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Components of Eukaryotic-like Protein Signaling Pathways in Mycobacterium tuberculosis
Abstract
Eukaryotic-like protein kinases have been identified recently in several prokaryotes by comparative studies of DNA sequences and Western blotting techniques using antiphosphoprotein antibodies. Examination of the Mycobacterium tuberculosis genome by means of PCR amplification with consensus primers, Southern hybridization, and comparative analysis of DNA sequences with genomic databases revealed the existence of at least seven eukaryotic-like protein kinases in this pathogen. In addition, we report the biochemical identification of phosphorylated proteins in M. tuberculosis. Taken together, these findings show that M. tuberculosis possesses elements of cell signaling similar to those observed in eukaryotic organisms. We suggest that some of these processes may play roles in the pathogenesis of M. tuberculosis.
... In the last decades, PKs have attracted interest as drug targets in a variety of diseases such as cancer [1], diabetes [2], Alzheimer [3], chronic inflammations [4], etc. Since abnormal PK actions in the human body are related to many diseases, human PKs have been widely studied because the interest in their use as therapeutic targets; however, the study of Mycobacterium tuberculosis serine/threonine PKs (MTPKs) involved in tuberculosis pathogenesis had its origins later [5], and at this moment there are many issues to investigate about these PKs. ...
... Mycobacterium tuberculosis encodes eleven putative eukaryotic-like MTPKs (PknA, PknB, PknD, PknE, PknF, PknG, PknH, PknI, PknJ, PknK, and PknL) [5][6][7], where nine of them are receptor type PKs and two others are cytoplasmatic. Experiments where proteins are phosphorylated in vitro confirm the presence of functional MTPKs 1 3 [8]. ...
In the last decades, human protein kinases (PKs) have been relevant as targets in the development of novel therapies against many diseases, but the study of Mycobacterium tuberculosis PKs (MTPKs) involved in tuberculosis pathogenesis began much later and has not yet reached an advanced stage of development. To increase knowledge of these enzymes, in this work we studied the structural features of MTPKs, with focus on their ATP-binding sites and their interactions with inhibitors. PknA, PknB, and PknG are the most studied MTPKs, which were previously crystallized; ATP-competitive inhibitors have been designed against them in the last decade. In the current work, reported PknA, PknB, and PknG inhibitors were extracted from literature and their orientations inside the ATP-binding site were proposed by using docking method. With this information, interaction fingerprints were elaborated, which reveal the more relevant residues for establishing chemical interactions with inhibitors. The non-crystallized MTPKs PknD, PknF, PknH, PknJ, PknK, and PknL were also studied; their three-dimensional structural models were developed by using homology modeling. The main characteristics of MTPK ATP-binding sites (the non-crystallized and crystallized MTPKs, including PknE and PknI) were accounted; schemes of the main polar and nonpolar groups inside their ATP-binding sites were constructed, which are suitable for a major understanding of these proteins as antituberculotic targets. These schemes could be used for establishing comparisons between MTPKs and human PKs in order to increase selectivity of MTPK inhibitors. As a key tool for guiding medicinal chemists interested in the design of novel MTPK inhibitors, our work provides a map of the structural elements relevant for the design of more selective ATP-competitive MTPK inhibitors.
... However, it is important to emphasize that these signaling enzymes have been studied extensively in other pathogenic and nonpathogenic bacteria. These include B. subtilis [16,43,[141][142][143], B. anthracis [17], M. tuberculosis [22,[144][145][146][147][148][149][150][151] and L. monocytogenes [27,28]. A brief summary of the role of eukaryotic-like signaling enzymes in these organisms is provided (also see [150]). ...
... The STK, BA-Stk1 and phosphatase BA-Stp1 have been shown to regulate virulence of B. anthracis [17] suggesting that virulence regulation by STKs is a conserved function in Gram-positive pathogens. M. tuberculosis, the causative agent of tuberculosis, encodes eleven STKs; however, only PknB possess the extracellular PASTA domains commonly found in the homologous STKs reviewed here [22,144,150,151]. Unlike the STKs reviewed in this aticle, PknB is essential in M. tuberculosis [145][146][147], and is the first prokaryotic STK whose crystal structure has been resolved [148,149]. ...
Living organisms adapt to the dynamic external environment for their survival. Environmental adaptation in prokaryotes is thought to be primarily accomplished by signaling events mediated by two-component systems, consisting of histidine kinases and response regulators. However, eukaryotic-like serine/threonine kinases (STKs) have recently been described to regulate growth, antibiotic resistance and virulence of pathogenic bacteria. This article summarizes the role of STKs and their cognate phosphatases (STPs) in Gram-positive cocci that cause invasive infections in humans. Given that a large number of inhibitors to eukaryotic STKs are approved for use in humans, understanding how serine/threonine phosphorylation regulates virulence and antibiotic resistance will be beneficial for the development of novel therapeutic strategies against bacterial infections.
... Interestingly, both these kinases have been shown to be required for the full virulence of these pathogens in mouse models. Previously, we have shown that M. tuberculosis encodes at least eight eukaryotic-like protein kinases (Gay and Davies, 1997). Furthermore, we have demonstrated that six proteins are phosphorylated in vitro (Gay and Davies, 1997), suggesting the presence of functional kinases in M. tuberculosis . ...
... Previously, we have shown that M. tuberculosis encodes at least eight eukaryotic-like protein kinases (Gay and Davies, 1997). Furthermore, we have demonstrated that six proteins are phosphorylated in vitro (Gay and Davies, 1997), suggesting the presence of functional kinases in M. tuberculosis . The completion of the M. tuberculosis genome sequencing project has provided a complete list of these eukaryotic-like protein kinases and phosphatases forming the M. tuberculosis PSTK family (Cole et al ., 1998). ...
The function of the Mycobacterium tuberculosis eukaryotic-like protein serine/threonine kinase PknG was investigated by gene knock-out and by expression and biochemical analysis. The pknG gene (Rv0410c), when cloned and expressed in Escherichia coli, encodes a functional kinase. An in vitro kinase assay of the recombinant protein demonstrated that PknG can autophosphorylate its kinase domain as well as its 30 kDa C-terminal portion, which contains a tetratricopeptide (TPR) structural signalling motif. Western analysis revealed that PknG is located in the cytosol as well as in mycobacterial membrane. The pknG gene was inactivated by allelic exchange in M. tuberculosis. The resulting mutant strain causes delayed mortality in SCID mice and displays decreased viability both in vitro and upon infection of BALB/c mice. The reduced growth of the mutant was more pronounced in the stationary phase of the mycobacterial growth cycle and when grown in nutrient-depleted media. The PknG-deficient mutant accumulates glutamate and glutamine. The cellular levels of these two amino acids reached approximately threefold of their parental strain levels. Higher cellular levels of the amine sugar-containing molecules, GlcN-Ins and mycothiol, which are derived from glutamate, were detected in the DeltapknG mutant. De novo glutamine synthesis was shown to be reduced by 50%. This is consistent with current knowledge suggesting that glutamine synthesis is regulated by glutamate and glutamine levels. These data support our hypothesis that PknG mediates the transfer of signals sensing nutritional stress in M. tuberculosis and translates them into metabolic adaptation.
