TEXT

Top Abstract Text References

Bacillary dysentery is a severe diarrheal disease affecting hundreds of millions of people worldwide, leading to more than 500,000 deaths annually (11). The disease is caused by four bacterial species comprising the genus Shigella, i.e., Shigella flexneri, S. dysenteriae, S. sonnei, and S. boydii. Shigella spp. are transmitted to their hosts via the fecal-oral route and infect the colonic epithelium. Subsequent cell destruction, inflammation, and ulceration of the colon are responsible for the bloody, mucoid diarrhea that is characteristic of the disease. In recent years much has been learned about the sophisticated virulence mechanisms that allow Shigella to invade epithelial cells and spread to neighboring cells (5). However, nothing is known about the first step in the infection process, colonization of the host.

Bacterial colonization of the host intestine is generally mediated by fimbrial adhesins (2, 3, 8, 10). However, it is not clear what role fimbriae play in the virulence of Shigella (22). Over the last decade, Escherichia coli and Salmonella spp. have been found to express a surface structure termed thin aggregative fimbriae or curli (14, 20, 21). In E. coli, curli mediate the formation of biofilms on inert surfaces (26). However, in Salmonella enterica serovar Typhimurium, curli mediate bacterial attachment to mouse intestinal cells in vitro (25), and the expression of curli at 37°C is a phase-variable characteristic that is essential for full virulence in mice (24). These findings demonstrate that curli probably have a role in the colonization of the mouse intestine by serovar Typhimurium. Furthermore, curli are capable of mediating bacterial binding to a wide variety of tissues (15), cell matrix proteins, and plasma proteins (13, 14) and may therefore have additional roles in virulence.

During investigations of a deletable chromosomal element encoding multiple antibiotic resistance in S. flexneri serotype 2a (17, 18), members of our group discovered a locus with high sequence similarity to the csg gene clusters encoding curli in E. coli and serovar Typhimurium. This preliminary finding prompted us to investigate the presence of csg loci in a variety of Shigella strains.

Restriction analysis and DNA sample sequencing of csg loci in Shigella. To test whether the csg locus was present in all four species of Shigella, oligonucleotide primers were designed for the PCR amplification of an internal portion of the csg locus. Primer 4477 was homologous to a 5'-terminal sequence of the csgE gene of E. coli K-12, while primer 4480 was homologous to a 3'-terminal sequence of csgA (Fig. 1). PCR amplification of the csg internal fragment from the E. coli control strain, DH5 (9), generated a DNA fragment of 2.4 kb, the length predicted from sequence analysis of the E. coli K-12 csg locus (GenBank accession no. X90754). Similarly, a 2.4-kb fragment was amplified from S. dysenteriae serotype 3 strain SBA1304. However, fragments of 3.6, 4.0, and 2.15 kb were amplified from S. flexneri serotype 2a (SBA1100), S. sonnei (SBA1302), and S. boydii serotype 3 (SBA1308), respectively. These results implied that the csg locus was present in all four species of Shigella but had acquired insertions or undergone internal duplications in S. flexneri and S. sonnei, while the S. boydii csg locus appeared to have undergone a deletion.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Deletion and insertion mutations in the csg loci of Shigella spp. Restriction maps of the internal portion of the csg locus, spanning the 5' terminus of csgE and the 3' terminus of csgA, are shown for four strains of Shigella. The positions of ISs and deletions in relation to the csg genes are indicated by dashed lines. The 250-bp deletion in the S. boydii (SBA1308) csg gene lies within a 350-bp region indicated by a horizontal bracket. The positions of promoters for the csgBAC and csgDEFG operons are indicated by bent arrows. The binding sites for primers are indicated by open arrows. Restriction sites include HindIII (H), PstI (P), and EcoRV (V). Primer sequences are as follows: 4477, 5'-GCGGCGAACAGAAATTCTGCC-3'; 4480, 5'-GTTACCAAAGCCAACCTGAGTCACG-3'; BAP1021, 5'-TCAGGATTCCGGTGGAAC-3'; BAP1022, 5'-TTAAGACTTTTCTGAAGAGG-3'; BAP1023, 5'-CAGCGAAAATACGGTTAAAACGC-3'; BAP1232, 5'-TCCTGCTCAAAGTATCCTGCC-3'); and BAP1233, GATTGCTGCAATTGCTGCTAC-3'.

Survey of insertion and deletion mutations in the csg locus of Shigella strains. Initial analysis of the four Shigella strains representing each species suggested that mutations within the csg locus are probably widespread phenomena. To test this hypothesis, 43 Shigella strains, representing a wide range of serotypes isolated over several years from patients in Australia and Japan, were surveyed by colony PCR with the primers BAP1021 and BAP1022, which flank the complete csg locus. The results (Table 1) demonstrated that insertions into the csg locus are widespread in Shigella spp. The size variation in the csg loci suggests that different types of insertion events have occurred. The smaller PCR products are consistent with the insertion of single IS elements, while the larger products (6.4 to 7.4 kb) are consistent with the insertion of multiple IS elements similar to those in S. dysenteriae serotype 3 strain SBA1304 and S. sonnei strain SBA1302 (Fig. 1). However, in many strains the csg locus was either partially deleted or not detected at all.

Significance of mutations in the csg loci of Shigella strains. The insertion and precise excision of IS elements into genes have been described as a possible mechanism for the control of gene expression. However, this is only likely to be significant when insertion and excision occur at high frequencies. For example, the expression of exopolysaccharide synthesis in Pseudomonas atlantica is mediated by an IS element that excises from the eps locus at frequencies as high as 0.5 (1). This generates genetically distinct subpopulations that are preadapted to environmental change (1). In contrast, precise excision of IS1 in E. coli occurs at frequencies of less than 10⁵ (12). Furthermore, since it appears that curli loci in Shigella are often interrupted by multiple IS elements, the restoration of curli expression by the simultaneous excision of more than one IS element seems unlikely. Rather than being involved in the control of curli expression, we propose that insertions into the csg locus are common because of a strong selection against the expression of curli in Shigella. This is supported by the finding that up to a quarter of Shigella strains may have partial or complete deletions of the csg locus.

Nucleotide sequence accession numbers. Nucleotide sequences of the sites of IS element insertion into the csg locus have been deposited into the GenBank database under the accession numbers AF237724, AF237725, AF237726, and AF237727.