Helicobacter pylori is a human bacterial pathogen responsible for chronic active gastritis [1], gastric and duodenal ulcer diseases [2], and is associated with the development of gastric carcinoma [3]. Almost half of the world’s population carries an H. pylori infection, and disease outcome depends on many factors, including bacterial genotype, host physiology, and diet. The primary factors associated with pathogenesis include the urease enzyme [4], the flagellins [5], the vacuolating toxin VacA ^[6][7][6,7], and the cytotoxin-associated protein CagA ^[8][9][8,9]. As with other pathogens, the ability to acquire metal ions such as iron (Fe²⁺) ^[10][11][10,11] and nickel (Ni²⁺) ^[12][13][14][12,13,14] also seems to contribute to the virulence of H. pylori. Moreover, the heat-shock response plays a fundamental role during infection, as it allows cells to adapt to hostile environmental conditions and survive during stress.

Since the first H. pylori genome, strain 26695, was sequenced in 1997 [15], over 2000 isolates have been sequenced and annotated [16], and a representative reference genome reports a median chromosome of 1.63254 Mb in length coding for a median of 1440 proteins. Genome analysis revealed an abundance of polymorphic genetic elements, many of which reside in factors that may be associated with virulence [17].

Despite the enormous amount of information deduced from the genomes, our knowledge of many aspects of the molecular mechanisms regulating the physiology and metabolism of H. pylori is still limited. Most of the efforts focused on the bacterial and pathological processes of direct clinical relevance. In contrast, studies of the basic molecular mechanisms and the regulatory genes involved in the physiology and expression of virulence genes are still limited, and more efforts are required for better elucidation.

2. Genome and Regulatory Functions

In 1997, Tomb and collaborators published the genome sequence of the H. pylori strain 26695 [15]. The second genome sequenced was that of H. pylori strain J99 [18]. Although H. pylori were expected to exhibit a significant degree of genomic and allelic diversity, the overall genomic organization, gene order, and predicted gene products of the two strains were remarkably similar [18]. Moreover, other H. pylori strains have been sequenced, including strains G27 [19], N6 [20], and others. The analysis of the H. pylori genome revealed that the genes encoding the basic transcriptional mechanism are very similar to those of other Gram-negative bacteria. Unlike most prokaryotes, the rpoB and rpoC genes encoding the β and β’ subunits of H. pylori RNA polymerase are fused into a single polypeptide. This peculiarity of gene fusion has also been observed in the closely related bacterium H. felis and the Wolinella species ^[21][22][21,22]. Overall, H. pylori codes for only three sigma factors, σ⁸⁰ (RpoD), σ⁵⁴ (RpoN), and σ²⁸ (FliA) (Table 1), and it does not possess homologs of the stationary phase sigma factor (σ^s) nor the heat-shock sigma factor (σ³²). The absence of these factors suggested that H. pylori has a different stress response mechanism from other bacteria. Moreover, its genome contains four histidine kinases with their cognate response regulators and two orphan response regulators [23]. Furthermore, there are only a few other transcriptional regulators in H. pylori, which resemble repressors from other systems (Table 1). The low abundance of regulators might reflect the adaptation of H. pylori to its very restricted niche in the mucus layer of the human stomach, and the lack of competition from other micro-organisms [24].

3. The Two-Component Systems of H. pylori

Two-component systems (TCSs) are widespread signal transduction devices that couple the perception of external stimuli with an adaptive intracellular response. These systems are generally composed of two proteins, a histidine kinase (HK) and a response regulator (RR), both of which are characterized by different functional domains. Sensing a chemical or physical stimulus at the N-terminal input domain, the HK catalyzes the autophosphorylation of a conserved His residue on its C-terminal effector domain. The phosphate group is then transferred to a highly conserved Asp residue on the N-terminal receiver domain of the RR, leading to its activation [42]. Currently, different phosphotransmission schemes are known [43], enlarging the possible RR activation pathways. In most cases, RRs are characterized by C-terminal effector DNA-binding domains and act as transcriptional regulators [44]. Other types of RRs show RNA-, protein- or ligand-binding domains or are proteins with enzymatic activity, thereby broadening their spectrum of action to a post-transcriptional and post-translational level ^{[44][45][46][47]}[44,45,46,47]. The H. pylori genome has evolved to better survive in the restricted colonization niche (i.e., the gastrointestinal tract), where it appears to be the dominant bacterial species and, therefore, with almost an absence of competition from other bacteria. Accordingly, efficient signaling networks have evolved to adequately express the factors necessary for survival in this harsh niche ^[48][49][48,49]. Compared to other bacteria, H. pylori encodes only four TCSs: FlgRS, ArsRS, CrdRS, and CheYA [15]. Two H. pylori histidine kinases (CrdS, ArsS) are likely transmembrane sensor proteins with a periplasmic input and a cytoplasmic transmitter domain. The atypical NtrB-like HK FlgS has revealed a cytoplasmic localization instead. Based on sequence similarities in their output domains, CrdR and ArsR RRs are classified as members of the OmpR subfamily, but FlgR is classified as an NtrC-like protein ^[23][50][23,50].