... The first STPK in M. tuberculosis was reported and characterized in 1997 as Mbk (mycobacterial kinase), which was later identified as PknD (35). In the same year, application of PCR amplification, Southern hybridization, and genome database screening, Av-Gay and Davies showed the presence of seven eukaryotic-like protein kinases in M. tuberculosis (36). The abundance of STPKs varies from 4 STPKs in M. leprae, 18 in M. smegmatis, 11 in M. bovis, 10 in M. avium and the highest number of STPKs, 24; in M. marinum (37). ...
The emergence of increasingly drug‐resistant Mycobacterium tuberculosis strains has become a crucial public health concern. In order to effectively treat tuberculosis, it is imperative to find newer drug targets, which are important for the in vivo bacterial survival and persistence. Phosphorylation based signaling cascades modulated by eukaryotic‐like serine/threonine protein kinases and phosphatase in M. tuberculosis, transduce extracellular stimuli to a cellular response ensuing pathogen's growth, persistence and pathogenesis. Of the 11 STPKs that M. tuberculosis genome encodes, three kinases, namely PknA, PknB and PknG and the sole serine/threonine phosphatase PstP are crucial for the intracellular survival of the bacteria. PknA and PknB regulates cell growth, cell wall synthesis and morphological changes during bacterial cell division; while PknG modulates metabolic changes in response to stress and aids in bacterial survival during latency like conditions. PstP functions to dephosphorylate STPKs and their substrates and hence is important at nearly all stages of infection. Here, we review the current knowledge on PstP, PknA, PknB and PknG based on the genetic, biochemical, and functional studies in M. tuberculosis physiopathology. We further explore the potential of these molecules as targets for therapeutic intervention and discuss the advancement made in the development of inhibitors against these targets.
... There is increasing supportive evidence indicating that in mycobacteria, Ser/Thr phosphorylation plays a critical role both in the physiology as well as the virulence of this intracellular pathogen (6)(7)(8)(9)(10)(11)(12)(13). Interestingly, mycobacteria have an unusually large repertoire of kinases for a bacterium, including 11 twocomponent system and 11 Serine/Threonine protein kinases (STPKs) (PknA-PknL) (14)(15)(16)(17). ...
Mycobacterial Ser/Thr kinases play a critical role in bacterial physiology and pathogenesis. Linking kinases to the substrates they phosphorylate in vivo, thereby elucidating their exact functions, is still a challenge. The aim of this work was to associate protein phosphorylation in mycobacteria with important subsequent macro cellular events by identifying the physiological substrates of PknG in Mycobacterium bovis BCG. The study compared the phosphoproteome dynamics during the batch growth of M. bovis BGC versus the respective PknG knock-out mutant (ΔPknG-BCG) strains. We employed TiO2 phosphopeptide enrichment techniques combined with label-free quantitative phosphoproteomics workflow on LC/MS/MS. The comprehensive analysis of label-free data identified 603 phosphopeptides on 307 phosphoproteins with high confidence. 55 phosphopeptides were differentially phosphorylated, of these, 23 phosphopeptides were phosphorylated in M. bovis BCG wild-type only and not in the mutant. These were further validated through targeted mass spectrometry assays (PRMs). Kinase-peptide docking studies based on a published crystal structure of PknG in complex with GarA revealed that the majority of identified phosphosites presented docking scores close to that seen in previously described PknG substrates, GarA, and ribosomal protein L13. Six out of the 23 phosphoproteins had higher docking scores than GarA, consistent with the proteins identified here being true PknG substrates. Based on protein functional analysis of the PknG substrates identified, this study confirms that PknG plays an important regulatory role in mycobacterial metabolism, through phosphorylation of ATP binding proteins and enzymes in the TCA cycle. This work also reinforces PknG's regulation of protein translation and folding machinery.
... MTB comprises 11 serine/threonine protein kinases (STPKs) [15] which probably function in signal transduction pathways and may direct important cellular decisions such as dormancy and cell division [16,17]. Out of the 11 proteins, 10 proteins (Rv0014c, Rv0015c, Rv0410c, Rv0931c, Rv1743, Rv1746, Rv2088, Rv2176, Rv2914c and Rv3080c) were found to be identical in both the strains in this study, while a single amino acid substitution (R607Q) was identified in one of the proteins of MTB H 37 Rv (Rv1266c) compared with H 37 Ra (MRA_1274). ...
Background
Mycobacterium tuberculosis (MTB) H37Ra is an attenuated tubercle bacillus closely related to the virulent type strain MTB H37Rv. In spite of extensive study, variation in virulence between the MTB H37Rv and MTB H37Ra strains is still to be understood. The difference in protein expression or structure due to mutation may probably be an important factor for the virulence property of MTB H37Rv strain.
Methods
In this study, a whole proteome comparison between these two strains was carried out using bioinformatics approaches to elucidate differences in their protein sequences.
Results
On comparison of whole proteome using NCBI standalone BLAST program between these two strains, 3759 identical proteins in both the strains out of 4003 proteins were revealed in MTB H37Rv and 4034 proteins were revealed in MTB H37Ra; 244 proteins of MTB H37Rv and 260 proteins of MTB H37Ra were found to be non-identical. A total of 172 proteins were identified with mutations (Insertions/deletions/substitutions) in MTB H37Ra while 53 proteins of MTB H37Rv and 85 proteins of MTB H37Ra were found to be distinct. Among 244 non-identical proteins, 19 proteins were reported to have an important biological function; In this study, mutation was shown in these proteins of MTB H37Ra.
Conclusion
This study reports the protein differences with mutations between MTB H37Rv and H37Ra, which may help in better understanding the pathogenesis and virulence properties of MTB H37Rv.
... PSTKs expressed by bacteria presumed to act by interfering with host cellular signalling mechanisms during development or pathogenesis [9,14]. Although M. tuberculosis PSTKs are phylogenetically dis- tinct [9,11,12], they contain all 12 distinct Hank's kinase subdomains [8,15]. Further genome analysis work showed that PSTK homologs are present in other mycobacteria such as Mycobacterium bovis Bacille Calmette Guerin (BCG) and Mycobacterium smegmatis [16,17] . ...
The protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) was found to inhibit the growth of two different mycobacterial strains, the slow-growing Mycobacterium bovis Bacille Calmette Guerin (BCG) and the fast-growing saprophyte Mycobacterium smegmatis mc2 155, in a dose-dependent manner. While screening for the effect of kinase inhibitors on mycobacterial growth, millimolar concentrations of H7 induced a 40% decrease in the growth of M. bovis BCG when measured as a function of oxidative phosphorylation. This H7-induced decrease in growth was shown to involve a 2-log fold decrease in the viable counts of M. smegmatis within a 48-h period and a 50% reduction in the number of BCG viable counts within a 10-day period. Micromolar concentrations of H7 compound induced a significant decrease in the activity of the Mycobacterium tuberculosis protein serine/threonine kinase (PSTK) PknB. The inhibition of mycobacterial growth as well as the inhibition of a representative M. tuberculosis protein serine/threonine kinase PknB suggests that conventional PSTK inhibitors can be used to study the role that the mycobacterial PSTK family plays in controlling bacterial growth.
... Such G proteins, particularly the heterotrimeric G proteins, are important in mammalian cell signalling (Gilman, 1987) and are parts of macrophage signalling (Makhlouf et al., 1996). Mycobacteria such as M. tuberculosis have recently been shown to possess components of eukaryotic-like protein signalling pathways comprising protein kinases such as ᮊ 1998 Blackwell Science Ltd, Molecular Microbiology, 28, 875-882 serine/threonine protein kinase (Av-Gay and Davies, 1997), which may play a role in the survival of M. tuberculosis in the macrophages. We have also reported the occurrence of proteins in M. tuberculosis H37Rv cells that cross-react with antibodies directed against mammalian heterotrimeric G protein subunits and have indeed observed that components of mammalian heterotrimeric G proteins form complexes with M. tuberculosis Ndk, directing its NTP-synthesizing specificity to GTP (Shankar et al., 1997b). ...
