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Identification of novel virulence determinants in Mycobacterium paratuberculosis by screening a library of
Sung Jae Shin1, Chia-Wei Wu, Howard Steinberg
1Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, 1656 Linden Drive, Madison, WI 53706-1581, USA.
Abstract:
Johne's disease, caused by Mycobacterium paratuberculosis infection, is a worldwide problem for the dairy industry and has a possible involvement in Crohn's disease in humans. To identify virulence determinants of this economically important pathogen, a library of 5,060 transposon mutants was constructed using Tn5367 insertion mutagenesis, followed by large-scale sequencing to identify disrupted genes. In this report, 1,150 mutants were analyzed and 970 unique insertion sites were identified. Sequence analysis of the disrupted genes indicated that the insertion of Tn5367 was more prevalent in genomic regions with G+C content (50.5 to 60.5%) lower than the average G+C content (69.3%) of the rest of the genome. Phenotypic screening of the library identified disruptions of genes involved in iron, tryptophan, or mycolic acid metabolic pathways that displayed unique growth characteristics. Bioinformatic analysis of disrupted genes identified a list of potential virulence determinants for further testing with animals. Mouse infection studies showed a significant decrease in tissue colonization by mutants with a disruption in the gcpE, pstA, kdpC, papA2, impA, umaA1, or fabG2_2 gene. Attenuation phenotypes were tissue specific (e.g., for the umaA1 mutant) as well as time specific (e.g., for the impA mutant), suggesting that those genes may be involved in different virulence mechanisms. The identified potential virulence determinants represent novel functional classes that could be necessary for mycobacterial survival during infection and could provide suitable targets for vaccine and drug development against Johne's and Crohn's diseases.
Insights
Researchers identified key genes in Mycobacterium paratuberculosis, the cause of Johne
Area of Science:
- Microbiology and Immunology
- Veterinary Medicine
- Gastroenterology
Background:
- Johne's disease, caused by Mycobacterium paratuberculosis, significantly impacts the global dairy industry.
- There is a potential link between Mycobacterium paratuberculosis infection and Crohn's disease in humans.
- Identifying virulence factors is crucial for understanding and combating this pathogen.
Purpose of the Study:
- To identify virulence determinants of Mycobacterium paratuberculosis through large-scale mutagenesis and sequencing.
- To investigate the role of specific disrupted genes in pathogen survival and colonization.
- To discover potential targets for novel vaccine and drug development.
Main Methods:
- Construction of a large-scale transposon mutant library (5,060 mutants) using Tn5367.
- High-throughput sequencing to identify disrupted genes and insertion sites.
- Phenotypic screening of mutants and subsequent mouse infection studies.
Main Results:
- Analysis of 1,150 mutants revealed 970 unique insertion sites, with preferential insertion in lower G+C content regions.
- Mutants with disruptions in iron, tryptophan, or mycolic acid metabolic pathways showed altered growth.
- Disruptions in gcpE, pstA, kdpC, papA2, impA, umaA1, or fabG2_2 genes significantly reduced tissue colonization in mice.
- Observed tissue-specific and time-specific attenuation phenotypes suggest diverse roles for these genes in virulence.
Conclusions:
- Several novel genes (gcpE, pstA, kdpC, papA2, impA, umaA1, fabG2_2) are identified as potential virulence determinants.
- These determinants are crucial for Mycobacterium paratuberculosis survival and colonization during infection.
- The identified genes represent promising targets for developing vaccines and therapeutics against Johne's and Crohn's diseases.
