[Show abstract][Hide abstract] ABSTRACT: Pathogenicity of the human pathogen Helicobacter pylori 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 coiled coil rich proteins (Ccrp) cytoskeletal elements on pathogenicity factors of H. pylori. Deletion of any of the ccrp resulted in a strongly decreased activity of the main pathogenicity factor urease. We further investigated their role using in vitro co-culture experiments with the human gastric adenocarcinoma cell line AGS modeling H. pylori - 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 ccrp deletion mutant. Furthermore, co-incubation with the ccrp59 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 H. pylori Ccrp proteins influence many cellular processes and are thereby crucial for the virulence of this human pathogen.
PLoS ONE 03/2015; 10(3):e0121463. DOI:10.1371/journal.pone.0121463 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Gram-negative, non-motile, rod shaped, facultative anaerobic and acid tolerant bacterial strain DKE6(T) was isolated from an acidic biofilm (pH 2.5) that was harvested in the pyrite mine 'Drei Kronen und Ehrt' in Germany. The strain grows optimally at pH 5.5, between 25°C and 30°C and with casein as carbon and energy source. Although a variety of sugars was tested as growth substrates, none of these supported growth of the strain. During casein consumption the DKE6(T) produces ammonium which leads to an alkalization of the medium. This is a possible strategy to raise the pH in direct vicinity of the cell and hence modulate the pH towards the growth optimum. The predominant fatty acids are iso-C(11:0) 3-OH, anteiso-C(15:0), iso-C(17:0) and iso-C(17:1) ω9c. The DNA G+C content is 66.6%. Strain DKE6(T) is not able to oxidize iron or thiosulfate. Iron reduction was detected. The strain shows 93.3% 16S rRNA gene sequence similarity to the closest related cultivated species Dokdonella koreensis and 93.2% or less to other type species of the Gammaproteobacteria. On the basis of physiological and biochemical data, the new strain is considered to represent a member of a novel genus in the class of the Gammaproteobacteria. We propose the name Metallibacterium scheffleri gen. nov., sp. nov., for the new isolate. The type strain is DKE6(T) (DSM 24874(T); JCM 17596(T)).
International Journal of Systematic and Evolutionary Microbiology 04/2013; 63(4):1499-1504. DOI:10.1099/ijs.0.042986-0 · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Of the various kinds of cell division, the most common mode is binary fission, the division of a cell into two morphological identical daughter cells. However in the case of asymmetric cell division, C. crescentus produces two morphological and functional distinct cell types. Here, we have studied cell cycle progression of the human pathogen Helicobacter pylori using a functional GFP-fusion of FtsZ protein and membrane staining. In small cells, representing newly divided cells, FtsZ localizes to a single cell pole. During the cell cycle, spiral intermediates are formed, until a FtsZ-ring is positioned with little precision, such that central as well as acentral rings can be observed. Daughter cells showed considerably different sizes, suggesting that H. pylori divides asymmetrically. FRAP analyses demonstrate that the H. pylori FtsZ-ring is about as dynamic as that of E. coli but that polar assemblies show less turnover. Strikingly our results demonstrate that H. pylori cell division follows a different route from that in E. coli and B. subtilis. It is also different from that in C. crescentus, where cytokinesis regulation proteins like MipZ play a role. Therefore this report provides the first cell biological analysis of FtsZ dynamics in the human pathogen H. pylori and even in ε-proteobacteria to our knowledge. In addition, analysis of the filament architecture of H. pylori and E. coli FtsZ filaments in the heterologous system of D. melanogaster S2 Schneider cells revealed that both have different filamentation properties in vivo suggesting a unique intrinsic characteristic of each protein.
Journal of bacteriology 01/2013; 195(7). DOI:10.1128/JB.01490-12 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Helicobacter pylori colonizes the stomachs of at least half of the world's human population. The role of the oral cavity in this colonization is not clear and there are, to date, no comprehensive data that clearly demonstrate the isolation of this bacterium from the oral cavity. The aim of this study was to evaluate the prevalence of H. pylori in the oral cavity of 15 patients who tested positive for H. pylori. A comprehensive dental examination of all patients was conducted. Samples were taken from supragingival and subgingival plaque, saliva, periapical exudates and tongue swabs. All samples were taken before the application of antibiotics. A total of 163 oral samples were investigated by PCR using two different H. pylori-specific primer pairs. A PCR inhibition control using a modified plasmid was always included for the most specific primer pair. In addition, a culture technique was used to confirm PCR results. Despite a PCR detection limit of 10(2) bacteria ml(-1), out of 14 patients, H. pylori could not be detected in any of the samples taken. In one patient, H. pylori-positive PCR signals were obtained in two samples using only one primer pair. H. pylori could not be cultivated from these two PCR-positive samples; therefore, no correlation to oral colonization status could be established. This study challenges the misleading preconception that H. pylori resides in the human oral cavity and suggests that this bacterium should be considered transient and independent of the oral status. To date, positive PCR results for H. pylori in the oral cavity have been overestimated and not critically interpreted in literature.
