A pneumococcal pilus influences virulence and host inflammatory responses.

Streptococcus pneumoniae (pneumococcus) is a major cause of morbidity and mortality world-wide. The initial event in invasive pneumococcal disease is the attachment of encapsulated pneumococci to epithelial cells in the upper respiratory tract. This work provides evidence that initial bacterial adhesion and subsequent ability to cause invasive disease is enhanced by pili, long organelles able to extend beyond the polysaccharide capsule, previously unknown to exist in pneumococci. These adhesive pili-like appendages are encoded by the pneumococcal rlrA islet, present in some, but not all, clinical isolates. Introduction of the rlrA islet into an encapsulated rlrA-negative isolate allowed pilus expression, enhanced adherence to lung epithelial cells, and provided a competitive advantage upon mixed intranasal challenge of mice. Furthermore, a pilus-expressing rlrA islet-positive clinical isolate was more virulent than a nonpiliated deletion mutant, and it out-competed the mutant in murine models of colonization, pneumonia, and bacteremia. Additionally, piliated pneumococci evoked a higher TNF response during systemic infection, compared with nonpiliated derivatives, suggesting that pneumococcal pili not only contribute to adherence and virulence but also stimulate the host inflammatory response.

Cloning, expression, purification, and characterization of Streptococcus pneumoniae IgA1 protease.

The IgA1 protease of Streptococcus pneumoniae is a Zn-metalloproteinase of 1964 amino acids that specifically cleaves the hinge region of IgA1, the predominant class of immunoglobulin present on mucosal membranes. This protease is associated to the bacterial cell surface via an N-terminal membrane anchor. Following proteolysis it is released in several forms of different molecular weight. Here, we describe the cloning, expression, and characterization of the enzymatic activity and immunogenicity of three fragments of IgA1 protease, including a large one lacking only the 103 N-terminal amino acids that constitute a typical prokaryotic signal sequence. Further, a proteolytically inactive mutant was generated by replacement of the glutamate residue with an alanine residue in the active site motif HExxH (1605-1609). This is the first report of recombinant active forms of S. pneumoniae IgA1 protease, which open the possibility of identifying specific inhibitors that could interfere with the mucosal colonization by pneumococcus. Moreover the inactive mutant could be considered as a candidate vaccine component.

"Cervia Working Group Report": guidelines on the diagnosis and treatment of Helicobacter pylori infection.

Different national attitudes exist between countries in Europe concerning eradication of Helicobacter pylori infection due to the wide differences in Helicobacter pylori prevalence, gastric cancer risk, bacterial resistance to antibiotics, health care systems and financial resources. The Cervia Working Group Report has been established in order to fill the gap in the absence of National Guidelines in Italy concerning the diagnosis and treatment of Helicobacter pylori infection. The recommendations made are, by and large, similar to the European Guidelines but differ slightly with regard to the "test-and-treat" approach to young dyspeptics without sinister symptoms. In the absence of a national validation of this strategy a case-by-case assessment of dyspepsia has been promoted, both at primary care and specialist level. Another area of partial disagreement concerns the eradication of Helicobacter pylori in patients undergoing long-term proton pump inhibitor treatment which has not been generally recommended as scientific evidence in support of this policy is at present rather weak.

The design of vaccines against Helicobacter pylori and their development.

