Disruption of Mycobacterium avium subsp. paratuberculosis-specific genes impairs in vivo fitness

Abstract

BackgroundMycobacterium avium subsp. paratuberculosis (MAP) is an obligate intracellular pathogen that infects many ruminant species. The acquisition of foreign genes via horizontal gene transfer has been postulated to contribute to its pathogenesis, as these genetic elements are absent from its putative ancestor, M. avium subsp. hominissuis (MAH), an environmental organism with lesser pathogenicity. In this study, high-throughput sequencing of MAP transposon libraries were analyzed to qualitatively and quantitatively determine the contribution of individual genes to bacterial survival during infection.ResultsOut of 52384 TA dinucleotides present in the MAP K-10 genome, 12607 had a MycoMarT7 transposon in the input pool, interrupting 2443 of the 4350 genes in the MAP genome (56%). Of 96 genes situated in MAP-specific genomic islands, 82 were disrupted in the input pool, indicating that MAP-specific genomic regions are dispensable for in vitro growth (odds ratio = 0.21). Following 5 independent in vivo infections with this pool of mutants, the correlation between output pools was high for 4 of 5 (R = 0.49 to 0.61) enabling us to define genes whose disruption reproducibly reduced bacterial fitness in vivo. At three different thresholds for reduced fitness in vivo, MAP-specific genes were over-represented in the list of predicted essential genes. We also identified additional genes that were severely depleted after infection, and several of them have orthologues that are essential genes in M. tuberculosis.ConclusionsThis work indicates that the genetic elements required for the in vivo survival of MAP represent a combination of conserved mycobacterial virulence genes and MAP-specific genes acquired via horizontal gene transfer. In addition, the in vitro and in vivo essential genes identified in this study may be further characterized to offer a better understanding of MAP pathogenesis, and potentially contribute to the discovery of novel therapeutic and vaccine targets.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-415) contains supplementary material, which is available to authorized users.

Analysis of Mycobacterium avium subsp. paratuberculosis mutant libraries reveals loci-dependent transposition biases and strategies for novel mutant discovery

“…Though a sensible genetic approach was used, the screening method is labour intensive to screen a representative mariner transposon library. More recently, a larger study was performed that collected 90 000 Himar1 mutants but identified insertions in approximately 12 000 out of 52 000 (ca 25 %) recognition sites (Wang et al, 2014). Wang et al (2014) attributed this low number of recognition sites represented to bottle-necking effects during DNA sample preparation or the sequencing stage.…”

“…More recently, a larger study was performed that collected 90 000 Himar1 mutants but identified insertions in approximately 12 000 out of 52 000 (ca 25 %) recognition sites (Wang et al, 2014). Wang et al (2014) attributed this low number of recognition sites represented to bottle-necking effects during DNA sample preparation or the sequencing stage. However, loci-dependent transposition biases are more likely to explain these results.…”

“…This procedure differs from prior screens where mutants were picked based on phenotypes that may be associated with virulence or libraries that were not comprehensive (Cavaignac et al, 2000;Harris et al, 1999;Scandurra et al, 2009;Shin et al, 2006;Wang et al, 2014).…”

Mycobacterium avium subspecies paratuberculosis: An Emerging Bacterial Disease of Global Public Health Significance

“…avium, and M. avium ssp. paratuberculosis that are shared by different animal species including primates (Pal,2007;Wang et al, 2014). These pathogens resist adverse environmental conditions (Verma, 2013).…”

“…Recently, M.avium subspecies paratuberculosis (MAP) has emerged as a major and successful animal pathogen with significant zoonotic, and public health concerns since first observed by Johne and Frothingham in 1895 (Pal,2007;OIE,2008).MAP is an intracellular pathogen that causes Johne's disease(JD), a chronic (2 to 5 years) intestinal inflammation in cattle, sheep, goats, and wild deer etc. species including primates (Pal,2007;Wang et al, 2014). JD is a chronic granulomatous bowel inflammatory condition in animals provoked by progressive weight loss resulting death.The World Organization for Animal Health (OIE) considers JD as a disease of major global importance.…”