Murali K. R. Tummuru

Helicobacter pylori picB, a homologue of the Bordetella pertussis toxin secretion protein, is required for induction of IL‐8 in gastric epithelial cells

Approximately 60% of Helicobacter pylori strains are cagA+ and this genotype is more frequently associated with duodenal ulcer disease. Although most wild‐type cagA+ strains are both cytotoxigenic and induce enhanced Interleukin‐8 (IL‐8) secretion in gastric epithelial cells, isogenic cagA− mutants retain full activity in these assays; thus, cagA appears to be a marker of enhanced virulence. Delineation of the nucleotide sequence of a 4 kb region upstream of cagA allowed the identification of 966 bp (picA) and 2655 bp (picB) open reading frames encoding 36 kDa and 101 kDa polypeptides, respectively. picA and picB constitute an operon in opposite orientation to cagA. The deduced picB product showed significant homology (26% identity and 50% similarity) with the Bordetella pertussis toxin secretion protein (PtlC). Of 55 H. pylori clinical isolates, the picA and picB segment was conserved exclusively in cagA+ strains and present in all isolates from patients with duodenal ulceration, versus 59% of isolates from patients with gastritis alone (P=0.01). Using gene‐replacement techniques, we constructed picA and picB mutant H. pylori strains and demonstrated that the picB gene product is involved in the induction of IL‐8 expression in gastric epithelial cells. Further, Northern blot hybridization and RT‐PCR data showed that picA and picB are co‐transcribed and an insertional mutation in picA ablates picB expression. These studies indicate a role of picA and picB in the induction of an inflammatory response in gastric epithelial cells either directly or by enabling secretion of an unidentified product, and suggest a mechanism for the overrepresentation of strains possessing these genes in patients with peptic ulceration.

High-frequency S-layer protein variation in Campylobacter fetus revealed by sapA mutagenesis.

Campylobacter fetus utilizes paracrystalline surface (S‐) layer proteins that confer complement resistance and that undergo antigenic variation to facilitate persistent mucosal colonization in ungulates. C. fetus possesses multiple homologues of sapA, each of which encode full‐length S‐layer proteins. Disruption of sapA by a gene targeting method (insertion of kanamycin (km) resistance) caused the loss of C. fetus cells bearing full‐length S‐layer proteins and their replacement by cells bearing a 50 kDa truncated protein that was not exported to the cell surface. After incubation of the mutants with serum, the survival rate was approximately 2 × 10‐2. Immunoblots of survivors showed that phenotypic reversion involving high‐level production of full‐length (98, 127 or 149 kDa) S‐layer proteins had occurred. Revertants were serum resistant but caused approximately 10‐fold less bacteraemia in orally challenged mice than did the wild‐type strain. Southern hybridizations of the revertants showed rearrangement of sapA homologues and retention of the km marker. These results indicate that there exists high‐frequency generation of C. fetus sapA antigenic variants, and that intracellular mechanisms acting at the level of DNA reciprocal recombination play key roles in this phenomenon.

DNA sequence conservation and diversity in transposable element IS605 of Helicobacter pylori.

Background. IS605, a transposable element‐like sequence identified in the virulence‐associated cag region of Helicobacter pylori reference strain NCTC11638, is unusual in containing two oppositely‐oriented open reading frames whose products are homologues of the single transposases of the unrelated elements, IS200 and IS1341.