Nucleoside diphosphate kinase (Ndk) is an important enzyme that generates nucleoside triphosphates (NTPs) or their deoxy derivatives by terminal phosphotransfer from an NTP such as ATP or GTP to any nucleoside diphosphate or its deoxy derivative. As NTPs, particularly GTP, are important for cellular macromolecular synthesis and signalling mechanisms, Ndk plays an important role in bacterial growth, signal transduction and pathogenicity. Specific examples of the role of Ndk in regulating growth, NTP formation and cell surface polysaccharide synthesis in two respiratory tract pathogens, Pseudomonas aeruginosa and Mycobacterium tuberculosis, are discussed.
... Each of the 10 prokaryotic genomes we inspected contained ORFs whose potential products exhibited sequence features characteristic of eukaryotic PKs and PPs. (Independent analysis of another prokaryotic genome, that of the bacterium M. pneumoniae G-37, indicated that it contained ORFs for a potential PK and a PPM family PP as well [88], and ORFs for seven potential PKs have been identi¢ed in Mycobacterium tuberculosis [89].) In the case of the ORFs for potential PPs, it appears that a clear majority predict products that contain the minimal essential set of primary sequence features necessary to support phosphohydrolase activity. ...
Inspection of the genomes for the bacteria Bacillus subtilis 168, Borrelia burgdorferi B31, Escherichia coli K-12, Haemophilus influenzae KW20, Helicobacter pylori 26695, Mycoplasma genitalium G-37, and Synechocystis sp PCC 6803 and for the archaeons Archaeoglobus fulgidus VC-16 DSM4304, Methanobacterium thermoautotrophicum delta H, and Methanococcus jannaschii DSM2661 revealed that each contains at least one ORF whose predicted product displays sequence features characteristic of eukaryote-like protein-serine/threonine/tyrosine kinases and protein-serine/threonine/tyrosine phosphatases. Orthologs for all four major protein phosphatase families (PPP, PPM, conventional PTP, and low molecular weight PTP) were present in the bacteria surveyed, but not all strains contained all types. The three archaeons surveyed lacked recognizable homologs of the PPM family of eukaryotic protein-serine/threonine phosphatases; and only two prokaryotes were found to contain ORFs for potential phosphatases from all four major families. Intriguingly, our searches revealed a potential ancestral link between the catalytic subunits of microbial arsenate reductases and the protein-tyrosine phosphatases; they share similar ligands (arsenate versus phosphate) and features of their catalytic mechanism (formation of arseno-versus phospho-cysteinyl intermediates). It appears that all prokaryotic organisms, at one time, contained the genetic information necessary to construct protein phosphorylation-dephosphorylation networks that target serine, threonine, and/or tyrosine residues on proteins. However, the potential for functional redundancy among the four protein phosphatase families has led many prokaryotic organisms to discard one, two, or three of the four.
... However, intracellular cAMP-mediated functions via cAMP receptor protein were not observed (34). The M. tuberculosis H37Rv genome sequence revealed eukaryotic-like protein kinases (7,35), which may be involved in the intracellular cAMP-mediated signaling pathways. An important extracellular function for cAMP from M. tuberculosis H37Rv may be the prevention of fusion between phagosomes and lysosomes. ...
Screening the Mycobacterium tuberculosis H37Rv genomic library for complementation of catabolic defect for cAMP-dependent expression of maltose operon produced the adenylyl cyclase gene (Mtb cya, (1997)) annotated later as Rv1625c (Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E., III, et al. (1998) Nature 393, 537-544). The deduced amino acid (aa) sequence (443 aa) encoded by Mtb cya contains a single hydrophobic domain of six transmembrane helices (152 aa) in the amino-terminal half of the protein. Flanking this domain are an arginine-rich (17%) amino-terminal cytoplasmic tail (46 aa) and a carboxyl-terminal cytoplasmic domain (245 aa) with extensive homology to the catalytic core of eukaryotic adenylyl cyclases. Site-directed mutagenesis of Arg(43) and Arg(44) to alanine/glycine showed a loss of adenylyl cyclase activity, whereas mutagenesis to lysine restored the activity. Hence it is proposed that the formation of the catalytic site in Mtb adenylyl cyclase requires an interaction between Arg(43) and Arg(44) residues in the distal cytoplasmic tail and the carboxyl-terminal cytoplasmic domain. Mtb adenylyl cyclase activity at the physiological concentration of ATP (1 mm) was 475 nmol of cAMP/min/mg of membrane protein in the presence of Mn(2+) but only 10 nmol of cAMP/min/mg of membrane protein in the presence of Mg(2+). The physiological significance of the activation of Mtb adenylyl cyclase by Mn(2+) is discussed in view of the presence of manganese transporter protein in mycobacteria and macrophages wherein mycobacteria reside.
... In prokaryotes, a two-component system consisting of a histidine kinase and its associated response regulator protein are used in sensing extracellular signals and coordinating intracellular events (Stock et al., 1989). In a number of prokaryotes Ser\Thr protein kinases also regulate development, response to stress conditions and pathogenicity (Av-Gay & Davies, 1997). Examples include regulation of antibiotic production in Streptomyces coelicolor (Matsumoto et al., 1994), spore production in Myxococcus xanthus (Munoz-Dorado et al., 1991) and transduction of environmental signals in Bacillus subtilis (Yang et al., 1996). ...
Pathogenesis of Mycobacterium tuberculosis is closely connected to its survival and replication within the host. Some pathogenic bacteria employ protein kinases that interfere with the cellular signalling network of host cells and promote bacterial survival. In this study, the pknF and pknG genes, which encode two putative protein kinases of M. tuberculosis H(37)Rv, protein kinase F (PknF) and protein kinase G (PknG), respectively, were cloned and expressed in Escherichia coli. Purified PknF phosphorylated the peptide substrate myelin basic protein (MBP) at serine and threonine residues, while purified PknG phosphorylated only at serine residues. The activity of the two kinases was abrogated by mutation of the codon for the predicted ATP-binding-site lysine residue. Southern blot analysis revealed that homologues of the genes encoding the two kinases are present in M. tuberculosis H(37)Ra and Mycobacterium bovis BCG, but not in Mycobacterium smegmatis. Immunoblot analysis of various cellular fractions of M. tuberculosis H(37)Rv revealed that PknF is a transmembrane protein and that PknG is predominantly a cytosolic enzyme. The present study should aid in elucidating the role of these protein kinases in the pathogenesis of mycobacteria.
... PSTKs expressed by bacteria presumed to act by interfering with host cellular signalling mechanisms during development or pathogenesis [9,14]. Although M. tuberculosis PSTKs are phylogenetically dis- tinct [9,11,12], they contain all 12 distinct Hank's kinase subdomains [8,15]. Further genome analysis work showed that PSTK homologs are present in other mycobacteria such as Mycobacterium bovis Bacille Calmette Guerin (BCG) and Mycobacterium smegmatis [16,17] . ...
The protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) was found to inhibit the growth of two different mycobacterial strains, the slow-growing Mycobacterium bovis Bacille Calmette Guerin (BCG) and the fast-growing saprophyte Mycobacterium smegmatis mc2 155, in a dose-dependent manner. While screening for the effect of kinase inhibitors on mycobacterial growth, millimolar concentrations of H7 induced a 40% decrease in the growth of M. bovis BCG when measured as a function of oxidative phosphorylation. This H7-induced decrease in growth was shown to involve a 2-log fold decrease in the viable counts of M. smegmatis within a 48-h period and a 50% reduction in the number of BCG viable counts within a 10-day period. Micromolar concentrations of H7 compound induced a significant decrease in the activity of the Mycobacterium tuberculosis protein serine/threonine kinase (PSTK) PknB. The inhibition of mycobacterial growth as well as the inhibition of a representative M. tuberculosis protein serine/threonine kinase PknB suggests that conventional PSTK inhibitors can be used to study the role that the mycobacterial PSTK family plays in controlling bacterial growth.
... The Yersinia STK (YpkA) is distinct from other prokaryotic homologues because kinase activation occurs only upon binding to host cytoplasmic factors such as actin (23,24). A number of STK have also been described in Mycobacterium tuberculosis (25)(26)(27). However, the phenotypes of mutations in these genes and their biological role in pathogenesis have yet to be reported. ...
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.
... Genomics and experimental rationale. An inspection of the available genome sequences of mycobacteria, corynebacteria, and streptomycetes (1,8,11,12,20) revealed a conserved operon, near the chromosomal origin of replication, comprising five or six genes that may be involved in signal transduction pathways and cell division. In M. tuberculosis, this operon, in which the termination codon of each gene overlaps the initiation codon of its follower, begins with Rv0019, coding for forkhead-associated (FHA) protein B. This is followed by an in-frame mycobacterial intergenic repetitive unit (42) which precedes pstP, encoding phosphoserine/threonine protein phosphatase (9), and the rodA and pbpA genes before the operon ends with the STPK genes, pknA and pknB (Fig. 1). ...
The receptor-like protein kinase PknB from Mycobacterium tuberculosis is encoded by the distal gene in a highly conserved operon, present in all actinobacteria, that may control cell shape and
cell division. Genes coding for a PknB-like protein kinase are also found in many more distantly related gram-positive bacteria.
Here, we report that the pknB gene can be disrupted by allelic replacement in M. tuberculosis and the saprophyte Mycobacterium smegmatis only in the presence of a second functional copy of the gene. We also demonstrate that eukaryotic Ser/Thr protein kinase
inhibitors, which inactivate PknB in vitro with a 50% inhibitory concentration in the submicromolar range, are able to kill
M. tuberculosis H37Rv, M. smegmatis mc2155, and Mycobacterium aurum A+ with MICs in the micromolar range. Furthermore, significantly higher concentrations of these compounds are required to
inhibit growth of M. smegmatis strains overexpressing PknB, suggesting that this protein kinase is the molecular target. These findings demonstrate that
the Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth and support the development of protein kinase
inhibitors as new potential antituberculosis drugs.
... Protein kinases and phosphatases form a signaling network within the cell and enable the cell to translate extra cellular signals to intra cellular responses [1]. In bacteria, the molecular system responsible for stimulus response coupling involves the histidine kinase (HK)–response regulator (RR) 1 two component systems (TCS) [2]. ...
PknL, a eukaryotic like serine threonine protein kinase from Mycobacterium tuberculosis, is predicted to be involved in transcriptional regulation and cell division. Attempts to clone and over-express the protein in Escherichia coli using pET43.1c as the vector were unsuccessful. The fusion protein was expressed as a truncated product and showed feeble autokinase activity. To overcome this technical glitch, the pknL ORF was cloned into a mycobacterial expression vector, pALACE and the purified His-tagged protein was evaluated for autokinase activity. Phosphorylation experiments with exogenous substrates like myelin basic protein (MBP) were performed. For the fast identification of protein phosphorylation sites, chromatographic methods of separating the [gamma-32P]phosphate radio labeled amino acids using thin-layer chromatography (TLC) on cellulose sheets was carried out. Thus, the activity of PknL was demonstrated using autophosphorylation and substrate phosphorylation experiments. Phospho amino acid determinations revealed that PknL was phosphorylated predominantly on serine and also on threonine residues. A single amino acid substitution of lysine to methionine in the active site completely abolished enzymatic action, thereby confirming the authenticity of the kinase function of the expressed protein.
Phase separation is involved in the organization and regulation of membraneless cellular components and their functions. We showed previously that the cytoplasmic regulatory module of the ABC transporter Rv1747 of Mycobacterium tuberculosis, which is involved in cell wall biosynthesis, is able to phase separate in vitro. Here we demonstrate that this regulatory module forms static foci and full length Rv1747 mobile ones dependent on the presence of the regulatory module in Mycobacterium smegmatis. Disrupting the key interactions by the FHA domains in this regulatory module, in particular FHA-1, and phosphorylated threonines in the linker between the FHA domains demolish the resulting foci in both constructs. Specifically, we show that the S47A single mutant and the S47A, S248A double mutant exhibit no foci formation while the S248A single mutant exhibits a mixed phenotype dominated by diminished clustering. Complementarily, the regulatory module of Rv1747 carrying phosphor-ablative mutations of all Thr/Ser to Ala in the linker also shows no sign of foci formation. In addition, charges of the intrinsically disordered linker also contribute to foci formation by the Rv1747 regulatory module, as the mutant carrying Arg/Lys to Ala mutations displays no foci in M. smegmatis. Collectively, these data strongly suggest that Rv1747 phase separates in M. smegmatis and highlight the role of phosphorylation and non-specific charge-charge interactions by the FHA domains and the ID linker in regulating phase separation of Rv1747 in M. smegmatis.
This is my Doctoral Thesis from which the paper: "Mycobacterium tuberculosis transporter MmpL7 is a potential substrate for kinase PknD" (Biochemical and Biophysical Research Communications 348 (2006) 6–12,) was published. As a researcher, I know that not always the manuscript of a Doctoral Thesis is easily available. But I think that its content is very important because not all the results are published as "papers". Most of the times, unexplained results or "not beautiful" results are unpublished and are forgotten. Nevertheless, these results could be very useful for other researchers working in similar areas. So, here is my work and I hope to be useful. The language is Spanish but if someone needs a translation of a particular text , I could do it in English. Thank you very much.
Mycobacterium tuberculosis contains 11 serine threonine kinases which includes PknA and
PknB. PknA is a secreted protein of M. tuberculosis. PknA is identified as one of the most
interacting protein in our protein-protein interaction study between M. tuberculosis and human.
Primary structure prediction and physicochemical analysis of PknA was carried out by
ProtParam. Comparative study of the secondary structure of PknA using GORIV and SOPMA
revealed a high percentage of alpha helix and coiled coils than beta sheets. In the present study,
we predicted PknA tertiary structure by homology modelling because its structure is not yet
revealed by any experimental methods. The structural analysis will help in the functional study
of the protein.
Probing protein extracts from exponentially growing and stationary phase cultures of Mycobacterium bovis BCG with anti-phospho amino acid antibodies revealed a 31-kDa anti-phospho threonine antibody-reactive protein specific to growing culture. The corresponding protein was purified via two-dimensional gel electrophoresis and identified via mass spectrometry to be malonyl coenzyme A:acyl carrier protein transacylase (MCAT), a component of the fatty acid biosynthetic pathway. MCAT tagged with histidine reacted with anti-phospho threonine antibody and was positive in an in-gel chemical assay for phospho proteins. Analysis of the growth phase dependence of MCAT-His phosphorylation and protein levels showed that phosphorylated MCAT-His can be detected only in growing culture. In contrast, MCAT-His protein level was growth phase-independent. These results suggest that MCAT may be a substrate of a protein kinase and phosphatase, and that aspects of fatty acid synthesis in tubercle bacilli are regulated by protein phosphorylation.
