|RATHNAIAH, GOVARDHAN - University Of Nebraska|
|ZINNIEL, DENISE - University Of Nebraska|
|GROHN, YRJO - Cornell University - New York|
|BARLETTA, RAUL - University Of Nebraska|
Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/15/2016
Publication Date: 2/16/2016
Citation: Rathnaiah, G., Bannantine, J.P., Bayles, D.O., Zinniel, D.K., Stabel, J.R., Grohn, Y.T., Barletta, R.G. 2016. Analysis of Mycobacterium avium subsp. paratuberculosis mutant libraries reveals loci-dependent transcription biases and strategies to novel mutant discovery. Microbiology. 162:633-641. doi: 10.1099/mic.0.000258.
Interpretive Summary: Johne's disease is one of the most significant health problems for the United States cattle and dairy industries, with annual losses estimated at $250 million to $1.5 billion. Moreover, the potential for Mycobacterium avium subsp. paratuberculosis (MAP) being a zoonotic and/or food-borne pathogen, as evidenced by its possible linkage to Crohn's disease, creates concerns for national and international trade. Even though the analysis of disease pathogenesis and the development of diagnostic tests and vaccines have progressed significantly, most diseased animals escape detection. With the completion of the MAP genome sequence, pathogenesis can now be addressed on a genome-wide scale. This study used genetic technology to identify molecular targets for the development of state of the art safe and effective vaccines against Johne’s disease. We discovered a transposon that can disrupt over 99% of all MAP genes. This is important in understanding which genes are essential for survival in the animal host. Those bacteria that cannot survive with a transposon insertion in gene x suggests that gene x is required for survival in the animal and thus a target for vaccine development.
Technical Abstract: Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in ruminants and it has been implicated as a cause of Crohn’s disease in humans. The generation of comprehensive random mutant banks by transposon mutagenesis is a fundamental wide genomic technology utilized to determine the role of MAP genes in pathogenesis. In this study, bioinformatics was applied to perform a comparative analysis of insertion sites for the classical mycobacterial transposons Tn5367 and Tn5370, both derived from the Mycobacterium smegmatis insertion sequence IS1096 and the mariner transposons carrying the Himar1 transposase. We determined that only mariner transposons provide a random representation of insertions in 99% of all MAP genes. Genome analysis showed that 710 (16.3%) of all ORFs do not possess IS1096 recognition sites, while only 37 (0.85%) of all ORFs do not have the recognition site for Himar1 insertions. Thus, a significant number of MAP genes remain underrepresented in Tn5367 or Tn5370 insertion libraries. Experimental confirmation was provided by the analysis of a pool of 109 Himar1 and 108 Tn5367 mutants. Sequencing of these reduced transposon mutant pools proved sufficient to identify six Himar1 insertion mutants in genes that lack IS1096 insertion sites. Most mutants had insertions in different MAP genes, indicating a random distribution among this sampling. In summary, Himar1 transposon mutants are necessary to obtain random mutant bank collections that could be used in the study of absolute and conditional (e.g., in vivo) gene essentiality.