3.1. FlgRS Control the Transcription of Flagellar Genes

H. pylori flagellar genes are organized in a hierarchal regulon depending on the sigma factor required for their transcription, as σ⁸⁰ (RpoD), σ⁵⁴ (RpoN), and σ²⁸ (FliA) govern the expression of early (class 1), intermediate (class 2 and class 3) and late (class 3) flagellar genes, respectively [27]. Due to its role in intermediate flagellar gene regulation, the TCS FlgRS has been shown to constitute an essential factor for H. pylori motility, hence, to host colonization [51]. Following stimulation, FlgS transfers its phosphate group to the cognate FlgR that directly interacts with RpoN, allowing the expression of its regulon. RpoN binding and subsequent activation were shown to occur in an enhancer-independent way, as FlgR lacks the typical C-terminal DNA-binding domain generally found in most σ⁵⁴-dependent activators [26]. Interestingly, in mutants lacking FlgS or FlgR, an increase in flaA level was observed, a class 3 flagellar gene whose transcription is under the FliA control, suggesting a possible inverse regulation [27]. However, an intracellular signal leading to FlgS autophosphorylation and consequent FlgR activation remains to be elucidated [52]. Tsang and coworkers ^{[53][54][55][56]}[53,54,55,56] hypothesized that different components of the flagellar export apparatus could coordinately operate to stimulate the autokinase activity of FlgS, as deletion of several of those proteins (FlhA, FliF, FliM, FliY, FliH, FliE, FlgB, FlgC) impaired the RpoN-mediated regulon. In the model they proposed, the correct assembly of the basal body and the Type 3 secretion system proteins of the flagellar apparatus generates a regulatory checkpoint for the on/off switch of FlgRS TCS. Moreover, they also demonstrated that the 25 N-terminus cytosolic residues of the export apparatus protein FlhA interact in vitro with FlgS via its C-terminal kinase domain. However, no increase in FlgS phosphorylation levels was observed in vitro in the presence of ATP [56]. Similar results were obtained in the close relative Campylobacter jejuni, where it was observed that the MS ring protein FliF and C ring protein FliG are necessary to activate FlgSR- and RpoN-dependent flagellar expression [57]. FlgS was further proposed to be a soluble pH sensor. In response to acidic (4.5) and strong acidic (2.5) cytoplasmic pH, FlgS mediates the transcription of an FlgR-independent regulon of over a hundred targets, comprising acid acclimation genes (ureA, ureB, ureI, ureF, ansB, rocF, amiE). FlgS is unable to bind directly to the promoters of the genes belonging to this large pH-responsive regulon. Furthermore, FlgR is not required for the regulation, and the factors that mediate the transcriptional response are still unknown. Most FlgR-independent FlgS-regulated genes (82%) are not present in the ArsS regulon, suggesting that FlgS and ArsS adopt different signaling pathways in response to gastric acidic pH. In addition, FlgS was found to be essential for H. pylori survival at pH 2.5 [58].