IntroductionRationale for Ser/Thr Protein Kinases and Protein Phosphatases as Drug TargetsDrug Target Validation by Genetic InactivationSTPK Mechanisms, Substrates, and FunctionsM. tuberculosis STPK InhibitorsConclusions and ProspectsReferences
Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding of the biology of this slow-growing pathogen and to help the conception of new prophylactic and therapeutic interventions. The genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high guanine + cytosine content that is reflected in the biased amino-acid content of the proteins. M. tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation.
PknB is a member of the newly discovered eukaryotic-like protein serine/threonine kinase (PSTK) family of proteins. The pknB gene was cloned and expressed in Escherichia coli. The active recombinant protein was purified and shown to be reactive with antiphosphoserine antibodies, as well as with antibodies to the phosphorylated eukaryotic Ser/Thr kinases mitogen-activated protein kinase kinase 3 and 6, P38, and Creb. In vitro kinase assays demonstrated that PknB is a functional kinase that is autophosphorylated on serine/threonine residues and is also able to phosphorylate the peptide substrate myelin basic protein. Analysis of pknB expression in Mycobacterium tuberculosis indicates the presence of pknB mRNA in (i) organisms grown in vitro in bacteriological media, (ii) a murine macrophage in vitro infection model, and (iii) in vivo alveolar macrophages from a patient with tuberculosis.
Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in eye, urinary tract, burn, and immunocompromised patients. We have cloned and characterized a serine/threonine (Ser/Thr) kinase and its cognate phosphoprotein phosphatase. By using oligonucleotides from the conserved regions of Ser/Thr kinases of mycobacteria, an 800-bp probe was used to screen P. aeruginosa PAO1 genomic library. A 20-kb cosmid clone was isolated, from which a 4.5-kb DNA with two open reading frames (ORFs) were subcloned. ORF1 was shown to encode Ser/Thr phosphatase (Stp1), which belongs to the PP2C family of phosphatases. Overlapping with the stp1 ORF, an ORF encoding Hank's type Ser/Thr kinase was identified. Both ORFs were cloned in pGEX-4T1 and expressed in Escherichia coli. The overexpressed proteins were purified by glutathione-Sepharose 4B affinity chromatography and were biochemically characterized. The Stk1 kinase is 39 kDa and undergoes autophosphorylation and can phosphorylate eukaryotic histone H1. A site-directed Stk1 (K86A) mutant was shown to be incapable of autophosphorylation. A two-dimensional phosphoamino acid analysis of Stk1 revealed strong phosphorylation at a threonine residue and weak phosphorylation at a serine residue. The Stp1 phosphatase is 27 kDa and is an Mn(2+)-, but not a Ca(2+)- or a Mg(2+)-, dependent Ser/Thr phosphatase. Its activity is inhibited by EDTA and NaF, but not by okadaic acid, and is similar to that of PP2C phosphatase.
In bacteria, extracellular signals are generally transduced into cellular responses via a two-component system. However, genome sequence data have now revealed the presence of 'eukaryotic-like' protein kinases and phosphatases. Mycobacterium tuberculosis appears to be unique among bacteria in that its genome contains 11 members of a newly identified protein kinase family. These M. tuberculosis eukaryotic-like protein kinases could be key regulators of metabolic processes, including transcription, cell development and interactions with host cells.
Cellular and humoral immunity induced by Mycobacterium tuberculosis has led to identification of newer vaccine candidates, but despite this, many questions concerning the protection against tuberculosis remain unanswered. Recent progress in this field has centered on T cell subset responses and cytokines that these cells secrete. There has been a steady progress in identification and characterization of several classes of major mycobacterial proteins which includes secretory/export proteins, cell wall associated proteins, heat shock proteins and cytoplasmic proteins. The protein antigens are now believed to represent the key protective immunity inducing antigens in the bacillus. In this review, various mycobacterial protein antigens of vaccination potential are compared for their efficacy in light of current immunological knowledge.
Eukaryotic cells predominantly use serine, threonine, and tyrosine phosphorylation in various intracellular signal transduction pathways. In contrast, prokaryotic organisms employ numerous "two-component" systems, in which signaling is achieved by transferring a phosphoryl group from phosphohistidine in the "sensor kinase" component to aspartate in the "response regulator" component. In the last several years, genetic screens and genome projects have identified sensor kinases and response regulators in lower eukaryotes and plants, revealing that eukaryotic organisms also make use of His-Asp phosphotransfer in a limited number of signaling pathways. Extensive studies in yeasts have demonstrated that a variation of the two-component system, a multistep "phosphorelay," is the prevailing mechanism among distantly related yeast species. In the budding yeast Saccharomyces cerevisiae, a His-Asp-His-Asp phosphorelay transmits osmotic stress signals to a mitogen-activated protein kinase (MAPK) cascade to induce adaptive responses. A phosphorelay in the fission yeast Schizosaccharomyces pombe, analogous to the S. cerevisiae phosphorelay, is responsible for MAPK activation in response to peroxide stress. Mammalian cells do not have any two-component or phosphorelay systems, although protein histidine kinases unrelated to the sensor kinase may be involved in cellular signaling. Because some phosphorelay proteins are essential for virulence of microbial pathogens, including the yeast fungus Candida albicans, novel antibiotics targeted to phosphorelays may be effective against eukaryotic pathogens without causing host cell damage.
Everything that we need to know about Mycobacterium leprae, a close relative of the tubercle bacillus, is encrypted in its genome. Inspection of the 3.27 Mb genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus identified 1,605 genes encoding proteins and 50 genes for stable RNA species. Comparison with the genome sequence of Mycobacterium tuberculosis revealed an extreme case of reductive evolution, since less than half of the genome contains functional genes while inactivated or pseudogenes are highly abundant. The level of gene duplication was approximately 34% and, on classification of the proteins into families, the largest functional groups were found to be involved in the metabolism and modification of fatty acids and polyketides, transport of metabolites, cell envelope synthesis and gene regulation. Reductive evolution, gene decay and genome downsizing have eliminated entire metabolic pathways, together with their regulatory circuits and accessory functions, particularly those involved in catabolism. This may explain the unusually long generation time and account for our inability to culture the leprosy bacillus.
We have identified a strain of Streptomyces in which aerial hyphae formation appears to be especially sensitive to inhibition by protein kinase inhibitors. Using this assay, a number of bacterial cultures have been screened and novel inhibitors of eukaryotic protein kinases have been identified. Since M. tuberculosis possesses multiple eukaryotic-like protein kinase genes, we tested the active kinase inhibitors for the inhibition of mycobacterial growth and obtained several potent compounds. This identifies a new biochemical class of antimycobacterial agents.
Adaptive gene expression in prokaryotes is mediated by protein kinases and phosphatases. These regulatory proteins mediate
phosphorylation of histidine or aspartate in two-component systems and serine/threonine or tyrosine in eukaryotic and eukaryote-like
protein kinase systems. The genome sequence of Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease, does not possess a defined tyrosine kinase. Nevertheless, it encodes for protein
tyrosine phosphatases. Here, we report that Map1985, is a functional low-molecular tyrosine phosphatase that is secreted intracellularly
upon macrophage infection. This finding suggests that Map1985 might contribute to the pathogenesis of Mycobacterium avium subsp. paratuberculosis by dephosphorylating essential macrophage signaling and/or adaptor molecules.
