Konstantinos S. Papadakos

Strategy To Characterize the Number and Type of Repeating EPIYA Phosphorylation Motifs in the Carboxyl Terminus of CagA Protein in Helicobacter pylori Clinical Isolates

ABSTRACT Cytotoxin-associated gene A (CagA) diversity with regard to EPIYA-A, -B, -C, or -D phosphorylation motifs may play an important role in Helicobacter pylori pathogenesis, and therefore determination of these motifs in H. pylori clinical isolates can become a useful prognostic tool. We propose a strategy for the accurate determination of CagA EPIYA motifs in clinical strains, based upon one-step PCR amplification using primers that flank the EPIYA coding region. We thus analyzed 135 H. pylori isolates derived from 75 adults and 60 children Greek patients. A total of 34 cases were found to be EPIYA PCR negative and were consequently verified as cagA negative by cagA-specific PCR, empty-site cagA PCR, and Western blotting. Sequencing of the remaining 101 PCR-positive amplicons confirmed that an accurate prediction of the number of EPIYA motifs on the basis of size distribution of the PCR products was feasible in all cases. Furthermore, our assay could identify closely related H. pylori subclones within the same patient, harboring different numbers of EPIYA repeats. The prevalence of CagA proteins with three EPIYA motifs (ABC) or four EPIYA motifs (ABCC) was the same within the adult and children groups. However, CagA species with more than four EPIYA motifs were observed exclusively within adults (8.6%), suggesting that CagA-positive strains may acquire additional EPIYA-C motifs throughout adulthood. Our strategy requires no initial cagA screening of the clinical isolates and can accurately predict the number of EPIYA repeats in single or multiple closely related subclones bearing different numbers of EPIYA motifs in their CagA, which may coexist within the same patient.

CagA and VacA Polymorphisms Do Not Correlate with Severity of Histopathological Lesions in Helicobacter pylori-Infected Greek Children

ABSTRACT The presence of various numbers of EPIYA tyrosine phosphorylation motifs in the CagA protein of Helicobacter pylori has been suggested to contribute to pathogenesis in adults. In this prospective study, we characterized H. pylori isolates from symptomatic children, with reference to the diversity of functional EPIYA motifs in the CagA protein and vacA isotypes, and assessed the potential correlation with the histopathological manifestations of the infection. We analyzed 105 H. pylori isolates from 98 children and determined the diversity of EPIYA motifs in CagA by amplification and sequencing of the 3′ variable region of the cagA gene as well as vacA isotypes for the signal, middle, and intermediate regions. CagA phosphorylation and levels of secreted IL-8 were determined following in vitro infection of AGS gastric epithelial cells. Histopathological evaluation of H. pylori colonization, activity, and severity of the associated gastritis was performed according to the updated Sydney criteria. EPIYA A (GLKN[ST]EPIYAKVNKKK), EPIYA B (Q[V/A]ASPEPIY[A/T]QVAKKVNAKI), and EPIYA C (RS[V/A]SPEPIYATIDDLG) motifs were detected in the ABC (46.6%) and ABCC (17.1%) combinations. No isolates harboring more than two EPIYA C motifs in CagA were found. The presence of isogenic strains with variable numbers of CagA EPIYA C motifs within the same patient was detected in seven cases. Occurrence of increasing numbers of EPIYA C motifs correlated strongly with presence of a high-vacuolation (s1 or s2/i1/m1) phenotype and age. A weak positive correlation was observed between vacuolating vacA genotypes and presence of nodular gastritis. However, CagA- and VacA-dependent pathogenicities were not found to contribute to severity of histopathology manifestations in H. pylori-infected children.

Laboratory investigation and phylogenetic analysis of enteroviruses involved in an aseptic meningitis outbreak in Greece during the summer of 2007