Journal of Medical Microbiology 04/2012; 61(Pt 8):1146-52. DOI:10.1099/jmm.0.043893-0 · 2.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We identified two additional genes of Helicobacter pyloriencoding Ccrp proteins. All four Ccrps have different multimerization and filamentation properties and different types of
smallest subunits and do not copurify, suggesting a system of individual Ccrp filaments. Despite the presence of morphologically
unaltered flagella, all ccrpmutants displayed significantly reduced motility.
Journal of bacteriology 06/2011; 193(17):4523-30. DOI:10.1128/JB.00231-11 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytochrome oxidases are perfect model substrates for analyzing the assembly of multisubunit complexes because the need for
cofactor incorporation adds an additional level of complexity to their assembly. cbb3-type cytochrome c oxidases (cbb3-Cox) consist of the catalytic subunit CcoN, the membrane-bound c-type cytochrome subunits CcoO and CcoP, and the CcoQ subunit, which is required for cbb3-Cox stability. Biogenesis of cbb3-Cox proceeds via CcoQP and CcoNO subcomplexes, which assemble into the active cbb3-Cox. Most bacteria expressing cbb3-Cox also contain the ccoGHIS genes, which encode putative cbb3-Cox assembly factors. Their exact function, however, has remained unknown. Here we analyzed the role of CcoH in cbb3-Cox assembly and showed that CcoH is a single spanning-membrane protein with an N-terminus-out-C-terminus-in (Nout-Cin) topology. In its absence, neither the fully assembled cbb3-Cox nor the CcoQP or CcoNO subcomplex was detectable. By chemical cross-linking, we demonstrated that CcoH binds primarily
via its transmembrane domain to the CcoP subunit of cbb3-Cox. A second hydrophobic stretch, which is located at the C terminus of CcoH, appears not to be required for contacting
CcoP, but deleting it prevents the formation of the active cbb3-Cox. This suggests that the second hydrophobic domain is required for merging the CcoNO and CcoPQ subcomplexes into the active
cbb3-Cox. Surprisingly, CcoH does not seem to interact only transiently with the cbb3-Cox but appears to stay tightly associated with the active, fully assembled complex. Thus, CcoH behaves more like a bona
fide subunit of the cbb3-Cox than an assembly factor per se.
Journal of bacteriology 10/2010; 192(24):6378-89. DOI:10.1128/JB.00988-10 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Author Summary
The human pathogen Helicobacter pylori lives in the hostile environment of the human stomach. H. pylori possesses a spiral shape and high motility that enable the bacterium to swim through the stomach lumen and to come into close contact with epithelial cells. High urease activity in the bacterium counterbalances the low pH within the stomach, in order to persist within the viscous mucus layer. In this work, we analysed the molecular basis of the spiral structure of H. pylori. We demonstrate that the helical cell shape depends on so called coiled coil rich proteins (Ccrp), which form extended filamentous structures in vitro and in vivo, and are differentially required for the maintenance of proper cell morphology. In most bacteria analysed so far, the actin-like protein MreB affects cell morphology. Contrarily, H. pylori MreB is not involved in the maintenance of cell shape, but affects the progression of the cell cycle. Mutant cells were highly elongated, characteristic for a delay in cell division, and contained non-segregated chromosomes. The persistence of H. pylori in the hostile environment of the human stomach depends on the activity of urease. Interestingly, mreB mutant cells displayed significantly reduced urease activity, revealing a novel connection between the cytoskeletal element and an enzyme, and thus with pathogenicity. These experiments show that H. pylori has a novel type of system setting up helical cell shape, which has not yet been described for any bacterium. Our work will allow studying H. pylori cell cycle and pathogenicity at a new visual level.
