Foto del docente

Davide Roncarati

Associate Professor

Department of Pharmacy and Biotechnology

Academic discipline: BIO/11 Molecular Biology


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.

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