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Infection and Immunity, July 2008, p. 3100-3115, Vol. 76, No. 7
0019-9567/08/$08.00+0     doi:10.1128/IAI.00015-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Use of Gene Dosage Effects for a Whole-Genome Screen To Identify Mycobacterium marinum Macrophage Infection Loci{triangledown}

Bonggoo Park,{ddagger} Selvakumar Subbian, Sahar H. El-Etr,{dagger} Suat L. G. Cirillo, and Jeffrey D. Cirillo*

Department of Microbial and Molecular Pathogenesis, Texas A&M University System Health Sciences Center, 467 Reynolds Medical Building, College Station, Texas 77843-1114

Received 13 December 2007/ Returned for modification 17 January 2008/ Accepted 17 April 2008

We recently identified two loci, mel1 and mel2, that affect macrophage infection by Mycobacterium marinum. The ability of these loci to confer enhanced infection in trans is presumably due to gene dosage effects since their presence on plasmids increases expression from five- to eightfold. Reasoning that this phenomenon would allow identification of other mycobacterial genes involved in macrophage infection, we conducted a screen of an M. marinum DNA library that provides 2.6-fold coverage of the entire genome for clones that affect macrophage infection. Our preliminary screen identified 76 plasmids that carry loci affecting macrophage infection. We eliminated plasmids that do not confer the expected phenotype when retransformed (70%), that have identical physical maps (5%), or that carry either of the mel1 or mel2 loci (14%) from further consideration. Four loci that confer enhanced infection (mel) and four that confer repressed infection (mrl) of macrophages were identified, and two of each group were chosen for detailed analysis. Saturating transposon mutagenesis was used to identify the loci responsible, and M. marinum mutants were constructed in the genes involved. We expect these genes to provide insight into how mycobacteria parasitize macrophages, an important component of innate immunity.

* Corresponding author. Mailing address: Department of Microbial and Molecular Pathogenesis, Texas A&M University System Health Sciences Center, 467 Reynolds Medical Building, College Station, TX 77843-1114. Phone: (979) 458-0778. Fax: (979) 845-3479. E-mail: jdcirillo{at}medicine.tamhsc.edu

{triangledown} Published ahead of print on 28 April 2008.

Editor: A. Camilli

{ddagger} Present address: Lawrence Livermore National Lab, Biosciences and Biotechnology Division, Livermore, CA 94550.

{dagger} Present address: Cedars-Sinai Medical Center, Immunology Research Institute, Los Angeles, CA 90048.

Infection and Immunity, July 2008, p. 3100-3115, Vol. 76, No. 7
0019-9567/08/$08.00+0     doi:10.1128/IAI.00015-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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