[Show abstract][Hide abstract] ABSTRACT: Here we describe that the Helicobacter pylori sensor kinase produced by HP1364 and the response regulator produced by HP1365 and designated CrdS and CrdR, respectively,
are both required for transcriptional induction of the H. pylori copper resistance determinant CrdA by copper ions. CrdRS-deficient mutants lacked copper induction of crdA expression and were copper sensitive. A direct role of CrdR in transcriptional regulation of crdA was confirmed by in vitro binding of CrdR to the crdA upstream region. A 21-nucleotide sequence located near the crdA promoter was shown to be required for CrdR binding.
Journal of Bacteriology 08/2005; 187(13):4683-8. DOI:10.1128/JB.187.13.4683-4688.2005 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Maintaining iron homeostasis is a necessity for all living organisms, as free iron augments the generation of reactive oxygen species like superoxide anions, at the risk of subsequent lethal cellular damage. The iron-responsive regulator Fur controls iron metabolism in many bacteria, including the important human pathogen Helicobacter pylori, and thus is directly or indirectly involved in regulation of oxidative stress defense. Here we demonstrate that Fur is a direct regulator of the H. pylori iron-cofactored superoxide dismutase SodB, which is essential for the defense against toxic superoxide radicals. Transcription of the sodB gene was iron induced in H. pylori wild-type strain 26695, resulting in expression of the SodB protein in iron-replete conditions but an absence of expression in iron-restricted conditions. Mutation of the fur gene resulted in constitutive, iron-independent expression of SodB. Recombinant H. pylori Fur protein bound with low affinity to the sodB promoter region, but addition of the iron substitute Mn2+ abolished binding. The operator sequence of the iron-free form of Fur, as identified by DNase I footprinting, was located directly upstream of the sodB gene at positions -5 to -47 from the transcription start site. The direct role of Fur in regulation of the H. pylori sodB gene contrasts with the small-RNA-mediated sodB regulation observed in Escherichia coli. In conclusion, H. pylori Fur is a versatile regulator involved in many pathways essential for gastric colonization, including superoxide stress defense.
Journal of Bacteriology 07/2005; 187(11):3687-92. DOI:10.1128/JB.187.11.3687-3692.2005 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Research in the last year has provided new insights into the function of the the cag-associated type IV secretion system and the vacuolating toxin VacA. A quite new aspect was disclosed by the finding that Helicobacter pylori in Mongolian gerbils colonizes a very distinct topology in the gastric mucous layer, obviously providing optimal conditions for long-term survival. Further research activities focused on H. pylori ammonia and metal metabolism as well as on bacterial stress defence mechanisms. Differential expression of approximately 7% of the bacterial genome was found at low pH suggesting that H. pylori has evolved a multitude of acid-adaptive mechanisms. VacA was shown to interrupt phagosome maturation in macrophage cell lines as well as to modulate and interfere with T lymphocyte immunological functions. Gastric mucosa as well as the H. pylori-infected epithelial cell line AGS strongly express IL-8 receptor A and B, which might contribute to the augmentation of the inflammatory response. Accumulating evidence implicates genetic variation in the inflammatory response to H. pylori in the etiology of the increased risk of gastric cancer after H. pylori infection. The chronic imbalance between apoptosis and cell proliferation is the first step of gastric carcinogenesis. In this regard, it was demonstrated that coexpression of two H. pylori proteins, CagA and HspB, in AGS cells, caused an increase in E2F transcription factor, cyclin D3, and phosphorylated retinoblastoma protein. Taken together, we now have a better understanding of the role of different virulence factors of H. pylori. There is still a lot to be learned, but the promising discoveries summarized here, demonstrate that the investigation of the bacterial survival strategies will give novel insights into pathogenesis and disease development.
