Two-component signalling pathways in bacteria

The orthodox HPK and hybrid HPK are different in many ways. However, the structure of the kinase catalytic core of both classes of HPK is similar. The kinase core is approximately 350 amino acids in length and consist of an ATP/ADP binding catalytic domain [20] and a dimerization domain. The kinase core direct kinase transphosphorylation and is responsible in binding ATP.

Domains of HPK

HPK contain sensing domain, linker domain and phosphotransfer domain which contain histidine. The sensing domain, which directly or indirectly detects environmental stimuli, is located at the N-terminal of HPK. Cytosolic sensing modules are also integrated into HPKs. Once such example is the adaptable PAS domain which monitors, depending on their associated cofactor, changes in oxygen, redox potential light and small ligands. [21] It was also observed that sensing domains share very little similarity in primary sequence, supporting the notion that each domain is specific in stimulus/ligand interactions.

In transmembrane HPK, a cytoplasmic linker connects the sensing domain to the cytoplasmic kinase core. The linker domain varies in length from 40 amino acids to more than 180. [22] While the linker domain is yet to be very well understood, Colin and his colleagues [23] had indicated the importance of linker domain for proper signal transduction. Some hybrid HPK has histidine containing phosphotransfer (HPts) domain. As its name imply, HPt domains contain a histidine residue, which participate in phosphoryl transfer reaction.  HPts also serve as specific communication modules between different proteins as it do not exhibit phosphatase or kinase activity. [24] With such wide diversity and functions, it can be said that HPK are very important components in the “communication” and survival of bacteria.

Response Regulator (RR)

Most RRs consist of a conserved N-terminal regulatory domain and a C-terminal effector domain. RR functions as phosphorylation-activated switches to effect the adaptive response. Responding to the stimuli, the RR catalyses a phosphoryl transfer from the phosphor-His of the HPK to a conserved Aspartate in its own regulatory domain without the assistance of HPK as small molecules such as acetyl phosphate and carbamoyl phosphate can serve as phosphodonors to the RRs [25]. In most prokaryotes, RRs are the terminal component of the pathway.

Domains of RR

Like HPK, RRs also contain several domains, namely the DNA-binding effector domain, effector domain and regulatory domain. Most of the RRs contain DNA-binding effector domains that act as transcription factors. [26] Some of the RRs however, have a C-terminal domain that functions as enzymes. One such example is the Dictyostelium cAMP phosphodiesterase RegA [22]

Similar to HPK, there is great diversity in effector domains and the majority of effector domains have DNA-binding activity. The function of effector domain is to activate and/or repress transcription of specific genes. However, the arrangement of binding sites, mechanism of transcriptional regulation and the recognition of DNA sequence differ from individual RR.

The regulatory domain of the RRs exists in equilibrium between the active and inactive state, with phosphorylation shifting the equilibrium to the active form. The regulatory domain is illustrated with chemotaxis protein CheY. The carboxylate side chains help to coordinate Mg2+ required for dephosphorylation and phosphoryl transfer. [27] CheY has a phosphorylation site in Asp 57 and two other highly conserved residues in Thr 87 and Lys 109. [28] The clusters of conserved residues also surround the regulatory domain active site. A further analysis of the protein structure revealed an octahedral coordination involving Asp12, Asp 57, three water molecules and the backbone oxygen of Asn 59. The coordination suggest a phosphoryl transfer through a bipyramidal pentavalent phosphorous transition state, and an intermediate that might be involved in the mechanism of autodephosphorylation. [29] The overall features of the regulatory domain are similar to that of CheY, with difference in orientation of helices and lengths and conformation of surface loops [30] 

Phosphorylation Activation

Effector domains are hugely diversified. However, with such great diversity of effector domains, how can then a conserved regulatory domain function to regulate many different activities by the effector domains. As mentioned earlier, the regulatory domains of RRs exist in equilibrium between the active and non-active form and activated via phosphorylation. This allow a different molecular surface that can help facilitate specific protein-DNA or protein-protein interaction with the help of inter or intra-molecular interaction regulating it.

Using crystal structure, Djordjevc and his colleagues [31] provide a structural basis that unphosphorylated regulatory domain inhibit effector domain activity. For example, the regulatory domain of chemotaxis methyl esterase CheB blocks the chemoreceptor from assessing the active site of the esterase and the regulation domain of transcription factor NarL blocks access of DNA to the regulation helix. [32] It was also observed that a repositioning of N and C terminal domains is involved for the activation induced by phosphorylation.

There are a number of different RR activation mechanisms. In some cases, the phosphorylated regulatory domains play an active role to promote dimerization, interaction with other proteins, DNA or higher order oligomerisation. [33]. In other cases, a period of inhibition is involved before the activation of the RR and activated by removing the N-terminal regulatory domain. [34] Some proteins combine these two mechanisms [35] but however phosphorylation does not necessarily lead to activation, as shown in “off” state in a phosphorylated SSK1 in yeast osmoregulation.

The conformation change induced by phosphorylation affects a large area of the regulatory domain. This provides an ample molecular surface for multiple protein-protein interactions as many RRs are involved in interaction with several different macromolecular targets through phosphorylation-modulation which include HPK, effector domains, other regulatory domain within dimmers, phosphatases.  Some components of the transcriptional machinery could also possibly be involved.

