Role of serine/threonine phosphatase (SP-STP) in Streptococcus pyogenes physiology and virulence

Reversible phosphorylation is the key mechanism regulating several cellular events in prokaryotes and eukaryotes. In prokaryotes, signal transduction is perceived to occur primarily via the two-component signaling system involving histidine kinases and cognate response regulators. Although an alternative regulatory pathway controlled by the eukaryote-type serine/threonine kinase (Streptococcus pyogenes serine/threonine kinase; SP-STK) has been shown to modulate bacterial growth, division, adherence, invasion, and virulence in group A Streptococcus (GAS; S. pyogenes), the precise role of the co-transcribing serine/threonine phosphatase (SP-STP) has remained enigmatic. In this context, this is the first report describing the construction and characterization of non-polar SP-STP mutants in two different strains of Type M1 GAS. The STP knock-out mutants displayed increased bacterial chain lengths in conjunction with thickened cell walls, significantly reduced capsule and hemolysin production, and restoration of the phenotypes postcomplementation. The present study also reveals important contribution of cognately regulated-reversible phosphorylation by SP-STK/SP-STP on two major response regulators of two-component systems, WalRK and CovRS. We also demonstrate a distinct role of SP-STP in terms of expression of surface proteins and SpeB in a strain-specific manner. Further, the attenuation of virulence in the absence of STP and its restoration only in the complemented strains that were generated by the use of a low copy plasmid and not by a high copy one emphasize not only the essential role of STP in virulence but also highlight the tightly regulated SP-STP/SP-STK-mediated cognate functions. SP-STP thus is an important regulator of GAS virulence and plays a critical role in GAS pathogenesis.     

Streptococcus pyogenes Ser/Thr Kinase-regulated Cell Wall Hydrolase Is a Cell Division Plane-recognizing and Chain-forming Virulence Factor

Cell division and cell wall synthesis are closely linked complex phenomena and play a crucial role in the maintenance and regulation of bacterial virulence. Eukaryotic-type Ser/Thr kinases reported in prokaryotes, including that in group A Streptococcus (GAS) (Streptococcus pyogenes Ser/Thr kinase (SP-STK)), regulate cell division, growth, and virulence. The mechanism of this regulation is, however, unknown. In this study, we demonstrated that SP-STK-controlled cell division is mediated under the positive regulation of secretory protein that possesses a cysteine and histidine-dependent aminohydrolases/peptidases (CHAP) domain with functionally active cell wall hydrolase activity (henceforth named as CdhA (CHAP-domain-containing and chain-forming cell wall hydrolase). Deletion of the CdhA-encoding gene resulted in severe cell division and growth defects in GAS mutants. The mutant expressing the truncated CdhA (devoid of the CHAP domain), although displayed no such defects, it became attenuated for virulence in mice and highly susceptible to cell wall-acting antibiotics, as observed for the mutant lacking CdhA. When CdhA was overexpressed in the wild-type GAS as well as in heterologous strains, Escherichia coli and Staphylococcus aureus, we observed a distinct increase in bacterial chain length. Our data reveal that CdhA is a multifunctional protein with a major function of the N-terminal region as a cell division plane-recognizing domain and that of the C-terminal CHAP domain as a virulence-regulating domain. CdhA is thus an important therapeutic target.     

An improved method to unravel phosphoacceptors in Ser/Thr protein kinase-phosphorylated substrates

Identification of the phosphorylated residues of bacterial Ser/Thr protein kinase (STPK) substrates still represents a challenging task. Herein, we present a new strategy allowing the rapid determination of phosphoacceptors in kinase substrates, essentially based on the dual expression of the kinase with its substrate in the surrogate E. coli, followed by MS analysis in a single-step procedure. The performance of this strategy is illustrated using two distinct proteins from Mycobacterium tuberculosis as model substrates, the GroEL2 and HspX chaperones. A comparative analysis with a standard method that includes mass spectrometry analysis of in vitro phosphorylated substrates is also addressed.     