[Show abstract][Hide abstract] ABSTRACT: Poxviruses have evolved various strategies to counteract the host immune response, one of which is based on the expression of soluble cytokine receptors. Using various biological assays, we detected a chicken interferon-gamma (chIFN-gamma)-neutralizing activity in supernatants of fowlpox virus (FPV)-infected cells that could be destroyed by trypsin treatment. Secreted viral proteins were purified by affinity chromatography using matrix-immobilized chIFN-gamma, followed by two-dimensional gel electrophoresis. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis indicated that the viral IFN-gamma-binding protein in question was encoded by the FPV gene 016. The chicken IFN-gamma binding and neutralizing activity of the recombinant FPV016 protein was confirmed using supernatants of cells infected with a recombinant vaccinia virus that lacked its own IFN-gamma-binding protein but instead expressed the FPV016 gene. The FPV016 gene product also neutralized the activity of duck and human IFN-gamma but failed to neutralize the activity of mouse and rat IFN-gamma. Unlike previously known cellular and poxviral IFN-gamma receptors, which all contain fibronectin type III domains, the IFN-gamma-binding protein of FPV contains an immunoglobulin domain. Remarkably, it exhibits no significant homology to any known viral or cellular protein. Because IFN-gamma receptors of birds have not yet been characterized at the molecular level, the possibility remains that FPV016 represents a hijacked chicken gene and that avian and mammalian IFN-gamma receptors have fundamentally different primary structures.
[Show abstract][Hide abstract] ABSTRACT: Mechanisms involved in maintaining cytoplasmic metal ion homeostasis play a central role in the adaptation of Helicobacter pylori to the changing gastric environment. An investigation of the global regulatory responses to copper ions by using RNA profiling
with a threshold factor of 4.0 revealed that copper induces transcription of 19 H. pylori genes and that only the ferritin gene pfr is repressed. The 57-fold copper induction identified the HP1326 gene encoding an H. pylori-specific protein as a candidate for a novel copper resistance determinant. The HP1326 gene is expressed as a monocistronic
unit, and two small HP1326 mRNAs are copper induced. The HP1326 protein is secreted and is required for copper resistance
maintained by cytoplasmic copper homeostasis, as H. pylori HP1326 mutants were copper sensitive and displayed increased copper induction of HP1326 transcription as well as elevated
copper repression of ferritin synthesis. The clear copper-sensitive phenotype displayed by H. pylori HP1327 and HP1328 mutants provides strong evidence that the HP1326 protein, together with the signal peptide site of the
H. pylori-specific protein HP1327, whose gene is located downstream from that encoding HP1326, and the CzcB and CzcA metal efflux system
component homologs HP1328 and HP1329, constitutes a novel type of copper efflux pump, as discussed below. The HP1329 gene
could not be inactivated, but the 14-fold transcriptional copper induction determined by RNA profiling points towards a function
of the encoded CzcA homolog in copper resistance. In summary, results from RNA profiling identified the novel H. pylori-specific copper resistance determinants CrdA (HP1326) and CrdB (HP1327), which are required for adaptation to copper-rich
Journal of Bacteriology 01/2003; 184(23):6700-8. DOI:10.1128/JB.184.23.6700-6708.2002 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We show here that Mg(2+) acquisition by CorA is essential for Helicobacter pylori in vitro, as corA mutants did not grow in media without Mg(2+) supplementation. Complementation analysis performed with an Escherichia coli corA mutant revealed that H. pylori CorA transports nickel and cobalt in addition to Mg(2+). However, Mg(2+) is the dominant CorA substrate, as the corA mutation affected neither cobalt and nickel resistance nor nickel induction of urease in H. pylori. The drastic Mg(2+) requirement (20 mM) of H. pylori corA mutants indicates that CorA plays a key role in the adaptation to the low-Mg(2+) conditions predominant in the gastric environment.
Infection and Immunity 08/2002; 70(7):3930-4. DOI:10.1128/IAI.70.7.3930-3934.2002 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The reactivity of the essential element iron necessitates a concerted expression of ferritins, which mediate iron storage in a nonreactive state. Here we have further established the role of the Helicobacter pylori ferritin Pfr in iron metabolism and gastric colonization. Iron stored in Pfr enabled H. pylori to multiply under severe iron starvation and protected the bacteria from acid-amplified iron toxicity, as inactivation of the pfr gene restricted growth of H. pylori under these conditions. The lowered total iron content in the pfr mutant, which is probably caused by decreased iron uptake rates, was also reflected by an increased resistance to superoxide stress. Iron induction of Pfr synthesis was clearly diminished in an H. pylori feoB mutant, which lacked high-affinity ferrous iron transport, confirming that Pfr expression is mediated by changes in the cytoplasmic iron pool and not by extracellular iron. This is well in agreement with the recent discovery that iron induces Pfr synthesis by abolishing Fur-mediated repression of pfr transcription, which was further confirmed here by the observation that iron inhibited the in vitro binding of recombinant H. pylori Fur to the pfr promoter region. The functions of H. pylori Pfr in iron metabolism are essential for survival in the gastric mucosa, as the pfr mutant was unable to colonize in a Mongolian gerbil-based animal model. In summary, the pfr phenotypes observed give new insights into prokaryotic ferritin functions and indicate that iron storage and homeostasis are of extraordinary importance for H. pylori to survive in its hostile natural environment.