3.2. ArsRS Regulates the Transcription of Genes Involved in Acid Resistance

It is commonly recognized that the acid-adaptive response of H. pylori is tightly controlled, with ArsRS TCS at the top of the list of acid-responsive transcriptional regulation ^[59][60][59,60]. The pH-responsive regulon of ArsRS includes genes involved in acid adaptation (ureAB, amidases), oxidative stress responses (katA, sodB), iron or nickel homeostasis transcriptional modulation (fur, nikR), genes encoding outer membrane proteins (including sabA, alpA, alpB, hopD, and horA), and three genes coding for acetone carboxylase subunits (acxA, acxB, and acxC) [61]. The environmental pH is detected directly by the N-terminal periplasmic domain of the HK ArsS, which autophosphorylates and transfers the phosphate group to its cognate RR ArsR. The TCS ArsRS appears to be negatively autoregulated, as an ArsR binding site was found downstream of the arsR transcriptional start site (TSS) ^[59][62][59,62]. The ArsR regulon is composed of two sets of target genes: (I) genes controlled by the unphosphorylated ArsR, at least one of which is essential for in vitro growth conditions, and (II) non-essential genes which are regulated by the phosphorylated ArsR in response to acidic pH. The observation that an ArsS-deficient mutant is unable to colonize mice further emphasizes the role of the ArsRS in the acid adaptation of H. pylori in the host [63]. When H. pylori is exposed to low pH, the transcription of ureAB and the ureIEFGH operon is induced. This transcriptional initiation is mediated mainly by the ArsRS two-component system, as the effect was largely reduced in an ArsS-deficient mutant ^[60][64][60,64]. It was also demonstrated that the pH- and Ni²⁺-dependent transcription of ureA is mediated by two independent regulatory mechanisms involving transcriptional activators (NikR and ArsR) that compete for interaction with partially overlapping binding sites (see NikR) [28]. In response to acidic pH and the presence of low amounts of urea substrate for urease, H. pylori has additional mechanisms for ammonia synthesis. Two aliphatic amidases, AmiE and AmiF, appear to play a key role in nitrogen metabolism. Both amidase genes are transcribed in response to acidic pH, a response predominantly controlled by the ArsRS regulatory network ^[65][66][65,66]. Moreover, a dual regulation of acxABC and fabD-pfs operons by the ArsRS TCS and the orphan response regulator HP1021 was observed, indicating a central role in the pathway that sees acetone as an alternative carbon source. In fact, mutagenesis on the acxABC operon was shown to hamper H. pylori’s capacity to colonize mice ^[67][68][67,68]. Recent data have shown that the ArsRS plays a crucial regulatory role even in the absence of a low-pH condition, suggesting that it might be responsive to other environmental stimuli besides low pH [61].

3.3. CrdRS Regulates the Transcription of Genes Important for Copper Homeostasis and Central Cellular Responses

As a cofactor of oxidases (such as cbb3-type cytochrome-c oxidase), copper is critical for respiration in H. pylori, as it promotes the bacterial colonization of mucosal surfaces. On the other hand, copper acts as a chemotactic factor that repels H. pylori motility ^[69][70][69,70]. Therefore, copper homeostasis plays a crucial role in adaptation to the gastric environment [63], making necessary a fine regulation of the H. pylori intracellular copper level [71]. H. pylori has developed sophisticated efflux techniques to avoid copper toxicity by upregulating several genes involved in copper homeostasis, including the copAP operon [71]. In the presence of copper, the sensor kinase CrdS phosphorylates the cognate regulator CrdR that promotes the transcription of crdAB and czcAB, resulting in the expression of a secreted copper resistance protein (CrdA) and a copper efflux complex (CrdB, CzcB, and CzcA) ^[29][72][29,72]. CrdB, CzcB, and CzcA usefully complete the copper resistance mediated by the metal export pump CopA (a P-type ATPase). H. pylori CrdS and CrdR mutants were not able to colonize mice, further supporting the hypothesis that the copper resistance is mainly guided by the TCS CrdRS [29]. As shown by Hung and coworkers [73], H. pylori 26695 crdS, but not flgS nor arsS, was significantly upregulated in the presence of NO (nitrosative) stress, suggesting that CrdRS governs the expression of the NO-dependent regulon. In addition, this TCS was seen to play a role in increasing iron uptake for bacterial proliferation by upregulation of the iron-scavenging systems. Thereby, CrdRS makes a critical contribution to survive the host’s innate immune response ^[73][74][75][73,74,75].

