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Characterization of genes differentially expressed within macrophages by virulent and attenuated Mycobacterium tuberculosis identifies candidate genes involved in intracellular growth

¹ Department of Pathology and Laboratory Medicine, University of British Columbia, 950 West 28th Avenue, Vancouver, BC V5Z 4H4, Canada
² Department of Pathology and Laboratory Medicine, University of British Columbia, Department of Cancer Genetics, BC Cancer Research Centre, 601 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada
³ Departments of Paediatrics and Pathology and Laboratory Medicine, University of British Columbia, Division of Infectious and Immunological Diseases, BC Children's Hospital, 950 West 28th Avenue, Vancouver, BC V5Z 4H4, Canada

Correspondence
Richard W. Stokes
rstokes{at}interchange.ubc.ca

To identify genes involved in the intracellular survival of Mycobacterium tuberculosis we compared the transcriptomes of virulent (H37Rv) and attenuated (H37Ra) strains during their interaction with murine bone-marrow-derived macrophages. Expression profiling was accomplished via the bacterial artificial chromosome fingerprint array (BACFA) technique. Genes identified with BACFA, and confirmed via qPCR to be upregulated in the attenuated H37Ra at 168 h post-infection, were frdB, frdC and frdD. Genes upregulated in the virulent H37Rv were pks2, aceE and Rv1571. Further qPCR analysis of these genes at 4 and 96 h post-infection revealed that the frd operon (encoding the fumarate reductase enzyme complex) is expressed at higher levels in the virulent H37Rv at earlier time points while the expression of aceE and pks2 is higher in the virulent strain throughout the course of infection. Assessment of frd transcripts in oxygen-limited cultures of M. tuberculosis H37Ra and H37Rv showed that the attenuated strain displayed a lag in frdA and frdB expression at the onset of microaerophilic culture, when compared to microaerophilic cultures of H37Rv and aerated cultures of H37Ra. Lastly, treatment of intracellular bacteria with a putative inhibitor of fumarate reductase resulted in a significant reduction of bacterial growth.