ABSTRACTcagA, a gene that codes for an immunodominant antigen, is present only in Helicobacter pylori strains that are associated with severe forms of gastroduodenal disease (type I strains). We found that the genetic locus that contains cagA (cag) is part of a 40-kb DNA insertion that likely was acquired horizontally and integrated into the chromosomal glutamate racemase gene. This pathogenicity island is f lanked by direct repeats of 31 bp. In some strains, cag is split into a right segment (cagI) and a left segment (cagII) by a novel insertion sequence (IS605). In a minority of H. pylori strains, cagI and cagII are separated by an intervening chromosomal sequence. Nucleotide sequencing of the 23,508 base pairs that form the cagI region and the extreme 3 end of the cagII region reveals the presence of 19 ORFs that code for proteins predicted to be mostly membrane associated with one gene (cagE), which is similar to the toxin-secretion gene of Bordetella pertussis, ptlC, and the transport systems required for plasmid transfer, including the virB4 gene of Agrobacterium tumefaciens. Transposon inactivation of several of the cagI genes abolishes induction of IL-8 expression in gastric epithelial cell lines. Thus, we believe the cag region may encode a novel H. pylori secretion system for the export of virulence determinants.

Helicobacter pylori has been associated with gastritis, peptic ulcer, and gastric adenocarcinoma. We report the nucleotide sequence and expression of an immunodominant antigen of H. pylori and the immune response to the antigen during disease. The antigen, named CagA (cytotoxin-associated gene A), is a hydrophilic, surface-exposed protein of 128 kDa produced by most clinical isolates. The size of the cagA gene and its protein varies in different strains by a mechanism that involves duplication of regions within the gene. Clinical isolates that do not produce the antigen do not have the gene and are unable to produce an active vacuolating cytotoxin. An ELISA to detect the immune response against a recombinant fragment of this protein detects 75.3% of patients with gastroduodenal diseass and 100% of patients with duodenal ulcer (P < 0.0005), suggesting that only bacteria harboring this protein are associated with disease.

The gram negative, microaerophilic bacterium Helicobacter I~Iori colonizes the human gastric mucosa and establishes a chronic infection that is tightly associated with atrophic gastritis, peptic ulcer, and gastric carcinoma. Cloning of the H. pylori cytotoxin gene shows that the protein is synthesized as a 140-kD precursor that is processed to a 94-kD fully active toxin. Oral administration to mice of the purified 94-kD protein caused ulceration and gastric lesions that bear some similarities to the pathology observed in humans. The cloning of the cytotoxin gene and the development of a mouse model of human gastric disease will provide the basis for the understanding of H. ~lori pathogenesis and the development of therapeutics and vaccines. The recently discovered, gram negative, microaerophilic bacterium Helicobacter pylori colonizes the human gastric mucosa and establishes a chronic infection that is tightly associated with atrophic gastritis, peptic ulcer, and gastric carcinoma (1-5). H. pylori infection is a worldwide problem, since in developing countries it affects over 80% of the population older than 20. Also in developed countries the infection is present in 20% of the population by the age of 30 rising to over 50% by the age of 60. Clinical isolates of H. pylori can be classified into two groups based on the presence or absence of the vacuolating cytotoxin (6, 7) whose expression is linked to a surface exposed immunodominant antigen (CagA) (8, 9). Since high titers of serum antibodies to the CagA protein are detected in all patients with duodenal ulcer (8) and most of those with gastric carcinoma (10, 11), it has been proposed that disease development requires infection with cytotoxin-producing strains.The cytotoxin causes massive vacuolation in several mammalian cell lines (6), and similar vacuoles have also been observed in the gastric epithelia of patients with active chronic gastritis associated with H. pylori infection (12), indicating that the cytotoxin can contribute significantly to the pathogenesis of gastritis. Cell vacuolation in vitro can be blocked and reversed by inhibitors of V-type ATPases and potentiated by inhibitors of the Na+-K + ATPase (13,14), suggesting that the mechanism of action of the toxin is due to aberrant cation transport within the target cells. The purified toxin has been described as a protein of ~87 kD that is found in the bacterial culture supernatants, and the sequence of the NH2-terminal 23 amino acids has been determined (7).Despite the epidemiological correlation between infection with cytotoxic strains and disease (8) and the in vitro evidence for the presence of a cytotoxin, the in vivo roles of infection and cytotoxin have not been established due to the lack of a suitable animal model. H. ~lori does not colonize the gastric mucosa of mice or other small laboratory animals.To overcome this limitation, we administered H. pylori extracts and purified cytotoxin orally to mice. Using this model, extracts from cytotoxic H. Ioylori strains and purified cytotoxin ind...

Type 1 diabetes is characterized by T cell-mediated autoimmune destruction of pancreatic ␤ cells. Several studies have suggested an association between Coxsackie enterovirus seroconversion and onset of disease. However, a direct link between ␤ cell viral infection and islet inflammation has not been established. We analyzed pancreatic tissue from six type 1 diabetic and 26 control organ donors. Immunohistochemical, electron microscopy, wholegenome ex vivo nucleotide sequencing, cell culture, and immunological studies demonstrated Coxsackie B4 enterovirus in specimens from three of the six diabetic patients. Infection was specific of ␤ cells, which showed nondestructive islet inflammation mediated mainly by natural killer cells. Islets from enterovirus-positive samples displayed reduced insulin secretion in response to glucose and other secretagogues. In addition, virus extracted from positive islets was able to infect ␤ cells from human islets of nondiabetic donors, causing viral inclusions and signs of pyknosis. None of the control organ donors showed signs of viral infection. These studies provide direct evidence that enterovirus can infect ␤ cells in patients with type 1 diabetes and that infection is associated with inflammation and functional impairment.Coxsackie B4 enterovirus ͉ type 1 diabetes

