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Identification of essential genes in Mycobacterium avium subsp. paratuberculosis genome for persistence in dairy
Razieh Eshraghisamani1, Amanda J Mirto2, Joyce Wang3
1Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada.
Abstract:
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 MAP's pathogenesis and potentially develop a live attenuated Johne's disease vaccine by knocking out these genes. MAP 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 MAP Tn mutants, with insertions in 56% of TA sites, interrupting 88% of genes, was created using a MycoMarT7 phagemid containing a mariner transposon. Six calves were inoculated with a high dose of a library of MAP mutants, 1011 CFUs, (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. Statistical analysis for the determination of essential genes was performed by a Hidden Markov Model (HMM), categorizing genes into essential genes that are devoid of insertions and growth-defect genes whose disruption impairs the growth of the organism. Sequence analysis identified 430 in vivo essential and 260 in vivo growth-defect genes. Gene ontology enrichment analysis of the in vivo essential and growth-defect genes with the highest reduction in the tissues revealed a high representation of genes involved in metabolism and respiration, cell wall and cell processing, virulence, and information pathway processes. This study has systematically identified essential genes for the growth and persistence of MAP in the natural host body.
Insights
Researchers identified essential genes for Mycobacterium avium subsp. paratuberculosis (MAP) survival in cattle. This discovery aids understanding of Johne's disease pathogenesis and could inform vaccine development.
Area of Science:
- Microbiology and Immunology
- Bacterial Pathogenesis
- Veterinary Medicine
Background:
- Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease in ruminants, requiring host cell entry and immune manipulation.
- The genetic basis for MAP's pathogenesis and survival within host cells is largely unknown.
- Identifying essential MAP genes is crucial for understanding disease mechanisms and developing control strategies, including potential live attenuated vaccines.
Purpose of the Study:
- To systematically identify genes essential for MAP's survival and growth within its natural host, ruminants.
- To understand the genetic requirements for MAP pathogenesis in dairy calves.
- To provide a foundation for developing targeted interventions against Johne's disease.
Main Methods:
- Genome-wide transposon mutagenesis was used to create a saturated library of MAP mutants.
- High-throughput sequencing and Hidden Markov Model (HMM) analysis were employed to identify essential genes in vivo.
- MAP mutants were inoculated into dairy calves, and bacterial populations were recovered from tissues after two months for genetic analysis.
Main Results:
- 430 in vivo essential genes and 260 in vivo growth-defect genes were identified in MAP.
- Genes involved in metabolism, respiration, cell wall processing, virulence, and information pathways were significantly represented among essential genes.
- This study provides a comprehensive catalog of genes critical for MAP's persistence in the host.
Conclusions:
- This research systematically identified key genes enabling MAP growth and persistence in dairy calves.
- The findings offer critical insights into MAP pathogenesis and host-pathogen interactions.
- The identified essential genes represent potential targets for novel therapeutic strategies and vaccine development against Johne's disease.
