Shigella Flexneri 2a Strain Research Articles

Three-way interactions among the Sfh, StpA and H-NS nucleoid-structuring proteins of Shigella flexneri 2a strain 2457T.

Shigella flexneri 2a strain 2457T has been found to express Sfh, a new member of the H-NS-like family of nucleoid-structuring proteins. With H-NS and its paralogue, StpA, this brings to three the number of these proteins expressed in this bacterium. This raises the possibility that three-way interactions may occur in S. flexneri among these proteins and between the proteins and each other's genes. Such three-way interactions among H-NS-like proteins have not been described previously. The expression of the sfh, stpA and hns genes was studied at the transcriptional and post-transcriptional levels. The Sfh protein displays growth phase-dependent regulation that distinguishes it from both H-NS and StpA. Like H-NS and StpA, Sfh can bind to its own promoter region, it negatively autoregulates transcription of its own gene, and when overexpressed all three proteins cross-repress transcription of each other's genes. The presence of highly conserved oligomerization domains within these molecules suggested the possibility of protein-protein interactions. Like H-NS and StpA, the purified Sfh protein forms homodimers in solution. Using the yeast two-hybrid assay we show that each of the three proteins also forms homodimers in vivo and, additionally, each protein can form heterodimers with either of its homologues. This raises the possibility that Sfh may modulate the activities of H-NS and StpA, and vice versa.

Comparative genome analysis of deleted genes in Shigella flexneri 2a strain 301

Comparative genome analysis is performed between Shigella flexneri 2a strain 301 and its close relatives, the non-pathogenic E. coli K-12 strain MG1655. Result shows that there are 136 DNA segments whose size is larger than 1000 bp absent from Shigella flexneri 2a strain 301, which is up to 717253 bp in total length. These deleted segments altogether contain 670 open reading frames (ORFs). Prediction of these ORFs indicates that there are 40% genes of unknown function. The other genes of definite functions encode metabolic enzymes, structure proteins, transcription regulatory factors and some elements correlated with horizontal transfer. Here we compare the complete genomic sequences of the two closely related species, which differ in pathogenic phenotype. To our knowledge, this not only reveals the difference of genomic sequence between the two important enteric pathogens for the first time, but also provides valuable clues to further researches in its process of physiological activity, pathogenesis and the evolution of enteric bacteria.

Lipopolysaccharide O antigen chains mask IcsA (VirG) in Shigella flexneri

Shigella flexneri 2a strain 2457T lipopolysaccharide (LPS) has O antigen (Oag) chains with two modal lengths (S-type and VL-type), and has IcsA apparently located at one pole on its cell surface. Treatment of Y serotype derivatives of 2457T and RMA696 (2457T wzz SF) with Sf6 tailspike protein (TSP) resulted in hydrolysis of Oag chains, and an increase in detection of IcsA by indirect immunofluorescence staining on both the lateral and polar regions of the cell surface. Newly synthesised IcsA expressed from a pBAD promoter in a S. flexneri Y strain was also detected on both the lateral and polar regions of the cell when incubated with TSP prior to immunofluorescence staining. We conclude that IcsA is actually located on both lateral and polar regions of the S. flexneri cell surface, and that LPS Oag chains mask the presence of IcsA by hindering its detection with antibodies. These results have implications for the mechanism of IcsA export. They suggest that while IcsA export is predominantly targeted to the old cell pole, it can also occur on the lateral regions of the cell surface.

Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a

The complete nucleotide sequence and organization of the large virulence plasmid pCP301 (termed by us) of Shigella flexneri 2a strain 301 were determined and analyzed. The result showed that the entire DNA sequence of pCP301 is composed of 221618 bp which form a circular plasmid. Sequence analysis identified 272 open reading frames (ORFs), among which, 194 correspond to the proteins described previously, 61 have low identity (<60%) to known proteins and the rest 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include the genes associated with bacterial virulence, the genes associated with regulation and the genes relating to plasmid maintenance, stability and DNA metabolism. Insertion sequence (IS) elements are 68 kb in length and account for 30 percent of complete sequence of the plasmid which indicates that gene multiple rearrangements of the pCP301 have taken place in Shigella flexneri evolution history. The research result is helpful for interpreting the pathogenesis of Shigella, as well as the genetics and evolution of the plasmid.

Comparative genome analysis of deleted genes in Shigella flexneri 2a strain 301

Comparative genome analysis of deleted genes in Shigella flexneri 2a strain 301

Regulation of the overlapping pic/set locus in Shigella flexneri and enteroaggregative Escherichia coli.

