shigella flexneri can be integrated into the surfaces of other gram-negative bacteria.
(20/922)
Incubation of intact Salmonella typhi Ty21a, Salmonella enterica serovar Typhimurium (Salmonella typhimurium) aroA or Escherichia coli DH5 alpha with membrane vesicles (MVs) derived from either Shigella flexneri M90T or Pseudomonas aeruginosa dsp89 resulted in a significant incorporation of vesicle antigens into the outer membrane of the bacteria; each recipient strain possessed a surface mosaic of new Shigella and Pseudomonas antigens intermixed with the native antigens of the Salmonella or Escherichia strains. Electron microscopy of preparations during the integration of vesicle antigens revealed that the MVs rapidly fused with the outer membrane of the host strains. Western blot analysis of host bacteria confirmed the integration of foreign antigens. Quantitative analysis for binding and fusion of antigens using an ELISA showed that approximately 78.7 +/- 12.8 ng of the Pseudomonas and 67.5 +/- 13.8 ng of the Shigella LPSs (microgram host protein)-1 were integrated into the Sal. typhimurium strain. Similar integrations of the Shigella or Pseudomonas vesicles were found with the E. coli or Sal. typhi strains. There was no loss of viability in the recipient bacteria after incorporation of the MVs, although vesicle antigens became diluted during continued growth as daughter cells shared the vesicle antigens. The new antigens were highly stable after being incorporated into recipient strains, being able to withstand storage of several months at 4 degrees C as well as several cycles of freezing and thawing. Since the recipient bacteria are common vaccine strains, the procedure described here offers a simple efficient means of introducing exogenous surface antigens, in their native form, into the outer membranes of Gram-negative bacteria for possible vaccine use. (+info)
Comparative value of colonic biopsy and intraluminal fluid culture for diagnosis of bacterial acute colitis in immunocompetent patients. Infectious Colitis Study Group.
(21/922)
We compared the yield of intraluminal fluid culture to that of biopsy specimens obtained during colonoscopy for the diagnosis of bacterial colitis in 93 immunocompetent patients with a recent episode of diarrhea and macroscopic lesions of colitis. Stool culture findings were also available for 68 patients. At least one bacterial pathogen was isolated from the biopsy specimen, intraluminal fluid, or stool from 48 patients (51.6%). Salmonella species, Clostridium difficile, Klebsiella oxytoca, Shigella species, and Campylobacter species were recovered from 16 (17.2%), 15 (16.1%), 8 (8.6%), 7 (7.5%), and 4 (4.3%) of the patients, respectively. One Shigella species and one K. oxytoca strain were isolated from biopsy specimens but not from intraluminal fluid, and intraluminal fluid was the only positive specimen in 12 cases (yielding 1 Salmonella species, 2 Shigella species, 2 K. oxytoca, and 7 C. difficile isolates). In nine cases out of 10, toxin B was detected only in intraluminal fluid. A correlation of 91.2% was observed between stool and intraluminal fluid cultures for Salmonella, Shigella, and Campylobacter species isolations. Culture of biopsy specimens adds little to the diagnosis of infectious colitis, and stools and intraluminal fluids appear to have comparable value. (+info)
Activation of the CDC42 effector N-WASP by the Shigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complex and bacterial actin-based motility.
