CPMK2, an SLT2-homologous mitogen-activated protein (MAP) kinase,is essential for pathogenesis of Claviceps purpurea on rye: evidence for a second conserved pathogenesis-related MAP kinase cascade in phytopathogenic fungi

Cpmk2, encoding a mitogen-activated protein (MAP) kinase from the ascomycete Claviceps purpurea, is an orthologue of SLT2 from Saccharomyces cerevisiae, the first isolated from a biotrophic, non-appressorium-forming pathogen. Deletion mutants obtained by a gene replacement approach show impaired vegetative properties (no conidiation) and a significantly reduced virulence, although they retain a limited ability to colonize the host tissue. Increased sensitivity to protoplasting enzymes indicates that the cell wall structure of the mutants may be altered. As the phenotypes of these mutants are similar to those observed in strains of the rice pathogen, Magnaporthe grisea, that have been deprived of their MAP kinase gene mps1, the ability of cpmk2 to complement the defects of delta mps1 was investigated. Interestingly, the C. purpurea gene, under the control of its own promoter, was able to complement the M. grisea mutant phenotype: transformants were able to sporulate and form infection hyphae on onion epidermis and were fully pathogenic on barley leaves. This indicates that, despite the differences in infection strategies, which include host and organ specificity, mode of penetration and colonization of host tissue, CPMK2/MPS1 defines a second MAP kinase cascade (after the Fus3p/PMK1 cascade) essential for fungal pathogenicity.     

Listeria monocytogenes, an invasive bacterium, stimulates MAP kinase upon attachment to epithelial cells.

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.     

Crystallization of MAP kinases

X-ray structural studies of MAP kinases and MAP kinase module components are elucidating how kinase activity is regulated and how specificity of signaling is conferred. In the past decade, MAP kinases have been crystallized in their active, phosphorylated forms or low-activity, unphosphorylated forms, as well as in the presence of binding partners such as docking peptides and inhibitors. Crystallization has been achieved via diverse strategies including control of phosphorylation, coding sequence modification, incorporation of tags for purification, and use of a variety of cell-types for protein expression. Recently, interest has been focused on use of crystallography for lead optimization in the development for pharmacological inhibitors on MAP kinases. Further, some success has been gained in crystallizing the MAP kinase activators MAP2Ks and MAP3K kinase domains. This review describes the key methods that have been utilized to crystallize MAP kinases and MAP kinase pathway components.     

Phosphorylation of MAP kinases by MAP/ERK involves multiple regions of MAP kinases.

Mitogen-activated protein (MAP) kinases are activated with great specificity by MAP/ERK kinases (MEKs). The basis for the specific activation is not understood. In this study chimeras composed of two MAP kinases, extracellular signal-regulated protein kinase 2 and p38, were assayed in vitro for phosphorylation and activation by different MEK isoforms to probe the requirements for productive interaction of MAP kinases with MEKs. Experimental results and modeling support the conclusion that the specificity of MEK/MAP kinase phosphorylation results from multiple contacts, including surfaces in both the N- and C-terminal domains.     

Specificity and specialization of congeneric monogeneans parasitizing cyprinid fish

Patterns and likely processes connected with evolution of host specificity in congeneric monogeneans parasitizing fish species of the Cyprinidae were investigated. A total of 51 Dactylogyrus species was included. We investigated (1) the link between host specificity and parasite phylogeny; (2) the morphometric correlates of host specificity, parasite body size, and variables of attachment organs important for host specificity; (3) the evolution of morphological adaptation, that is, attachment organ; (4) the determinants of host specificity following the hypothesis of specialization on more predictable resources considering maximal body size, maximal longevity, and abundance as measures of host predictability; and (5) the potential link between host specificity and parasite diversification. Host specificity, expressed as an index of host specificity including phylogenetic and taxonomic relatedness of hosts, was partially associated with parasite phylogeny, but no significant contribution of host phylogeny was found. The mapping of host specificity into the phylogenetic tree suggests that being specialist is not a derived condition for Dactylogyrus species. The different morphometric traits of the attachment apparatus seem to be selected in connection with specialization of specialist parasites and other traits favored as adaptations in generalist parasites. Parasites widespread on several host species reach higher abundance within hosts, which supports the hypothesis of ecological specialization. When separating specialists and generalists, we confirmed the hypothesis of specialization on a predictable resource; that is, specialists with larger anchors tend to live on fish species with larger body size and greater longevity, which could be also interpreted as a mechanism for optimizing morphological adaptation. We demonstrated that ecology of host species could also be recognized as an important determinant of host specificity. The mapping of morphological characters of the attachment organ onto the parasite phylogenetic tree reveals that morphological evolution of the attachment organ is connected with host specificity in the context of fish relatedness, especially at the level of host subfamilies. Finally, we did not find that host specificity leads to parasite diversification in congeneric monogeneans.     

Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation

The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay.     

Intracellular Theileria annulata Promote Invasive Cell Motility through Kinase Regulation of the Host Actin Cytoskeleton

The intracellular, protozoan Theileria species parasites are the only eukaryotes known to transform another eukaryotic cell. One consequence of this parasite-dependent transformation is the acquisition of motile and invasive properties of parasitized cells in vitro and their metastatic dissemination in the animal, which causes East Coast Fever (T. parva) or Tropical Theileriosis (T. annulata). These motile and invasive properties of infected host cells are enabled by parasite-dependent, poorly understood F-actin dynamics that control host cell membrane protrusions. Herein, we dissected functional and structural alterations that cause acquired motility and invasiveness of T. annulata-infected cells, to understand the molecular basis driving cell dissemination in Tropical Theileriosis. We found that chronic induction of TNFα by the parasite contributes to motility and invasiveness of parasitized host cells. We show that TNFα does so by specifically targeting expression and function of the host proto-oncogenic ser/thr kinase MAP4K4. Blocking either TNFα secretion or MAP4K4 expression dampens the formation of polar, F-actin-rich invasion structures and impairs cell motility in 3D. We identified the F-actin binding ERM family proteins as MAP4K4 downstream effectors in this process because TNFα-induced ERM activation and cell invasiveness are sensitive to MAP4K4 depletion. MAP4K4 expression in infected cells is induced by TNFα-JNK signalling and maintained by the inhibition of translational repression, whereby both effects are parasite dependent. Thus, parasite-induced TNFα promotes invasive motility of infected cells through the activation of MAP4K4, an evolutionary conserved kinase that controls cytoskeleton dynamics and cell motility. Hence, MAP4K4 couples inflammatory signaling to morphodynamic processes and cell motility, a process exploited by the intracellular Theileria parasite to increase its host cell''s dissemination capabilities.     

Developmentally regulated expression of a mitogen-activated protein kinase in Xenopus laevis.

Mitogen-activated protein (MAP) kinases are activated in somatic cells in response to many extracellular stimuli and in oocytes during meiotic maturation. We have examined the tissue specificity of expression of a MAP kinase (Xp42) in adult and larval Xenopus laevis. MAP kinase RNA and protein were abundant in the nervous system and lymphoid tissues and were readily detected in most other organs. A remarkably high level of RNA was detected in ovary. Fractionation of oocytes showed that MAP kinase RNA is expressed at the highest level in small oocytes, suggesting that it is a maternal RNA that is stored for early embryogenesis. The levels of MAP kinase RNA and protein did not change from the time of fertilization through to late blastula. The results are consistent with functions for MAP kinases in signal transduction in embryonic as well as adult cells.     

Selective regulation of MAP kinase signaling by an endomembrane phosphatidylinositol 4-kinase

Multiple MAP kinase pathways share components yet initiate distinct biological processes. Signaling fidelity can be maintained by scaffold proteins and restriction of signaling complexes to discreet subcellular locations. For example, the yeast MAP kinase scaffold Ste5 binds to phospholipids produced at the plasma membrane and promotes selective MAP kinase activation. Here we show that Pik1, a phosphatidylinositol 4-kinase that localizes primarily to the Golgi, also regulates MAP kinase specificity but does so independently of Ste5. Pik1 is required for full activation of the MAP kinases Fus3 and Hog1 and represses activation of Kss1. Further, we show by genetic epistasis analysis that Pik1 likely regulates Ste11 and Ste50, components shared by all three MAP kinase pathways, through their interaction with the scaffold protein Opy2. These findings reveal a new regulator of signaling specificity functioning at endomembranes rather than at the plasma membrane.     

Docking domains and substrate-specificity determination for MAP kinases

Signalling specificity in eukaryotic cells is maintained by several mechanisms. One mechanism by which mitogen-activated protein (MAP) kinases ensure their specificity of action is by interacting with their substrates through docking domains. These docking domains recruit the kinases to the correct substrates and enhance their fidelity and efficiency of action. Additional specificity determinants in the substrates serve to enhance the specificity of substrate phosphorylation by MAP kinases further.     

