1.4. La medición en la Gestión de Proyectos de Software
1.4.1. Antecedentes y estado actual
Lone Rasmussen1, Mette Elena Skindersoe2, Karen Krogfelt2
1Department of Clinical Microbiology, Copenhagen, Denmark, 2Department of Microbiological Surveillance and Research, Copenhagen, Denmark
Objectives: Studies have shown that different naturally occurring compounds inhibit the binding of Helicobacter pylori to the human gastric mucosa and different types of human cell lines. To enable identification of novel anti-adhesive compounds
and/or plant extracts we designed a quantitative high throughput adhesion assay and screened several compounds. Methods:
Four different assays where developed and tested-the most effective one is used here. A 96 well plate were coated with AGS cells and grown to confluent growth ON, wash, for Displacement assay incubation with H. pylori 1h allowing bacteria to adhere, add anti-adhesive extract and incubate 1h, wash three times, cell lysis with 0,05% saponin, add alamarBlue-with the active substance resazurin, which in itself is non-fluorescent but is metabolized to the fluorescent compound resorufin.
We show that by adding resazurin to H. pylori adhering to lysed cells, fluorescence intensities can be used to quantify H.
pylori adhesion. Three different assays, with varying orders of compound/bacteria adding where used in the screening:
Displacement, Competition (mix of bacteria/compounds incubated 1 hour before adding to cells) and Blocking (compounds incubate 1 hour, add bacteria and incubate 1 hour). Results: The anti-adhesive effect on 1 H. pylori strain (SS1) is shown, as there was no difference among tested strains. Part of the results is shown here. Conclusion: We developed a quantitative, high throughput, assay which enable identification of novel anti-adhesive compounds. Three compounds almost completely inhibited the adhesion of H. pylori to AGS cells, regardless of the assay order, Displacement, Competition or Blocking.
P69. Significance of Campylobacter IgA and IgG Antibodies in Reactive Arthritis
Christine Reichhuber1, Gottfried Mauff2, Christina Nölting1, Kerstin Schenon3, Erwin Soutschek11Mikrogen GmbH, Neuried, Germany, 2LADR GmbH MVZ Laboratory, Neuruppin, Germany, 3LADR GmbH MVZ Laboratory, Geesthacht, Germany
Introduction: After campylobacter infections reactive arthritis gains increasing importance. Detection of antibodies may characterize post-infectious sequelae. Recombinant immunodominant PEP-antigens, peptidoglycan-associated lipoprotein (OMP18) and ATP/GTP binding protein P39 were described to avoid cross reactivity. Materials: One-hundred patients with enteritis for a mean of 11.3 days (range 1 to 121 days) and campylobacter stool cultures were recruited. Serum samples of 84 patients with all study criteria were collected within a mean of 28,7 days (range 8 to 129 days) after receiving stool samples and investigated by commercial campylobacter ELISA (Mikrogen Campylobacter ELISA IgG, IgA). A questionaire of participating medical practitioners registered complaints of mono- or oligo-arthritis in patients. Results: 23/84 patients had no campylobacter IgG antibodies by recomLine assay (Mikrogen). Sixteen patients reported for arthritic joint and/or muscular fatigue symptoms, 13 had positive scores by recomLine IgG immunoblot, in recomLine IgA only two had a positive score.
All patients were tested in parallel for antibodies by recomLine yersinia immunoblot. Except for four patients the majority had positive IgG results, only two a positive IgA result. None of campylobacter IgA positive patients were yersinia IgA positive.
Impact of Research: From the presented data it may be concluded that campylobacter IgA antibodies are not found in patients with ReA. Since IgA is considered as one of the primary humoral defense mechanisms at the intestinal mucosa, lack of IgA immune response in infected patients could be one of the requirements for further extraintestinal progression and thus an signal for developing sequelae.
P70. The Role of Putative RNA Degradation Proteins in C. jejuni Virulence
Mark Reuter1, Paula Periago2, Fran Mulholland1, Arnoud van Vliet11Institute of Food Research, Norwich, UK, 2Technical University of Cartagena (UPCT), Cartagena, Spain
Over the last ten years, there have been nearly 500,000 confirmed cases of Campylobacter in the UK, with numbers of cases steadily increasing (HPA). The molecular mechanisms of virulence for Campylobacter are still poorly understood, partly due to the lack of tractable virulence models for this organism. In this work, we describe the use of an invertebrate virulence model (Galleria mellonella) to identify a highly interconnected and regulated putative RNA degradation system, involved in C. jejuni virulence. We have previously defined the roles of PAS-domain signalling proteins in energy taxis. A search of the C. jejuni genome revealed the presence of a further PAS-domain linked regulatory protein - Cj1387c. Proteomic analysis of a cj1387c mutant showed that the adjacent protein, Cj1388 was over-expressed, suggesting that Cj1387c represses expression of Cj1388. The Cj1388 protein comprises an Endoribonuclease L-PSP domain, which is proposed to inhibit protein synthesis by mRNA cleavage. The cj1388 mutant is highly attenuated in the Galleria virulence model, while the cj1387c mutant is moderately attenuated. The cj1387c and cj1388 mutants showed wild-type growth, motility, aerotolerance, and peroxide stress response, suggesting that these phenotypes do not confound the virulence phenotype. Further bioinformatic searching of the C. jejuni genome revealed another Endoribonuclease L-PSP domain protein, Cj0327. In the Protein Interaction network, both Cj1388 and Cj0327 are highly connected to proteins that are in turn highly connected in the protein interaction network. In summary, we have identified a novel C. jejuni virulence determinant and continue to characterise this system.
