Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae and pathogenic Neisseria, the random switching of the modA gene, associated with a phase-variable type III restriction modification (R-M) system, controls expression of a phase-variable regulon of genes (a “phasevarion”), via differential methylation of the genome in the modA ON and OFF states. Phase-variable type III R-M systems are also found in Helicobacter pylori, suggesting that phasevarions may also exist in this key human pathogen. Phylogenetic studies on the phase-variable type III modH gene revealed that there are 17 distinct alleles in H. pylori, which differ only in their DNA recognition domain. One of the most commonly found alleles was modH5 (16% of isolates). Microarray analysis comparing the wild-type P12modH5 ON strain to a P12ΔmodH5 mutant revealed that six genes were either up- or down-regulated, and some were virulence-associated. These included flaA, which encodes a flagella protein important in motility and hopG, an outer membrane protein essential for colonization and associated with gastric cancer. This study provides the first evidence of this epigenetic mechanism of gene expression in H. pylori. Characterisation of H. pylori modH phasevarions to define stable immunological targets will be essential for vaccine development and may also contribute to understanding H. pylori pathogenesis.

Enterotoxigenic Escherichia coli (ETEC) is an enteric pathogen that causes cholera-like diarrhea in humans and animals. ETEC secretes a heat-labile enterotoxin (LT), which resembles cholera toxin, but the actual mechanism of LT secretion is presently unknown. We have identified a previously unrecognized type II protein secretion pathway in the prototypic human ETEC strain, H10407 (serotype O78:H11). The genes for this pathway are absent from E. coli K-12, although examination of the K-12 genome suggests that it probably once possessed them. The secretory pathway bears significant homology at the amino acid level to the type II protein secretory pathway required by Vibrio cholerae for the secretion of cholera toxin. With this in mind, we determined whether the homologous pathway of E. coli H10407 played a role in the secretion of LT. To this end, we inactivated the pathway by inserting a kanamycinresistance gene into one of the genes (gspD) of the type II secretion pathway by homologous recombination. LT secretion by E. coli H10407 and the gspD mutant was assayed by enzyme immunoassay, and its biological activity was assessed by using Y-1 adrenal cells. This investigation showed that the protein secretory pathway is functional and necessary for the secretion of LT by ETEC. Our findings have revealed the mechanism for the secretion of LT by ETEC, which previously was unknown, and provide further evidence of close biological similarities of the LT and cholera toxin. Enterotoxigenic Escherichia coli (ETEC) is an enteric pathogen that causes watery cholera-like diarrhea in animals and humans (1). Infections with ETEC pose a major health problem in developing countries, accounting for more than 200 million cases of diarrhea and approximately 380,000 deaths annually among children under 5 years of age (2). ETEC is also the most common cause of diarrhea among travelers from industrialized to less developed countries, including military troops on deployment (2).ETEC secrete at least one of two types of enterotoxins, known as heat-labile (LT) and heat-stable enterotoxin, respectively (1, 3). LT is an 84-kDa multimeric protein comprised of a single A subunit and a pentamer of identical B subunits. The pentameric B subunit mediates binding to G M1 ganglioside on intestinal epithelial cells, after which the toxin is internalized and processed. The free A subunit then catalyses the ADP-ribosylation of G s␣ , a GTP-binding regulatory protein, leading to activation of adenylate cyclase, production of excessive amounts of cAMP, disruption of electrolyte transport across the intestinal lumen, and diarrhea (4). Prostaglandins and neurotransmitters of the enteric nervous system also play a role in the induction of fluid secretion by LT (5, 6). Although a great deal is known about the structure and biological activity of LT, little is known about the mechanism of its secretion by ETEC.LT is structurally and biologically related to cholera toxin (CT), the major virulence determinant of Vibrio cholerae, with over 77% nucleotide (4) an...