Kinome analysis of Toll-like receptor signaling in bovine monocytes.

The Toll-like receptors (TLRs) are a family of pathogen recognition receptors that alert the host to the presence of microbial challenge. Each TLR responds to a specific microbial associated ligand. For example, TLR4 is activated by lipopolysaccharide (LPS), whereas TLR9 responds to microbial DNA (CpGs). In this report signal transduction responses of bovine monocytes to stimulation with LPS and CpG are described through a bovine-specific peptide array. In addition to confirming activation of the defined TLR pathway in bovine cells, unique phosphorylation events not previously attributed to TLR signaling are described and validated. For example, array data predicts phosphorylation of Tyr40 of Etk in response to LPS, but not CpG, stimulation as well as the activation of oxidative burst in CpG, but not LPS. This investigation confirms interspecies conservation of the TLR pathway in bovine as well as providing insight into the complexity and mechanisms of TLR signaling.

Genome to kinome: species-specific peptide arrays for kinome analysis.

Tools for conducting high-throughput kinome analysis do not exist for many species. For example, two commonly used techniques for monitoring phosphorylation events are phosphorylation-specific antibodies and peptide arrays. The majority of phosphorylation-specific antibodies are for human or mouse targets, and the construction of peptide arrays relies on information from phosphorylation databases, which are similarly biased toward human and mouse data. This is a substantial obstacle because many species other than mouse represent important biological models. On the basis of the observation that phosphorylation events are often conserved across species with respect to their relative positioning within proteins and their biological function, we demonstrate that it is possible to predict the sequence contexts of phosphorylation events in other species for the production of peptide arrays for kinome analysis. Through this approach, genomic information can be rapidly used to create inexpensive, customizable, species-specific peptide arrays for high-throughput kinome analysis. We anticipate that these arrays will be valuable for investigating the conservation of biological responses across species, validating animal models of disease, and translating research to clinical applications.

Microarray analysis of Streptococcus pneumoniae gene expression changes to human lung epithelial cells.

Streptococcus pneumoniae infection starts from the respiratory tract where interaction with host epithelial cells occurs. To gain more insights on pneumococcal pathogenesis, an oligonucleotide (oligo)-based microarray was used to investigate gene expression changes of one serotype 3 encapsulated pathogenic S. pneumoniae strain 82 and one unencapsulated avirulent S. pneumoniae strain R6 upon exposure to human lung epithelial cells (A549) for 1 and 3 h, respectively. We observed that genes associated with many functional categories were differentially regulated in strain 82, such as genes in pathogenesis, cell envelope, transcription, translation, transport, metabolism, and unknown functions. In contrast, few genes were changed in strain R6 except for genes in ribonucleotide biosynthesis and unknown functions. Quantitative real-time PCR analysis confirmed the microarray results for most of the genes tested. To further characterize functions of the selected genes, knockout mutants were constructed in strain R6. We demonstrated that 2 genetic loci, SP_2170 (AdcB, zinc ABC transporter) and SP_0157 (hypothetical protein), were involved in adherence to A549 cells. These data suggest that divergent gene expression changes occur in S. pneumoniae pathogenic and avirulent strains during interaction with human lung epithelial cells. Some of those genes are involved in pneumococcal pathogenesis.

Comparative approaches to the investigation of responses to stress and viral infection in cattle.

Fatal bovine respiratory disease (BRD) is a major cause of financial losses in the cattle industry. A variety of stressors have been implicated as contributing to disease severity. However, it has proven difficult to determine the role these individual factors may play in the final outcome of this disease complex. The objective of the present investigation was to obtain proteomic, metabonomic, and elemental profiles of bovine serum samples from stressed and control animals before and after a primary viral infection to determine if these profiles could distinguish between responses to stressors and viral infection. Multivariate analysis revealed distinct differential trends in the distribution profile of proteins, metabolites, and elements following a stress response both before and after primary viral infection. A group of acute phase proteins, metabolites, and elements could be specifically linked to either a stress response (decreased serum amyloid A and Cu, increased apolipoprotein CIII, amino acids, LDL, P, and Mo) or a primary viral respiratory infection (increased apolipoprotein A1, haptoglobin, glucose, amino acids, LDL and Cu, decreased Lipid, and P). Thus, combined OMICS analysis of serum samples revealed that multimethod analysis could be used to discriminate between the complex biological responses to stress and viral infection.

Molecular analyses of disease pathogenesis: application of bovine microarrays.

The molecular analysis of disease pathogenesis in cattle has been limited by the lack of availability of tools to analyze both host and pathogen responses. These limitations are disappearing with the advent of methodologies such as microarrays that facilitate rapid characterization of global gene expression at the level of individual cells and tissues. The present review focuses on the use of microarray technologies to investigate the functional pathogenomics of infectious disease in cattle. We discuss a number of unique issues that must be addressed when designing both in vitro and in vivo model systems to analyze host responses to a specific pathogen. Furthermore, comparative functional genomic strategies are discussed that can be used to address questions regarding host responses that are either common to a variety of pathogens or unique to individual pathogens. These strategies can also be applied to investigations of cell signaling pathways and the analyses of innate immune responses. Microarray analyses of both host and pathogen responses hold substantial promise for the generation of databases that can be used in the future to address a wide variety of questions. A critical component limiting these comparative analyses will be the quality of the databases and the complete functional annotation of the bovine genome. These limitations are discussed with an indication of future developments that will accelerate the validation of data generated when completing a molecular characterization of disease pathogenesis in cattle.