Identifying mycobacterium tuberculosis virulence determinants - new technologies for a difficult problem: response

DesJardin and Schlesinger correctly acknowledge the potential, and the possible pitfalls, of new technologies for the identification of Mycobacterium tuberculosis virulence factors, and we appreciate the positive statements in the commentary. However, we would like to comment specifically on aspects of the PCR-based strategies discussed, as methods based on nucleic acid amplification will be crucial to the study of mycobacterial virulence in human disease, firstly using cultured primary human macrophages and, subsequently, by analysis of infected animal and human tissues.The method described by Plum and Clark-Curtiss1xInduction of Mycobacterium avium gene expression following phagocytosis by human macrophages. Plum, G. and Clark-Curtiss, J.E. Infect. Immun. 1994; 62: 476–483PubMedSee all References is a subtractive hybridization approach, in which complementary cDNAs prepared from Mycobacterium avium grown in human macrophages and broth cultures are hybridized and removed, leaving behind cDNAs unique to phagocytosed bacilli. We described an alternative approach2xIdentification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS). Graham, J.E. and Clark-Curtiss, J.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11554–11559Crossref | PubMed | Scopus (342)See all References, selective capture of transcribed sequences (SCOTS), in which bacterial cDNAs unique to M. tuberculosis growing in human macrophages are selected by differential hybridization to bacterial genomic DNA that has been pre-hybridized (blocked) with both total cDNA from broth-grown bacilli and rDNA fragments.The instability of RNA presents a serious challenge. With SCOTS, we attempted to obtain reproducible RNA expression patterns by directly isolating bacterial RNA from infected macrophages, thereby avoiding changes associated with separating host cells and microorganisms. To reduce the biases inherent in amplifying complex cDNA mixtures, SCOTS uses random priming of both cDNA strands to generate a library of short fragments (200–400 bp) that have been shown to be amplified without significant bias within a limited number of cycles3xUnbiased amplification of a highly complex mixture of DNA fragments by ‘lone linker’-tagged PCR. Ko, M.S. et al. Nucleic Acids Res. 1990; 18: 4293–4294Crossref | PubMed | Scopus (44)See all References, 4xPCR amplification of megabase DNA with tagged random primers (T-PCR). Grothues, D. et al. Nucleic Acids Res. 1993; 21: 1321–1322Crossref | PubMed | Scopus (63)See all References. This also provides a variety of different sequence contexts in which any particular sequence can be amplified, providing redundancy in representation of the original RNAs. Additionally, we used between three and ten parallel PCR reactions for each amplification. We acknowledge that the composition of the RNA pool might have an effect on reverse transcription, but consider it unlikely that increasing the level of potential target mRNA orders of magnitude (by PCR) while decreasing the complexity of the pool to a similar extent (via several rounds of SCOTS) would lead to the inability to obtain a specific cDNA. Nevertheless, we agree that repeated sampling of RNA and further analysis of additional cDNAs is important. So far, in an analysis of shorter duration macrophage infections (J.E. Graham and J.E. Clark-Curtiss, unpublished), we have obtained comparable complex hybridization patterns and cDNAs for previously described M. tuberculosis genes2xIdentification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS). Graham, J.E. and Clark-Curtiss, J.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11554–11559Crossref | PubMed | Scopus (342)See all References.The same obstacles to determining the role of differentially expressed genes exist for other, more resource-intensive, genome-wide analysis methods, including microarray hybridization. It seems likely to us, given the variation in abundance of individual microbial RNAs within an ‘ocean’ of rRNA, that amplification methods which also equalize representation of mRNAs as cDNA, a fundamental aspect of SCOTS, will be necessary before microarray methods can be fully applied to the analysis of in vivo prokaryotic gene expression. Having identified, for the first time, M. tuberculosis genes expressed in response to interaction with human cells, we also look forward to the application of other recently described techniques to this difficult problem, and hope that the timely release of our initial findings will facilitate further analyses and selection of candidate targets for new treatment strategies.