Regulation of bacterial phosphorelay systems

Abstract

In terms of biomass, bacteria are the most successful organisms on earth. This is partly attributed to their tremendous adaptive capabilities, which allows them to sense and rapidly organise responses to changing environmental stimuli. Using complex signalling mechanisms, bacteria can relay cellular information to fine-tune their metabolism, maintain homeostasis, and trigger virulence processes during infection. Across all life, protein phosphorylation represents the most abundant signalling mechanism, which is controlled by a versatile class of enzymes called protein kinases and their cognate phosphatases. For many years, histidine kinase (HK)-containing two-component systems (TCSs) were considered the canonical instruments of bacterial sensing. However, advances in metagenomics has since proven that bacterial phosphorelay is in fact orchestrated by a functionally diverse array of integrated protein kinase types, including Ser, Thr, Tyr and Arg-targeting enzymes. In this review, we provide an up-to-date appraisal of bacterial kinase signalling, with an emphasis on how these sensing pathways are regulated to modulate kinase output. Finally, we explore how selective kinase inhibitors may be exploited to control infections and combat the looming health emergency of multidrug resistant bacteria.

Fig. 1. Structural similarities between eukaryotic and prokaryotic Ser/Thr kinases. Crystal structures of (A) human PKA in complex with ATP (PDB: 4WB5), (B) M. tuberculosis PknG in complex with ADP (4Y0X) and (C) Bacillus atypical protein kinase, CotH, in complex with AMP. Insets show ATP/AMP coordinating amino acid residues (white sticks). α-Helices are shaded blue, β-strands are shaded red, loops are shaded grey, Mg²⁺ ions are represented as turquoise spheres and ATP/AMP is represented by green sticks. (D) Sequence alignment of PKA with PknG and CotH (using MUSCLE). Canonical amino acid residues (or functional equivalents) involved in ATP binding or phosphate transfer are colour coordinated. Note that the atypical kinase, CotH, aligns poorly due to its highly diverged sequence.

Fig. 2. Overview of bacterial kinase signal transduction mechanisms. (A) Asymmetrical TCS kinase state: classical two component signalling systems (TCSs) respond to extracellular (through periplasmic receptor) or cytosolic (through HAMP/PAS/GAF) sensor domains, leading to dimerization. In a multistep phosphorelay, the catalytic CA domains phosphorylate, in trans, a conserved His in the DHp domain. The phosphate is then shuttled (dashed arrows) to a conserved Asp residue in cognate response regulators (RRs). RRs dimerize and bind to a specific DNA binding domain (DBD) to elicit transcription of target genes. Symmetrical TCS phosphatase state: in the absence of stimuli, the CA domain undergoes a conformation switch to a phosphatase-active state, dephosphorylating RRs in cis, releasing phosphate into the cytosol. (B) Simplified schematic of a B. subtilis eSTK, PrkC, responding to extracytosolic stimuli via PASTA sensor domains. During stationary phase, binding of muropeptides to PASTA repeats induces dimerization of PrkC TM and eSTK domains, leading to in trans autophosphorylation and subsequent phosphorylation of a range of target cytosolic proteins. Substrates include, but are not limited to, GpsB (a cell division protein), YkwC (an oxidoreductase) and CpgA (a GTPase). PrkC also regulates the WalRK TCS by directly phosphorylating the WalR RR, influencing expression of genes relating to cell wall biosynthesis.

Fig. 3. Cross-talk between GacS/A and other TCSs in P. aeruginosa. (1) Periplasmic or cytosolic sensor domains respond to environmental or cellular signals that cause GacS homodimerization. The ND and CA domains facilitate autophosphorylation of a conserved His in the DHp domain. GacS contains an additional receiver (D1) domain, which becomes phosphorylated by intramolecular phosphotransfer. The phosphate is subsequently shuttled to the H2 domain and finally GacA, the RR. (2) LadS positively regulates GacS function, enhancing kinase activity and supporting chronic infection characteristics. (3) RetS forms a complex with GacS, inhibiting its kinase activity thereby suppressing GacA phosphorylation, leading to enhanced expression of acute virulence factors. (4) PA1611 binds to RetS, preventing its inhibitory interaction with GacS. By sequestering RetS, PA1611 allows GacS to maintain its kinase activity, facilitating GacA phosphorylation and promoting chronic infection pathways.