Transposon next generation sequencing of Mycobacterium avium paratuberculosis

Identification of essential genes in Mycobacterium avium subsp. paratuberculosis genome for persistence in dairy calves:To cause disease Mycobacterium avium subsp. paratuberculosis needs to enter mammalian cells, arrest phagosomal maturation and manipulate the host immune system. The genetic basis of the bacterial capacity to achieve these outcomes remains largely unknown. Identifying these genes would allow us to gain a deeper understanding of mycobacterium avium paratuberculosis pathogenesis.Mycobacterium avium paratuberculosis genes demonstrated to be essential for colonization in the natural host, ruminants, are unknown. Genome-wide transposon mutagenesis and high-throughput sequencing were combined to evaluate the essentiality of each coding region in the bacterial genome to survive in dairy calves.A saturated library of 3,852 transpososn mutants, with insertions in 56percent of TA sites, interrupting 88 percent of genes, was created using a MycoMarT7 phagemid containing a mariner transposon. Six calves were inoculated with a high dose of a library of mycobacterium avium paratuberculosis mutants (input) at 2 weeks of age. Following 2 months of incubation, MAP cells were isolated from the ileum, jejunum, and their associated lymph nodes of calves, resulting in approximately 100,000 colonies grown on solid media across 6 animals (output). Targeted next-generation sequencing was used to identify the disrupted genes in all the mutants in the input pool and the output pool recovered from the tissues to identify in vivo essential genes.