P71. The modulatory effects of H. pylori infection in the DNA repair mechanisms
Juliana Santos1,2, Victor de Almeida1, Marcelo Ribeiro1,21Sao Francisco University Medical School Clinical Pharmacology and Gastroenterology Unit Laboratory of
Microbiology and Molecular Biology, Braganca Paulista/SP, Brazil, 2UNICAMP, Universidade Estadual de Campinas, Genetics and Molecular Biology Post Graduation Program, Campinas/SP, Brazil
The bacterial pathogen H. pylori chronically infects the human gastric mucosa and is the leading risk factor for the development of gastric cancer. The development of gastric cancer is a complex, multistep process involving multiple genetic and epigenetic alterations. Since there are evidences indicating that the bacteria may promote gastric carcinogenesis compromising the integrity and stability of their host’s genome, we evaluated the modulatory effects of H. pylori infection in the DNA repair mechanisms. AGS cells and the H. pylori strain 26695 were employed for co-culture experiments for 4, 12h. To evaluate the effects of infection in the DNA repair mechanisms, it was analyzed the expression of 84 genes using the Human DNA Repair PCR Array (QIAGEN). The methylation pattern of 22 genes was also studied using the Human DNA Repair DNA Methylation PCR Array (QIAGEN). Lastly, the expression of 84 miRNAs was evaluated by means of Human Cancer microRNA PCR Array (QIAGEN). The in silico network analysis was performed using MetaCore v6.13 to characterize the biological pathways connecting miRNA-mRNA pairs. Our data indicated that 48% of the genes were significantly down-regulated after H. pylori infection. On the other hand, 25% of the miRNAs were up-regulated by the bacteria. Hypermethylation was observed among 14% of the genes. In summary, the in silico analysis pointed that H. pylori infection could affect the genome integrity impacting the efficiency of the following DNA repair mechanisms: nucleotide excision repair, mismatch repair, non-homologous end joining repair, and non-homologous recombinational repair.
P72. Gene expression profiling during heat-shock response of Campylobacter (C.) jejuni,
C. coli and C. lariCarolin Riedel1, Greta Gölz1, Konrad U. Förstner2, Cynthia M. Sharma2, Thomas Alter1
1Institute of Food Hygiene, Freie Universität Berlin, Berlin, Germany, 2ZINF Research Center for Infectious Diseases, University of Würzburg, Würzburg, Germany
Although Campylobacter species lack typical stress response mechanisms and sigma factors, they are able to survive in the environment and overcome the barriers along the food chain. The response of C. jejuni to temperatures above the physiological range is sufficiently characterized. Proteomic analyses and gene expression studies revealed an increased expression of common heat-shock genes and synthesis of corresponding proteins. However survival strategies of C. coli and C. lari are still largely unexplored. This study was conducted to examine the response to heat-shock of these two Campylobacter species in comparison to C. jejuni. First, survival rates at 37 °C, 42 °C, 46 °C and 50 °C were mesured. In our study C. jejuni showed a better fitness compared to isolates of C. coli and C. lari. Gene expression studies by real time qPCR revealed an increased expression of different heat-shock genes (dnaK, dnaJ, grpE, groEL, groES, clpB) in C. jejuni, which correlates with earlier studies. The involvement of these chaperones in the heat-shock response of C. coli could be demonstrated as well. In contrast, our results for C. lari suggest alternative heat-shock response mechanisms, since the analyzed known heat-shock genes did not show an altered expression. RNA-Seq data for C. jejuni, C. coli and C. lari provide an explicit global insight in the diverse transcriptome changes as a consequence of temperature upshift from 37 °C to 46 °C.
P73. Functional analysis of Helicobacter polysaccharide lyases
Pradeep Kondadi, Marja-Liisa Hänninen, Mirko RossiFaculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland
Aim: Many bacterial organisms produce polysaccharide lyases (PL) in order to facilitate their invasion of host tissues or to use host polysaccharides as a carbon source. Helicobacter heilmannii s.l. type-2 are the only epsilon-proteobacterial species possessing putative PL genes. The aim of this study was the functional annotation of Helicobacter PLs. Methods: Polysaccharide lyase genes from H. bizzozeronii CIII-1GEN (HBZC1_15820) and H. felis CS1 (Hfelis_14140) were amplified and cloned in pet28b+. His-tag was fused at the N-terminal terminus of the protein. Both proteins were overexpressed in Rosetta(DE3) pLysS E. coli strain and purified using His-Mag Agarose Beads. Capability in degradation of hyaluronic acid (HA), chondroitin sulphate A (CA) and C (CC), dermatan sulphate (DS) and xanthan gum (XG) was evaluated using acid agar method. Major
Findings: Both Helicobacter PLs belong to PL family 8, have similar protein domains and do not harbour signal peptide.
However, they share only 54% of amino acid identity. Only the recombinant (r-)H. bizzozeronii PL showed activity on 3 out of 5 polysaccharides tested, while no lyase activity were observed for r-H. felis PL. r-H. bizzozeronii PL was able to degrade CA and CC and partially DS. Main conclusion: The H. bizzozeronii PL acts as chondroitinase sulphate ABC lyase. Impact of the research: It has been observed that the chief cells of the canine gastric mucosa produce sulphated-moco-saccharide which is depleted on ulcerogenic regimen. The potential role of chondroitinase activity of H. bizzozeronii PL in the pathogenesis of gastric disease of dogs is under investigation.