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Phage infection, transfection and transformation of Mycobacterium avium complex and Mycobacterium paratuberculosis
Ellen M Foley-Thomas1, Diana L Whipple2, Luiz E Bermudez3
1Department of Veterinary and Biomedical Sciences, Center for Biotechnology, University of Nebraska, Lincoln, NE 68583-0905, USA.
Abstract:
Mycobacterium avium complex strains and Mycobacterium paratuberculosis are closely related intracellular pathogens affecting humans and animals. M. avium complex infections are a leading cause of morbidity and mortality in AIDS patients, and M. paratuberculosis is the agent of Johne's disease in ruminants. Genetic manipulation of these micro-organisms would facilitate the understanding of their pathogenesis, the construction of attenuated vaccine strains and the development of new drugs and treatment methods. This paper describes the replication of mycobacterial shuttle phasmids and plasmids, and the expression of the firefly luciferase reporter gene in M. avium complex and M. paratuberculosis. The mycobacteriophage TM4 propagated on M. smegmatis or M. paratuberculosis plaqued at the same efficiency on these two mycobacterial hosts. Screening of M. avium complex and M. paratuberculosis clinical isolates with TM4-derived luciferase reporter phages demonstrated that the majority of these isolates were susceptible to TM4. Conditions for introduction of DNA were determined by transfection of M. paratuberculosis with TM4 DNA and applied to isolate kanamycin-resistant transformants of M. avium complex and M. paratuberculosis with Escherichia coli-Mycobacterium shuttle plasmids. Recombinant plasmids were recovered from transformants without apparent loss of DNA sequences. These results provide the basis for the genetic manipulation of these pathogenic mycobacterial species.
Insights
Researchers developed methods for genetic manipulation of Mycobacterium avium complex and Mycobacterium paratuberculosis. These advancements enable new strategies for understanding disease, creating vaccines, and developing treatments for these important pathogens.
Area of Science:
- Microbiology
- Genetics
- Pathogen Research
Background:
- Mycobacterium avium complex (MAC) and Mycobacterium paratuberculosis are related intracellular pathogens.
- MAC infections cause significant illness and death in AIDS patients.
- M. paratuberculosis causes Johne's disease in ruminants.
Purpose of the Study:
- To establish methods for genetic manipulation of MAC and M. paratuberculosis.
- To facilitate research into pathogenesis, vaccine development, and novel therapeutics.
Main Methods:
- Replication of mycobacterial shuttle phasmids and plasmids.
- Expression of firefly luciferase reporter gene in MAC and M. paratuberculosis.
- Transfection of M. paratuberculosis with mycobacteriophage TM4 DNA.
- Isolation of kanamycin-resistant transformants using E. coli-Mycobacterium shuttle plasmids.
Main Results:
- Mycobacteriophage TM4 showed efficient plaque formation on both M. avium complex and M. paratuberculosis.
- Most clinical isolates of MAC and M. paratuberculosis were susceptible to TM4.
- Successful introduction of DNA and recovery of recombinant plasmids from transformants without sequence loss.
Conclusions:
- Developed foundational techniques for genetic manipulation of pathogenic mycobacteria.
- Opened avenues for further research on MAC and M. paratuberculosis pathogenesis and control.
- Provided tools for potential development of attenuated vaccines and new treatments.