A Legionella effector kinase is activated by host inositol hexakisphosphate.

The transfer of a phosphate from ATP to a protein substrate, a modification known as protein phosphorylation, is catalyzed by protein kinases. Protein kinases play a crucial role in virtually every cellular activity. Recent studies of atypical protein kinases have highlighted the structural similarity of the kinase superfamily despite notable differences in primary amino acid sequence. Here, using a bioinformatics screen, we searched for putative protein kinases in the intracellular bacterial pathogen Legionella pneumophila and identified the type 4 secretion system effector Lpg2603 as a remote member of the protein kinase superfamily. Employing an array of biochemical and structural biology approaches, including in vitro kinase assays and isothermal titration calorimetry, we show that Lpg2603 is an active protein kinase with several atypical structural features. Importantly, we found that the eukaryote-specific host signaling molecule inositol hexakisphosphate (IP6) is required for Lpg2603 kinase activity. Crystal structures of Lpg2603 in the apo-form and when bound to IP6 revealed an active-site rearrangement that allows for ATP binding and catalysis. Our results on the structure and activity of Lpg2603 reveal a unique mode of regulation of a protein kinase, provide the first example of a bacterial kinase that requires IP6 for its activation, and may aid future work on the function of this effector during Legionella pathogenesis.

The secreted protein kinase CstK from Coxiella burnetii influences vacuole development and interacts with the GTPase-activating host protein TBC1D5.

The intracellular bacterial pathogen Coxiella burnetii is the etiological agent of the emerging zoonosis Q fever. Crucial to its pathogenesis is type 4b secretion system-mediated secretion of bacterial effectors into host cells that subvert host cell membrane trafficking, leading to the biogenesis of a parasitophorous vacuole for intracellular replication. The characterization of prokaryotic serine/threonine protein kinases in bacterial pathogens is emerging as an important strategy to better understand host-pathogen interactions. In this study, we investigated CstK (for Coxiella Ser/Thr kinase), a protein kinase identified in C. burnetii by in silico analysis. We demonstrate that this putative protein kinase undergoes autophosphorylation on Thr and Tyr residues and phosphorylates a classical eukaryotic protein kinase substrate in vitro This dual Thr-Tyr kinase activity is also observed for a eukaryotic dual-specificity Tyr phosphorylation-regulated kinase class. We found that CstK is translocated during infections and localizes to Coxiella-containing vacuoles (CCVs). Moreover, a CstK-overexpressing C. burnetii strain displayed a severe CCV development phenotype, suggesting that CstK fine-tunes CCV biogenesis during the infection. Protein-protein interaction experiments identified the Rab7 GTPase-activating protein TBC1D5 as a candidate CstK-specific target, suggesting a role for this host GTPase-activating protein in Coxiella infections. Indeed, CstK co-localized with TBC1D5 in noninfected cells, and TBC1D5 was recruited to CCVs in infected cells. Accordingly, TBC1D5 depletion from infected cells significantly affected CCV development. Our results indicate that CstK functions as a bacterial effector protein that interacts with the host protein TBC1D5 during vacuole biogenesis and intracellular replication.

Disruption of the dimerization interface of the sensing domain in the dimeric heme-based oxygen sensor AfGcHK abolishes bacterial signal transduction.

The heme-based oxygen sensor protein AfGcHK is a globin-coupled histidine kinase in the soil bacterium Anaeromyxobacter sp. Fw109-5. Its C-terminal functional domain exhibits autophosphorylation activity induced by oxygen binding to the heme-Fe(II) complex located in the oxygen-sensing N-terminal globin domain. A detailed understanding of the signal transduction mechanisms in heme-containing sensor proteins remains elusive. Here, we investigated the role of the globin domain's dimerization interface in signal transduction in AfGcHK. We present a crystal structure of a monomeric imidazole-bound AfGcHK globin domain at 1.8 Å resolution, revealing that the helices of the WT globin dimer are under tension and suggesting that Tyr-15 plays a role in both this tension and the globin domain's dimerization. Biophysical experiments revealed that whereas the isolated WT globin domain is dimeric in solution, the Y15A and Y15G variants in which Tyr-15 is replaced with Ala or Gly, respectively, are monomeric. Additionally, we found that although the dimerization of the full-length protein is preserved via the kinase domain dimerization interface in all variants, full-length AfGcHK variants bearing the Y15A or Y15G substitutions lack enzymatic activity. The combined structural and biophysical results presented here indicate that Tyr-15 plays a key role in the dimerization of the globin domain of AfGcHK and that globin domain dimerization is essential for internal signal transduction and autophosphorylation in this protein. These findings provide critical insights into the signal transduction mechanism of the histidine kinase AfGcHK from Anaeromyxobacter.

