Molecular Genetic Analysis of Mycobacterium Avium Subsp. Paratuberculosis (MAP) and Related Mycobacterial Pathogens

NON-TECHNICAL SUMMARY: Mycobacterium avium subsp. paratuberculosis (MAP), one of the slowest growing mycobacteria, is the etiologic agent of paratuberculosis (Johne?s disease, JD), in ruminants. JD has a significant economic impact and worldwide distribution. JD has lower prevalence in beef herds but still has a negative impact on trade. MAP has the potential to be a zoonotic and/or food-borne pathogen, as evidenced by its possible linkage to Crohn?s disease. Other pathogenic mycobacteria include Mycobacterium tuberculosis, the agent of human tuberculosis, and Mycobacterium bovis that causes bovine tuberculosis, a zoonotic disease transmissible from ruminants and wildlife to human hosts. Bovine tuberculosis is classified as a category I Program disease requiring immediate reporting. Though M. bovis infections are rare, they could become major concerns for trade requiring quarantine and onerous expenses for containment. Our work is directed at the generation of vaccines, identification of new drug targets, and development of new drugs to control and treat mycobacterioses. Our major interest will focus on the veterinary pathogen MAP. We have pioneered the molecular technologies used to manipulate MAP and have most molecular procedures in place. Moreover, recently established collaborations will allow us to implement cutting edge technologies and obtain complementary expertise in immunology, chemistry, and biochemistry. We have also the necessary institutional facilities to work with the BSL3 pathogen M. tuberculosis. Technical problems may still arise, but we are confident these will be readily overcome. Furthermore, a solid conceptual foundation has been developed for the experimental objectives proposed. Thus, we expect their outcomes will lead to novel findings. In MAP research, we expect that, in conjunction with industry, our attenuated strains will be further developed into a marketed vaccine product to control JD. We also expect to develop new diagnostic tests to differentiate infected from vaccinated animals. Our research on metabolic pathways is directed to the identification of new drug targets against tuberculosis. The established collaborations are expected to lead to the development of actual candidate drugs.

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APPROACH: Objective 1: We plan to apply TraSH mutagenesis to analyze attenuating mutations in Mycobacterium avium subsp. paratuberculosis (MAP) and, eventually, in related pathogens such as additional members of the M. avium group and virulent M. bovis. We plan to determine the mechanism of their attenuation in bovine macrophages, construct deletion mutant strains, and conduct complementation analysis. These strains will also serve as controls of attenuated strains for TraSH mutagenesis experiments. Large pools of transposon mutants will be generated and subjected to selection pressures under different experimental conditions. Differentially-labeled RNA probes, complementary to the chromosomal DNA flanking each transposon insertion site, will be prepared from the mutants recovered under each condition and their representation will be compared by microarray hybridization. Objective 2: We plan to continue the analysis of PPE proteins in MAP, and expand to other pathogenic mycobacteria. The focus will be the construction and characterization of deletion mutants in MAP1152, MAP1153 and MAP1155; the corresponding PPE proteins will be overproduced in Escherichia coli. In this objective, we plan to continue our studies on the PPE proteins in the MAP1152 MAP1156 cluster, others PPE proteins encoded elsewhere in the genome. Using a panel of epidemiologically relevant strains, we plan to perform comparative bioinformatic and sequencing analyses of the coding sequences of PPE proteins to determine those that are most conserved. Based on these results, deletion mutants will be constructed and their interactions with BM will be characterized. In addition, selected PPE proteins will be overproduced in recombinant E. coli and the purified proteins will be subjected to tests of humoral and cellular immunity. At first, this project will focus on the MAP1152-MAP1156 cluster already identified as relevant to MAP pathogenesis. Objective 3: In this objective, we will analyze metabolic pathways in M. smegmatis and M. tuberculosis crucial for cell physiology in vitro and in vivo. We will focus on the mechanisms underlying redox homeostasis in M. tuberculosis latency and the role of L-alanine dehydrogenase. We will also analyze the effects of potential inhibitors including the anti-mycobacterial agent DCS, novel inhibitors of M. tuberculosis growth and D-alanine ligase, fatty acid analogs, cationic detergents, and polyketide derivatives. Studies on latency and redox homeostasis will be focused on L-alanine dehydrogenase (Ald) and related enzymes. Metabolomic and proteomic analyses will be performed on wildtype and mutant strains grown in various media under aerobic and anaerobic (e. g, similar condition to that of dormant bacilli) conditions. For new compounds, we will determine minimal inhibitory concentrations (MIC), bactericidal activities and survival curves (e.g., polyketides, fatty acid analogs, cationic detergents, etc). For putative inhibitors of D-alanine ligase, IC50 values will be determined by enzyme assays.