BACKGROUND Aseptic meningitis is the most commonly observed CNS infection and is mainly attributed to Non-Polio Enteroviruses (EV). OBJECTIVE Identification and genetic analysis of the EV involved in the recent aseptic meningitis outbreak which occurred in Greece, during the summer of 2007. STUDY DESIGN In total, 213 CSF and faecal samples were examined for EV presence by culture, while enteroviral RNA detection was performed by nucleic acid sequence-based amplification assay (NASBA). EV strains were typed by seroneutralization, as well as nested RT-PCR followed by VP1-2A gene partial sequencing. Phylogenetic analysis was carried out for the identification of the genetic relatedness among the isolated EV strains. RESULTS EV detection rate in CSF and faecal samples was 43.9% and 70.8%, respectively. EV serotyping and VP1 region analysis revealed the predominance of echovirus 4 (ECV4) serotype and the circulation of ECV6, 9, 14, 25, Coxsackie A6, A15, A24 and Coxsackie B1 serotypes. All ECV4 isolates presented a 98.7% similarity in nucleotide sequence, with a Spanish ECV4 strain, isolated during a meningitis outbreak in 2006. CONCLUSIONS It is the first time that ECV4 is associated with an aseptic meningitis outbreak in Greece, during which 9 different EV serotypes were co-circulating. All Greek ECV4 isolates were closely related to the Spanish ECV4 strain. Genetic analysis of the VP1 gene can significantly contribute to the revelation of the endemic EV strains circulation pattern and their phylogenetic relationship with enteroviruses involved in epidemics of distant geographical areas at different time periods.

CagA and VacA polymorphisms are associated with distinct pathological features in Helicobacter pylori-infected adults with peptic ulcer and non-peptic ulcer disease.

ABSTRACT Polymorphic variability in Helicobacterpylori factors CagA and VacA contributes to bacterial virulence. The presence of one CagA EPIYA-C site is an independent risk factor for gastroduodenal ulceration (odds ratio [OR], 4.647; 95% confidence interval [CI], 2.037 to 10.602), while the presence of the vacA i1 allele is a risk factor for increased activity (OR, 5.310; 95% CI, 2.295 to 12.287) and severity of gastritis (OR, 3.862; 95% CI, 1.728 to 8.632).

Presence of Terminal EPIYA Phosphorylation Motifs in Helicobacter pylori CagA Contributes to IL-8 Secretion, Irrespective of the Number of Repeats

CagA protein contributes to pro-inflammatory responses during H. pylori infection, following its intracellular delivery to gastric epithelial cells. Here, we report for the first time in an isogenic background, on the subtle role of CagA phosphorylation on terminal EPIYA-C motifs in the transcriptional activation and expression of IL-8. We utilized isogenic H. pylori mutants of P12 reference strain, expressing CagA with varying number of EPIYA-C motifs and the corresponding phosphorylation defective EPIFA-C motifs while preserving intact the CM multimerization motifs. These mutants had been previously closely scrutinized in terms of type IV secretion system functionality, CagA translocation and its subsequent phosphorylation. Following infection of gastric epithelial cell lines, transcriptional activation of IL-8 gene and secreted IL-8 levels were found to be strictly dependent upon the functionality of the EPIYA-C phosphorylation motifs, as EPIFA-C phosphorylation-deficient CagA expression failed to induce full IL-8 transcriptional activity. Interestingly, levels of IL-8 gene activation and of secreted IL-8 were the same, irrespective of the number of EPIYA-C terminal repeats. We monitored IkBα phosphorylation and confirmed CagA involvement in NF-kB activation. Furthermore, we observed that presence of EPIYA-C functional phosphorylation motifs contributed to NF-kB activation. NF-kB upstream signaling events, such as early ERK1/2 and AKT activation were confirmed to be independent of EPIYA-C phosphorylation. On the contrary, use of TAK1 specific inhibitor 5Z-7-Oxozeaenol resulted in complete arrest of IL-8 secretion, in a dose-dependent manner, irrespective of CagA status. H. pylori-infected TAK1-/- mouse embryonic fibroblasts (MEFs) failed to induce NF-kB activity, unlike the respective control MEFs. CagA and TAK1 were found to immunoprecipitate together, irrespective of CagA EPIYA-C status, thus confirming earlier reports of TAK1 and CagA protein interaction. Our data suggest that CagA may potentially interfere with TAK1 activity during NF-kB activation for IL-8 induction in early H. pylori infection.