Deletion of pknG Abates Reactivation of Latent Mycobacterium tuberculosis in Mice.

Eradication of tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), has been a challenge due to its uncanny ability to survive in a dormant state inside host granulomas for decades. Mtb rewires its metabolic and redox regulatory networks to survive in the hostile hypoxic and nutrient-limiting environment, facilitating the formation of drug-tolerant persisters. Previously, we showed that protein kinase G (PknG), a virulence factor required for lysosomal escape, aids in metabolic adaptation, thereby promoting the survival of nonreplicating mycobacteria. Here, we sought to investigate the therapeutic potential of PknG against latent mycobacterium. We show that inhibition of PknG by AX20017 reduces mycobacterial survival in in vitro latency models such as hypoxia, persisters, and nutrient starvation. Targeting PknG enhances the bactericidal activity of the frontline anti-TB drugs in peritoneal macrophages. Deletion of pknG resulted in 5- to 15-fold-reduced survival of Mtb in chronically infected mice treated with anti-TB drugs. Importantly, in the Cornell mouse model of latent TB, the deletion of pknG drastically attenuated Mtb's ability to resuscitate after antibiotic treatment compared with wild-type and complemented strains. This is the first study to investigate the sterilizing activity of pknG deletion and inhibition for adjunct therapy against latent TB in a preclinical model. Collectively, these results suggest that PknG may be a promising drug target for adjunct therapy to shorten the treatment duration and reduce disease relapse.

Structure, function, and mechanism of the core circadian clock in cyanobacteria.

Circadian rhythms enable cells and organisms to coordinate their physiology with the cyclic environmental changes that come as a result of Earth's light/dark cycles. Cyanobacteria make use of a post-translational oscillator to maintain circadian rhythms, and this elegant system has become an important model for circadian timekeeping mechanisms. Composed of three proteins, the KaiABC system undergoes an oscillatory biochemical cycle that provides timing cues to achieve a 24-h molecular clock. Together with the input/output proteins SasA, CikA, and RpaA, these six gene products account for the timekeeping, entrainment, and output signaling functions in cyanobacterial circadian rhythms. This Minireview summarizes the current structural, functional and mechanistic insights into the cyanobacterial circadian clock.

The domain architecture of PtkA, the first tyrosine kinase from Mycobacterium tuberculosis, differs from the conventional kinase architecture.

The discovery that MptpA (low-molecular-weight protein tyrosine phosphatase A) from Mycobacterium tuberculosis (Mtb) has an essential role for Mtb virulence has motivated research of tyrosine-specific phosphorylation in Mtb and other pathogenic bacteria. The phosphatase activity of MptpA is regulated via phosphorylation on Tyr128 and Tyr129 Thus far, only a single tyrosine-specific kinase, protein-tyrosine kinase A (PtkA), encoded by the Rv2232 gene has been identified within the Mtb genome. MptpA undergoes phosphorylation by PtkA. PtkA is an atypical bacterial tyrosine kinase, as its sequence differs from the sequence consensus within this family. The lack of structural information on PtkA hampers the detailed characterization of the MptpA-PtkA interaction. Here, using NMR spectroscopy, we provide a detailed structural characterization of the PtkA architecture and describe its intra- and intermolecular interactions with MptpA. We found that PtkA's domain architecture differs from the conventional kinase architecture and is composed of two domains, the N-terminal highly flexible intrinsically disordered domain (IDDPtkA) and the C-terminal rigid kinase core domain (KCDPtkA). The interaction between the two domains, together with the structural model of the complex proposed in this study, reveal that the IDDPtkA is unstructured and highly dynamic, allowing for a "fly-casting-like" mechanism of transient interactions with the rigid KCDPtkA This interaction modulates the accessibility of the KCDPtkA active site. In general, the structural and functional knowledge of PtkA gained in this study is crucial for understanding the MptpA-PtkA interactions, the catalytic mechanism, and the role of the kinase-phosphatase regulatory system in Mtb virulence.

