Strains, plasmids and primers used in this study.

<p>Strains, plasmids and primers used in this study.</p

A) Western blot using Ccrp59 specific antiserum, strains as indicated above the lanes.

<p>Equal amounts of protein were loaded onto each lane. B) Fluorescent micrographs of FM4-64 membrane stained cells of KE88-3887 (KE) wild type, KE-59PCAT and KE-59PCAT-K. C) DIC and fluorescent micrographs of FM4-64 membrane stained cells KE-59PCAT-K2. D) Analysis of Ccrp59 production in KE88-3887 (KE) wild type, KE-59PCAT, KE-59PCAT-K and KE-59PCAT-K2 using Ccrp59 specific antiserum Equal amounts of protein were loaded onto each lane. E) Western blot analysis of CagA in KE88-3887 (KE) wild type, KE-59PCAT, KE-59PCAT-K and KE-59PCAT-K2. White scale bar 2 μm.</p

Coiled Coil Rich Proteins (Ccrp) Influence Molecular Pathogenicity of <i>Helicobacter pylori</i>

<div><p>Pathogenicity of the human pathogen <i>Helicobacter pylori</i> relies on its capacity to adapt to a hostile environment and to escape the host response. Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its contribution to virulence. In this study we have explored the influence of <u>c</u>oiled <u>c</u>oil <u>r</u>ich <u>p</u>roteins (Ccrp) cytoskeletal elements on pathogenicity factors of <i>H</i>. <i>pylori</i>. Deletion of any of the <i>ccrp</i> resulted in a strongly decreased activity of the main pathogenicity factor urease. We further investigated their role using <i>in vitro</i> co-culture experiments with the human gastric adenocarcinoma cell line AGS modeling <i>H</i>. <i>pylori</i> - host cell interactions. Intriguingly, host cell showed only a weak “scattering/hummingbird” phenotype, in which host cells are transformed from a uniform polygonal shape into a severely elongated state characterized by the formation of needle-like projections, after co-incubation with any <i>ccrp</i> deletion mutant. Furthermore, co-incubation with the <i>ccrp59</i> mutant resulted in reduced type IV secretion system associated activities, e.g. IL-8 production and CagA translocation/phosphorylation. Thus, in addition to their role in maintaining the helical cell shape of <i>H</i>. <i>pylori</i> Ccrp proteins influence many cellular processes and are thereby crucial for the virulence of this human pathogen.</p></div

A) AGS cells were co-cultured with <i>H</i>. <i>pylori</i> wild type (KE) or the indicated mutants, and IL-8 production was analyzed by ELISA as described in Methods.

<p>Results shown are the averages of four independent experiments and expressed as a percentage relative to wild-type (set at 100%; no error bar); error bars denote standard deviations. Exact percentage values are indicated above the bars. Three asterisks indicate a significant difference with a P value below 0.001 as determined by Student's t test. B) Kinetics of IL-8 production shown as absolute values of <i>H</i>. <i>pylori</i> wild type (G27), <i>ccrp59</i> mutant (G27-CAT) and <i>cagE</i> mutant (G27ΔcagE), respectively; error bars denote standard deviations.</p

A) Analysis of CagA expression and CagA tyrosine phosphorylation in the <i>ccrp59</i> mutant.

<p>Bacterial lysates from <i>H</i>. <i>pylori</i> wild type KE and <i>ccrp59</i> mutant KE-59PCAT were prepared respectively. Each sample that consisted of equivalent amounts of protein was subjected to immunoblotting assay using antiserum against CagA. AGS cells were not infected or infected with these strains at an MOI of 100 for 4h and subjected to immunoblotting analysis using specific antibody against phosphorylated CagA (Cag-P). B) Control experiment showing that CagA of the <i>ccrp59</i> mutant can be phosphorylated in vitro by mixing <i>H</i>. <i>pylori</i> lysates with AGS cell lysates.</p

Western blot analysis of CagY and CagT in KE and KE-59PCAT respectively.

<p>Western blot analysis of CagY and CagT in KE and KE-59PCAT respectively.</p

A) Urease activity of strain KE88-3887 wild-type and <i>ccrp</i> mutants.

<p>Activity is expressed as percentage relative to wild-type in unsupplemented medium (set at 100%; no error bar). Results shown are the averages of six independent experiments; error bars denote standard deviations. Exact percentage values are indicated above the bars. Asterisks indicate a significant difference in urease activity between <i>ccrp</i> mutants and wild-type <i>H</i>. <i>pylori</i> (the P value was <0.001, as determined by Student's t test). B) Western blot using urease specific antiserum and strains as indicated above the lanes. Equal amounts of protein were loaded onto each lane. C) Urease activity of the wild-type strain and the <i>ccrp59</i> mutant in unsupplemented and nickel-supplemented media (as indicated in μM) shown as a percentage relative to wild-type in unsupplemented medium (set at 100%; no error bar). Results shown are the averages of six independent growth experiments; error bars denote standard deviations. Exact percentage values are indicated above the bars. Asterisks indicate a significant difference in urease activity between ccrp mutants and wild-type <i>H</i>. <i>pylori</i> (the P value was <0.001, as determined by Student's t test).</p

A) AGS cells co-incubated either with wild-type <i>H</i>. <i>pylori</i> cells (KE), <i>ccrp</i> deletion mutants as indicated or uninfected (AGS).

<p>Co-incubation was performed at MOI of 100 for 4 h. Cells were visualized by phase-contrast microscopy (BZ-9000E (KEYENCE) microscope) to assess AGS cell morphology. Scale bar, 20 μm. B) Quantification of the percentage of elongated cells from (A). All samples were examined in triplicate in at least three independent experiments. Data are presented as mean value of three independent experiments. For each strain between 1830 and 2800 cells were counted and evaluated. Exact percentage values are indicated above the bars. Asterisks indicate a significant difference between the <i>ccrp</i> mutants and wild-type <i>H</i>. <i>pylori</i> (the P value was <0.001, as determined by Student's t test). C) Bacterial adherence analysis in AGS cells infected with KE88-3887 or <i>ccrp</i> deletion mutants as indicated. AGS cells were infected with <i>H</i>. <i>pylori</i> for 3 h and 6 h, respectively. The number of cfu per cell was determined as described in experimental procedures and normalized to ml.</p

Quantification of CagT localization in immunostaining micrographs.

<p>Quantification of CagT localization in immunostaining micrographs.</p