Most strains of Shigella flexneri 2a and enteroaggregative Escherichia coli carry a highly conserved chromosomal locus which encodes a 109-kDa secreted mucinase (called Pic) and, on the opposite strand in overlapping fashion, an oligomeric enterotoxin called ShET1, encoded by the setA and setB genes. Here, we characterize the genetic regulation of these overlapping genes. Our data suggest that pic and the setBA loci are transcribed as complementary 4-kb mRNA species. The major pic promoter is maximally activated at 37 degrees C in exponential growth phase. Our data suggest that the setB gene is transcribed from a promoter which lies more than 1.5 kb upstream of the setB structural gene; setA may be transcribed via readthrough of the setB transcript and possibly by its own promoter. The long leader of the setB gene provides a strong silencing effect on setB transcription. The signals which provide relief from setB silencing are not clear, but significant induction is observed in a continuous anaerobic culture of human fecal bacteria, suggesting that some complex characteristics of the human intestine act to lift repression of setB expression. Our studies provide the first insights into the mechanisms affecting expression of this unusual virulence locus.

Construction of a trivalent candidate vaccine against Shigella species with DNA recombination

In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, could stably express CtxB, but its growth in LB medium depended on the presence of diaminopimelic acid (DAP). Then form I plasmid of Shigella sonnei strain S7 was labeled with strain T32 asd gene and mobilized into FWL01. Thus a trivalent candidate oral vaccine strain, designed as FSW01, was constructed. In this candidate strain, a balanced-lethal system was constituted between the host strain and the form I plasmid expressing S. sonnei O antigen. Therefore the candidate strain can express stably not only its own O antigen but also CtxB and O antigen of S. sonnei in the absence of any antibiotic. Experiments showed that FSW01 did not invade HeLa cells or cause keratoconjunctivitis in guinea pigs. However, rabbits immunized FSW01 can elicit significant immune responses. In mice and rhesus monkey models, vaccinated animals were protected against the challenges of wild S. flexneri 2a strain 2457T and S. sonnei strain S9.

Nested deletions of the SRL pathogenicity island of Shigella flexneri 2a.

In this study, we determined the boundaries of a 99-kb deletable element of Shigella flexneri 2a strain YSH6000. The element, designated the multiple-antibiotic resistance deletable element (MRDE), had recently been found to contain a 66-kb pathogenicity island (PAI)-like element (designated the SRL PAI) which carries the Shigella resistance locus (SRL), encoding resistance determinants to streptomycin, ampicillin, chloramphenicol, and tetracycline. The YSH6000 MRDE was found to be flanked by two identical IS91 elements present at the S. flexneri homologs of the Escherichia coli genes putA and mdoA on NotI fragment D. Sequence data from two YSH6000-derived MRDE deletants, YSH6000T and S2430, revealed that deletion of the MRDE occurred between the two flanking IS91 elements, resulting in a single IS91 element spanning the two original IS91 loci. Selection for the loss of tetracycline resistance confirmed that the MRDE deletion occurred reproducibly from the same chromosomal site and also showed that the SRL PAI and the SRL itself were capable of independent deletion from the chromosome, thus revealing a unique set of nested deletions. The excision frequency of the SRL PAI was estimated to be 10(-5) per cell in the wild type, and mutation of a P4-like integrase gene (int) at the left end of the SRL PAI revealed that int mediates precise deletion of the PAI.

Genetic organization of the she pathogenicity island in Shigella flexneri 2a

In this study we report the complete nucleotide sequence and genetic organization of the she pathogenicity island (PAI) of Shigella flexneri 2a strain YSH6000T. The 46 603 bp she PAI is situated adjacent to the 3′ terminus of the pheV tRNA gene and includes an imperfect direct repeat of the 3′-terminal 22 bp of thepheV gene at the right boundary of the PAI. The she PAI carries a bacteriophage P4-like integrase gene within thepheV -proximal boundary of the PAI, intact and truncated mobile genetic elements, plasmid-related sequences, open reading frames exhibiting high sequence similarity to those found on the locus of enterocyte effacement (LEE) PAI of enterohemorrhagicEscherichia coli (EHEC), and the SHI-2 PAI of S. flexneri and several other open reading frames of unknown function. The she PAI also encodes two autotransporter proteins, including SigA, a cytopathic protease that contributes to intestinal fluid accumulation and Pic, a protease with mucinase, and hemagglutinin activities. In addition, an open reading frame (orf) termed sap, has high sequence similarity to the gene encoding Antigen 43, a surface-located autotransporter protein of E. coli. The ShET1 enterotoxin genes, associated predominantly with S. flexneri 2a strains, are also located on the she PAI.