Protection of the general stress response σS factor by the CrsR regulator allows a rapid and efficient adaptation of Shewanella oneidensis.

To cope with environmental stresses, bacteria have evolved various strategies, including the general stress response (GSR). GSR is governed by an alternative transcriptional σ factor named σS (RpoS) that associates with RNA polymerase and controls the expression of numerous genes. Previously, we have reported that posttranslational regulation of σS in the aquatic bacterium Shewanella oneidensis involves the CrsR-CrsA partner-switching regulatory system, but the exact mechanism by which CrsR and CrsA control σS activity is not completely unveiled. Here, using a translational gene fusion, we show that CrsR sequesters and protects σS during the exponential growth phase and thus enables rapid gene activation by σS as soon as the cells enter early stationary phase. We further demonstrate by an in vitro approach that this protection is mediated by the anti-σ domain of CrsR. Structure-based alignments of CsrR orthologs and other anti-σ factors identified a CsrR-specific region characteristic of a new family of anti-σ factors. We found that CrsR is conserved in many aquatic proteobacteria, and most of the time it is associated with CrsA. In conclusion, our results suggest that CsrR-mediated protection of σS during exponential growth enables rapid adaptation of S. oneidensis to changing and stressful growth conditions, and this ability is probably widespread among aquatic proteobacteria.

A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA.

Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial Penicillin-binding-protein And Serine/Threonine kinase-Associated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to ß-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to ß-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various ß-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate ß-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition.

Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction.

The heme-based oxygen sensor histidine kinase AfGcHK is part of a two-component signal transduction system in bacteria. O2 binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH- and -CN- complexes of AfGcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. Here, we determined the crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN- and inactive 5-coordinate Fe(II) forms, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry to characterize the conformations of the active and inactive forms of full-length AfGcHK in solution, we investigated the intramolecular signal transduction mechanisms. Major differences between the active and inactive forms were observed on the heme-proximal side (helix H5), at the dimerization interface (helices H6 and H7 and loop L7) of the globin domain and in the ATP-binding site (helices H9 and H11) of the kinase domain. Moreover, separation of the sensor and kinase domains, which deactivates catalysis, increased the solvent exposure of the globin domain-dimerization interface (helix H6) as well as the flexibility and solvent exposure of helix H11. Together, these results suggest that structural changes at the heme-proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of AfGcHK. We conclude that AfGcHK functions as an ensemble of molecules sampling at least two conformational states.

In Silico Screen and Structural Analysis Identifies Bacterial Kinase Inhibitors which Act with ß-Lactams To Inhibit Mycobacterial Growth.

New tools and concepts are needed to combat antimicrobial resistance. Actinomycetes and firmicutes share several eukaryotic-like Ser/Thr kinases (eSTK) that offer antibiotic development opportunities, including PknB, an essential mycobacterial eSTK. Despite successful development of potent biochemical PknB inhibitors by many groups, clinically useful microbiologic activity has been elusive. Additionally, PknB kinetics are not fully described, nor are structures with specific inhibitors available to inform inhibitor design. We used computational modeling with available structural information to identify human kinase inhibitors predicted to bind PknB, and we selected hits based on drug-like characteristics intended to increase the likelihood of cell entry. The computational model suggested a family of inhibitors, the imidazopyridine aminofurazans (IPAs), bind PknB with high affinity. We performed an in-depth characterization of PknB and found that these inhibitors biochemically inhibit PknB, with potency roughly following the predicted models. A novel X-ray structure confirmed that the inhibitors bound as predicted and made favorable protein contacts with the target. These inhibitors also have antimicrobial activity toward mycobacteria and nocardia. We demonstrated that the inhibitors are uniquely potentiated by ß-lactams but not antibiotics traditionally used to treat mycobacteria, consistent with PknB's role in sensing cell wall stress. This is the first demonstration in the phylum actinobacteria that some ß-lactam antibiotics could be more effective if paired with a PknB inhibitor. Collectively, our data show that in silico modeling can be used as a tool to discover promising drug leads, and the inhibitors we discovered can act with clinically relevant antibiotics to restore their efficacy against bacteria with limited treatment options.

The General Stress Response σS Is Regulated by a Partner Switch in the Gram-negative Bacterium Shewanella oneidensis.

