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Open Access Highly Accessed Research article

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

Joyce Wang1, Justin R Pritchard2, Louis Kreitmann3, Alexandre Montpetit4 and Marcel A Behr135*

Author Affiliations

1 Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, QC H3A 2B4, Canada

2 Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115, USA

3 Department of Medicine, McGill University, 1650 Cedar Avenue, Montreal, QC H3G 1A4, Canada

4 McGill University and Génome Québec Innovation Centre, 740 Dr. Penfield Avenue, Montreal H3A 0G1, QC, Canada

5 McGill International TB Centre, 1650 Cedar Avenue, Montreal, QC H3G 1A4, Canada

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BMC Genomics 2014, 15:415  doi:10.1186/1471-2164-15-415

Published: 31 May 2014

Abstract

Background

Mycobacterium 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.

Results

Out 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.

Conclusions

This 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.

Keywords:
Mycobacterium avium; M. avium subsp. paratuberculosis; Transposon insertion sequencing; Horizontal gene transfer; Mycobacterial pathogenesis