Regulation of Histidine Kinase Activities

The activities of HPK are regulated directly or indirectly by stimuli. HPK determine the level of RR phosphorylation by autophosphorylation and RR phosphatase activity. In many systems, the RR phosphatase activity is regulated [15, 36] instead of autophosphorylation. There are however regulations that occur exclusively for autophosphorylation as not all HPK possess phosphatase activity. [37] Typically in transmembrane HPKs, physical stimuli are detected by sensing domain. The sensing domain may also bind directly to ligands. There are more complex indirect detection of signals through interaction with other protein components. For example, an auxiliary protein PII regulates the RR phosphatase activities of the soluble HPK NtrB. The autophosphorylation activity of chemotaxis HPK CheA, which forms a complex with an adaptor protein CheW and chemoreceptors, is either inhibited or activated by signals transmitted from chemoreceptors. [38] HPK activities can also be regulated by intrinsic domains. For example, the turgor sensor KdpD contains an auxiliary ATP-binding domain where ATP binding is essential for phosphatase activities instead of hydrolysis. [39]

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Regulation of Response Regulator Dephosphorylation

RR dephosphorylation is influenced by phosphatase activities of HPKs that are regulated by many different mechanisms. In most cases, the phosphatase mechanism appear to be a reversal of phosphotransfer and do not require the H box His of the HPK but in some cases ATP is the stimulant by phosphatase activity. [15] RR dephosphorylation can also be effected by auxiliary proteins. For example, an auxiliary protein CheZ oligomerises with phospho-CheY and accelerates its dephosphorylation in bacterial chemotaxis. [40]

Beside regulations of HPK and RR dephosphorylation, there are also a small number of additional regulatory mechanisms involved in specific systems. In some systems, competition for the phosphoryl groups influences the activation of different branches of the signalling pathway. [41] The process of phosphotransfer itself can be regulated; for instance, by physically interacting with the autophosphorylation site, the C-terminal Asp-containing domain can regulate the phosphotransfer ability of the kinase core. [42] Control of gene expression itself also regulates the level of RR such that the phosphorylated RR functions as an activator or repressor of the operon encoding the TCS proteins themselves. [43]

Phosphotransfer system and phosphorelay system

The majority of TCS have a simplistic phosphotransfer design which uses a single phosphoryl transfer to activate a RR protein to elicit a response. There are also more elaborate versions of TCS which have multiple phosphotransfer pathways known as phosphorelays. [44] The basic design of the phosphorelay has five phosphoryl transfer reactions. The multiple domains in phosphorelay systems might provide an alternative pathway to transfer phosphoryl. In HPK ArcB, either Hisitidine containing domain or HPt can be phosphorylated from ATO and donate the phosphoryl groups to the RR ArcA. Tsuzuki and his colleagues [24] also suggest that there might be different pathways for aerobic and anaerobic conditions.

The signalling pathways can also be integrated into cellular networks. For instance in Bacillus subtilis, each characterised TCS appear to interface with at least one other phosphotransfer pathway. [45] One example of such integration is between the pathways controlling the sporulation (KinA-B/Spo0A), aerobic and anaerobic respiration (ResE/ResD) and the utilisation of phosphate (PhoR/PhoP). The phosphate utilisation and respiration co-regulate with one another while phosphor-PhoP is required for ResD expression and vice versa. [46] Furthermore, once the cell commits to sporulation, respiration and phosphate utilisation become down regulated. Phospho-Spo0A is thus a negative regulator of both phospho-PhoP and phosphor-ResD and hence become mutually exclusive with both responses. [46]

Many studies involving different systems of TCS had been studied over the 20 plus years since TCS were first described. The irony is that it took scientists a very long time to discover so important and widespread among bacteria. There are two possible reasons, the early work focus on prokaryote gene regulation revolved around non-covalent interactions of specific proteins with small molecules and that the phosphorylated response regulator in inherent instable with chemotaxis CheY of a half life of few seconds.  With increasing technology, the biochemical activities, 3D structure of the conserved protein are well known. However, the stimuli sensed by the HPKs and the molecular mechanism of the signal transmission across the membrane from the sensing domain have yet to be determine. Similarly, the mechanism for conformational changes in regulatory domain to activate the effector domain has yet to be known. However, there is a potentially exciting application of TCS in antimicrobial drug and control of virulence factor in pathogens.

Application of TCS for antimicrobial therapy

TCS are appealing for antimicrobial therapy for several reasons. Factors that encode for, regulate, or facilitate the transfer of antibacterial or antibiotic resistance may also contribute to the bacterium's shift to the pathogenic state. Virulence factors contribute to the ability of the microorganism to survive and grow at the site of infection and to its pathogenicity. TCSs are widespread in bacteria and absent in mammals. Therefore, the most attractive reason is that TCS are used by pathogenic bacteria to control virulence factors. There are several well-characterised virulence systems such as Salmonella typhimurium PhoP/PhoQ system and Agrobacteria tumefaciens VirA/VirG system.  Interestingly, some bacteria have used TCS to regulate resistance to antibiotics as well. [47] Selective inhibition can be achieved by targeting specific HPKs or RRs, while most TCS are non essential, there is interdependence among the systems. Hence slowing down of intracellular network may be a way to effect a cellular shutdown.

Many companies, including Johnson & Johnson, initiated screening programs to identify TCS inhibitors. Chemical library screens yielded leads in the form of triphenylalkyl derivatives, diaryltriazole analogs, salicylanilides, bisphenols, cyclohexenes and benzoxazines. Unfortunately, poor selectivity and safety drawbacks had not result in a successful product yet and so far, research has shown that resistance to vancomycin is regulated by the sensor kinase VanS and its response regulator VanR in reaction to some as yet unknown signal.[48]  Few publications now describe compounds that inhibit two-component systems, One of the natural inhibitors of TCS is unsaturated fatty acids being noncompetitive inhibitors of ATP-dependent autophosphorylation of the histidine protein kinase KinA involved in the regulation of sporulation in Bacillus. subtilis [49] and a synthetic inhibitor being found in the TCS that regulate virulence expression factors in [50] Pseudomonas aeruginosa alginate biosynthetic pathway.

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