Systems kinomics for characterizing host responses to high-consequence pathogens at the NIH/NIAID Integrated Research Facility-Frederick

Abstract

Currently, there is a paucity of information regarding the molecular pathogenesis for many high-consequence pathogens (HCPs) that pose threats to both national and international public health. In spite of this, investigations of the molecular pathogenesis for many HCPs have been limited to gross pathological changes in animal models or global analysis of gene expression. Further, questions remain regarding the ability of animal models of disease to recapitulate human molecular pathogenesis or act as predictors of therapeutic efficacy. Thus, it is likely that medical countermeasure development for HCPs will rely on identifying therapeutic targets that are uniquely modulated during HCP infection. It is also appreciated that many cellular processes can be regulated independently of changes in transcription or translation through phosphorylation events. Cellular kinases, individually or collectively (the kinome), play critical roles in regulating complex biology, underlie various malignancies, and represent high-priority drug targets. The growing interest in kinases in both basic and translational research has driven efforts to develop technologies that enable characterization of phosphorylation-mediated signal transduction. To this end, enhanced technical capabilities at the IRF-Frederick provide the unique capability for characterizing host responses to HCP insult during the course of infection and identify novel targets for therapeutic intervention.

Keywords: filovirus; high-consequence pathogens; kinase; kinome; orthopoxvirus; signaling pathways.

Figure 1

Development and refinement of animal models of human infectious diseases at the IRF‐Frederick. Animal models of infectious disease are characterized and refined through the concerted efforts of the Medical Imaging, Pathology/Comparative Medicine and Clinical core groups at the IRF‐Frederick. Lastly, the molecular virology expertise at the IRF‐Frederick, including systems biology and traditional virology/molecular biology approaches, provides additional capabilities for characterization of high‐consequence pathogens and, ultimately, aid in the refinement of animal infection models.

Figure 2

Overview of kinome peptide array experimental procedure. Host cells/tissues are infected in vitro or in vivo with pathogen of interest (I). Following infection for the desired time course, cells/tissues are isolated (pelleting by centrifugation or homogenized, respectively) followed by cell lysis (II). Cell debris is removed by high‐speed centrifugation, and supernatants are spotted on the kinome arrays (III). Arrays are incubated to allow for phosphotransfer from activated kinases in the supernatants to the specific peptide targets on the arrays followed by washing, staining, and imaging. (IV). Data are extracted from the spot intensities, and fold‐change differences in phosphorylation are derived using PIIKA followed by functional network analysis (ex. InnateDB or Ingenuity Pathway Analysis) (V).

Figure 3

Designing species‐specific kinome peptide arrays for characterizing host responses to infection. Kinase phosphorylation targets within host proteins are identified based upon homology with previously characterized phosphorylation sites (human, mouse, etc.) through bioinformatic analyses. Peptides representing phosphorylation sites from the species of interest are synthesized and covalently linked to microscope slides to produce kinome peptide arrays that encompass hundreds to thousands of kinase targets.

Figure 4

Model of outcome‐based molecular characterization of high‐consequence pathogens. For many high‐consequence pathogens, there is a paucity of information regarding molecular disease mechanisms. Thus, molecular approaches for characterization of these diseases at the IRF‐Frederick focus on molecular processes within the host and pathogen. This model provides output data in regard to the molecular characterization of infectious disease pathogenesis, identification of biomarkers associated with disease, and novel insight into potential therapeutic intervention strategies.