Δ guaBA attenuated Shigella flexneri 2a strain CVD 1204 as a Shigella vaccine and as a live mucosal delivery system for fragment C of tetanus toxin

The Δ guaBA Shigella flexneri 2a vaccine candidate, CVD 1204, was evaluated as a delivery system for the non-toxic C-terminal of tetanus toxin (fragment C), either as a polypeptide expressed in the bacteria or as a DNA vaccine. CVD 1204 was transformed with plasmid pTET nir15 which encodes the fragment C gene ( tetC) under the control of the inducible prokaryotic nir15 promoter or a DNA vaccine plasmid pcDNA3 tetC which encodes tetC under the eukaryotic hCMV promoter. Guinea pigs immunised intranasally (i.n.) with either recombinant strain mounted a secretory immune response against S. flexneri 2a Lipopolysaccharide (LPS) and were protected against ocular challenge with wild-type S. flexneri 2a. Both strains were effective in eliciting a serum IgG response against fragment C in guinea pigs following i.n. immunisation. Furthermore, serum from guinea pigs immunised with CVD 1204(pTET nir15) contained tetanus toxin neutralising antibodies. These results demonstrate that this S. flexneri 2a vaccine candidate can serve as a vehicle for the delivery of foreign antigens to the systemic immune system while retaining its capacity to serve as a mucosal Shigella vaccine.

Killed oral Shigella vaccine made from Shigella flexneri 2a protects against challenge in the rabbit model of shigellosis

The protective efficacy of an orally administered heat‐killed virulent strain of Shigella flexneri 2a (5 weekly oral doses) was evaluated in 25 rabbits (14 immunized and 11 non‐immunized controls) against challenge with the same strain of Shigella using the rabbit model of shigellosis. All 11 non‐immunized rabbits developed bloody diarrhoea following challenge and 6 (54%) died. None of the 14 immunized rabbits developed diarrhoea (all had pellet stools) but 3 (21%) died from causes not associated with diarrhoea. Protection from diarrhoea and dysentery following oral immunization with a killed Shigella species was 100% and highly significant. Death following challenge was 2.5‐fold higher in the non‐immunized group (p= 0.115) but was not significant. These promising results suggest that further studies should be undertaken to develop a killed oral vaccine against shigellosis.

Killed oral Shigella vaccine made from Shigella flexneri 2a protects against challenge in the rabbit model of shigellosis.

The protective efficacy of an orally administered heat-killed virulent strain of Shigella flexneri 2a (5 weekly oral doses) was evaluated in 25 rabbits (14 immunized and 11 non-immunized controls) against challenge with the same strain of Shigella using the rabbit model of shigellosis. All 11 non-immunized rabbits developed bloody diarrhoea following challenge and 6 (54%) died. None of the 14 immunized rabbits developed diarrhoea (all had pellet stools) but 3 (21%) died from causes not associated with diarrhoea. Protection from diarrhoea and dysentery following oral immunization with a killed Shigella species was 100% and highly significant. Death following challenge was 2.5-fold higher in the non-immunized group (p = 0.115) but was not significant. These promising results suggest that further studies should be undertaken to develop a killed oral vaccine against shigellosis.

Mechanism of resistance of Shigella flexneri 2a resistant to new quinolone antibiotics

A Shigella flexneri 2a strain, which did not respond clinically or in laboratory tests to treatment with new quinolone derivatives, was isolated for the first time in Japan from a patient admitted for diarrhea to a Tokyo hospital. The mechanism of resistance was examined by sequencing the quinolone resistance determining region (QRDR) of the gyrA (a quinolone target enzyme) gene and by comparing the active efflux mechanisms of two strains isolated from this patient (before hospitalization and after tosufloxacin treatment) and one strain isolated from a patient with secondary infection with that of the standard strain ATCC 29903. DNA sequencing revealed that two amino acid substitutions, namely, Ser (TCG)-83-->Leu (TTG) and Asp (GAC)-87-->Gly (GGC), had occurred in the gyrA of all 3 strains isolated from the patients. Examination of the accumulation of antibiotics in these 3 strains revealed that the strain isolated after tosufloxacin treatment had the highest resistance to tosufloxacin, and exhibited decreased accumulation of tosufloxacin in the bacterial cells discharged after 5 days of treatment with this antibiotic. However, accumulation was restored by addition of a proton-pump inhibitor. These results suggest that the strains isolated from the inpatient and the patient with secondary infection acquired resistance due to dual gyrA mutation induced by treatment with new quinolone antibiotics. Furthermore, in addition to this dual mutation, the active efflux mechanism also appears to be associated with resistance in bacteria that have been exposed to tosufloxacin.