Proteome‐wide B and T cell epitope repertoires in outer membrane proteins of Mycobacterium avium subsp. paratuberculosis have vaccine and diagnostic relevance: a holistic approach

Mycobacterium avium subsp. paratuberculosis (MAP) is an etiological agent of chronic inflammation of the intestine among ruminants and humans. Currently, there are no effective vaccines and sensitive diagnostic tests available for its control and detection. For this, it is of paramount importance to identify the MAP antigens, which may be immunologically recognized by the host immune system. To address this challenge, we performed identification of the immunogenic epitopes in the MAP outer membrane proteins (OMPs). We have previously identified 57 MAP proteins as OMPs [Rana A, Rub A, Akhter Y. 2014. Molecular BioSystems, 10:2329–2337] and have evaluated them for the epitope selection and analysis employing a computational approach. Thirty‐five MAP OMPs are reported with nine‐mer peptides showing high binding affinity to major histocompatibility complex (MHC) class I molecules and 28 MAP OMPs with 15‐mer peptides of high binding affinity for MHC class II molecules. The presence of MHC binding epitopes indicates the potential cell‐mediated immune response inducing capacity of these MAP OMPs in infected host. To further investigate the humoral response inducing properties of OMPs of MAP, we report potential B cell epitopes based on the sequences of peptide antigens and their molecular structures. We also report 10 proteins having epitopes for both B and T cells representing potential candidates which may invoke both humoral and cellular immune responses in the host. These findings will greatly accelerate and expedite the formulation of effective and cost‐efficient vaccines and diagnostic tests against MAP infection. Copyright © 2015 John Wiley & Sons, Ltd.     

The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.     

Geographical variation in host specificity of fleas (Siphonaptera) parasitic on small mammals: the influence of phylogeny and local environmental conditions

The evolution of host specificity remains a central issue in the study of host‐parasite relationships. Here we tackle three basic questions about host specificity using data on host use by fleas (Siphonaptera) from 21 geographical regions. First, are the host species exploited by a flea species no more than a random draw from the locally available host species, or do they form a taxonomically distinct subset? Using randomization tests, we showed that in the majority of cases, the taxonomic distinctness (measured as the average taxonomic distances among host species) of the hosts exploited by a flea is no different from that of random subsets of hosts taken from the regional pool. In the several cases where a difference was found, the taxonomic distinctness of the hosts used by a flea was almost always lower than that of the random subsets, suggesting that the parasites use hosts within a narrower taxonomic spectrum than what is available to them. Second, given the variation in host specificity among populations of the same flea species, is host specificity truly a species character? We found that host specificity measures are repeatable among different populations of the same flea species: host specificity varies significantly more among flea species than within flea species. This was true for both measures of host specificity used in the analyses: the number of host species exploited, and the index measuring the average taxonomic distinctness of the host species and its variance. Third, what causes geographical variation in host specificity among populations of the same flea species? In the vast majority of flea species, neither of our two measures of host specificity correlated with either the regional number of potential host species or their taxonomic distinctness, or the distance between the sampled region and the center of the flea's geographical range. However, in most flea species host specificity correlated with measures of the deviation in climatic conditions (precipitation and temperature) between the sampled region and the average conditions computed across the flea's entire range. Overall, these results suggest that host specificity in fleas is to a large extent phylogenetically constrained, while still strongly influenced by local environmental conditions.     

Combining phylogenetic and ecological information into a new index of host specificity

Host specificity has 2 independent facets: the extent to which different host species are used by a parasite, and the phylogenetic distances among these hosts. Although the number of host species exploited by a parasite commonly is used as a measure of host specificity, it fails to capture ecological and phylogenetic differences among hosts. Here, a new index of host specificity, S(TD)*, is developed and illustrated. This index measures the average taxonomic distinctness among the host species used by a parasite, weighted for the parasite's prevalence in the different hosts. For a given number of host species, the index approaches its minimum value when a parasite achieves high prevalence in a few closely related host species, and the index approaches its highest value when a parasite reaches its highest prevalence values in distantly related host species. Simple hypothetical examples are used to demonstrate the index's computation and some of its properties. The new index is influenced independently both by the taxonomic (or phylogenetic) affinities of a set of host species and by the distribution of prevalence values among these hosts. A single value cannot truly capture all the nuances of a phenomenon as complex as host specificity; nevertheless, the proposed index incorporates the features of specificity that are most relevant to parasitologists and will be a useful tool for comparative studies.     