Helicobacter pylori is a gram negative, spiral, microaerophylic bacterium that infects the stomach of more than 50% of the human population worldwide. It is mostly acquired during childhood and, if not treated, persists chronically, causing chronic gastritis, peptic ulcer disease, and in some individuals, gastric adenocarcinoma and gastric B cell lymphoma. The current therapy, based on the use of a proton-pump inhibitor and antibiotics, is efficacious but faces problems such as patient compliance, antibiotic resistance, and possible recurrence of infection. The development of an efficacious vaccine against H. pylori would thus offer several advantages. Various approaches have been followed in the development of vaccines against H. pylori, most of which have been based on the use of selected antigens known to be involved in the pathogenesis of the infection, such as urease, the vacuolating cytotoxin (VacA), the cytotoxin-associated antigen (CagA), the neutrophil-activating protein (NAP), and others, and intended to confer protection prophylactically and/or therapeutically in animal models of infection. However, very little is known of the natural history of H. pylori infection and of the kinetics of the induced immune responses. Several lines of evidence suggest that H. pylori infection is accompanied by a pronounced Th1-type CD4(+) T cell response. It appears, however, that after immunization, the antigen-specific response is predominantly polarized toward a Th2-type response, with production of cytokines that can inhibit the activation of Th1 cells and of macrophages, and the production of proinflammatory cytokines. The exact effector mechanisms of protection induced after immunization are still poorly understood. The next couple of years will be crucial for the development of vaccines against H. pylori. Several trials are foreseen in humans, and expectations are that most of the questions being asked now on the host-microbe interactions will be answered.

Cellular responses induced after contact with Helicobacter pylori.

Contact-dependent activation of the cag organelle, a type IV secretion system of Helicobacter pylori, promotes translocation of CagA into the host cell. CagA is an immunodominant antigen of H. pylori, encoded by cag. It is thought to be associated with severe clinical outcomes, but has an unclear role in pathogenesis. Now we know that CagA is injected into the host and is tyrosine-phosphorylated by a membrane-associated eukaryotic tyrosine kinase. After activation, CagA induces morphological changes in the host, as well as actin reorganization, variations in the cell cycle and autocrine effects. Subversion of cell control may ultimately lead to cellular damage and to increased risks for gastric cancer development. cag instability contributes to long-term persistence within the host by attenuating bacterial virulence. We still do not know if additional factors are co-translocated with CagA and we do not know their specific mechanisms of action, but there is a strong experimental evidence that indicates that cag is the major player in the host-pathogen relationship.

Composition and gene expression of the cag pathogenicity island in Helicobacter pylori strains isolated from gastric carcinoma and gastritis patients in Costa Rica.

The composition and in vitro expression of the cag pathogenicity island genes in a group of Helicobacter pylori strains obtained from patients suffering from chronic gastritis-associated dyspepsia (n = 26) or gastric carcinoma (n = 17) were analyzed. No significant difference in the distribution of the 10 studied regions was found between the cases and the controls. Nine strains did not harbor any of the selected regions: eight (30.8%) isolated from patients with gastritis only and one (5.9%) from a patient with gastric carcinoma. No association was found between the number of repeated sequences at the 3' end of the cagA gene or the presence of tyrosine phosphorylation motifs and the clinical origin of the strains. The virB10 homolog gene was the sole gene studied to be significantly expressed more often in cancer strains than in gastritis strains (P = 0.03).

Helicobacter pylori activates mitogen-activated protein kinase cascades and induces expression of the proto-oncogenes c-fos and c-jun.

Helicobacter pylori is an etiological agent in the development of mucosa-associated lymphoid tissue lymphoma and gastric adenocarcinoma. Patients infected with H. pylori carry a 3-6-fold increased risk of developing cancer compared with uninfected individuals. H. pylori strains expressing the cytotoxin-associated antigen A (CagA) are more frequently associated with the development of neoplasia than cagA-negative strains. However, the molecular mechanism by which H. pylori causes neoplastic transformation remains unclear. Here we report that exposure of gastric epithelial cells to H. pylori induces activation of the transcription factor activator protein 1. Activation of the proto-oncogenes c-fos and c-jun is strongly induced. We show that H. pylori activates the ERK/MAP kinase cascade, resulting in Elk-1 phosphorylation and increased c-fos transcription. H. pylori strains that do not express CagA or that are mutated in cag genes encoded by the CagI pathogenicity island do not induce activator protein 1, MAP kinase activity, or c-fos or c-jun activation. Proto-oncogene activation may represent a crucial step in the pathomechanism of H. pylori induced neoplasia.

The alleles of the bft gene are distributed differently among enterotoxigenic Bacteroides fragilis strains from human sources and can be present in double copies.