(22/922)
To propel itself in infected cells, the pathogen Shigella flexneri subverts the Cdc42-controlled machinery responsible for actin assembly during filopodia formation. Using a combination of bacterial motility assays in platelet extracts with Escherichia coli expressing the Shigella IcsA protein and in vitro analysis of reconstituted systems from purified proteins, we show here that the bacterial protein IcsA binds N-WASP and activates it in a Cdc42-like fashion. Dramatic stimulation of actin assembly is linked to the formation of a ternary IcsA-N-WASP-Arp2/3 complex, which nucleates actin polymerization. The Arp2/3 complex is essential in initiation of actin assembly and Shigella movement, as previously observed for Listeria monocytogenes. Activation of N-WASP by IcsA unmasks two domains acting together in insertional actin polymerization. The isolated COOH-terminal domain of N-WASP containing a verprolin-homology region, a cofilin-homology sequence, and an acidic terminal segment (VCA) interacts with G-actin in a unique profilin-like functional fashion. Hence, when N-WASP is activated, its COOH-terminal domain feeds barbed end growth of filaments and lowers the critical concentration at the bacterial surface. On the other hand, the NH(2)-terminal domain of N-WASP interacts with F-actin, mediating the attachment of the actin tail to the bacterium surface. VASP is not involved in Shigella movement, and the function of profilin does not require its binding to proline-rich regions. (+info)
Treatment with succinic anhydride improves the immunogenicity of Shigella flexneri type 2a O-specific polysaccharide-protein conjugates in mice.
(23/922)
Seroepidemiological data and a clinical trial with a Shigella sonnei O-specific polysaccharide (O-SP)-Pseudomonas aeruginosa recombinant exoprotein A (rEPA) conjugate provide evidence that a critical level of immunoglobulin G (IgG) lipopolysaccharide (LPS) antibodies in serum confers protection against shigellosis. We evaluated the immunogenicity of conjugates whose carrier proteins and O-SPs were treated with succinic anhydride (SA), which reacts with amino groups at neutral pH to form amide-linked carboxyls (succinylation). Conjugates were synthesized with either of two genetically inactivated medically useful toxins, the diphtheria protein CRM9 or rEPA, bound to the O-SP of Shigella flexneri type 2a. Conjugates composed of the succinylated protein, succinylated O-SP, or both succinylated components were administered to mice by a clinically relevant scheme, and their levels of serum IgG anti-LPS and anti-proteins were assayed 7 days after the second and third injections. CRM9 served as a more immunogenic carrier than rEPA. Conjugates composed of succinylated components were more immunogenic than the conjugates composed of the native components. SA treatment of both the carrier protein and the O-SP did not confer an advantage over the succinylated protein alone. Conjugates prepared with native proteins, in general, elicited slightly higher levels of IgG protein antibodies than conjugates composed of the SA-treated proteins. (+info)
Global burden of Shigella infections: implications for vaccine development and implementation of control strategies.
(24/922)
Few studies provide data on the global morbidity and mortality caused by infection with Shigella spp.; such estimates are needed, however, to plan strategies of prevention and treatment. Here we report the results of a review of the literature published between 1966 and 1997 on Shigella infection. The data obtained permit calculation of the number of cases of Shigella infection and the associated mortality occurring worldwide each year, by age, and (as a proxy for disease severity) by clinical category, i.e. mild cases remaining at home, moderate cases requiring outpatient care, and severe cases demanding hospitalization. A sensitivity analysis was performed to estimate the high and low range of morbid and fatal cases in each category. Finally, the frequency distribution of Shigella infection, by serogroup and serotype and by region of the world, was determined. The annual number of Shigella episodes throughout the world was estimated to be 164.7 million, of which 163.2 million were in developing countries (with 1.1 million deaths) and 1.5 million in industrialized countries. A total of 69% of all episodes and 61% of all deaths attributable to shigellosis involved children under 5 years of age. The median percentages of isolates of S. flexneri, S. sonnei, S. boydii, and S. dysenteriae were, respectively, 60%, 15%, 6%, and 6% (30% of S. dysenteriae cases were type 1) in developing countries; and 16%, 77%, 2%, and 1% in industrialized countries. In developing countries, the predominant serotype of S. flexneri is 2a, followed by 1b, 3a, 4a, and 6. In industrialized countries, most isolates are S. flexneri 2a or other unspecified type 2 strains. Shigellosis, which continues to have an important global impact, cannot be adequately controlled with the existing prevention and treatment measures. Innovative strategies, including development of vaccines against the most common serotypes, could provide substantial benefits. (+info)