Raul Barletta

JD caused by Mycobacterium avium subsp. paratuberculosis (MAP) is one of the most significant problems in animal health, especially for the dairy industry. Moreover, the potential linkage with Crohn’s disease makes MAP a concern as a zoonotic and/or food-borne pathogen. We developed a novel approach to comprehensively identify MAP essential genes, and mine its genome for virulence determinants encoded by non-essential genes involved in intracellular (macrophage) survival or required for infection of calves. Mariner transposon mutagenesis coupled with new generation sequencing technologies for high-resolution phenotypic profiling (Tn-seq) and Transposon Site Hybridization (TraSH) will be applied to analyze fully random mutant banks representing genomic regions that are likely significantly underrepresented from current collections. We hypothesize that these approaches will result in mining of the MAP genome for virulence determinant discovery, independently of their constitutive or regulated expression. We are currently applying these studies to develop MAP live-attenuated vaccines with DIVA capabilities. The overall impact is the embodiment of a JD vaccine with DIVA capabilities. Achievement of this goal would be of significant importance in ameliorating the negative impact of JD on national and international trade.

Re-emergence of TB is caused by multiple drug-resistant strains and the need to develop new drug therapies and other control strategies. In this aspect, my research on anti-TB drug development and biodefense focuses on D-alanine racemase and D-alanine ligase, the presumed lethal target of D-cycloserine, an analog of D-alanine. This enzyme forms the critical dipeptide D-alanyl-D-alanine, an essential building block of peptidoglycan. Institutional grants allowed my laboratory to develop a "proof of concept" in the model system Mycobacterium smegmatis while a National Institutes of Health grant permitted us to further pursue the development of D-alanine ligase as a target for rational drug design. My laboratory analyzed the crystal structure of this drug target by completing collaborations with Dr. James Sacchettini (Department of Biochemistry and Biophysics, Texas A&M University). These research studies resulted in various publications and the granting of a US patent.

Ongoing research:

1. DIVA grant objectives. (1) Generate attenuated unmarked in-frame deletion mutants with DIVA capabilities. New unmarked deletion mutants in MAP_1152 and MAP_1156 from MAP wild type strain K-10 will be generated. We have demonstrated that the original marked DMAP52 and DMAP56, already available in our laboratories, are attenuated in bovine macrophages. These mutants will be complemented and tested for invasion and intracellular survival in bovine macrophages. (2) Test novel antigens for DIVA capabilities. Recombinant capture antigens MAP_1152 and MAP_1156 have been shown to differentiate between seropositive and seronegative samples. Herein, we will expand the humoral immunity test to a greater number of samples from JD natural and experimentally infected animals, and develop tests of cellular immunity. (3) Assess the immunogenicity and pathogenicity of unmarked mutants and their complemented strains in calves.
2. Expand drug target search to additional essential steps in peptidoglycan biosynthesis and D-alanine metabolism such as L-alanine dehydrogenase.
3. Determine the essential roles of D-alanine ligase and D-alanine racemase in the physiology of Mycobacterium tuberculosis (MTB). Search for novel inhibitors of the MTB D-alanine ligase that could serve as novel anti-tuberculosis drugs.

Teaching and training program:

My teaching commitments involve pathogenic microbiology courses: I am the main instructor of Basic (VBMS424/824) and Advanced (VBMS951) Infectious Diseases, and contribute to lectures in Pathogenic Microbiology (VBMS441/841) and VMED686. These courses are an essential component to instruct both undergraduate, graduate and professional veterinary students interested in the molecular basis of infectious diseases. The Advanced course is also geared to provide elements of proposal writing to advanced graduate students. Regarding training, 3 senior personnel, 4 post-doctoral fellows, and 15 graduate students (5 PhD and 10 MS) trained under my supervision. Finally, I work with undergraduate students in the UNL UCARE program, Honors Thesis and Independent Research.