Enterotoxigenic Bacteroides fragilis (ETBF) strains are associated with diarrheal disease in children. These strains produce a zinc metalloprotease enterotoxin, or fragilysin, that can be detected by a cytotoxicity assay with HT-29 cells. Recently, three different isoforms or variants of the enterotoxin gene, designated bft-1, bft-2, and bft-3, have been identified and sequenced. We used restriction fragment length polymorphism analysis of the PCR-amplified enterotoxin gene to detect the isoforms bft-1 and bft-2 or bft-3 borne by ETBF. By sequencing the portion of the bft gene corresponding to the mature toxin in some strains and applying allele-specific PCR for strains categorized as bft-2 or bft-3, we found in our collection two strains harboring bft-3, a variant that had been described for isolates from East Asia. Analysis of 66 ETBF strains from different sources showed that bft-1 is the most frequent allele, being present in 65% of isolates; it is largely predominant in isolates from feces of adults, while bft-2 is present in isolates from feces of children. This association is statistically significant (P, 0.0064). Sixteen strains were examined by Southern hybridization using, as probes, the bft and second metalloprotease genes, both included in a pathogenicity islet. Five strains were found to harbor double copies of both genes, suggesting that the whole islet was duplicated. Four of these strains, harboring bft-1 (three strains) or bft-2 (one strain), were found to produce a large amount of biologically active toxin, as determined by a cytotoxicity assay with HT-29 cells. The strains harboring bft-3, either in a single copy or in double copies, produced the smallest amount of toxin in our collection.

Tyrosine phosphorylation of the Helicobacter pylori CagA antigen after cag-driven host cell translocation.

Helicobacter pylori strains associated with severe tissue damage and inflammation possess a unique genetic locus, cag, containing 31 genes originating from a distant event of horizontal transfer and retained as a pathogenicity island. The cag system is an Helicobacter-specific type IV secretion engine involved in cellular responses like induction of pedestals, secretion of IL-8, and phosphorylation of proteic targets. It has previously been reported that cocultivation of epithelial cells with Helicobacter pylori triggers signal transduction and tyrosine phosphorylation of a 145-kDa putative host cell protein. Herein, we demonstrate that this protein is not derived from the host but rather is the bacterial immunodominant antigen CagA, a virulence factor commonly expressed in peptic ulcer disease and thought to be an orphan of a specific biological function. Thus, CagA is delivered into the epithelial cells by the cag type IV secretion system where it is phosphorylated on tyrosine residues by an as yet unidentified host cell kinase and wired to eukaryotic signal transduction pathways and cytoskeletal plasticity.

Seropositivity to Helicobacter pylori and CagA protein in schoolchildren of different ages living in urban and rural areas in southern Estonia.

OBJECTIVE: To evaluate Helicobacter pylori and CagA seropositivity in a non-selected group of schoolchildren in southern Estonia, with reference to previous studies where high seroprevalence to H. pylori (87%) and anti-CagA positivity (63%) in an adult population from the same region were found. STUDY POPULATION: A total of 421 schoolchildren selected haphazardly from a random population (n = 1018, ages 9, 12 or 15 years) and living in urban or rural areas. METHODS: H. pylori status was determined by evaluation of IgG antibodies against cell surface proteins of H. pylori, strain CCUG 17874, using standard ELISA. Anti-CagA IgGs were determined by ELISA using a recombinant fragment of CagA (CCUG 17874) as solid-phase antigen. Absorbance values > 0.3 (405 nm) were taken as a CagA-positive result based on a study of 25 sera from H. pylori-negative children. RESULTS: Of the 421 subjects, 235 (56%) were H. pylori-ELISA positive, and 109 out of the 235 (46%) were anti-CagA positive. Neither H. pylori nor CagA positivity were significantly different in girls and boys, or in children aged 9, 12 or 15 years. The H. pylori prevalence rate (118/181, 65%) as well as CagA positivity (64/181, 35%) in rural areas were higher compared with those in towns (117/240, 49% and 54/240, 22%, respectively; P = 0.001 and P = 0.005). CONCLUSION: Of schoolchildren living in southern Estonia, 56% were seropositive to H. pylori. Half of them had anti-CagA antibodies. Schoolchildren living in rural areas were infected significantly more often with CagA-seropositive strains compared with those living in towns.