Bacterial lipopolysaccharide (LPS) is a potent activator of antibacterial responses by macrophages. Following LPS stimulation, the tyrosine phosphorylation of several proteins is rapidly increased in macrophages, and this event appears to mediate some responses to LPS. We now report that two of these tyrosine phosphoproteins of 41 and 44 kDa are isoforms of mitogen-activated protein (MAP) kinase. Each of these proteins was reactive with anti-MAP kinase antibodies and comigrated with MAP kinase activity in fractions eluted from a MonoQ anion-exchange column. Following LPS stimulation, column fractions containing the tyrosine phosphorylated forms of p41 and p44 exhibited increased MAP kinase activity. Inhibition of LPS-induced tyrosine phosphorylation of these proteins was accompanied by inhibition of MAP kinase activity. Additionally, induction of p41/p44 tyrosine phosphorylation and MAP kinase activity by LPS appeared to be independent of activation of protein kinase C, even though phorbol esters also induced these responses. These results demonstrate that LPS induces the tyrosine phosphorylation and activation of at least two MAP kinase isozymes. Since MAP kinases appear to modulate cellular processes in response to extracellular signals, these kinases may be important targets for LPS action in macrophages.
We have produced a cell line which lacks the protein tyrosine kinase
JAK1 and is completely defective in interferon response. Complementation of
this mutant with JAK1 restored the response, establishing the requirement for
JAK1 in both the interferon-& alpha;/& beta; and -& gamma; signal
transduction pathways. The reciprocal interdependence between JAK1 and Tyk2
activities in the interferon-& alpha; pathway, and between JAK1 and JAK2 in
the interferon-& gamma; pathway, may reflect a requirement for these
kinases in the correct assembly of interferon receptor complexes.
Bacteria continuously adapt to changes in their environment. Responses are largely controlled by signal transduction systems that contain two central enzymatic components, a protein kinase that uses adenosine triphosphate to phosphorylate itself at a histidine residue and a response regulator that accepts phosphoryl groups from the kinase. This conserved phosphotransfer chemistry is found in a wide range of bacterial species and operates in diverse systems to provide different regulatory outputs. The histidine kinases are frequently membrane receptor proteins that respond to environmental signals and phosphorylate response regulators that control transcription. Four specific regulatory systems are discussed in detail: chemotaxis in response to attractant and repellent stimuli (Che), regulation of gene expression in response to nitrogen deprivation (Ntr), control of the expression of enzymes and transport systems that assimilate phosphorus (Pho), and regulation of outer membrane porin expression in response to osmolarity and other culture conditions (Omp). Several additional systems are also examined, including systems that control complex developmental processes such as sporulation and fruiting-body formation, systems required for virulent infections of plant or animal host tissues, and systems that regulate transport and metabolism. Finally, an attempt is made to understand how cross-talk between parallel phosphotransfer pathways can provide a global regulatory curcuitry.
Mycobacterium intracellulare is a slow-growing pathogenic mycobacterium closely related to Mycobacterium avium. In contrast to Mycobacterium tuberculosis and Mycobacterium bovis BCG, M. intracellulare has received little attention as a model species for studies of mycobacterial molecular biology and genetics. This study shows that M. intracellulare 1403 (ATCC 35761) can be transformed by electroporation with high frequencies (up to 10(6) transformants per microgram of DNA), using plasmids pYT937 and pMH94 as replicative and integrative vectors, respectively. We also describe an experimental system that we used to study DNA recombination in M. intracellulare. First, an integrative plasmid was introduced into M. intracellulare 1403. A nonreplicative, nonintegrative plasmid having homology with the integrated plasmid was then introduced, and the resultant recombinants were analyzed to distinguish between events of homologous and illegitimate recombination. No illegitimate recombination occurred; in all recombinants, a single crossover between homologous regions of the two plasmids was noted. During subsequent growth of a recombinant clone, a spontaneous deletion occurred that resulted in a gene replacement on the chromosome of M. intracellulare 1403. The ability to construct site-specific mutations in M. intracellulare will provide novel insights into the biology of slow-growing mycobacteria.
Protein tyrosine phosphorylation is an important regulatory mechanism for many cellular processes in eucaryotic cells. During the invasion of the gram-positive pathogen, Listeria monocytogenes, into host epithelial cells, two host proteins become tyrosine phosphorylated. We have identified these major tyrosine phosphorylated species to be two isoforms of mitogen-activated protein (MAP) kinase, the 42 and 44 kDa MAP kinases. This activation begins within 5 to 15 min of bacterial infection. The tyrosine kinase inhibitor, genistein, blocks invasion as well as the tyrosine phosphorylation of these MAP kinases. Using cytochalasin D to block bacterial internalization but not adhesion, we showed that bacterial adherence rather than uptake is required for MAP kinase activation. Internalin mutants, which are unable to adhere efficiently to host cells, do not trigger MAP kinase activation. Other invasive bacteria, including enteropathogenic Escherichia coli (EPEC), and E. coli expressing Yersinia enterocolitica invasion, were not observed to activate MAP kinase during invasion into cultured epithelial cells. These results suggest that L. monocytogenes activates MAP kinase during invasion and a MAP kinase signal transduction pathway may be involved in mediating bacterial uptake.
The success of Mycobacterium species as pathogens depends on their ability to maintain an infection inside the phagocytic vacuole of the macrophage. Although the bacteria are reported to modulate maturation of their intracellular vacuoles, the nature of such modifications is unknown. In this study, vacuoles formed around Mycobacterium avium failed to acidify below pH 6.3 to 6.5. Immunoelectron microscopy of infected macrophages and immunoblotting of isolated phagosomes showed that Mycobacterium vacuoles acquire the lysosomal membrane protein LAMP-1, but not the vesicular proton-adenosine triphosphatase (ATPase) responsible for phagosomal acidification. This suggests either a selective inhibition of fusion with proton-ATPase-containing vesicles or a rapid removal of the complex from Mycobacterium phagosomes.
An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis, was constructed by using a twin-pronged approach. Pulsed-field gel electrophoretic analysis enabled cleavage sites for Asn I and Dra I to be positioned on the 4.4-Mb circular chromosome, while, in parallel, clones from two cosmid libraries were ordered into contigs by means of fingerprinting and hybridization mapping. The resultant contig map was readily correlated with the physical map of the genome via the landmarked restriction sites. Over 165 genes and markers were localized on the integrated map, thus enabling comparisons with the leprosy bacillus, Mycobacterium leprae, to be undertaken. Mycobacterial genomes appear to have evolved as mosaic structures since extended segments with conserved gene order and organization are interspersed with different flanking regions. Repetitive sequences and insertion elements are highly abundant in M. tuberculosis, but the distribution of IS6110 is apparently nonrandom.
