Franck Cantet

Which test to use to detect Helicobacter pylori infection in patients with low-grade gastric mucosa-associated lymphoid tissue lymphoma?

Helicobacter pylori is involved in the pathogenesis of lymphoma of the gastric mucosa-associated lymphoid tissue (MALT). Because gastric MALT lymphoma is a rare disease, few studies comparing the accuracy of diagnostic tests in this group of patients have been carried out, and only a limited number of tests (essentially histological) were performed. The aim of our study was to compare the results of four different diagnostic methods used to detect H. pylori (histology, culture, polymerase chain reaction, and serology) in a prospective multicenter study. A patient was considered to be H. pylori positive if culture or histology was positive. During the period 1995–2000, a total of 90 patients with low-grade gastric MALT lymphoma were enrolled. Results for the four tests were available for 56 patients (62.2%). Among these patients, the four tests were concordant in 35 cases (62.5%), i.e., were positive in 19 cases (33.9%) and negative in 16 patients (17.8%). Histology (39/40 positive, 97.5%) and serology (38/40 positive, 95.0%) were the most sensitive tests. Polymerase chain reaction (PCR) and culture were positive in 52.5% and 50%, respectively. The cagA gene was detected in 47.4% of the strains.

Characterization of the genes rdxA and frxA involved in metronidazole resistance in Helicobacter pylori

Metronidazole (Mtz) resistance in Helicobacter pylori has been found to be associated with mutations in rdxA, a gene encoding an oxygen-insensitive NADPH nitroreductase, and enhanced by mutations in frxA, a gene encoding a NAD(P)H-flavin oxidoreductase. The roles of these two genes in Mtz resistance in H. pylori were examined in this study. The rdxA and frxA genes were sequenced in nine pairs of strains isolated from biopsies obtained from patients before and after failed eradication treatments which included Mtz and resulted in the appearance of resistant strains. Metronidazole resistance could be explained in seven of these pairs of strains by mutations in rdxA and frxA. However, in one pair of strains, rdxA was identical in the susceptible and resistant strains, and only changes in frxA were observed; and in another pair, neither rdxA nor frxA were different in the susceptible and resistant strains. Sequencing of the upstream region of frxA and of the recA gene in the latter pair of strains did not reveal any mutations. To establish whether mutations in frxA alone could be involved in Mtz resistance, a resistant Escherichia coli strain transformed with the frxA of a Mtz susceptible H. pylori strain was rendered susceptible, and transformation with a mutated H. pylori frxA gene under the same conditions did not change the resistant E. coli phenotype. The results suggested that a Mtz resistance phenotype may arise in H. pylori without mutations in rdxA or frxA, or with mutations only in frxA.

Identification of OmpA, a Coxiella burnetii protein involved in host cell invasion, by multi-phenotypic high-content screening.

Coxiella burnetii is the agent of the emerging zoonosis Q fever. This pathogen invades phagocytic and non-phagocytic cells and uses a Dot/Icm secretion system to co-opt the endocytic pathway for the biogenesis of an acidic parasitophorous vacuole where Coxiella replicates in large numbers. The study of the cell biology of Coxiella infections has been severely hampered by the obligate intracellular nature of this microbe, and Coxiella factors involved in host/pathogen interactions remain to date largely uncharacterized. Here we focus on the large-scale identification of Coxiella virulence determinants using transposon mutagenesis coupled to high-content multi-phenotypic screening. We have isolated over 3000 Coxiella mutants, 1082 of which have been sequenced, annotated and screened. We have identified bacterial factors that regulate key steps of Coxiella infections: 1) internalization within host cells, 2) vacuole biogenesis/intracellular replication, and 3) protection of infected cells from apoptosis. Among these, we have investigated the role of Dot/Icm core proteins, determined the role of candidate Coxiella Dot/Icm substrates previously identified in silico and identified additional factors that play a relevant role in Coxiella pathogenesis. Importantly, we have identified CBU_1260 (OmpA) as the first Coxiella invasin. Mutations in ompA strongly decreased Coxiella internalization and replication within host cells; OmpA-coated beads adhered to and were internalized by non-phagocytic cells and the ectopic expression of OmpA in E. coli triggered its internalization within cells. Importantly, Coxiella internalization was efficiently inhibited by pretreating host cells with purified OmpA or by incubating Coxiella with a specific anti-OmpA antibody prior to host cell infection, suggesting the presence of a cognate receptor at the surface of host cells. In summary, we have developed multi-phenotypic assays for the study of host/pathogen interactions. By applying our methods to Coxiella burnetii, we have identified the first Coxiella protein involved in host cell invasion.

