Ben Adler

A dual transcriptional activation system for the 230 kb plasmid genes coding for virulence‐associated antigens of Shigella flexneri

The expression of plasmid‐encoded, invasion‐related antigens lpa b, c and d of Shigella flexneri was found to be positively regulated at transcriptional level by a 33kD protein produced by the previously defined, virulence‐associated Region 1 on the SalI fragment B of the 230 kb invasion plasmid. The gene (designated virB) was identified and its nucleotide sequence determined. No Ipa b or c was produced in the absence of an intact virB gene although lower levels of d were produced. The previously reported regulatory activity of the virF gene some 30 kb distance away was shown to act exclusively through virB. In contrast, the activation of the virG gene necessary for intercellular spread occurred directly by virF without the requirement for virB. This study thus ascribes a critical function to a previously recognized, but functionally undefined, virulence locus on the large invasion plasmid of S. flexneri. The virF gene appears to have a central role in activation of the 230kb plasmid‐encoded virulence genes.

The sigA gene which is borne on the she pathogenicity island of Shigella flexneri 2a encodes an exported cytopathic protease involved in intestinal fluid accumulation.

ABSTRACT In this study, the sigA gene situated on theshe pathogenicity island of Shigella flexneri2a was cloned and characterized. Sequence analysis showed thatsigA encodes a 139.6-kDa protein which belongs to the SPATE (serine protease autotransporters of Enterobacteriaceae) subfamily of autotransporter proteins. The demonstration that SigA is autonomously secreted from the cell to yield a 103-kDa processed form and possesses a conserved C-terminal domain for export from the cell were consistent with the autotransporter pathway of secretion. Functional analysis showed that SigA is a secreted temperature-regulated serine protease capable of degrading casein. SigA was cytopathic for HEp-2 cells, suggesting that it may be a cell-altering toxin with a role in the pathogenesis ofShigella infections. SigA was at least partly responsible for the ability of S. flexneri to stimulate fluid accumulation in ligated rabbit ileal loops.

Functional organization and nucleotide sequence of virulence Region‐2 on the large virulence plasmid in Shigella flexneri 2a

The 7kb virulence Region‐2 of the large (virulence) plasmid in Shigella flexneri 2a encodes several proteins required for invasion of intestinal epithelial cells. Insertion and deletion mutagenesis, DNA subcloning and SDS‐polyacrylamide gel electro‐phoresis of proteins synthesized in minicells demonstrated five genes in this region. They encode 24, 18, 62 (lpaB), 41 (lpaC) and 37 (lpaD)‐kiloDalton (kD) proteins. Complementation of Tn5‐induced mutations in Region‐2 with the above plasmid constructs indicated that Region‐2 consists of two operons and that the three lpa proteins are essential for the virulence phenotype. The transcriptional organization determined by Northern blotting, S1 nuclease protection and the effect of Tn5 insertions on expression of the lpa proteins revealed that Region‐2 has three promoters that transcribe RNAs of 4.0, 4.5 and 7.5kb. The 4.0 kb RNA was the transcript for the operon encoding the 24, 18 kD, lpaB and C proteins and the 4.5 kb RNA for the ipsD gene. In addition, the full‐length RNA of 7.5 kb which covers Region‐2 supplemented full expression of the lpa proteins. The 7663 nucleotides of Region‐2 were determined to confirm the five open reading frames encoding 23655, 17755, 62168, 41077 and 36660 Dalton proteins, respectively, and their regulatory sequences.

Curli loci of Shigella spp.

ABSTRACT An unstable chromosomal element encoding multiple antibiotic resistance in Shigella flexneri serotype 2a was found to include sequences homologous to the csg genes encoding curli in Escherichia coli and Salmonella enterica serovar Typhimurium. As curli have been implicated in the virulence of serovar Typhimurium, we investigated thecsg loci in all four species of Shigella. DNA sequencing and PCR analysis showed that the csg loci of a wide range of Shigella strains, of diverse serotypes and different geographical distributions, were almost universally disrupted by deletions or insertions, indicating the existence of a strong selective pressure against the expression of curli. Strains of enteroinvasive E. coli (EIEC), which share virulence traits with Shigella spp. and cause similar diseases in humans, also possessed insertions or deletions in the csg locus or were otherwise unable to produce curli. Since the production of curli is a widespread trait in environmental isolates of E. coli, our results suggest that genetic lesions that abolish curli production in the closely related genus Shigella and in EIEC are pathoadaptive mutations.

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.

The immunogenic SigA enterotoxin of Shigella flexneri 2a binds to HEp-2 cells and induces fodrin redistribution in intoxicated epithelial cells.

Background We have previously shown that the enterotoxin SigA which resides on the she pathogenicity island (PAI) of S. flexneri 2a is an autonomously secreted serine protease capable of degrading casein. We have also demonstrated that SigA is cytopathic for HEp-2 cells and plays a role in the intestinal fluid accumulation associated with S. flexneri infections. Methods/Principal Findings In this work we show that SigA binds specifically to HEp-2 cells and degrades recombinant human αII spectrin (α-fodrin) in vitro, suggesting that the cytotoxic and enterotoxic effects mediated by SigA are likely associated with the degradation of epithelial fodrin. Consistent with our data, this study also demonstrates that SigA cleaves intracellular fodrin in situ, causing its redistribution within cells. These results strongly implicate SigA in altering the cytoskeleton during the pathogenesis of shigellosis. On the basis of these findings, cleavage of fodrin is a novel mechanism of cellular intoxication for a Shigella toxin. Furthermore, information regarding immunogenicity to SigA in infected patients is lacking. We studied the immune response of SigA from day 28 post-challenge serum of one volunteer from S. flexneri 2a challenge studies. Our results demonstrate that SigA is immunogenic following infection with S. flexneri 2a. Conclusions This work shows that SigA binds to epithelial HEp-2 cells as well as being able to induce fodrin degradation in vitro and in situ, further extending its documented role in the pathogenesis of Shigella infections.