Seroprevalence of cytotoxin-associated gene A positive Helicobacter pylori strains in Changle, an area with very high prevalence of gastric cancer in south China.

BACKGROUND: Helicobacter pylori, especially the CagA-positive strains, are closely associated with peptic ulcers and gastric cancers. We performed a large scale gastric cancer screening project and examined the prevalence of H. pylori and CagA-positive strains in Changle, China, an area with one of the World's highest gastric cancer mortality. We also compared the prevalence with that in Hong Kong which has one-tenth of the gastric cancer mortality of that in Changle. METHODS: A total of 2424 subjects in Changle and 523 subjects in Hong Kong had endoscopic examination and venesection. Sera were tested for anti-H. pylori antibody and anti-CagA antibody and correlated with endoscopic findings. RESULTS: In Changle, 80. 9% of the subjects were H. pylori carriers. Out of 551 carriers, 408 (74%) were positive for anti-CagA antibody. A total of 76% and 87% of the asymptomatic and gastric cancer patients were positive for anti-CagA antibody, respectively (P > 0.05). Compared to Hong Kong, there was a significantly (P < 0.0001) higher prevalence of CagA-positive strains in asymptomatic subjects in Changle (76%) than in Hong Kong (28%), but not in peptic ulcers or gastric cancers. CONCLUSIONS: Subjects in Changle had a high prevalence of H. pylori infection and a high prevalence of the CagA-positive strains. The contrast in the prevalence of CagA-positive strains, in asymptomatic subjects in two areas with differing gastric cancer mortality, supports the pathogenic role of CagA-positive strains in gastric carcinogenesis.

Activation of activator protein 1 and stress response kinases in epithelial cells colonized by Helicobacter pylori encoding the cag pathogenicity island.

Helicobacter pylori interacts with the apical membrane of the gastric epithelium and induces a number of proinflammatory cytokines/chemokines. The subsequent infiltration of macrophages and granulocytes into the mucosa leads to gastric inflammation accompanied by epithelial degeneration. Gastric diseases, e.g. peptic ulcer or gastric adenocarcinoma, are more common among people infected with H. pylori strains producing VacA (vacuolating cytotoxin A) and possessing a cag (cytotoxin-associated antigen A) pathogenicity island. For the induction of the cytokine/chemokine genes in response to H. pylori, we studied the signaling leading to the nuclear activation of the early response transcription factor activator protein 1 (AP-1). We found that H. pylori strains carrying the pathogenicity island induce activation of AP-1 and nuclear factor kappaB. In contrast to the wild type or an isogenic strain without the vacA gene, isogenic H. pylori strains with mutations in certain cag genes revealed only weak AP-1 and nuclear factor kappaB activation. In respect to the molecular components that direct AP-1 activity, our results indicate a cascade of the cellular stress response kinases c-Jun N-terminal kinase, MAP kinase kinase 4, and p21-activated kinase, and small Rho-GTPases including Rac1 and Cdc42, which contributes to the activation of proinflammatory cytokines/chemokines induced by H. pylori encoding the cag pathogenicity island.

Patients younger than 40 years with gastric carcinoma: Helicobacter pylori genotype and associated gastritis phenotype.