Helicobacter pylori CagA protein induces factors involved in the epithelial to mesenchymal transition (EMT) in infected gastric epithelial cells in an EPIYA‐ phosphorylation‐dependent manner

As a result of Helicobacter pylori adhesion to gastric epithelial cells, the bacterial effector cytotoxin‐associated gene A (CagA) is translocated intracellularly, and after hierarchical tyrosine phosphorylation on multiple EPIYA motifs, de‐regulates cellular polarity and contributes to induction of an elongation and scattering phenotype that resembles the epithelial to mesenchymal transition (EMT). Stromelysin‐1/matrix metalloproteinase‐3 (MMP‐3) has been reported to induce a sequence of molecular alterations leading to stable EMT transition and carcinogenesis in epithelial cells. To identify the putative role of CagA protein in MMP‐3 induction, we exploited an experimental H. pylori infection system in gastric epithelial cell lines. We utilized isogenic mutants expressing CagA protein with variable numbers of EPIYA and phosphorylation‐deficient EPIFA motifs, as well as cagA knockout and translocation‐deficient cagE knockout strains. Increased levels of MMP‐3 transcriptional activation were demonstrated by quantitative real time‐PCR for strains with more than two terminal EPIYA phosphorylation motifs in CagA. MMP‐3 expression in total cell lysates and the corresponding culture supernatants was associated with CagA expression and translocation and was dependent on CagA phosphorylation. A CagA EPIYA phosphorylation‐dependent increase in gelatinase and caseinolytic activity was also detected in culture supernatants by zymography. A significant increase in the transcriptional activity of the mesenchymal markers Vimentin, Snail and ZEB1 and the stem cell marker CD44 was observed in the case of CagA containing phosphorylation‐functional EPIYA motifs. Our data suggest that CagA protein induces EMT through EPIYA phosphorylation‐dependent up‐regulation of MMP‐3. Moreover, no significant increase in EMT and stem cell markers was observed following infection with H. pylori strains that cannot effectively translocate CagA protein.

Expansion of European vacA and cagA alleles to East-Asian Helicobacter pylori strains in Cambodia.

Helicobacter pylori infection is associated with gastric cancer (GC). The highest incidence rates have been described in Asia, but regional variations exist that do not match the distribution of infection prevalence rates. The aim of the study was to examine the possible contribution of H. pylori virulence factors to geographic differences in the incidence of GC across East and Southeast Asia. We studied 66 isolates from Cambodian patients that had previously been assigned to two genetic populations based on sequences of seven housekeeping genes, namely hpEurope (n = 34, 51.5%) and hpEastAsia, subpopulation hspEAsia (n = 32, 48.5%). These strains were characterized with respect to vacA polymorphism and cagA status by PCR, and the CagA C-terminal region was sequenced. We also sequenced the complete cagA gene from 10 hpEurope and 10 hspEAsia strains chosen at random. The cagA gene was present in 92.4% of the 66 isolates and was mainly of Western type (n = 36, 59.0%). hspEAsia strains carrying East-Asian CagA and the m1-type vacA allele (15.2%) were less frequent among the 66 Cambodian isolates than reported in East Asian countries, a finding that might partly explain the intermediate incidence of GC in Cambodia, and by extension, in Southeast Asia (except for Vietnam). The observed high prevalence of s1a alleles (34.4%) and Western CagA (28.1%) among hspEAsia strains indicates frequent introgression of European vacA and cagA alleles into East Asian H. pylori strains. This expansion might have severe consequences for individual disease outcome.

A mutagenesis method for the addition and deletion of highly repetitive DNA regions: the paradigm of EPIYA motifs in the cagA gene of Helicobacter pylori.

CagA protein of Western origin Helicobacter pylori isolates contains at its carboxyl‐terminal end repeating types of EPIYA motifs, depending on the surrounding sequence, which dictate hierarchic tyrosine phosphorylation. To produce, in an isogenic background, mutant strains expressing CagA protein with variable numbers of EPIYA‐C terminal motifs, we have adopted a mutagenesis assay using a megaprimer approach.

Molecular and phylogenetic analysis of Greek measles 2010 strains

Although elimination of measles virus (MV) by 2010 was a revised target, a new epidemic has been ongoing in Greece and other European countries. The purpose of this study was the molecular and phylogenetic analysis of the Greek MV circulating strain. Twenty-four MV strains isolated from clinical samples during the 2010 outbreak were genotyped and studied in terms of nucleotide variation and phylogeny. All of the detected viruses were of the D4 genotype, which is circulating in Greece in the Roma population of Bulgarian nationality, the Greek Roma population and the Greek non-minority population, as well as in other EU countries. Phylogenetic analysis revealed that these viruses belonged to subgroup 4 of D4 MV strains. It is essential to continue epidemiological surveillance of measles in Greece to monitor the transmission pattern of the virus and the effectiveness of measles immunization, which eventually will lead to its elimination.