vacB, a novel chromosomal gene required for expression of virulence genes on the large plasmid of Shigella flexneri

Abstract

Shigellae, the causative agents of bacillary dysentery, are capable of adhering to and invading epithelial cells and spreading into adjacent cells. A chromosomal mutant of ShigeUla flexneri 2a YSH6000 with reduced invasive capacity was isolated by TnS insertion mutagenesis. The linkage of the mutant phenotype to the TnS insertion was determined by P1 phage transduction. The site of the TnS insertion was assigned to a NotI chromosomal restriction map, confirming that the virulence-associated locus, designated vacB, is a new locus on the chromosome. In the vacB mutant, production of the four plasmid-encoded virulence antigens, IpaB, -C, and -D and VirG, decreased to a low level compared with that in the wild type. In contrast, levels of transcription of the operons for virG, ipa, region-3.4, region-5, virF, and virB on the large plasmid, as determined by Northern dot blotting, were unaffected in the vacB mutant. Furthermore, transcriptional activation of the ipa operon by exploiting a tac promoter could not restore the vacB mutant to production of the same levels of the IpaB, -C, and -D proteins as those in the wild type, indicating that the vacB locus is involved in expression of the vir genes on the large plasmid at the posttranscriptional level. Cloning followed by nucleotide sequencing of the vacB region showed it to contain a 2, Shigellae cause bacillary dysentery in humans and monkeys. The essential early steps in infection comprise bacterial adherence and invasion of colonic epithelial cells followed by intracellular bacterial multiplication and spread of invading bacteria to adjacent cells (10). Seven separate virulence-associated DNA segments have been identified on the large 230-kb plasmid of Shigella flexneri 2a. In a 31-kb DNA sequence, five contiguous segments, designated virB, ipaBCD (ipa), and region-3, -4, and -5, were characterized. The virB gene acts as a positive regulator for the expression of the other four virulence regions (1); the ipa operon encodes three invasion-associated antigens, IpaB, -C, and -D; and region-3, -4, and -5 are required for both adherence to epithelial cells and excretion of the Ipa proteins (20a). The virG gene, outside the cluster, encodes a 116-kDa immunogenic, surface-exposed outer membrane protein and is es-

The RNase R from Campylobacter jejuni Has Unique Features and Is Involved in the First Steps of Infection

“…11). The results are in agreement with what has been reported for Shigella flexneri and E. coli EIEC, in which the ⌬rnr mutant strains were shown to be less invasive (50). These results indicate that RNase R, similarly to what was shown for polynucleotide phosphorylase (10), is an important protein for the first steps of infection of C. jejuni.…”

Modulation of Yersinia Type Three Secretion System by the S1 Domain of Polynucleotide Phosphorylase

“…In a transposon mutagenesis screen, a Shigella flexneri was isolated that displayed reduced host cell invasion. The disruption was mapped to a locus that was later identified as the gene (vacB/rnr) encoding RNaseR (27,28). More pertinent to this study and as mentioned in the introduction, a pnp mutant of Salmonella enterica was found to have a disregulated TTSS (12).…”

RNase R is a highly unstable protein regulated by growth phase and stress

“…In vivo, RNase R plays an important role in degradation of mRNAs with extensive secondary structures (Chen and Deutscher 2005) and in the quality control of rRNA and tRNA Deutscher 2002, 2003;Li et al 2002;S Chebolu, C Kim, E Quesada, and MP Deutscher, unpubl.). RNase R is also required for the expression of virulence genes in Shigella and enteroinvasive strains of E. coli (Tobe et al 1992). It also associates with the tmRNASmpB complex, which releases stalled ribosomes via a process known as trans-translation (Karzai and Sauer 2001;Withey and Friedman 2003).…”