A eukaryotic type serine/threonine kinase and phosphatase in Streptococcus agalactiae reversibly phosphorylate an inorganic pyrophosphatase and affect growth,cell segregation,and virulence

Protein phosphorylation is essential for the regulation of cell growth, division, and differentiation in both prokaryotes and eukaryotes. Signal transduction in prokaryotes was previously thought to occur primarily by histidine kinases, involved in two-component signaling pathways. Lately, bacterial homologues of eukaryotic-type serine/threonine kinases and phosphatases have been found to be necessary for cellular functions such as growth, differentiation, pathogenicity, and secondary metabolism. The Gram-positive bacteria Streptococcus agalactiae (group B streptococci, GBS) is an important human pathogen. We have identified and characterized a eukaryotic-type serine/threonine protein kinase (Stk1) and its cognate phosphatase (Stp1) in GBS. Biochemical assays revealed that Stk1 has kinase activity and localizes to the membrane and that Stp1 is a soluble protein with manganese-dependent phosphatase activity on Stk1. Mutations in these genes exhibited pleiotropic effects on growth, virulence, and cell segregation of GBS. Complementation of these mutations restored the wild type phenotype linking these genes to the regulation of various cellular processes in GBS. In vitro phosphorylation of cell extracts from wild type and mutant strains revealed that Stk1 is essential for phosphorylation of six GBS proteins. We have identified the predominant endogenous substrate of both Stk1 and Stp1 as a manganese-dependent inorganic pyrophosphatase (PpaC) by liquid chromatography/tandem mass spectrometry. These results suggest that these eukaryotic-type enzymes regulate pyrophosphatase activity and other cellular functions of S. agalactiae.     

Proteomic identification of M. tuberculosis protein kinase substrates: PknB recruits GarA, a FHA domain-containing protein, through activation loop-mediated interactions

Genes for functional Ser/Thr protein kinases (STPKs) are ubiquitous in prokaryotic genomes, but little is known about their physiological substrates and their actual involvement in bacterial signal transduction pathways. We report here the identification of GarA (Rv1827), a Forkhead-associated (FHA) domain-containing protein, as a putative physiological substrate of PknB, an essential Ser/Thr protein kinase from Mycobacterium tuberculosis. Using a global proteomic approach, GarA was found to be the best detectable substrate of the PknB catalytic domain in non-denatured whole-cell protein extracts from M. tuberculosis and the saprophyte Mycobacterium smegmatis. Enzymological and binding studies of the recombinant proteins demonstrate that docking interactions between the activation loop of PknB and the C-terminal FHA domain of GarA are required to enable efficient phosphorylation at a single N-terminal threonine residue, Thr22, of the substrate. The predicted amino acid sequence of the garA gene, including both the N-terminal phosphorylation motif and the FHA domain, is strongly conserved in mycobacteria and other related actinomycetes, suggesting a functional role of GarA in putative STPK-mediated signal transduction pathways. The ensuing model of PknB-GarA interactions suggests a substrate recruitment mechanism that might apply to other mycobacterial kinases bearing multiple phosphorylation sites in their activation loops.     

Novel substrates of Mycobacterium tuberculosis PknH Ser/Thr kinase

PknH Ser/Thr protein kinase of Mycobacterium tuberculosis controls the expression of a variety of cell wall related enzymes and regulates the in vivo growth in mice. Therefore, we predicted that the PknH kinase could phosphorylate several substrates controlling different metabolic and physiological pathways. Using a bioinformatic approach, we identified 40 potential substrates. Two substrates were shown to be phosphorylated by recombinant PknH kinase in vitro. Point mutation studies verified that substrates are phosphorylated at the in silico-predicted sites. Kinetic studies revealed a similar relative-phosphorylation rate (V(max)) of PknH towards two new substrates and the only previously known substrate, EmbR. Unlike the EmbR protein, the Rv0681 and DacB1 proteins do not contain an FHA domain and are possible participants of new signaling pathways mediated by the PknH kinase in M. tuberculosis.     

