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Identification of Mycobacterium marinum macrophage infection mutants
Parmod K Mehta1, Amit K Pandey, Selvakumar Subbian
1Department of Microbial and Molecular Pathogenesis, Texas A&M University Health Sciences Center, 471 Reynolds Medical Building, College Station, TX 77843, USA.
Abstract:
Mycobacterium marinum is an important pathogen of humans, amphibians and fish. Most pathogenic mycobacteria, including M. marinum, infect, survive and replicate primarily intracellularly within macrophages. We constructed a transposon mutant library in M. marinum using Tn5367 delivered by phage transduction in the shuttle phasmid phAE94. We screened 529 clones from the transposon library directly in macrophage infection assays. All clones were screened for their ability to initially infect macrophages as well as survive and replicate intracellularly. We identified 19 mutants that fit within three classes: class I) defective for growth in association with macrophages (42%), class II) defective for macrophage infection (21%) and class III) defective for infection of and growth in association with macrophages (37%). Although 14 of the macrophage infection mutants (Mim) carry insertions in genes that have not been previously identified, five are associated with virulence of mycobacteria in animal models. These observations confirm the utility of mutant screens directly in association with macrophages to identify new virulence determinants in mycobacteria. We complemented four of the Mim mutants with their M. tuberculosis homologue, demonstrating that secondary mutations are not responsible for the observed defect in macrophage infection. The genes we identified provide insight into the molecular mechanisms of macrophage infection by M. marinum.
Insights
Researchers identified new virulence factors in Mycobacterium marinum by screening mutants directly in macrophage infection assays. This approach revealed key genes essential for bacterial survival and replication within host cells.
Area of Science:
- Microbiology
- Pathogen Research
- Immunology
Background:
- Mycobacterium marinum is a significant pathogen affecting humans, fish, and amphibians.
- Pathogenic mycobacteria, including M. marinum, primarily infect and replicate within macrophages.
- Understanding M. marinum's interaction with macrophages is crucial for developing effective treatments.
Purpose of the Study:
- To identify novel virulence determinants of Mycobacterium marinum.
- To investigate the molecular mechanisms underlying M. marinum's intracellular survival and replication in macrophages.
- To validate the use of direct macrophage infection screens for discovering mycobacterial virulence factors.
Main Methods:
- Construction of a transposon mutant library in M. marinum using Tn5367.
- Screening of 529 mutants for defects in macrophage infection, survival, and intracellular growth.
- Complementation of selected mutants with homologous genes from Mycobacterium tuberculosis.
Main Results:
- Identification of 19 mutants with defects in macrophage interaction, categorized into three classes.
- Class I: defective growth associated with macrophages (42%).
- Class II: defective macrophage infection (21%).
- Class III: defective infection and growth (37%).
- 14 novel macrophage infection mutants (Mim) and 5 previously known virulence-associated genes were identified.
- Complementation confirmed that observed defects were due to specific mutations.
Conclusions:
- Direct screening of M. marinum mutants in macrophage infection assays is an effective strategy for identifying new virulence factors.
- The identified genes provide novel insights into the molecular basis of mycobacterial pathogenesis within macrophages.
- This study enhances our understanding of host-pathogen interactions and potential therapeutic targets for mycobacterial infections.
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