Davide Roncarati

In vivo dissection of the Helicobacter pylori Fur regulatory circuit by genome-wide location analysis.

Iron homeostasis is particularly important in pathogenic bacteria, which need to compete with the host for this essential cofactor. In Helicobacter pylori, a causative agent of several gastric pathologies, iron uptake and storage genes are regulated at the transcriptional level by the ferric uptake regulator Fur. The regulatory circuit of Fur has recently come under focus because of an intimate interlink with a broader regulatory network governing metal homeostasis, acidic response, and virulence. To dissect the Fur regulatory circuit and identify in vivo targets of regulation, we developed a genome-wide location analysis protocol which allowed the identification of 200 genomic loci bound by Fur as well as the investigation of the binding efficiency of the protein to these loci in response to iron. Comparative analysis with transcriptomes of wild-type and fur deletion mutant strains allowed the distinction between targets associated with Fur regulation and genes indirectly influenced by the fur mutation. The Fur regulon includes 59 genes, 25 of which appear to be positively regulated. A case study conducted by primer extension analysis of two oppositely regulated genes, hpn2 and flaB, suggests that negative regulation as well as positive regulation occurs at the transcriptional level. Furthermore, the results revealed the existence of 13 Fur targeted loci within polycistronic operons, which were associated with transcript deregulation in the fur mutant strain. This study provides a systematic insight of Fur regulation at the genome-wide level in H. pylori and points to regulatory functions extending beyond the classical Fur repression paradigm.

A novel phase variation mechanism in the meningococcus driven by a ligand-responsive repressor and differential spacing of distal promoter elements.

Phase variable expression, mediated by high frequency reversible changes in the length of simple sequence repeats, facilitates adaptation of bacterial populations to changing environments and is frequently important in bacterial virulence. Here we elucidate a novel phase variable mechanism for NadA, an adhesin and invasin of Neisseria meningitidis. The NadR repressor protein binds to operators flanking the phase variable tract and contributes to the differential expression levels of phase variant promoters with different numbers of repeats likely due to different spacing between operators. We show that IHF binds between these operators, and may permit looping of the promoter, allowing interaction of NadR at operators located distally or overlapping the promoter. The 4-hydroxyphenylacetic acid, a metabolite of aromatic amino acid catabolism that is secreted in saliva, induces NadA expression by inhibiting the DNA binding activity of the repressor. When induced, only minor differences are evident between NadR-independent transcription levels of promoter phase variants and are likely due to differential RNA polymerase contacts leading to altered promoter activity. Our results suggest that NadA expression is under both stochastic and tight environmental-sensing regulatory control, both mediated by the NadR repressor, and may be induced during colonization of the oropharynx where it plays a major role in the successful adhesion and invasion of the mucosa. Hence, simple sequence repeats in promoter regions may be a strategy used by host-adapted bacterial pathogens to randomly switch between expression states that may nonetheless still be induced by appropriate niche-specific signals.

Growth Phase and Metal-Dependent Transcriptional Regulation of the fecA Genes in Helicobacter pylori

Balancing metal uptake is essential for maintaining a proper intracellular metal concentration. Here, we report the transcriptional control exerted by the two metal-responsive regulators of Helicobacter pylori, Fur (iron-dependent ferric uptake regulator) and NikR (nickel-responsive regulator), on the three copies of the fecA genes present in this species. By monitoring the patterns of transcription throughout growth and in response to nickel, iron, and a metal chelator, we found that the expression of the three fecA genes is temporally regulated, responds to metals in different ways, and is selectively controlled by either one of the two regulators. fecA1 is expressed at a constant level throughout growth, and its expression is iron sensitive; the expression of fecA2 is mainly off, with minor expression coming up in late exponential phase. In contrast, the expression of fecA3 is maximal in early exponential phase, gradually decreases with time, and is repressed by nickel. The direct roles of Fur and NikR were studied both in vitro, by mapping the binding sites of each regulator on the promoter regions via DNase I footprinting analysis, and in vivo, by using primer extension analyses of the fecA transcripts in fur and nikR deletion strains. Overall, the results show that the expression of each fecA gene is finely tuned in response to metal availability, as well as during the bacterial growth phase, suggesting specific and dedicated functions for the three distinct FecA homologues.