Crystal structure of HPr kinase/phosphatase from Mycoplasma pneumoniae

HPr kinase/phosphatase (HPrK/P) modifies serine 46 of histidine-containing protein (HPr), the phosphorylation state of which is the control point of carbon catabolite repression in low G+C Gram-positive bacteria. To understand the structural mechanism by which HPrK/P carries out its dual, competing activities we determined the structure of full length HPrK/P from Mycoplasma pneumoniae (PD8 ID, 1KNX) to 2.5A resolution. The enzyme forms a homo-hexamer with each subunit containing two domains connected by a short loop. The C-terminal domain contains the well-described P-loop (Walker A box) ATP binding motif and takes a fold similar to phosphoenolpyruvate carboxykinase (PEPCK) from Escherichia coli as recently described in other HPrK/P structures. As expected, the C-terminal domain is very similar to the C-terminal fragment of Lactobacillus casei HPrK/P and the C-terminal domain of Staphylococcus xylosus HPrK/P; the N-terminal domain is very similar to the N-terminal domain of S.xylosus HPrK/P. Unexpectedly, the N-terminal domain resembles UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate ligase (MurE), yet the function of this domain is unclear. We discuss these observations as well as the structural significance of mutations in the P-loop and HPrK/P family sequence motif.     

Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC,a Ser/Thr kinase from Bacillus subtilis

We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases. In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing the kinase and a short juxtamembrane region. This fragment, which we designate PrkCc, undergoes autophosphorylation in E.coli. PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction. PrkC also displays kinase activity with myelin basic protein. Using high mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray tandem mass spectrometry, we identified seven phosphorylated threonine and one serine residue in PrkCc. All the corresponding residues were replaced by systematic site-directed mutagenesis and the purified mutant proteins were tested for in vitro kinase activity. Single and multiple replacement of four threonine residues, clustered between residues 162 and 167 in a putative activation loop, substantially reduced kinase activity and the effect was clearly additive. Replacement of the other three threonine residues, clustered between residues 290 and 320, had relatively little effect on activity. In contrast, substitution of Ser214, which is conserved in closely related receptor kinase-like bacterial proteins, independently affected activity and may represent a novel regulatory mechanism. When projected onto a 3D structure of PrkC modelled on the structure of known Hanks kinases, the first cluster of phospho-threonine residues falls precisely in the activation loop, controlling the access of substrate and ATP to the catalytic site of many eukaryotic receptor kinases, whereas the second cluster is located in the juxtamembrane region. These results indicate that regulation of PrkC kinase activity (and presumably autophosphorylation) includes a conserved activation loop mechanism. The juxtamembrane phospho-threonine residues may be essential, for example for the recruitment of other proteins necessary for a PrkC signalling cascade or for coupling to other signalling pathways. This is the first structure-function analysis of a bacterial receptor-like kinase of the Hanks family.     

Sensor domain of the Mycobacterium tuberculosis receptor Ser/Thr protein kinase, PknD, forms a highly symmetric beta propeller

Diverse pathogenic bacteria produce transmembrane receptor Ser/Thr protein kinases (STPKs), but little is known about the signals mediated by these "eukaryotic-like" proteins. To explore the basis for signaling in the bacterial STPK receptor family, we determined the structure of the sensor domain of Mycobacterium tuberculosis PknD. In two crystal forms, the PknD sensor domain forms a rigid, six-bladed beta-propeller with a flexible tether to the transmembrane domain. The PknD sensor domain is the most symmetric beta-propeller structure described. All residues that vary most among the blade subdomains cluster in the large "cup" motif, analogous to the ligand-binding surface in many beta-propeller proteins. These results suggest that PknD binds a multivalent ligand that signals by changing the quaternary structure of the intracellular kinase domain.     

Recognition of peptidoglycan and β-lactam antibiotics by the extracellular domain of the Ser/Thr protein kinase StkP from Streptococcus pneumoniae

The eukaryotic-type serine/threonine kinase StkP from Streptococcus pneumoniae is an important signal-transduction element that regulates the expression of numerous pneumococcal genes. We have expressed the extracellular C-terminal domain of StkP kinase (C-StkP), elaborated a three-dimensional structural model and performed a spectroscopical characterization of its structure and stability. Biophysical experiments show that C-StkP binds to synthetic samples of the cell wall peptidoglycan (PGN) and to β-lactam antibiotics, which mimic the terminal portions of the PGN stem peptide. This is the first experimental report on the recognition of a minimal PGN unit by a PASTA-containing kinase, suggesting that non-crosslinked PGN may act as a signal for StkP function and pointing to this protein as an interesting target for β-lactam antibiotics.     