Quantification of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae in French Mediterranean coastal lagoons.

Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae are human pathogens. Little is known about these Vibrio spp. in the coastal lagoons of France. The purpose of this study was to investigate their incidence in water, shellfish and sediment of three French Mediterranean coastal lagoons using the most probable number-polymerase chain reaction (MPN-PCR). In summer, the total number of V. parahaemolyticus in water, sediment, mussels and clams collected from the three lagoons varied from 1 to >1.1 × 10³ MPN/l, 0.09 to 1.1 × 10³ MPN/ml, 9 to 210 MPN/g and 1.5 to 2.1 MPN/g, respectively. In winter, all samples except mussels contained V. parahaemolyticus, but at very low concentrations. Pathogenic (tdh- or trh2-positive) V. parahaemolyticus were present in water, sediment and shellfish samples collected from these lagoons. The number of V. vulnificus in water, sediment and shellfish samples ranged from 1 to 1.1 × 10³ MPN/l, 0.07 to 110 MPN/ml and 0.04 to 15 MPN/g, respectively, during summer. V. vulnificus was not detected during winter. V. cholerae was rarely detected in water and sediment during summer. In summary, results of this study highlight the finding that the three human pathogenic Vibrio spp. are present in the lagoons and constitute a potential public health hazard.

Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development

Significance The biogenesis of a replicative vacuole is an essential step of Coxiella burnetii infections and involves the hijack of several host membrane trafficking pathways. Here we describe Coxiella vacuolar protein B (CvpB) as a Coxiella effector that interacts with phosphoinositides on host cell membranes and manipulates phosphatidylinositol 3-phosphate [PI(3)P] metabolism for optimal Coxiella-containing vacuole (CCV) development. This is achieved by perturbing the activity of the phosphatidylinositol 5-kinase PIKfyve, leading to an enrichment of PI(3)P on CCV membranes, which is required for the autophagy machinery to mediate CCV homotypic fusion. The importance of this process is highlighted by a homotypic fusion defect between CCVs in cells infected with CvpB Coxiella mutants, which translates into an attenuated virulence in the insect model Galleria mellonella. The Q fever bacterium Coxiella burnetii replicates inside host cells within a large Coxiella-containing vacuole (CCV) whose biogenesis relies on the Dot/Icm-dependent secretion of bacterial effectors. Several membrane trafficking pathways contribute membranes, proteins, and lipids for CCV biogenesis. These include the endocytic and autophagy pathways, which are characterized by phosphatidylinositol 3-phosphate [PI(3)P]-positive membranes. Here we show that the C. burnetii secreted effector Coxiella vacuolar protein B (CvpB) binds PI(3)P and phosphatidylserine (PS) on CCVs and early endosomal compartments and perturbs the activity of the phosphatidylinositol 5-kinase PIKfyve to manipulate PI(3)P metabolism. CvpB association to early endosome triggers vacuolation and clustering, leading to the channeling of large PI(3)P-positive membranes to CCVs for vacuole expansion. At CCVs, CvpB binding to early endosome- and autophagy-derived PI(3)P and the concomitant inhibition of PIKfyve favor the association of the autophagosomal machinery to CCVs for optimal homotypic fusion of the Coxiella-containing compartments. The importance of manipulating PI(3)P metabolism is highlighted by mutations in cvpB resulting in a multivacuolar phenotype, rescuable by gene complementation, indicative of a defect in CCV biogenesis. Using the insect model Galleria mellonella, we demonstrate the in vivo relevance of defective CCV biogenesis by highlighting an attenuated virulence phenotype associated with cvpB mutations.

Helicobacter pylori infection in patients consulting gastroenterologists in France : prevalence is linked to gender and region of residence