Regulated site‐specific recombination of the she pathogenicity island of Shigella flexneri

Summary The she pathogenicity island (PAI) is a chromosomal, laterally acquired, integrative element of Shigella flexneri that carries genes with established or putative roles in virulence. We demonstrate that spontaneous, precise excision of the element from its integration site in the 3′ terminus of the pheV tRNA gene is mediated by an integrase gene (int) and a gene designated rox (regulator of excision), both of which are carried on the she PAI. Integrase‐mediated excision occurs via recombination between a 22u2003bp sequence at the 3′ terminus of pheV and an imperfect direct repeat at the pheV‐distal boundary of the PAI. Excision leads to the formation of a circular episomal form of the PAI, reminiscent of circular excision intermediates of other mobile elements that are substrates for lateral transfer processes such as conjugation, packaging into phage particles and recombinase‐mediated integration into the chromosome. The circle junction consists of the pheV‐proximal and pheV‐distal boundaries of the PAI converging on a sequence identical to 22u2003bp at the 3′ terminus of pheV. The isolated circle was transferred to Escherichia coli where it integrated specifically into phe tRNA genes, as it does in S. flexneri, independently of recA. We also demonstrate that Rox stimulates, but is not essential for, excision of the she PAI in an integrase‐dependent manner. However, Rox does not stimulate excision by activating the transcription of the she PAI integrase gene, suggesting that it has an excisionase function similar to that of a related protein from the P4 satellite element of phage P2.

Molecular Epidemiology of the SRL Pathogenicity Island

ABSTRACT The Shigella resistance locus (SRL), which is carried on the SRL pathogenicity island (PAI) in Shigella flexneri 2a YSH6000, mediates resistance to the antibiotics streptomycin, ampicillin, chloramphenicol, and tetracycline. In the present study, we investigated the distribution and structural variation of the SRL and the SRL PAI in 71 Shigella isolates and 28 other enteric pathogens by PCR and Southern analysis. The SRL and SRL-related loci, although absent from the other enteric pathogens evaluated in this study, were found to be present in a number of Shigella isolates. SRL PAI markers were also present in the majority of strains carrying the SRL and SRL-related loci. PCR linkage studies with six of these strains demonstrated that the SRL is carried on elements similar in structure and organization to the YSH6000 SRL PAI, consistent with the hypothesis that the SRL PAI may be involved in the spread of multiple-antibiotic resistance in these strains.

Distribution and structural variation of the she pathogenicity island in enteric bacterial pathogens

Shigella flexneri serotype 2a carries a chromosomal pathogenicity island (PAI), termed the she PAI, that has been implicated in the pathogenesis of diarrhoeal disease. The complete nucleotide sequence and genetic organisation of the she PAI of S. flexneri 2a strain YSH6000T was determined recently. In the current study the distribution and structure of the she PAI was investigated by PCR and Southern analysis in 65 isolates of enteric pathogens including Shigella spp., enterohaemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), Yersinia enterocolitica and Salmonella enterica serovar Typhimurium. The study showed that the she PAI has undergone a variety of structural changes, defined by the presence or absence of specific marker genes in the PAI. The she PAI or structural variants of this element were found in all species of Shigella as well as in EIEC, EHEC and EPEC. No evidence of the PAI was found in Y. enterocolitica or Sal. Typhimurium. The structural form of the she PAI that exists in strain YSH6000T was present in all strains of S. flexneri serotype 2a and in some strains of S. flexneri serotypes 2b and 3c. Variants of the PAI that were missing one or more marker regions were found in all species of Shigella and in pathogenic strains of E. coli. In all strains, the PAIs have inserted into either pheV or a phe tRNA gene in another location on the chromosome. It was concluded that the she PAI is one of several closely related genetic elements that have disseminated throughout Shigella and pathogenic strains of E. coli and diverged into distinct stuctural forms.

Regulated site-specific recombination of the she pathogenicity island of Shigella flexneri.

Summary The she pathogenicity island (PAI) is a chromosomal, laterally acquired, integrative element of Shigella flexneri that carries genes with established or putative roles in virulence. We demonstrate that spontaneous, precise excision of the element from its integration site in the 3′ terminus of the pheV tRNA gene is mediated by an integrase gene (int) and a gene designated rox (regulator of excision), both of which are carried on the she PAI. Integrase‐mediated excision occurs via recombination between a 22u2003bp sequence at the 3′ terminus of pheV and an imperfect direct repeat at the pheV‐distal boundary of the PAI. Excision leads to the formation of a circular episomal form of the PAI, reminiscent of circular excision intermediates of other mobile elements that are substrates for lateral transfer processes such as conjugation, packaging into phage particles and recombinase‐mediated integration into the chromosome. The circle junction consists of the pheV‐proximal and pheV‐distal boundaries of the PAI converging on a sequence identical to 22u2003bp at the 3′ terminus of pheV. The isolated circle was transferred to Escherichia coli where it integrated specifically into phe tRNA genes, as it does in S. flexneri, independently of recA. We also demonstrate that Rox stimulates, but is not essential for, excision of the she PAI in an integrase‐dependent manner. However, Rox does not stimulate excision by activating the transcription of the she PAI integrase gene, suggesting that it has an excisionase function similar to that of a related protein from the P4 satellite element of phage P2.