Analysis of publicly available genomes of Streptococcus pneumoniae has led to the identification of a new genomic element containing genes typical of gram-positive pilus islets (PIs). Here, we demonstrate that this genomic region, herein referred to as PI-2 (consisting of pitA, sipA, pitB, srtG1, and srtG2) codes for a second functional pilus in pneumococcus. Polymerization of the PI-2 pilus requires the backbone protein PitB as well as the sortase SrtG1 and the signal peptidase-like protein SipA. Presence of PI-2 correlates with the genotype as defined by multilocus sequence typing and clonal complex (CC). The PI-2-positive CCs are associated with serotypes 1, 2, 7F, 19A, and 19F, considered to be emerging serotypes in both industrialized and developing countries. Interestingly, strains belonging to CC271 (where sequence type 271 is the predicted founder of the CC) contain both PI-1 and PI-2, as revealed by genome analyses. In these strains both pili are surface exposed and independently assembled. Furthermore, in vitro experiments provide evidence that the pilus encoded by PI-2 of S. pneumoniae is involved in adherence. Thus, pneumococci encode at least two types of pili that play a role in the initial host cell contact to the respiratory tract and are potential antigens for inclusion in a new generation of pneumococcal vaccines.

Colonization of the mucosa of the stomach and the duodenum by Helicobacter pylori is the major cause of acute and chronic gastroduodenal pathologies in humans. Duodenal ulcer formation strongly correlates with the expression of an antigen (CagA) that is usually coexpressed with the vacuolating cytotoxin (VacA), a protein that causes ulceration in the stomach of mice. However, the relationship between these two virulence factors is unknown. To define whether CagA and VacA are coexpressed in all clinical isolates and their relationships, we collected 43 clinical isolates of H. pylori and studied their genetic and phenotypic properties. Based on this analysis, most of the strains could be classified into two major types. Type I bacteria had the gene coding for CagA and expressed the CagA protein and the vacuolating cytotoxin. Type II bacteria did not have the gene coding for CagA and did not express either the CagA protein or the vacuolating cytotoxin. Type I and type II bacteria represented 56 and 16%, respectively, of the 43 clinical isolates, while the remaining 28% had an intermediate phenotype, expressing CagA independently of VacA or vice versa. This finding shows that although it is present in most cytotoxic strains, CagA is not necessary for the expression of the vacuolating cytotoxin.

Helicobacter pylori persistently colonizes the stomach of the majority of the world's population and is a tremendous medical burden due to its causal role in diverse gastric maladies. Since the stomach is a constantly changing environment, successful colonization of H. pylori within this niche requires regulation of bacterial gene expression to cope with the environmental fluctuations. In H. pylori, the ferric uptake regulator (Fur) has been shown to play an intricate role in adaptation of the bacterium to two conditions known to oscillate within the gastric mucosa: iron limitation and low pH. To extend our knowledge of the process of regulation and adaptation in H. pylori, we show that Fur is required for efficient colonization of the Mongolian gerbil: the mutant strain exhibits a 100-fold increase in the 50% infectious dose, as well as a 100-fold defect in competitive colonization, when coinfected with wild-type bacteria. Furthermore, we used DNA microarrays to identify genes whose expression was altered in a Fur-deficient strain. We show that the Fur regulon of H. pylori consists of approximately 30 genes, most of which have been previously annotated as acid stress associated. Finally, we investigate the role of Fur in acid-responsive modulation of gene expression and show that a large number of genes are aberrantly expressed in the Fur mutant specifically upon acid exposure. This fact likely explains the requirement for this regulator for growth and colonization in the stomach.

SllmmRryThe adult liver is an organ without constitutive lymphoid components. Therefore, any intrahepatic T cell found in chronic hepatitis should have migrated to the liver after infection and inflammation. Because of the little information available on the differences between intrahepatic and peripheral T cells, we used recombinant proteins of the hepatitis C virus (HCV) to establish specific T cell lines and clones from liver biopsies of patients with chronic hepatitis C and compared them with those present in peripheral blood mononuclear cells (PBMC). We found that the protein nonstructural 4 (NS4) was able to stimulate CD4 + T cells isolated from liver biopsies, whereas with all the other HCV proteins we consistently failed to establish liver-derived T cell lines from 16 biopsies. We then compared NS4-specific T cell clones obtained on the same day from PBMC and liver of the same patient. We found that the 22 PBMC-derived T cell clones represent, at least, six distinct clonal populations that differ in major histocompatibility complex restriction and response to superantigens, whereas the 27 liver-derived T cell clones appear all identical, as further confirmed by cloning and sequencing of the T cell receptor (TCK) variable and hypervariable regions. Remarkably, none of the PBMC-derived clones has a TCK identical to the liver-derived clone, and even with polymerase chain reaction oligotyping we did not find the liver-derived clonotypic TCK transcript in the PBMC, indicating a preferential intrahepatic localization of these T cells. Functionally, the liver-derived T cells provided help for polyclonal immunoglobulin (Ig)A production by B cells in vitro that is 10-fold more effective than that provided by the PBMC-derived clones, whereas there is no difference in the help provided for IgM and IgG production. Altogether these results demonstrate that the protein Ng4 is highly immunogenic for intrahepatic CD4 + T cells primed by HCV in vivo, and that there can he compartmentalization of some NS4-specific CD4 + T cells to the liver of patients with chronic hepatitis C.