6 research outputs found
Role of the Helicobacter pylori outer-membrane proteins AlpA and AlpB in colonization of the guinea pig stomach
The human gastric pathogen Helicobacter pylori expresses several putative
outer-membrane proteins (OMPs), but the role of individual OMPs in
colonization of the stomach by H. pylori is still poorly understood. The
role of four such OMPs (AlpA, AlpB, OipA and HopZ) in a guinea pig model
of H. pylori infection has been investigated. Single alpA, alpB, hopZ and
oipA isogenic mutants were constructed in the guinea pig-adapted,
wild-type H. pylori strain GP15. Guinea pigs were inoculated
intragastrically with the wild-type strain, single mutants or a mixture of
the wild-type and a single mutant in a 1: 1 ratio. Three weeks after
infection, H. pylori could be isolated from stomach sections of all
animals that were infected with the wild-type, the hopZ mutant or the oipA
mutant, but from only five of nine (P = 0.18) and one of seven (P = 0.02)
animals that were infected with the alpA or alpB mutants, respectively.
The hopZ and oipA mutants colonized the majority of animals that were
inoculated with the strain mixture, whereas alpA and alpB mutants could
not be isolated from anim
Role of the rdxA and frxA genes in oxygen-dependent metronidazole resistance of Helicobacter pylori
Almost 50 % of all Helicobacter pylori isolates are resistant to
metronidazole, which reduces the efficacy of metronidazole-containing
regimens, but does not make them completely ineffective. This discrepancy
between in vitro metronidazole resistance and treatment outcome may
partially be explained by changes in oxygen pressure in the gastric
environment, as metronidazole-resistant (MtzR) H. pylori isolates become
metronidazole-susceptible (MtzS) under low oxygen conditions in vitro. In
H. pylori the rdxA and frxA genes encode reductases which are required for
the activation of metronidazole, and inactivation of these genes results
in metronidazole resistance. Here the role of inactivating mutations in
these genes on the reversibility of metronidazole resistance under low
oxygen conditions is established. Clinical H. pylori isolates containing
mutations resulting in a truncated RdxA and/or FrxA protein were selected
and incubated under anaerobic conditions, and the effect of these
conditions on the MICs of metronidazole, amoxycillin, clarithromycin and
tetracycline, and cell viability were determined. While anaerobiosis had
no effect on amoxycillin, clarithromycin and tetracycline resistance, all
isolates lost their metronidazole resistance when cultured under anaerobic
conditions. This loss of metronidazole resistance also occurred in the
presence of the protein synthesis inhibitor chloramphenicol. Thus,
factor(s) that activate metronidazole under low oxygen tension are not
specifically induced by low oxygen conditions, but are already present
under microaerophilic conditions. As there were no significant differences
in cell viability between the clinical isolates, it is likely that neither
the rdxA nor the frxA gene participates in the reversibility of
metronidazole resistance
NikR mediates nickel-responsive transcriptional induction of urease expression in Helicobacter pylori
The important human pathogen Helicobacter pylori requires the abundant
expression and activity of its urease enzyme for colonization of the
gastric mucosa. The transcription, expression, and activity of H. pylori
urease were previously demonstrated to be induced by nickel
supplementation of growth media. Here it is demonstrated that the HP1338
protein, an ortholog of the Escherichia coli nickel regulatory protein
NikR, mediates nickel-responsive induction of urease expression in H.
pylori. Mutation of the HP1338 gene (nikR) of H. pylori strain 26695
resulted in significant growth inhibition of the nikR mutant in the
presence of supplementation with NiCl(2) at > or =100 microM, whereas the
wild-type strain tolerated more than 10-fold-higher levels of NiCl(2).
Mutation of nikR did not affect urease subunit expression or urease enzyme
activity in unsupplemented growth media. However, the nickel-induced
increase in urease subunit expression and urease enzyme activity observed
in wild-type H. pylori was absent in the H. pylori nikR mutant. A similar
lack of nickel responsiveness was observed upon removal of a 19-bp
palindromic sequence in the ureA promoter, as demonstrated by using a
genomic ureA::lacZ reporter gene fusion. In conclusion, the H. pylori NikR
protein and a 19-bp operator sequence in the ureA promoter are both
essential for nickel-responsive induction of urease expression in H.
pylori
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
The role of the ferric uptake regulator (Fur) in regulation of Helicobacter pylori iron uptake
Background. Availability of the essential nutrient iron is thought to vary greatly in the gastric mucosa, and thus the human gastric pathogen Helicobacter pylori requires regulatory responses to these environmental changes. Bacterial iron-responsive regulation is often mediated by Ferric Uptake Regulator (Fur) homologs, and in this study we have determined the role of H. pylori Fur in regulation of H. pylori iron uptake. Methods. Wild-type H. pylori and fur mutant derivatives were compared after growth in ironrestricted and iron-replete conditions. Iron-uptake was measured using 55Fe-labeled iron, whereas gene expression was mon
Iron-responsive regulation of the Helicobacter pylori iron-cofactored superoxide dismutase SodB is mediated by Fur.
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