Engineered deltaguaB-A deltavirG Shigella flexneri 2a strain CVD 1205: construction, safety, immunogenicity, and potential efficacy as a mucosal vaccine.

Shigella flexneri 2a strain CVD 1204, which was constructed by introducing a specific, in-frame deletion mutation in the guaB-A operon, was compared with deltaaroA strain CVD 1201. CVD 1204 was less invasive for HeLa cells than CVD 1201, whereas following invasion, the abilities of the two mutants to proliferate intracellularly were similarly impaired. The reduction in invasiveness was independent of the guanine auxotrophic phenotype and fully recovered when the chromosomal deletion mutation in CVD 1204 was repaired. Following inoculation of the conjunctival sac of guinea pigs (Serény test) at high doses (10(9) CFU per eye), both strains evoked minimal, short- lived conjunctival inflammation, which was significantly milder with strain CVD 1204. Double mutant deltaguaB-A deltavirG (also called icsA) strain CVD 1205 induced, after a single intranasal dose, high mucosal immunoglobulin A antilipopolysaccharide titers, which were significantly boosted further following a second dose of vaccine given 14 days later. Upon Serény test challenge with wild-type S. flexneri 2a, CVD 1205-vaccinated animals were significantly protected against keratoconjunctivitis (zero of eight vaccinees versus five of seven controls, P = 0.03; vaccine efficacy, 100%). CVD 1205 is an attractive candidate for human clinical trials.

Further characterization of delta aroA delta virG Shigella flexneri 2a strain CVD 1203 as a mucosal Shigella vaccine and as a live-vector vaccine for delivering antigens of enterotoxigenic Escherichia coli.

The use of attenuated delta aroA delta virG Shigella flexneri 2a strain CVD 1203 as a live vector for enterotoxigenic Escherichia coli (ETEC) antigens is reported. CVD 1203 alone or expressing colonization factor antigen fimbriae and CS3 fibrillae of ETEC was given to guinea pigs and mice, orogastrically (o.g.) or intranasally (i.n.). CVD 1203 given i.n. elicited high titers of antilipopolysaccharide (anti-LPS) immunoglobulin A (IgA) and was protective in guinea pigs against a homologous conjunctival challenge. Whereas a strong IgA response against colonization factor antigen CS3, and Shigella LPS was detected in tears and serum of guinea pigs after o.g. or i.n. immunization, the i.n. route elicited significantly higher antibody titers. A strong serum IgG response was also observed against the ETEC antigens, although no serum anti-LPS IgG response was detected. The immune response in mice followed a pattern similar to that in guinea pigs, and the difference between the responses after o.g. and i.n. administration was even more remarkable.

Shigella enterotoxin 1: an enterotoxin of Shigella flexneri 2a active in rabbit small intestine in vivo and in vitro.

Culture filtrates of Shigella flexneri 2a strain M4243 grown in iron-depleted medium, caused significant fluid accumulation in rabbit ileal loops. Also, when tested in Ussing chambers, a greater rise in potential difference and short circuit current was seen with such filtrates compared with the medium control. Analogous filtrates from two M4243 derivatives lacking the 140-MD invasiveness plasmid (either M4243avir or BS103) retained 60-65% of the wild-type enterotoxic activity. Ultrafiltration and gel exclusion size fractionation of M4243 filtrate revealed that the activity was approximately 60 kD. SDS-PAGE performed on this fraction showed 18 bands, 5 of which reacted with human convalescent sera. Genes encoding this enterotoxin, named ShET1 for Shigella enterotoxin 1, were cloned from the S. flexneri 2a chromosome, and two separate open reading frames of 534 and 186 bp were sequenced. These observations suggest that S. flexneri 2a elaborates two distinct enterotoxins: ShET1, encoded by genes located on the chromosome, and ShET2, encoded by a gene on the 140-MD invasiveness plasmid. ShET1, which is composed of two distinct subunits and is elaborated in vivo, where it elicits an immune response, may be important in the pathogenesis of diarrheal illness due to S. flexneri 2a.