Infection and Immunity 08/2002; 70(7):3923-9. DOI:10.1128/IAI.70.7.3923-3929.2002 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The only known niche of the human pathogen Helicobacter pylori is the gastric mucosa, where large fluctuations of pH occur, indicating that the bacterial response and resistance to acid
are important for successful colonization. One of the few regulatory proteins in the H. pylori genome is a homologue of the ferric uptake regulator (Fur). In most bacteria, the main function of Fur is the regulation
of iron homeostasis. However, in Salmonella enterica serovar Typhimurium, Fur also plays an important role in acid resistance. In this study, we determined the role of the H. pylori Fur homologue in acid resistance. Isogenic fur mutants were generated in three H. pylori strains (1061, 26695, and NCTC 11638). At pH 7 there was no difference between the growth rates of mutants and the parent
strains. Under acidic conditions, growth of the fur mutants was severely impaired. No differences were observed between the survival of the fur mutant and parent strain 1061 after acid shock. Addition of extra iron or removal of iron from the growth medium did not
improve the growth of the fur mutant at acidic pH. This indicates that the phenotype of the fur mutant at low pH was not due to increased iron sensitivity. Transcription of fur was repressed in response to low pH. From this we conclude that Fur is involved in the growth at acidic pH of H. pylori; as such, it is the first regulatory protein implicated in the acid resistance of this important human pathogen.
Infection and Immunity 03/2002; 70(2):606-11. DOI:10.1128/IAI.70.2.606-611.2002 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The nickel-containing enzyme urease is an essential colonization factor of the gastric pathogen Helicobacter pylori, as it allows the bacterium to survive the acidic conditions in the gastric mucosa. Although urease can represents up to 10% of the total protein content of H. pylori, expression of urease genes is thought to be constitutive. Here it is demonstrated that H. pylori regulates the expression and activity of its urease enzyme as a function of the availability of the cofactor nickel. Supplementation of brucella growth medium with 1 or 100 microM NiCl(2) resulted in up to 3.5-fold-increased expression of the urease subunit proteins UreA and UreB and up to 12-fold-increased urease enzyme activity. The induction was specific for nickel, since the addition of cadmium, cobalt, copper, iron, manganese, or zinc did not affect the expression of urease. Both Northern hybridization studies and a transcriptional ureA::lacZ fusion demonstrated that the observed nickel-responsive regulation of urease is mediated at the transcriptional level. Mutation of the HP1027 gene, encoding the ferric uptake regulator (Fur), did not affect the expression of urease in unsupplemented medium but reduced the nickel induction of urease expression to only twofold. This indicates that Fur is involved in the modulation of urease expression in response to nickel. These data demonstrate nickel-responsive regulation of H. pylori urease, a phenomenon likely to be of importance during the colonization and persistence of H. pylori in the gastric mucosa.
Infection and Immunity 09/2001; 69(8):4891-7. DOI:10.1128/IAI.69.8.4891-4897.2001 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Homologs of the ferric uptake regulator Fur and the iron storage protein ferritin play a central role in maintaining iron homeostasis in bacteria. The gastric pathogen Helicobacter pylori contains an iron-induced prokaryotic ferritin (Pfr) which has been shown to be involved in protection against metal toxicity and a Fur homolog which has not been functionally characterized in H. pylori. Analysis of an isogenic fur-negative mutant revealed that H. pylori Fur is required for metal-dependent regulation of ferritin. Iron starvation, as well as medium supplementation with nickel, zinc, copper, and manganese at nontoxic concentrations, repressed synthesis of ferritin in the wild-type strain but not in the H. pylori fur mutant. Fur-mediated regulation of ferritin synthesis occurs at the mRNA level. With respect to the regulation of ferritin expression, Fur behaves like a global metal-dependent repressor which is activated under iron-restricted conditions but also responds to different metals. Downregulation of ferritin expression by Fur might secure the availability of free iron in the cytoplasm, especially if iron is scarce or titrated out by other metals.
Journal of Bacteriology 12/2000; 182(21):5948-53. DOI:10.1128/JB.182.21.5948-5953.2000 · 2.81 Impact Factor