Transcriptional Regulation of Stress Response and Motility Functions in Helicobacter pylori Is Mediated by HspR and HrcA

The hrcA and hspR genes of Helicobacter pylori encode two transcriptional repressor proteins that negatively regulate expression of the groES-groEL and hrcA-grpE-dnaK operons. While HspR was previously shown to bind far upstream of the promoters transcribing these operons, the binding sites of HrcA were not identified. Here, we demonstrate by footprinting analysis that HrcA binds to operator elements similar to the so-called CIRCE sequences overlapping both promoters. Binding of HspR and HrcA to their respective operators occurs in an independent manner, but the DNA binding activity of HrcA is increased in the presence of GroESL, suggesting that the GroE chaperonin system corepresses transcription together with HrcA. Comparative transcriptome analysis of the wild-type strain and hspR and hrcA singly and doubly deficient strains revealed that a set of 14 genes is negatively regulated by the action of one or both regulators, while a set of 29 genes is positively regulated. While both positive and negative regulation of transcription by HspR and/or HrcA could be confirmed by RNA primer extension analyses for two representative genes, binding of either regulator to the promoters could not be detected, indicating that transcriptional regulation at these promoters involves indirect mechanisms. Strikingly, 14 of the 29 genes which were found to be positively regulated by HspR or HrcA code for proteins involved in flagellar biosynthesis. Accordingly, loss of motility functions was observed for HspR and HrcA single or double mutants. The possible regulatory intersections of the heat shock response and flagellar assembly are discussed.

Dual Control of Helicobacter pylori Heat Shock Gene Transcription by HspR and HrcA

The HspR repressor regulates transcription of the groESL, hrcA-grpE-dnaK, and cbpA-hspR-orf operons of Helicobacter pylori. Here we show that two of the HspR-regulated operons, namely, the groESL and dnaK operons, encoding the major cellular chaperone machineries are also regulated by the H. pylori homologue of the HrcA repressor. Similarly to the hspR mutation, deletion of the hrcA gene also leads to complete derepression of the Pgro and Phrc promoters. The presence of both HspR and HrcA is therefore necessary for regulated transcription from these promoters. HrcA binds directly to Pgro and Phrc, likely contacting two inverted repeats with similarity to the CIRCE motif, which are present on both promoters. HrcA regulation is, however, shown to depend on binding of the HspR protein, since deletion of the HspR-binding site of the Pgro promoter leads to loss of heat inducibility of this promoter. In contrast, transcription from the Pcbp promoter is regulated solely by HspR. HspR is also shown to form oligomers in vivo through a stretch of hydrophobic repeats between amino acid positions 66 and 97. The implications of these findings for the elucidation of the networks regulating heat shock gene expression in H. pylori are discussed.

FeON-FeOFF: the Helicobacter pylori Fur regulator commutates iron-responsive transcription by discriminative readout of opposed DNA grooves

Most transcriptional regulators bind nucleotide motifs in the major groove, although some are able to recognize molecular determinants conferred by the minor groove of DNA. Here we report a transcriptional commutator switch that exploits the alternative readout of grooves to mediate opposite output regulation for the same input signal. This mechanism accounts for the ability of the Helicobacter pylori Fur regulator to repress the expression of both iron-inducible and iron-repressible genes. When iron is scarce, Fur binds to DNA as a dimer, through the readout of thymine pairs in the major groove, repressing iron-inducible transcription (FeON). Conversely, on iron-repressible elements the metal ion acts as corepressor, inducing Fur multimerization with consequent minor groove readout of AT-rich inverted repeats (FeOFF). Our results provide first evidence for a novel regulatory paradigm, in which the discriminative readout of DNA grooves enables to toggle between the repression of genes in a mutually exclusive manner.

