Among the most important and widespread human pathogens, the
gram-negative bacterium Helicobacter pylori is able to
colonize the gastrointestinal tract and the presence of the
microorganism in the stomach has been associated with the
development of different gastric pathologies. Several factors allow
H. pylori to produce a persistent and efficacious infection:
in this context, chaperone proteins play a crucial role, primarly
because of their general function in protection of the pathogen
against the particularly hostile envinronment of the human stomach.
Two transcriptional regulators, HrcA and HspR, control the
expression of the major heat-shock proteins in H. pylori,
through a fine and complex regulatory network that include both
transcriptional and post-transcriptional mechanisms. Another key
feature, involved in the control of virulence as well as of other
essential cellular processes, is the regulation of metals
homeostasis. In particular, two transcriptional regulators, Fur and
NikR, control gene expression in response to iron and nickel,
respectively.
Our aim is to dissect the regulatory circuits described above by
integrating molecular, biochemical and genomic approaches, in order
to obtain a complete and detailed view of the system of interest.
Results obtained are anticipated to shed light on different
mechanisms that allow H. pylori to survive in the gastric
niche and to spread efficiently in the human population.
H. pylori , a gram-negative spiral-shaped microaerophilic
bacterium, was isolated by a gastric biopsy and described for the
first time in 1983 by R. Warren and B. Marshall. This pathogen
colonizes the human gastric mucosa and is recognized as the
causative agent of several pathologies of the gastro-intestinal
tract, such as chronic active gastritis, gastric and duodenal
ulcers, and is considered a risk factor for the development of
adenocarcinoma. Various bacterial factors contribute to the process
of infection and colonization of the gastric epithelium including
urease, the flagellar apparatus, the vacuolating toxin VacA, the
cytotoxin-associated protein CagA and various mechanisms of
molecular mimicry that allow the bacterium to elude the host immune
response; two other very important bacterial factors are the
regulation of metal homeostasis and the chaperone proteins.
The genome sequence of different clinical isolates provided
insight into the bacterium biology, and many efforts focused on
aspects of direct pathological relevance, identifying several
virulence factors such as urease , flagellins, the vacuolating
toxin VacA, and the cytotoxin-associated protein CagA. However,
studies of basic molecular mechanisms underlying virulence
regulatory control are still at the beginning, and the effective
role of most of the regulatory genes remains to be fully
elucidated. Genes coding for the basic transcriptional machinery
are found, suggesting that the transcription process in H.
pylori is similar to that of other Gram-negative bacteria. By
contrast, the stationary phase sigma factor (sigma-S) and the
heat-shock sigma factor (sigma-32) are missing, and only a few
transcriptional regulators are annotated. This aspect is of primary
relevance for bacterial infection, as expression of virulence
factors is often triggered at the transcriptional level by stress
conditions, including essential metal cofactor(s) limitation,
unfavourable pH conditions, as well as osmotic and oxidative
stress.
Heat-shock proteins regulation
The heat shock proteins of H. pylori have been studied in
some detail both because of their general role in protection of the
pathogen from the hostile environment of the human stomach and
because of their involvement in specific pathogenic processes. In
particular, the GroEL and GroES homologues of H. pylori are
considered important modulators of the stability and activity of
the urease enzyme, which protects the bacteria from the low pH of
the stomach lumen, and both DnaK and GroEL are thought to
contribute to the adherence of the bacteria to sulfated glycolipids
on the surface of epithelial cells. Expression of heat shock genes
is generally tightly regulated, with a basal level ensuring
cellular functions under normal growth conditions and a strong
induction occurring after exposure to a variety of environmental
stresses, including heat shock, osmotic or acidic shock, ethanol
treatment, exposure to heavy metals, etc. Although this stress
response is universally conserved throughout both the prokaryotic
and eukaryotic world, the basic molecular mechanisms differ
considerably between different species. Positive regulation is
observed in Escherichia coli and most other gram-negative
bacteria, where a specialized sigma factor (sigma-32) induces the
transcription of heat shock genes under stress conditions. In
Bacillus subtilis and a variety of other gram-positive and
gram-negative bacteria regulation is negative, involving a
specialized transcriptional repressor (HrcA), which binds to an
inverted repeat in the promoter regions of heat shock genes under
nonstressed conditions but not under stressed conditions. A variant
of this mechanism is active in Streptomyces spp., in which
HspR, a transcriptional repressor not related to HrcA, controls
transcription of the dnaK operon. Sequencing of the H.