Here, we show that a partner-switching system of the aquatic Proteobacterium Shewanella oneidensis regulates post-translationally σS (also called RpoS), the general stress response sigma factor. Genes SO2118 and SO2119 encode CrsA and CrsR, respectively. CrsR is a three-domain protein comprising a receiver, a phosphatase, and a kinase/anti-sigma domains, and CrsA is an anti-sigma antagonist. In vitro, CrsR sequesters σS and possesses kinase and phosphatase activities toward CrsA. In turn, dephosphorylated CrsA binds the anti-sigma domain of CrsR to allow the release of σS This study reveals a novel pathway that post-translationally regulates the general stress response sigma factor differently than what was described for other proteobacteria like Escherichia coli We argue that this pathway allows probably a rapid bacterial adaptation.

Regulation of Ergothioneine Biosynthesis and Its Effect on Mycobacterium tuberculosis Growth and Infectivity.

Ergothioneine (EGT) is synthesized in mycobacteria, but limited knowledge exists regarding its synthesis, physiological role, and regulation. We have identified Rv3701c from Mycobacterium tuberculosis to encode for EgtD, a required histidine methyltransferase that catalyzes first biosynthesis step in EGT biosynthesis. EgtD was found to be phosphorylated by the serine/threonine protein kinase PknD. PknD phosphorylates EgtD both in vitro and in a cell-based system on Thr(213). The phosphomimetic (T213E) but not the phosphoablative (T213A) mutant of EgtD failed to restore EGT synthesis in a ΔegtD mutant. The findings together with observed elevated levels of EGT in a pknD transposon mutant during in vitro growth suggests that EgtD phosphorylation by PknD negatively regulates EGT biosynthesis. We further showed that EGT is required in a nutrient-starved model of persistence and is needed for long term infection of murine macrophages.

Protein kinase A (PknA) of Mycobacterium tuberculosis is independently activated and is critical for growth in vitro and survival of the pathogen in the host.

The essential mycobacterial protein kinases PknA and PknB play crucial roles in modulating cell shape and division. However, the precise in vivo functional aspects of PknA have not been investigated. This study aims to dissect the role of PknA in mediating cell survival in vitro as well as in vivo. We observed aberrant cell shape and severe growth defects when PknA was depleted. Using the mouse infection model, we observe that PknA is essential for survival of the pathogen in the host. Complementation studies affirm the importance of the kinase, juxtamembrane, and transmembrane domains of PknA. Surprisingly, the extracytoplasmic domain is dispensable for cell growth and survival in vitro. We find that phosphorylation of the activation loop at Thr(172) of PknA is critical for bacterial growth. PknB has been previously suggested to be the receptor kinase, which activates multiple kinases, including PknA, by trans-phosphorylating their activation loop residues. Using phospho-specific PknA antibodies and conditional pknB mutant, we find that PknA autophosphorylates its activation loop independent of PknB. Fluorescently tagged PknA and PknB show distinctive distribution patterns within the cell, suggesting that although both kinases are known to modulate cell shape and division, their modes of action are likely to be different. This is supported by our findings that expression of kinase-dead PknA versus kinase-dead PknB in mycobacterial cells leads to different cellular phenotypes. Data indicate that although PknA and PknB are expressed as part of the same operon, they appear to be regulating cellular processes through divergent signaling pathways.

Streptococcus agalactiae capsule polymer length and attachment is determined by the proteins CpsABCD.

The production of capsular polysaccharides (CPS) or secreted exopolysaccharides is ubiquitous in bacteria, and the Wzy pathway constitutes a prototypical mechanism to produce these structures. Despite the differences in polysaccharide composition among species, a group of proteins involved in this pathway is well conserved. Streptococcus agalactiae (group B Streptococcus; GBS) produces a CPS that represents the main virulence factor of the bacterium and is a prime target in current vaccine development. We used this human pathogen to investigate the roles and potential interdependencies of the conserved proteins CpsABCD encoded in the cps operon, by developing knock-out and functional mutant strains. The mutant strains were examined for CPS quantity, size, and attachment to the cell surface as well as CpsD phosphorylation. We observed that CpsB, -C, and -D compose a phosphoregulatory system where the CpsD autokinase phosphorylates its C-terminal tyrosines in a CpsC-dependent manner. These Tyr residues are also the target of the cognate CpsB phosphatase. An interaction between CpsD and CpsC was observed, and the phosphorylation state of CpsD influenced the subsequent action of CpsC. The CpsC extracellular domain appeared necessary for the production of high molecular weight polysaccharides by influencing CpsA-mediated attachment of the CPS to the bacterial cell surface. In conclusion, although having no impact on cps transcription or the synthesis of the basal repeating unit, we suggest that these proteins are fine-tuning the last steps of CPS biosynthesis (i.e. the balance between polymerization and attachment to the cell wall).