Conserved autophosphorylation pattern in activation loops and juxtamembrane regions of Mycobacterium tuberculosis Ser/Thr protein kinases

The identification of phosphorylation sites in proteins provides a powerful tool to study signal transduction pathways and to establish interaction networks involving signaling elements. Using different strategies to identify phosphorylated residues, we report here mass spectrometry studies of the entire intracellular regions of four 'receptor-like' protein kinases from Mycobacterium tuberculosis (PknB, PknD, PknE, and PknF), each consisting of an N-terminal kinase domain and a juxtamembrane region of varying length (26-100 residues). The enzymes were observed to incorporate different numbers of phosphates, from five in PknB up to 11 in PknD or PknE, and all detected sites were dephosphorylated by the cognate mycobacterial phosphatase PstP. Comparison of the phosphorylation patterns reveals two recurrent clusters of pThr/pSer residues, respectively, in their activation loops and juxtamembrane regions, which have a distinct effect on kinase activity. All studied kinases have at least two conserved phosphorylated residues in their activation loop and mutations of these residues in PknB significantly decreased the kinase activity, whereas deletion of the entire juxtamembrane regions in PknB and PknF had little effect on their activities. These results reinforce the hypothesis that mycobacterial kinase regulation includes a conserved activation loop mechanism, and suggest that phosphorylation sites in the juxtamembrane region might be involved in putative kinase-mediated signaling cascades.     

Protein kinase and phosphatase signaling in Mycobacterium tuberculosis physiology and pathogenesis

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), evades the antimicrobial defenses of the host and survives within the infected individual through a complex set of strategies. These include active prevention of host cellular killing processes as well as overwhelming adaptive gene expression. In the past decade, we have gained an increased understanding of how mycobacteria not only have the ability to adapt to a changing host environment but also actively interfere with the signaling machinery within the host cell to counteract or inhibit parts of the killing apparatus employed by the macrophage. Mtb is able to sense its environment via a set of phospho-signaling proteins which mediate its response and interaction with the host in a coordinated manner. In this review, we summarize the current knowledge about selected Mtb serine, threonine, and tyrosine kinase and phosphatase signaling proteins, focusing on the protein kinases, PknG and PtkA, and the protein phosphatase, PtpA.     

Cloning and characterization of the pknA gene from Streptomyces coelicolor A3(2), coding for the Mn2+ dependent protein Ser/Thr kinase

A gene pknA, coding for an eukaryotic-type protein Ser/Thr kinase, was cloned from the Streptomyces coelicolor A3(2) chromosome. The PknA protein kinase, containing the C-terminal eukaryotic-type kinase domain with an N-terminal extension, was expressed in Escherichia coli and Streptomyces lividans. The affinity purified MBP-PknA fusion protein was assayed for kinase activity that showed its ability to autophosphorylate in vitro in the presence of [gamma-32P]ATP. The activity was Mn2+ dependent. The preautophosphorylated kinase phosphorylated at least two proteins (sizes 30 and 32 kDa) in the S. coelicolor J1501 cell-free extracts of all developmental stages. The larger of them was also phosphorylated in vitro by an endogenous protein kinase in late stages extracts, but not earlier. Although Mn2+ dependent protein phosphorylation has previously been described in Streptomyces, this is the first report of a gene encoding such an enzyme in this genus.     