pylori genome revealed the absence of a heat shock sigma factor
sigma-32 and the presence of homologues of both the B.
subtilis HrcA and the Streptomyces HspR heat shock
repressors. In this context, we were able to demonstrate that
transcription of the three major H. pylori chaperone
encoding operons is negatively regulated by the action of one or
both repressors. In particular, transcription of the
cbpA-hspR-helicase operon is repressed solely by HspR, while
transcription of the groES-groEL and hrcA-grpE-dnaK
operons is negatively regulated by both HrcA and HspR repressors:
moreover both regulators are necessary for repression of the two
coregulated operons. In vitro DNase I footprinting experiments
allowed the identification of the architectural organization of the
HspR and HrcA binding sites within the three promoters: our data
indicated that the two repressors, at coregulated promoters, bind
two distinct operators, separated by 27 and 18 bps (on groE
and hrcA promoters respectively), without directly
interacting and in an indipendent manner. This regulatory network
appears even more complex if we take into account also the role
played by the GroESL chaperonin. In other bacterial species, the
binding activity of the heat shock repressors is stimulated by the
chaperone systems that they control. Our results suggested that
GroESL directly interacts with HrcA, and possibly with HspR, to
increase their DNA binding affinities for the operators,
contributing to the transcriptional repression of the regulated
promoters. According to a “titration model” proposed for the B.
subtilis HrcA repressor, GroE might interact with H.
pylori HrcA to aid its folding and enhance its DNA binding
activity, thereby efficiently assisting in the repression of
transcription of the target promoters. In the presence of stress
stimuli, the GroE chaperonin would be titrated away by increasing
levels of misfolded proteins, relieving HrcA transcriptional
repression of the heat shock promoters. In parallel to the
dissection of heat shock genes regulatory network, we investigated
the genome-wide regulatory functions of HrcA and HspR by
transcriptome and phenotipic trait analysis of singly or doubly
deficient strains. We found that 43 genes were up- or
down-regulated at least 1.5-fold in the double-mutant strain
(hrcA -hspR -) or in one of the single-mutant
strains: fourteen of 43 genes were up-regulated, while 29 genes
were down-regulated. Intriguingly, the majority of these positively
regulated genes belong to the class of alternative sigma-54 and
sigma-28 transcribed promoters, and 14 of the 29 down-regulated
genes code for proteins involved in regulation and assembly of the
flagellar apparatus. Accordingly, loss of motility functions was
observed for both mutants, and transcription of the flaB
gene was down-regulated both in single mutants and in the
hspR -hrcA double mutant. No binding of HrcA and/or
HspR was observed on the promoter, suggesting that positive
regulation of this gene is due to indirect mechanisms. Although the
possibility was not investigated further, we speculated that
induction of chaperone proteins alters the assembly of the
flagellar apparatus and/or increases the activity of specialized
anti-sigma factors, such as FlgM, which in turn establishes
negative feedback for the programmed transcription of flagellar and
motility genes.
We are also inetersted in the structural characterization of the
two heat-shock genes' repressors HrcA and HspR, in a collaboration
project with the structural biology group run by Professor Zanotti
of the University of Padova.
Regulation of metal homeostasis
The role of iron as an essential element in processes such as
electron transport, energy metabolism and DNA synthesis in bacteria
is well documented, and genes involved in iron metabolism in H.
pylorihave been shown to be important for pathogenesis. Another
important metal for H. pyloriis nickel and its activating
role in the two nickel-containing enzymes urease and hydrogenase,
both required for efficient colonization. The urease metalloenzyme
allows buffering of the high acidic environment through the
conversion of readily available exogenous urea to ammonium and
bicarbonate, while the hydrogenase enzyme allows efficient
colonization of the stomach through breakdown of its
energy-yielding substrate hydrogen that is freely available in the
gastric niche.