BACKGROUND: In the general population, Helicobacter pylori (H. pylori), particularly the cagA positive strain, has been associated with intestinal-type gastric carcinoma. Gastric carcinomas are rarely observed in patients age < or = 40 years. Host-related factors have been thought to be more important than environmental agents in these early-onset cancers. The aim of this study was to ascertain the possible role of H. pylori infection and that of cagA positive strains in the development of gastric carcinoma in these young patients. METHODS: In this case-control study, 105 gastric carcinoma patients (male-to-female ratio = 1.1; mean age, 34.4 years; range, 16-40 years) and an equal number of controls (matched for gender and age) were retrospectively selected from the same geographic area. The phenotypes of gastritis and H. pylori were histologically assessed, and the presence of the ureC gene, which is indicative of H. pylori infection, and the cagA genotype were determined by polymerase chain reaction. Gastric carcinoma risk was calculated by both univariate and multivariate statistical methods, taking into account the cancer phenotype, the gastritis phenotype detected in both patients and controls, and the H. pylori genotype. RESULTS: For 74 diffuse and 31 intestinal gastric carcinomas, multivariate logistic regression analysis produced results consistent with those of univariate statistical tests, showing a significant association between gastric carcinoma and both H. pylori infection (odds ratio [OR] = 2.79; 95% confidence interval [CI] = 1.52-5.11) and cagA positive status (OR = 2.94; 95% CI = 1.56-5.52). CONCLUSIONS: In young Italian patients with gastric carcinoma, the significant association with cagA positive H. pylori infection suggests that the bacterium has an etiologic role in both diffuse-type and intestinal-type gastric carcinoma.

Immune response to a recombinant fragment of the CagA protein of Helicobacter pylori in blood donors and patients with gastric cancer: relation to ABO(H) blood group phenotype, stage of the disease and tumor morphology.

IgG immune response to CagA was evaluated by enzyme-linked imunosorbent assay (ELISA) using a recombinant fragment of CagA as antigen in 171 patients with gastric cancer and 298 blood donors to determine whether it could be related to the ABO(H) blood group phenotype, stage of cancer or tumor morphology. The CagA-ELISA showed a good specificity (93.5%) and sensitivity (88.5%) as compared with immunoblotting for blot CagA-negative and -positive donors. The Helicobacter pylori seropositive blood group A donors revealed the lowest proportion (37.6%) of strong responders to CagA: A

Helicobacter pylori virulence and genetic geography.

Isolated for the first time in 1982 from human gastric biopsy, Helicobacter pylori is responsible for gastritis, peptic ulcer, and gastric cancer. A pathogenicity island acquired by horizontal transfer, coding for a type IV secretion system, is a major determinant of virulence. The infection is now treated with antibiotics, and vaccines are in preparation. The geographic distribution suggests coevolution of man and Helicobacter pylori.

cagA positive and negative Helicobacter pylori strains are simultaneously present in the stomach of most patients with non-ulcer dyspepsia: relevance to histological damage.

BACKGROUND/AIMS: Infection with Helicobacter pylori strains harbouring the cagA gene (cagA+) is associated with an increased risk of developing peptic ulcer and gastric cancer. The aim of this study was to assess whether H pylori isolates with different cagA status were present in patients with non-ulcer dyspepsia, and whether a variable cagA status is relevant to histological gastric mucosal damage and glandular cell proliferation. METHODS: Well separated H pylori colonies (between 2 and 25) from primary plates, per gastric area, for each of 19 patients with non-ulcer dyspepsia were examined for cagA by hybridisation. Western blotting was used to examine both representative colonies for CagA expression and the patients' sera for antibody response to CagA. Glandular gastric cell proliferation was assessed immunohistochemically. RESULTS: Of the 747 colonies examined, 45.3% were cagA+. All colonies from four patients were cagA+, and all colonies from two patients were cagA-. In 13 patients (68%) both cagA+ and cagA- colonies were found. CagA expression of isolates corresponded to their cagA status. H pylori strains with different CagA molecular masses were present in three patients. Results based on all 19 patients studied showed that the prevalence of cagA+ colonies in areas with mucosal atrophy associated or not with intestinal metaplasia (67.9%) was significantly higher than in normal mucosa (44.7%) and mucosa from patients with chronic gastritis (44.0%) (p < 0.001). High levels of cell proliferation were associated with histological atrophy with or without intestinal metaplasia, but not with the possession of cagA by organisms colonising the same mucosal sites. CONCLUSIONS: Most patients with nonulcer dyspepsia are infected by both cagA+ and cagA- H pylori colonies. The cagA status of infecting organisms may play a role in the development of atrophy and intestinal metaplasia.