Serine/Threonine Protein Kinases from Bacteria, Archaea and Eukarya Share a Common Evolutionary Origin Deeply Rooted in the Tree of Life.

The main family of serine/threonine/tyrosine protein kinases present in eukarya was defined and described by Hanks et al. in 1988 (Science, 241, 42-52). It was initially believed that these kinases do not exist in bacteria, but extensive genome sequencing revealed their existence in many bacteria. For historical reasons, the term "eukaryotic-type kinases" propagated in the literature to describe bacterial members of this protein family. Here, we argue that this term should be abandoned as a misnomer, and we provide several lines of evidence to support this claim. Our comprehensive phylostratigraphic analysis suggests that Hanks-type kinases present in eukarya, bacteria and archaea all share a common evolutionary origin in the lineage leading to the last universal common ancestor (LUCA). We found no evidence to suggest substantial horizontal transfer of genes encoding Hanks-type kinases from eukarya to bacteria. Moreover, our systematic structural comparison suggests that bacterial Hanks-type kinases resemble their eukaryal counterparts very closely, while their structures appear to be dissimilar from other kinase families of bacterial origin. This indicates that a convergent evolution scenario, by which bacterial kinases could have evolved a kinase domain similar to that of eukaryal Hanks-type kinases, is not very likely. Overall, our results strongly support a monophyletic origin of all Hanks-type kinases, and we therefore propose that this term should be adopted as a universal name for this protein family.

Do Shoot the Messenger: PASTA Kinases as Virulence Determinants and Antibiotic Targets.

All domains of life utilize protein phosphorylation as a mechanism of signal transduction. In bacteria, protein phosphorylation was classically thought to be mediated exclusively by histidine kinases as part of two-component signaling systems. However, it is now well appreciated that eukaryotic-like serine/threonine kinases (eSTKs) control essential processes in bacteria. A subset of eSTKs are single-pass transmembrane proteins that have extracellular penicillin-binding-protein and serine/threonine kinase-associated (PASTA) domains which bind muropeptides. In a variety of important pathogens, PASTA kinases have been implicated in regulating biofilms, antibiotic resistance, and ultimately virulence. Although there are limited examples of direct regulation of virulence factors, PASTA kinases are critical for virulence due to their roles in regulating bacterial physiology in the context of stress. This review focuses on the role of PASTA kinases in virulence for a variety of important Gram-positive pathogens and concludes with a discussion of current efforts to develop kinase inhibitors as novel antimicrobials.

Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues.

Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD, and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

Protein-tyrosine phosphorylation interaction network in Bacillus subtilis reveals new substrates, kinase activators and kinase cross-talk.

Signal transduction in eukaryotes is generally transmitted through phosphorylation cascades that involve a complex interplay of transmembrane receptors, protein kinases, phosphatases and their targets. Our previous work indicated that bacterial protein-tyrosine kinases and phosphatases may exhibit similar properties, since they act on many different substrates. To capture the complexity of this phosphorylation-based network, we performed a comprehensive interactome study focused on the protein-tyrosine kinases and phosphatases in the model bacterium Bacillus subtilis. The resulting network identified many potential new substrates of kinases and phosphatases, some of which were experimentally validated. Our study highlighted the role of tyrosine and serine/threonine kinases and phosphatases in DNA metabolism, transcriptional control and cell division. This interaction network reveals significant crosstalk among different classes of kinases. We found that tyrosine kinases can bind to several modulators, transmembrane or cytosolic, consistent with a branching of signaling pathways. Most particularly, we found that the division site regulator MinD can form a complex with the tyrosine kinase PtkA and modulate its activity in vitro. In vivo, it acts as a scaffold protein which anchors the kinase at the cell pole. This network highlighted a role of tyrosine phosphorylation in the spatial regulation of the Z-ring during cytokinesis.