3.4. CheY1Y2A Two-Component System

A functional chemotactic response is an essential determinant for H. pylori swimming motility and hence host colonization [76]. The H. pylori signaling cascade responding to a chemotactic signal mostly resembles that of the two archetypes E. coli and Salmonella enterica serovar Typhimurium [77]. As chemotaxis regulation by the CheAY TCS does not involve transcription responses, this essential topic is beyond the scope herein, but will be briefly summarized hereafter. More detailed descriptions are reported in recent reviews ^[78][79][78,79]. The fundamental proteins involved in signal transduction from chemoreceptors to the flagellar switch are CheAY2, which consist of the CheA HK fused with the CheY-homolog receiver domain CheY2, and the CheY1 RR. In the absence of the specific ligand of the chemoreceptor, the HK CheA actively phosphorylates itself, and then the phosphate is transferred to an aspartate residue of the CheY1 RR. In turn, CheY1-P directly interacts with the flagellar motor and induces a clockwise rotation of the flagella, inducing a random reorientation in the space of the bacteria (tumbling behavior). Conversely, the binding of the ligand to the chemoreceptor inhibits the activity of CheA. Hence CheY1 remains unphosphorylated, and its interaction with the flagellar motor induces a counterclockwise rotation, prompting the bacteria to swim straight. In vitro, CheA transfers its phosphate group to CheY1 and CheY2, and CheY1 can transfer the phosphate back to CheA. It has been proposed that CheY2 interferes with the phosphate flow between CheA and CheY1, functioning as a phosphate sink. Three CheV phosphatases (homolog to E. coli CheW) target CheA-P and dephosphorylate the protein before phosphotransfer to CheY, but the former activity is less efficient than the latter [30]. Moreover, in a study conducted by Terry and coworkers [80], the ORF HP0170 was identified as a CheZ homolog that specifically directs the dephosphorylation of CheY.

3.5. Orphan Regulators

The genome of H. pylori encodes two DNA-binding regulatory proteins, named HP1021 and HP1043, that are defined as “orphan” regulators. They have been classified as response regulators based on sequence similarity to members of this protein family, but no histidine kinase, which is able to phosphorylate response regulators HP1021 and HP1043, has been identified. Considering that the phosphorylation receiver domains of both regulators show profound deviations from the consensus sequence and the experimental evidence that both proteins can bind in vitro target DNA without phosphorylation, the current hypothesis is that HP1021 and HP1043 may exert their regulatory function in the absence of receiver phosphorylation ^[81][82][81,82]. HP1043 proved to be essential for bacterial growth. The hp1043 gene cannot be inactivated by allelic exchange, nor the amount of protein modulated, supporting its role as a central regulator of H. pylori fundamental cellular processes ^[82][83][82,83]. Indeed, a recent study based on chromatin immunoprecipitation-sequencing approach (ChIP-seq) identified genome-wide the HP1043 in vivo binding sites [31]. This analysis identified 37 highly reproducible binding sites, ~90% of which are located in promoter regions, consistent with HP1043 functioning as a canonical transcriptional regulator. Gene ontology analysis of the identified targets revealed that HP1043 exerts a pleiotropic function, directly regulating all the fundamental processes in the cell, including translation, transcription, replication, energy metabolism, and virulence. To date, little is known about the environmental signals that regulate HP1043 expression and activity. A first observation proved that hp1043 transcription is growth-phase regulated, with the amount of transcript dropping in the late stationary phase [83]. A recent report shows a significant increase in hp1043 transcription following exposure of stationary cultures to urea, low pH, metals, biofilm, and AGS gastric epithelial cells [84]. In addition, HP1043 might be indirectly involved in response to oxidative stress since treatment of growing cells with redox-active compounds determined a decrease in protein levels and a reduction in the transcripts of regulated genes [85]. The inactivation of the HP1021 orphan response regulator leads to severe growth defects, but it is not essential for H. pylori viability [81]. A microarray-based whole genome transcriptional profiling study demonstrated that HP1021 controls the expression of 79 genes involved in different cellular processes, including transcription, translation, metabolic pathways, and synthesis of cofactors [67]. HP1021 has also been proposed to take part in the regulation of H. pylori chromosome replication. It has been shown that HP1021 binds to the oriC site and precludes the interaction of the DnaA replication initiator protein with this site, thereby inhibiting DNA unwinding [86]. As for HP1043, the environmental signals that trigger HP1021 activity have remained elusive for many years. A recently published paper proposed that HP1021 is a redox switch protein controlling the oxidative stress response in H. pylori [87]. In detail, HP1021 possesses several cysteine residues that are sensitive, both in vitro and in vivo, to oxidation. Moreover, the DNA-binding activity of HP1021 depends on redox conditions, and it regulates the transcription of specific genes in an oxygen-dependent manner.