Only two transcriptional regulators involved in metal
homeostasis have been identified in H. pylori: a homologue
of the bacterial Ferric uptake regulator protein Fur, and a
homologue of the NikR repressor, which controls expression of a
nickel permease in E. coli. Regulators mediating
environmental responses include also four histidine kinases with
their cognate response regulators as well as two essential orphan
response regulators. However, the target genes regulated by these
two-component systems are largely unknown.
The low abundance of regulators identified in the genome
has been speculated to reflect the adaptation of H. pylorito
its very restricted niche in the mucus layer of the human stomach,
and the lack of competition from other micro-organisms. However,
H. pyloriseems to use complex mechanisms to control gene
transcription. Examples are represented by the heat shock regulon,
which is controlled by the combined action of the HspR and HrcA
repressors, and by the Fur regulator that controls both
iron-induced and iron-repressed genes through complex repression
and derepression mechanisms. Fur has also been implicated in acid
resistance and nickel induction of the urease genes ureABand
its target genes have been shown to respond to metal signals other
than iron, indicating that its regulatory role may expand outside
that solely of iron metabolism. Notably, nickel induction of the
urease gene was also proposed to be under NikR transcriptional
control. Moreover, transcriptome studies identified a series of
genes deregulated in nikRdeletion mutants which have
recently been found deregulated in fur deletion mutants. Two
regulators are, therefore, involved in controlling gene expression
in a metal-dependent fashion, and they are implicated in the
regulation of overlapping sets of genes in H. pylori,
including urease. Recently, it has been demonstrated that Fur and
NikR can bind independently at distinct operators and also compete
for overlapping operators in some coregulated gene promoter. In
addition, a NikR-Fur double mutant is attenuated in the mouse
model, emphasizing the link between response to acidity, metal
metabolism and virulence of this gastric pathogen.
Given its importance in acid stress resistance, transcription of
the urease ureABgenes is unsurprisingly also induced by
acidic pH, together with other genes encoding components of
alternative pathways for the production of ammonia. Low pH is
thought to increase the solubility and therefore the intracellular
availability of nickel ions, it has thus been speculated that NikR
might act as a master regulator of acid adaptation by directly
mediating acid-induced transcription of ureABand by
controlling the transcription of other pH-regulated genes via a
regulatory cascade involving the Fur repressor. However, it has
been clearly demonstrated that acid induction of transcription of
ureABrequires the ArsRS two-component system. This system
comprises the essential OmpR-like response regulator ArsR and the
non-essential histidine kinase ArsS and controls transcription of
several H. pylori-specific genes in response to acidic pH.
Therefore, the two metal-responsive transcriptional regulators NikR
and Fur, as well as the essential two-component response regulator
ArsR mediate the acid response in H. pylori. In support of
this hypothesis, it has been reported that NikR e ArsR regulators
bind overlapping sites localized upstream of the urease promoter,
suggesting a complex mechanism of transcriptional regulation in
response to nickel and pH.
Despite these works, the molecular mechanisms of NikR and ArsR
mediated gene regulation remain to be established. For example, it
is not clear whether the role of H. pyloriNikR is solely as
a nickel-responsive repressor of gene transcription as with the
NikR protein of E. coli, or whether it may also activate
gene transcription. Also in the case of Fur, where the mode of
action has been studied more in detail, the mechanism by which
affinity varies for different operators, according to the iron
status of the regulator, remains elusive. Moreover, the direct
regulatory targets of both, Fur, NikR and ArsR have to be verified,
in order to dissect authentic transcriptional control from indirect
pleiotropic effects or intermediary regulatory circuits. Therefore,
our aim is to carry out a deeper analysis of these H.
pyloriregulators to unravel at the molecular level their mode
of action as well as their responses to environmental signals.
Detailed information of regulatory control is fundamental to
understand how the interplay between NikR, Fur and ArsR circuits
allow H. pylorito establish successful infection in the
gastric niche and become pathogenic.
