Transcriptional profiling of Helicobacter pylori Fur- and iron-regulated gene expression

Intracellular iron homeostasis is a necessity for almost all living organisms, since both iron restriction and iron overload can result in cell death. The ferric uptake regulator protein, Fur, controls iron homeostasis in most Gram-negative bacteria. In the human gastric pathogen Helicobacter pylori, Fur is thought to have acquired extra functions to compensate for the relative paucity of regulatory genes. To identify H. pylori genes regulated by iron and Fur, we used DNA array-based transcriptional profiling with RNA isolated from H. pylori 26695 wild-type and fur mutant cells grown in iron-restricted and iron-replete conditions. Sixteen genes encoding proteins involved in metal metabolism, nitrogen metabolism, motility, cell wall synthesis and cofactor synthesis displayed iron-dependent Fur-repressed expression. Conversely, 16 genes encoding proteins involved in iron storage, respiration, energy metabolism, chemotaxis, and oxygen scavenging displayed iron-induced Fur-dependent expression. Several Fur-regulated genes have been previously shown to be essential for acid resistance or gastric colonization in animal models, such as those encoding the hydrogenase and superoxide dismutase enzymes. Overall, there was a partial overlap between the sets of genes regulated by Fur and those previously identified as growth-phase, iron or acid regulated. Regulatory patterns were confirmed for five selected genes using Northern hybridization. In conclusion, H. pylori Fur is a versatile regulator involved in many pathways essential for gastric colonization. These findings further delineate the central role of Fur in regulating the unique capacity of H. pylori to colonize the human stomach

A Novel System of Cytoskeletal Elements in the Human Pathogen Helicobacter pylori

Pathogenicity of the human pathogen Helicobacter pylori relies upon its capacity to adapt to a hostile environment and to escape from the host response. Therefore, cell shape, motility, and pH homeostasis of these bacteria are specifically adapted to the gastric mucus. We have found that the helical shape of H. pylori depends on coiled coil rich proteins (Ccrp), which form extended filamentous structures in vitro and in vivo, and are differentially required for the maintenance of cell morphology. We have developed an in vivo localization system for this pathogen. Consistent with a cytoskeleton-like structure, Ccrp proteins localized in a regular punctuate and static pattern within H. pylori cells. Ccrp genes show a high degree of sequence variation, which could be the reason for the morphological diversity between H. pylori strains. In contrast to other bacteria, the actin-like MreB protein is dispensable for viability in H. pylori, and does not affect cell shape, but cell length and chromosome segregation. In addition, mreB mutant cells displayed significantly reduced urease activity, and thus compromise a major pathogenicity factor of H. pylori. Our findings reveal that Ccrp proteins, but not MreB, affect cell morphology, while both cytoskeletal components affect the development of pathogenicity factors and/or cell cycle progression

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

Strains, plasmids and primers used in this study.

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

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

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) 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