Background Because of limited data on the epidemiology of Helicobacter pylori in France, the prevalence of this infection by region and its associated risk factors were studied between 1995 and 1997 among patients consulting a representative sample of gastroenterologists by region. Method A cross-sectional study was performed. Patients consulting gastroenterologists for whatever reason were screened for H. pylori infection determined by specific salivary IgG. A questionnaire was filled out by the gastroenterologist. A multivariate analysis was performed with all relevant variables. Results 3153 patients were included. The mean age was 48.5 years; 51.8% were women. After stratification by patients consulting for upper digestive tract (UDT) and non-UDT symptoms, H. pylori infection was found to be more prevalent, in both groups, for characteristics such as being born in a developing country, overcrowding during childhood, and primary educational level. Interestingly, gender (odds ratio ORUDT for women = 0.7 [95% CI 0.5–0.8] and ORnon−UDT for women = 0.6 [95% CI 0.5–0.8]) and living in a region other than the south-west (ORUDT varying from 1.5 to 2.0 and ORnon−UDT varying from 1.3 to 2.1, depending on the region) was associated with the odds of prevalent infection. Conclusion These findings show (1) that gender deserves more attention in the epidemiology of H. pylori and (2) a regional disparity in France regarding H. pylori infection.

Cytoskeletal Rearrangements Induced by Helicobacter pylori Strains in Epithelial Cell Culture

The relationship between Helicobacter pylori adherence, cytotoxin production, and modification of the cytoskeletal structure was investigated by studying the effects of 12 H. pylori strains cocultured with Hep-2 epithelial cells. Bacterial strains were isolated from patients with peptic ulcer disease or nonulcer dyspepsia. Presence of the cag pathogenicity island and vacA subtypes of the strains were determined as was the production of vacuolating cytotoxin. We found that cytoskeletal rearrangements, as observed by confocal microscopy after double staining of the bacteria and the cell actin with Texas red and fluorescein-conjugated phalloidin, respectively, occurred essentially when the strains were cytotoxin producers and that the supernatants alone could also lead to these modifications.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Discriminatory power of RAPD, PCR-RFLP and southern blot analyses of ureCD or ureA gene probes on Helicobacter pylori isolates.

The genetic diversity of 33 Nigerian Helicobacter pylori isolates were studied using RAPD, PCR-RFLP and Southern blot analysis of ureA or ureCD gene probes. RAPD was able to distinguish the following number of isolates using the primers 3880 : 5ʹ-AAGAGCCCGT-3ʹ (28), 3881 :5ʹ-AACGCGCAAC-3ʹ (33) and OPH8 :5ʹ-GAAACACCCC-3ʹ (25). Southern blot analysis using the ureCD probe was also able to distinguish the 12 isolates tested into ten different patterns. The PCR-RFLP technique distinguished all 33 isolates into six types. In conclusion, considering typeability, discriminatory power, and convenience, RAPD with the 3881 primer was considered the most useful technique.

Multiple substrate usage of Coxiella burnetii to feed a bipartite-type metabolic network

The human pathogen Coxiella burnetii causes Q-fever and is classified as a category B bio-weapon. Exploiting the development of the axenic growth medium ACCM-2, we have now used 13C-labeling experiments and isotopolog profiling to investigate the highly diverse metabolic network of C. burnetii. To this aim, C. burnetii RSA 439 NMII was cultured in ACCM-2 containing 5 mM of either [U-13C3]serine, [U-13C6]glucose, or [U-13C3]glycerol until the late-logarithmic phase. GC/MS-based isotopolog profiling of protein-derived amino acids, methanol-soluble polar metabolites, fatty acids, and cell wall components (e.g., diaminopimelate and sugars) from the labeled bacteria revealed differential incorporation rates and isotopolog profiles. These data served to decipher the diverse usages of the labeled substrates and the relative carbon fluxes into the core metabolism of the pathogen. Whereas, de novo biosynthesis from any of these substrates could not be found for histidine, isoleucine, leucine, lysine, phenylalanine, proline and valine, the other amino acids and metabolites under study acquired 13C-label at specific rates depending on the nature of the tracer compound. Glucose was directly used for cell wall biosynthesis, but was also converted into pyruvate (and its downstream metabolites) through the glycolytic pathway or into erythrose 4-phosphate (e.g., for the biosynthesis of tyrosine) via the non-oxidative pentose phosphate pathway. Glycerol efficiently served as a gluconeogenetic substrate and could also be used via phosphoenolpyruvate and diaminopimelate as a major carbon source for cell wall biosynthesis. In contrast, exogenous serine was mainly utilized in downstream metabolic processes, e.g., via acetyl-CoA in a complete citrate cycle with fluxes in the oxidative direction and as a carbon feed for fatty acid biosynthesis. In summary, the data reflect multiple and differential substrate usages by C. burnetii in a bipartite-type metabolic network, resembling the overall topology of the related pathogen Legionella pneumophila. These strategies could benefit the metabolic capacities of the pathogens also as a trait to adapt for replication under intracellular conditions.