GTPase activity of mycobacterial FtsZ is impaired due to its transphosphorylation by the eukaryotic-type Ser/Thr kinase, PknA

FtsZ, a homolog of eukaryotic tubulin, is involved in the process of cell division, particularly in septum formation in bacteria. The primary amino acid sequences of this protein are fairly conserved in prokaryotes. We observed that a eukaryotic-type Ser/Thr protein kinase, PknA from Mycobacterium tuberculosis, when expressed in Escherichia coli exhibited cell elongation due to a defect in septum formation. We found that FtsZ either from Escherichia coli (eFtsZ) or from M. tuberculosis (mFtsZ) was phosphorylated on co-expression with PknA. Consistent with these observations, solid phase binding and in vitro kinase assays revealed the ability of PknA to interact with mFtsZ protein and also to phosphorylate it. We, therefore, ascertained mFtsZ as a substrate of PknA. Furthermore, the phosphorylated mFtsZ exhibited impairment in its GTP hydrolysis and polymerization abilities. Thus, our results highlighted the ability of PknA to phosphorylate as well as to regulate the functionality of FtsZ, the protein central to cell division throughout the bacterial lineage.     

Serine/threonine kinase activity in the putative histidine kinase-like ethylene receptor NTHK1 from tobacco

A histidine kinase-based signaling system has been proposed to function in ethylene signal transduction pathway of plants and one ethylene receptor has been found to possess His kinase activity. Here we demonstrate that a His kinase-like ethylene receptor homologue NTHK1 from tobacco has serine/threonine (Ser/Thr) kinase activity, but no His kinase activity. Evidence obtained by analyzing acid/base stability, phosphoamino acid and substrate specificity of the phosphorylated kinase domain, supports this conclusion. In addition, mutation of the presumptive phosphorylation site His (H378) to Gln did not affect the kinase activity whereas deletion of the ATP-binding domain eliminated it, indicating that the conserved His (H378) is not required for the kinase activity and this activity is intrinsic to the NTHK1-KD. Moreover, confocal analysis of NTHK1 expression in insect cells and plant cells suggested the plasma membrane localization of the NTHK1 protein. Thus, NTHK1 may represent a distinct Ser/Thr kinase-type ethylene receptor and function in an alternative mechanism for ethylene signal transduction.     

Identification of residues which regulate activity of the STE20-related kinase hMINK

Activity of the STE20-related kinase hMINK was investigated. hMINK was expressed widely, though not ubiquitously, in human tissues; highest levels being found in haematopoietic tissues but also in brain, placenta, and lung. Mutagenesis revealed that T(191) and Y(193) in the substrate recognition loop of the catalytic domain were critical for kinase activity against exogenous substrates and autophosphorylation. A mutation on T(187) showed reduced enzymatic activity against exogenous substrates but retained autophosphorylation activity. Phosphorylation was confirmed by the use of a phospho-specific T(187) antibody. hMINK activated the JNK signal transduction pathway and optimal JNK activation occurred when the C-terminus was deleted. In addition, overexpression of the C-terminal domain devoid of kinase activity also resulted in significant activation of the JNK pathway. These data suggest that hMINK requires an activation step that dissociates the C terminal, thereby freeing the catalytic domain to interact with substrates. Models for receptor-mediated activation of hMINK are discussed.     

Identification and characterization of protein phosphatase 2C activation by ceramide

Ceramide is a bioactive sphingolipid with many associated biological outcomes, yet there is a significant gap in our current understanding of how ceramide mediates these processes. Previously, ceramide has been shown to activate protein phosphatase (PP) 1 and 2A. While continuing this line of work, a late fraction from a Mono-Q column was consistently observed to be activated by ceramide, yet PP1 and PP2A were undetectable in this fraction. Proteomic analysis of this fraction revealed the identity of the phosphatase to be PP2Cγ/PPM1G. This was consistent with our findings that PP2Cγ 1-eluted in a high salt fraction due to its strongly acidic domain, and 2-was insensitive to okadaic acid. Further characterization was performed with PP2Cα, which showed robust activation by C(6)-ceramide. Activation was specific for the erythro conformation of ceramide and the presence of the acyl chain and hydroxyl group at the first carbon. In order to demonstrate more physiological activation of PP2Cα by ceramide, phospho-p38δ was utilized as substrate. Indeed, PP2Cα induced the dephosphorylation of p38δ only in the presence of C(16)-ceramide. Taken together, these results show that the PP2C family of phosphatases is activated by ceramide, which may have important consequences in mediating the biological effects of ceramide.