A putative two-component system, mtrA-mtrB, was isolated from M. tuberculosis H37Rv by using phoB from Pseudomonas aeruginosa as a hybridization probe. The predicted gene product of mtrA displayed high similarity with typical response regulators, including AfsQ1, PhoB, PhoP, and OmpR. The predicted gene product of mtrB displayed similarities with the histidine protein kinases AfsQ2, PhoR, and EnvZ and other members of this class of proteins. Expression analysis in the T7 system showed that mtrA encoded a polypeptide with an apparent molecular mass of 30 kDa. MtrA was overproduced, purified, and demonstrated to participate in typical phosphotransfer reactions using a heterologous histidine protein kinase, CheA, as a phosphoryl group donor. Mycobacterium bovis BCG, harboring an mtrA-gfp (green fluorescent protein cDNA) transcriptional fusion, was used to monitor mtrA expression in infected J774 monolayers. Flow cytometric and fluorescence microscopic analyses indicated that the mtrA promoter was activated upon entry and incubation in J774 macrophages. In contrast, the hsp60-gfp fusion displayed no change in expression under the growth conditions tested. These results suggest a potential role for mtrA in adaptation of the M. tuberculosis complex organisms to environmental changes which may include intracellular conditions.
Tyrosine phosphorylation is an extremely rare event in prokaryotes, occurring almost exclusively in multicellular eukaryotes. We have identified, for the first time, by the use of antiphosphotyrosine monoclonal antibody and Western blot (immunoblot) analysis, two tyrosine-phosphorylated membrane proteins in the multicellular prokaryote Myxococcus xanthus. The pattern of tyrosine phosphorylation was shown to change during development, indicating a possible role for this regulatory modification during two stages of development, i.e., aggregation and sporulation. Furthermore, the altered pattern of tyrosine phosphorylation observed in a variety of signaling mutants was shown to differ from that observed in the wild type, suggesting further the possible involvement of tyrosine phosphorylation during the development program.
Using phosphotyrosine-specific antibodies, we demonstrate that in several Streptomyces spp. a variety of proteins are phosphorylated on tyrosine residues. Tyrosine phosphorylation was found in a number of Streptomyces species including Streptomyces lividans, Streptomyces hygroscopicus and Streptomyces lavendulae. Each species exhibited a unique pattern of protein tyrosine phosphorylation. Moreover, the patterns of tyrosine phosphorylation varied during the growth phase and were also influenced by culture conditions. We suggest that metabolic shifts during the complex growth cycle of these filamentous bacteria, and possibly secondary metabolic pathways, may be controlled by the action of protein tyrosine kinases and phosphatases, as has been demonstrated in signal transduction pathways in eukaryotic organisms.
The selective enzyme inhibitors genistein and Ro 31-8220 were used to assess the importance of protein tyrosine kinase (PTK) and protein kinase C (PKC), respectively, in N-formyl-methionyl-leucyl-phenylalanine (FMLP) induced generation of superoxide anion and thromboxane B(2) (TXB(2)) in guinea-pig alveolar macrophages (AM). Genistein (3-100 muM) dose dependently inhibited FMLP (3 nM) induced superoxide generation in non-primed AM and TXB(2) release in non-primed or in lipopolysaccharide (LPS) (10 ng/ml) primed AM to a level > 80% but had litle effect up to 100 muM on phorbol myristate acetate (PMA) (10 nM) induced superoxide release. Ro 31-8220 inhibited PMA induced superoxide generation (IC(50) 0.21 +/- 0.10 muM) but had no effect on or potentiated (at 3 and 10 muM) FMLP responses in non-primed AM. In contrast, when present during LPS priming as well as during FMLP challenge Ro 31-8220 (10 muM) inhibited primed TXB(2) release by > 80%. The results indicate that PTK activation is required for the generation of these inflammatory mediators by FMLP in AM. PKC activation appears to be required for LPS priming but not for transducing the FMLP signal; rather, PKC activation may modulate the signal by a negative feedback mechanism.
A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) is essential for aerial mycelium formation and streptomycin (Sm) production in Streptomyces griseus. A protein Ser/Thr kinase (AfsK), the product of the Streptomyces coelicolor A3(2) afsK gene, controlling secondary metabolism in this strain, reversed the aerial mycelium-negative phenotype of an A-factor-deficient mutant strain, S. griseus HH1, and induced sporulation without affecting A-factor productivity or Sm production. A mutant AfsK protein lacking kinase activity failed to induce aerial mycelium formation which indicates the importance of the kinase activity for suppression in S. griseus. These data suggest that a Ser/Thr kinase functionally similar to S. coelicolor A3(2) AfsK plays a regulatory role in aerial mycelium formation in S. griseus, either as a member in the A-factor regulatory network or independently of this network
Publisher Summary This chapter describes an alternative homology cloning approach that uses degenerate oligodeoxynucleotides ("oligos") as hybridization probes. The oligo probes are designed to recognize target sequences that encode short stretches of six to nine highly conserved amino acid residues found within the catalytic domains. Such probes are ideally suited for the purpose of identifying a wide variety of novel protein kinases from a single library screen. Virtually all of the codon possibilities for a conserved stretch can be included in the probe mixture, thereby assuring that many different protein kinase genes (cDNAs) are recognized. False positives are reduced by targeting two or three conserved stretches and selecting further characterization only those clones that are identified by all of the probe mixtures. This approach is successful in identifying both novel protein-serine/threonine kinases and novel protein-tyrosine kinases. Degenerate oligodeoxynucleotides can also be used to prime the polymerase chain reaction and thus identify novel protein kinases through selective gene amplification.
Mycobacterium tuberculosis and Mycobacterium leprae, the causative agents of tuberculosis and leprosy, respectively, produce large quantities of lipoarabinomannan (LAM), a highly immunogenic, cell wall-associated glycolipid. This molecule has been previously reported to be a potent inhibitor of gamma interferon-mediated activation of murine macrophages. Studies of the mechanism by which this mycobacterial glycolipid down-regulates macrophage effector functions provide evidence that LAM acts at several levels and that it can (i) scavenge potentially cytotoxic oxygen free radicals, (ii) inhibit protein kinase C activity, and (iii) block the transcriptional activation of gamma interferon-inducible genes in human macrophage-like cell lines. These results suggest that LAM can inhibit macrophage activation and triggering and cytocidal activity and that it may represent a chemically defined virulence factor contributing to the persistence of mycobacteria within mononuclear phagocytes.
The principal efferent role of the macrophage in acquired resistance to intracellular pathogens depends on activation by T-cell lymphokines, primarily gamma interferon (IFN-gamma). However, mouse macrophages that are heavily burdened with Mycobacterium leprae are refractory to activation by IFN-gamma and are thus severely compromised in their capacity for both enhanced microbicidal and tumoricidal activities. We report here that lipoarabinomannan (LAM), a highly immunogenic lipopolysaccharide that is a prominent component of the cell walls of M. leprae and M. tuberculosis, was a potent inhibitor of IFN-gamma-mediated activation of mouse macrophages in vitro. Inhibition of macrophage activation by LAM required preincubation for approximately 24 h, resulting in uptake of LAM into cytoplasmic vacuoles of macrophages. Intact LAM was necessary to inhibit IFN-gamma-mediated activation, as this property was lost when the acyl side chains were removed from LAM by mild alkaline hydrolysis. In addition, LAM was an abundant constituent of macrophages isolated from lepromatous granulomas of M. leprae-infected nude mice and likely contributed to the defective activation of granuloma macrophages by IFN-gamma.
The analysis of protein phosphorylation in the bacterium Escherichia coli showed that, while most phosphoproteins are modified at serine and/or threonine residues, one of them is modified exclusively at tyrosine. This particular protein which has a molecular weight of 54,500 and a pHi value of 5.6 is found associated with the membrane/ribosome fraction of the cell.