Induction of matrix metalloproteinases and TLR2 and 6 in murine colon after oral exposure to Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP) is suspected to be a causative agent in Crohn's disease. Recent evidence suggests that MAP can induce the expression of Matrix Metalloproteinases (MMPs), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within the present study, we analysed whether oral MAP exposure can induce colonic MMP expression in vivo. In MAP exposed mice MAP and spheroplasts were visualized in intramucosal leukocyte aggregates. MAP exposed mice exhibited a higher colonic expression of Mmp-2, -9, -13, -14, Timp-1, Tlr2, Tlr6, Il-1β, and Tnf-α. Cell clusters of MMP-9 positive cells adjacent to intramucosal leukocyte aggregates and CD45(+) leukocytes were identified as the major cellular sources of MMP-9. Enhanced TLR2 expression was visualized on the luminal side of colonic enterocytes. Although MAP exposure did not lead to macroscopic intestinal inflammation, the observed MAP spheroplasts in intramucosal leukocyte aggregates together with increased colonic expression of toll-like receptors, pro-inflammatory cytokines, and MMPs upon MAP exposure represents a part of the host immune response towards MAP.     

Enzymatic activity and substrate specificity of mitogen-activated protein kinase p38alpha in different phosphorylation states

The mitogen-activated protein (MAP) kinases are essential signaling molecules that mediate many cellular effects of growth factors, cytokines, and stress stimuli. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Down-regulation of MAP kinase activity can be initiated by multiple serine/threonine phosphatases, tyrosine-specific phosphatases, and dual specificity phosphatases (MAP kinase phosphatases). This would inevitably lead to the formation of monophosphorylated MAP kinases. However, the biological functions of these monophosphorylated MAP kinases are currently not clear. In this study, we have prepared MAP kinase p38alpha, a member of the MAP kinase family, in all phosphorylated forms and characterized their biochemical properties. Our results indicated the following: (i) p38alpha phosphorylated at both Thr-180 and Tyr-182 was 10-20-fold more active than p38alpha phosphorylated at Thr-180 only, whereas p38alpha phosphorylated at Tyr-182 alone was inactive; (ii) the dual-specific MKP5, the tyrosine-specific hematopoietic protein-tyrosine phosphatase, and the serine/threonine-specific PP2Calpha are all highly specific for the dephosphorylation of p38alpha, and the dephosphorylation rates were significantly affected by different phosphorylated states of p38alpha; (iii) the N-terminal domain of MPK5 has no effect on enzyme catalysis, whereas deletion of the MAP kinase-binding domain in MKP5 leads to a 370-fold decrease in k(cat)/K(m) for the dephosphorylation of p38alpha. This study has thus revealed the quantitative contributions of phosphorylation of Thr, Tyr, or both to the activation of p38alpha and to the substrate specificity for various phosphatases.     

Association of CNK1 with Rho guanine nucleotide exchange factors controls signaling specificity downstream of Rho

BACKGROUND: Rho is a small GTPase that controls signal transduction pathways in response to a large number of extracellular stimuli. With over 15 potential Rho target proteins identified to date, however, it is not clear how distinct signaling outputs can be generated downstream of a particular stimulus. RESULTS: Several of the known Rho targets are structurally reminiscent of scaffold proteins, which are generally thought to play an important role in controlling signaling specificity. Here, we show that the Rho target CNK1 is a scaffold protein that interacts with Net1 or p115RhoGEF, two Rho-specific guanine nucleotide exchange factors (GEFs), as well with MLK2 and MKK7, two of the kinase components in the JNK MAP kinase cascade. CNK1 acts cooperatively with the two GEFs to activate JNK MAP kinase, but not other Rho-mediated pathways. In HeLa cells, serum or sphingosine-1-phosphate stimulate Rho-dependent activation of the JNK MAP kinase cascade, and this requires endogenous CNK1. CONCLUSIONS: We conclude that CNK1 couples a subset of Rho exchange factors to activation of the JNK MAP kinase pathway and that signaling specificity is achieved through complexes containing both upstream activators and downstream targets of Rho.