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Infection and Immunity, June 2002, p. 2787-2795, Vol. 70, No. 6
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.6.2787-2795.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Mycobacterium tuberculosis Genes Induced during Infection of Human Macrophages{dagger}

Eugenie Dubnau,1 Patricia Fontán,1 Riccardo Manganelli,1,2 Sonia Soares-Appel,1 and Issar Smith1*

TB Center, The Public Health Research Institute, Newark, New Jersey 07103,1 Department of Histology and Microbiology and Medical Biotechnologies, Medical School, University of Padua, 63 35121 Padua, Italy2

Received 27 September 2001/ Returned for modification 23 November 2001/ Accepted 21 February 2002

We identified Mycobacterium tuberculosis genes preferentially expressed during infection of human macrophages using a promoter trap adapted for this pathogen. inhA encodes an enoyl-acyl carrier protein reductase that is required for mycolic acid biosynthesis (A. Quemard et al., Biochemistry 34:8235-8241, 1995) and is a major target for isoniazid (INH) in mycobacterial species (A. Banerjee et al., Science 263:227-230, 1994). Since overexpression of inhA confers INH resistance in Mycobacterium smegmatis (Banerjee et al., Science 263:227-230, 1994), we designed a promoter trap based on this gene. A library of clones, containing small fragments of M. tuberculosis DNA cloned upstream of inhA in a plasmid vector, was electroporated into M. tuberculosis, and the resulting culture was used to infect the human monocytic THP-1 cell line. Selection was made for clones surviving INH treatment during infection but retaining INH sensitivity on plates. The DNA upstream of inhA was sequenced in each clone to identify the promoter driving inhA expression. Thirteen genes identified by this method were analyzed by quantitative reverse transcription-PCR (R. Manganelli et al., Mol. Microbiol. 31:715-724, 1999), and eight of them were found to be differentially expressed from cultures grown in macrophages compared with broth-grown cultures. Several of these genes are presumed to be involved in fatty acid metabolism; one potentially codes for a unique DNA binding protein, one codes for a possible potassium channel protein, and the others code for proteins of unknown function. Genes which are induced during infection are likely to be significant for survival and growth of the pathogen; our results lend support to the view that fatty acid metabolism is essential for the virulence of M. tuberculosis.

* Corresponding author. Mailing address: Public Health Research Institute, 225 Warren Street, Newark, NJ 07103. Phone: (973) 972-9150. Fax: (973) 972-9150. E-mail: smitty{at}phri.org.

Editor: S. H. E. Kaufmann

{dagger} Publication no. 71 from the TB Center.

Infection and Immunity, June 2002, p. 2787-2795, Vol. 70, No. 6
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.6.2787-2795.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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