In Depth Analysis of the Helicobacter pylori cag Pathogenicity Island Transcriptional Responses

The severity of symptoms elicited by the widespread human pathogen Helicobacter pylori is strongly influenced by the genetic diversity of the infecting strain. Among the most important pathogen factors that carry an increased risk for gastric cancer are specific genotypes of the cag pathogenicity island (cag-PAI), encoding a type IV secretion system (T4SS) responsible for the translocation of the CagA effector oncoprotein. To date, little is known about the regulatory events important for the expression of a functional cag-T4SS. Here we demonstrate that the cag-PAI cistrons are subjected to a complex network of direct and indirect transcriptional regulations. We show that promoters of cag operons encoding structural T4SS components display homogeneous transcript levels, while promoters of cag operons encoding accessory factors vary considerably in their basal transcription levels and responses. Most cag promoters are transcriptionally responsive to growth-phase, pH and other stress-factors, although in many cases in a pleiotropic fashion. Interestingly, transcription from the Pcagζ promoter controlling the expression of transglycolase and T4SS stabilizing factors, is triggered by co-culture with a gastric cell line, providing an explanation for the increased formation of the secretion system observed upon bacterial contact with host cells. Finally, we demonstrate that the highly transcribed cagA oncogene is repressed by iron limitation through a direct apo-Fur regulation mechanism. Together the results shed light on regulatory aspects of the cag-PAI, which may be involved in relevant molecular and etiological aspects of H. pylori pathogenesis.

The HrcA repressor is the thermosensor of the heat-shock regulatory circuit in the human pathogen Helicobacter pylori.

Bacteria exploit different strategies to perceive and rapidly respond to sudden changes of temperature. In Helicobacter pylori the response to thermic stress is transcriptionally controlled by a regulatory circuit that involves two repressors, HspR and HrcA. Here we report that HrcA acts as a protein thermometer. We demonstrate that temperature specifically modulates HrcA binding to DNA, with a complete and irreversible temperature‐dependent loss of DNA binding activity at 42°C. Intriguingly, although the reduction of HrcA binding capability is not reversible in vitro, transcriptional analysis showed that HrcA exerts its repressive influence in vivo, even when the de novo repressor synthesis is blocked after the temperature challenge. Accordingly, we demonstrate the central role of the chaperonine GroESL in restoring the HrcA binding activity, lost upon heat challenge. Together our results establish HrcA as a rare example of intrinsic temperature sensing transcriptional regulator, whose activity is post‐transcriptionally modulated by the GroESL chaperonine.

A Convenient and Robust In Vivo Reporter System To Monitor Gene Expression in the Human Pathogen Helicobacter pylori

ABSTRACT Thirty years of intensive research have significantly contributed to our understanding of Helicobacter pylori biology and pathogenesis. However, the lack of convenient genetic tools, in particular the limited effectiveness of available reporter systems, has notably limited the toolbox for fundamental and applied studies. Here, we report the construction of a bioluminescent H. pylori reporter system based on the Photorhabdus luminescens luxCDABE cassette. The system is constituted of a promoterless lux acceptor strain in which promoters and sequences of interest can be conveniently introduced by double homologous recombination of a suicide transformation vector. We validate the robustness of this new lux reporter system in noninvasive in vivo monitoring of dynamic transcriptional responses of inducible as well as repressible promoters and demonstrate its suitability for the implementation of genetic screens in H. pylori.

CbpA Acts as a Modulator of HspR Repressor DNA Binding Activity in Helicobacter pylori

The ability of pathogens to cope with disparate environmental stresses is a crucial feature for bacterial survival and for the establishment of a successful infection and colonization of the host; in this respect, chaperones and heat shock proteins (HSPs) play a fundamental role in host-pathogen interactions. In Helicobacter pylori, the expression of the major HSPs is tightly regulated through dedicated transcriptional repressors (named HspR and HrcA), as well as via a GroESL-dependent posttranscriptional feedback control acting positively on the DNA binding affinity of the HrcA regulator itself. In the present work we show that the CbpA chaperone also participates in the posttranscriptional feedback control of the H. pylori heat shock regulatory network. Our experiments suggest that CbpA specifically modulates HspR in vitro binding to DNA without affecting HrcA regulator activity. In particular, CbpA directly interacts with HspR, preventing the repressor from binding to its target operators. This interaction takes place only when HspR is not bound to DNA since CbpA is unable to affect HspR once the repressor is bound to its operator site. Accordingly, in vivo overexpression of CbpA compromises the response kinetics of the regulatory circuit, inducing a failure to restore HspR-dependent transcriptional repression after heat shock. The data presented in this work support a model in which CbpA acts as an important modulator of HspR regulation by fine-tuning the shutoff response of the regulatory circuit that governs HSP expression in H. pylori.