Mycobacterium lepraemurium and M. microti (causal agent of vole tuberculosis) were isolated from tissues of experimentally infected mice and used to infect normal mouse peritoneal macrophage cultures. The cellular response to these bacteria up to 4 days after infection was studied quantitatively by electron microscopy. Prelabeling with ferritin was used to facilitate observation of fusion between secondary lysosomes in the cells and phagosomes containing the bacteria. All bacteria were intraphagosomal, and a high proportion of them was morphologically "intact." Nearly all phagosomes containing morphologically damaged (presumed nonviable) bacteria also contained ferritin, having fused with secondary lysosomes. Fusion of lysosomes had also occurred with most phagosomes containing intact M. lepraemurium but was infrequent with phagosomes containing intact M. microti. This tendency of multiplying mycobacteria of the tubercle type to avoid contact with lysosomal contents has already been reported for M. tuberculosis strain H37Rv. The different intracellular circumstances of the parasites may reflect different means of intracellular survival.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Crude cell extracts from three strains of Mycobacterium tuberculosis were analyzed for the presence of proteins possessing phosphorylated tyrosine residues. A protein migrating at approximately 55 kDa was detected using an antiphosphotyrosine monoclonal antibody. In addition, less predominant bands were observed between 50 kDa and 60 kDa. That M. tuberculosis contains specific tyrosine phosphorylated proteins implies that M. tuberculosis has tyrosine kinase activity. Examination of other, non-pathogenic mycobacterium species yielded no major antiphosphotyrosine reactive proteins. This suggests that the antiphosphotyrosine reactive protein is specific to M. tuberculosis strains. These results provide evidence that M. tuberculosis contains an antiphosphotyrosine reactive protein.
The levels of tyrosine phosphorylation required for cell growth and differentiation are achieved through the coordinated action of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Depending upon the cellular context, these two types of enzymes may either antagonize or cooperate with each other during the signal transmission process. An imbalance between these enzymes may impair normal cell growth, leading to cellular transformation. Both PTKs and PTPs have evolved to a level of structural diversity that allows them to regulate many cellular processes. This review will focus on several specific examples that highlight the interplay between PTPs and PTKs in cell signaling.
Recent studies on the pathogenic mechanisms of several bacterial genera, including the Yersinia, Salmonella and Escherichia, have revealed novel strategies of infection that involve the signal transduction processes of eukaryotic cells. These model systems are reviewed here, with emphasis on the role of tyrosine phosphorylation in these bacterial-host cell interactions.
Central to understanding the pathogenesis of tuberculosis is the interaction between the pathogen and mononuclear phagocytes. A key question about that interaction is whether Mycobacterium tuberculosis exerts an effect on phagolysosome fusion. We have reexamined the dynamics of phagolysosome fusion and its effect on intracellular bacterial replication in M. tuberculosis-infected macrophages by performing an extensive study at the electron microscopic level. Thoria-labelled murine and human macrophages were infected with a virulent (H37Rv) or avirulent (H37Ra) strain of M. tuberculosis or with Mycobacterium bovis BCG vaccine for times ranging from 2 h to 7 days. In all cases, by 2 h postinfection, approximately 85% of the bacteria clearly resided in fused vacuoles. However, at 4 days postinfection, fusion levels for viable H37Rv and H37Ra were reduced by half, whereas the fusion profiles of BCG and of heat-killed H37Rv and H37Ra were unchanged. A comparison of the numbers of bacteria per fused and nonfused vacuoles suggests both a net transfer of bacteria out of fused vacuoles and preferential bacterial multiplication in nonfused vacuoles. H37Rv and H37Ra appeared to bud from the phagolysosomes into tightly apposed membrane vesicles that did not fuse with secondary lysosomes. In some cases, no such membrane was seen and the bacteria appeared to be free in the cytoplasm. Only viable H37Rv showed a significant increase in bacterial counts during the course of infection. Thus, both of the attenuated strains we examined differed from the virulent strain H37Rv in their abilities to replicate successfully within macrophages, but each diverged from H37Rv at a different point in the process. Viable tubercle bacilli H37Rv and H37Ra had the capacity to escape from fused vesicles as the infection progressed; BCG did not. After extrusion from the phagolysosome, H37Rv, but not H37Ra, was able to multiply. These results suggest a novel mechanism by which virulent M. tuberculosis eludes the microbicidal mechanisms of macrophages by escaping from fused phagolysosomes into nonfused vesicles or the cytoplasm.
Myxococcus xanthus contains a large family of genes encoding eukaryotic-like serine/threonine kinases. Among them, two genes, pkn5 and pkn6, are divergently located on the chromosome and share a 46 bp promoter region between their transcription initiation sites, as determined by RNA protection. Pkn5, consisting of 380 amino acid residues, is a soluble protein in the cytoplasm, while Pkn6, consisting of 710 amino acid residues, is a transmembrane protein. Its membrane topology was determined using the Pkn6-PhoA fusion protein in Escherichia coli, which has a single transmembrane domain with the N-terminal domain in the cytoplasm and the C-terminal domain outside the cytoplasmic membrane. Both proteins, when expressed in E. coli, were autophosphorylated: Pkn5 only at Ser, and Pkn6 at both Ser and Thr. In M. xanthus, both genes are expressed constitutively throughout the life cycle, with slight increases at an early stage of development. Most strikingly, a pkn5-deletion strain forms fruiting bodies much faster than the wild-type strain, while a pkn6-deletion strain develops slower than the wild-type strain. These results, together with the fact that the pkn5-deletion strain is able to form fruiting bodies on semi-rich media, suggest that Pkn5 and Pkn6 have reciprocal roles in M. xanthus growth and development. Furthermore, Pkn6 may be a transmembrane sensor of external signals for development, while Pkn5 is a kinase that negatively regulates M. xanthus development.
Bacteria play host to a wide range of protein phosphorylation-dephosphorylation systems (Fig. 1). As little as five years ago the known systems were thought to be late-emerging and absolutely prokaryote specific. Today we know that most protein kinases and protein phosphatases are descended from a set of common, and possibly quite ancient, prototypes. Prokaryote- and eukaryote-specific protein kinases and protein phosphatases are rare and represent exceptions, not the rule as previously thought. Commonality suggests that a dynamic and versatile regulatory mechanism was first adapted to the modulation of protein function as early if not earlier than more "basic" mechanisms such as allosterism, etc. The existence of common molecular themes confirms that the microbial world offers a unique, largely untapped library and a powerful set of tools for the understanding of a regulatory mechanism which is crucial to all organisms, tools whose diversity and experimental malleability will provide new avenues for exploring and understanding key modes of cellular regulation.
Protein Ser, Thr and Tyr kinases play essential roles in signal transduction in organisms ranging from yeast to mammals, where they regulate a variety of cellular activities. During the last few years, a number of genes that encode eukaryotic-type protein kinases have also been identified in four different bacterial species, suggesting that such enzymes are also widespread in prokaryotes. Although many of them have yet to be fully characterized, several studies indicate that eukaryotic-type protein kinases play important roles in regulating cellular activities of these bacteria, such as cell differentiation, pathogenicity and secondary metabolism. A model based on the possible coupling between two-component systems and eukaryotic-type protein kinases is proposed to explain the function of eukaryotic-type protein kinases in bacterial signalling in the light of studies in bacteria, as well as in plants and yeast. These two groups of eukaryotes possess signal-transduction pathways involving both two-component systems and eukaryotic protein kinases.