Construction and characterization of attenuated delta aroA delta virG Shigella flexneri 2a strain CVD 1203, a prototype live oral vaccine.

We engineered an oral Shigella vaccine prototype that can invade intestinal epithelial cells but cannot undergo extensive intracellular replication or extend to adjacent epithelial cells. Strain CVD 1203, derived from wild-type Shigella flexneri 2a by introducing deletions in chromosomal aroA and invasion plasmid virG, was highly attenuated in the Sereny test. Two 10(9)-CFU orogastric doses (2 weeks apart) stimulated production of secretory immunoglobulin A antibodies to S. flexneri 2a and protected against conjunctival sac challenge with virulent S. flexneri 2a.

The large virulence plasmid of Shigella.

Invasion of human colonic epithelial cells constitutes one of the earliest steps in the pathogenesis of dysentery caused by Shigella species and enteroinvasive strains of Escherichia coli (EIEC). Following the invasion of target cells by virulent bacteria, their subsequent multiplication, intracellular movement, and intercellular spread result in a focus of Shigella infection that is characterized by severe desquamation and ulceration of the mucosa. The first indication that invasion of colonic epithelial cells was critical in the development of the dysenteric syndrome was presented by Labrec et al. (1964) who showed that a virulent, translucent colony morphology strain of Shigella flexneri 2a (2457T) was able to penetrate colonic epithelia in both rhesus monkeys and opiated guinea pigs, whereas an avirulent opaque variant (24570) did not. However, it was not until the early 1980s and the pioneering work of Sansonetti and colleagues that the essential role of a large (100–140MDa) plasmid, found in all virulent strains of Shigella and EEC, was established for the invasion phenotype (Kopecko et al. 1980; Sansonetti et al. 1981,1982,1983b; Harris et al. 1982). Subsequent rapid progress in the study of virulence-related (vir) genes carried by this large invasion plasmid has been complemented by the development of recombinant DNA technology, in vitro assay systems for the various virulence phenotypes, and advances in cellular and molecular biology. As will be reviewed in this chapter, our knowledge of the role of the large plasmid in determining the virulence properties of Shigella has accumulated substantially over the last few years; however, a full characterization of the vir genes has not yet been realized.KeywordsShigella FlexneriLarge PlasmidvirB GeneInvasion PlasmidipaH GeneThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Differences in blood group B-specific mucinase activity between virulent and avirulent Shigella flexneri 2a strains

Based on our previous findings we postulate that the production of blood group B-degrading mucinase by Shigella flexneri 2a is related to virulence. The virulent S. flexneri 2a strain M4243 produced a blood group B mucinase which decreased the blood group B reactivity of germfree mouse mucins by a factor of 16 and the B reactivity of human saliva by a factor of 32. Avirulent S. flexneri 2a B-1, serologically similar, but not genetically identical to the M4243 strain, failed to degrade the blood group reactivity. The mucin-degrading ability of S. flexneri 2a M4243 harboring a large virulence-conferring 140 MDa plasmid was then compared with a genetically similar large plasmid-free avirulent S. flexneri 2a M4243A 1. Virulent S. flexneri M4243 grew in human salivary mucins while the genetically identical avirulent M4243A 1 did not. Supernatant of virulent M4243 culture decreased the blood B reactivity of salivary mucins by a factor of 32 while the avirulent M4243a 1 had no effect. Eleven of 12 colonies of the transconjugant hybrid Escherichia coli K12 (7300-1-5) containing the shigella PWR 110 plasmid and chromosomal markers decreased the blood group B reactivity by a factor of 4-32, and two of four colonies of the E. coli strain 7262 containing only the plasmid reduced the B reactivity by a factor of 4–16. These findings suggest that blood group B-specific mucinase production may be related to S. flexneri virulence.

Hemagglutinating properties of Shigella dysenteriae type 1 and other Shigella species

Strains of Shigella dysenteriae type 1 cultured in Casamino Acids-yeast extract broth medium in the presence of 1 mM calcium chloride at 37 degrees C for 22 h induced hemagglutination of erythrocytes that was inhibited by N-acetylneuraminic acid, N-acetylneuramin-lactose, and alpha 1-glycoprotein. The hemagglutination was heat labile, and the absence of cell-surface appendages suggested a nonfimbrial adhesin(s). Under the same conditions, strains of Shigella flexneri (types 1a, 1b, 2a, and 2b) showed N-acetylneuraminic acid-resistant hemagglutination of erythrocytes.