Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization

Abstract

Tuberculosis is a chronic infectious disease that is transmitted by cough-propelled droplets that carry the etiologic bacterium, Mycobacterium tuberculosis. Although currently available drugs kill most isolates of M. tuberculosis, strains resistant to each of these have emerged, and multiply resistant strains are increasingly widespread. The growing problem of drug resistance combined with a global incidence of seven million new cases per year underscore the urgent need for new antituberculosis therapies. The recent publication of the complete sequence of the M. tuberculosis genome has made possible, for the first time, a comprehensive genomic approach to the biology of this organism and to the drug discovery process. We used a DNA microarray containing 97% of the ORFs predicted from this sequence to monitor changes in M. tuberculosis gene expression in response to the antituberculous drug isoniazid. Here we show that isoniazid induced several genes that encode proteins physiologically relevant to the drug's mode of action, including an operonic cluster of five genes encoding type II fatty acid synthase enzymes and fbpC, which encodes trehalose dimycolyl transferase. Other genes, not apparently within directly affected biosynthetic pathways, also were induced. These genes, efpA, fadE23, fadE24, and ahpC, likely mediate processes that are linked to the toxic consequences of the drug. Insights gained from this approach may define new drug targets and suggest new methods for identifying compounds that inhibit those targets.

Figure 1

INH-induced mRNA expression profiles monitored by microarray hybridization analysis. (a) A subarray containing spots corresponding to 203 different M. tuberculosis ORFs illustrates the INH-induced gene response profile. Included were genes thought to encode components of the mycolic acid pathway. The expression response to a 4-hr treatment with INH is shown as a pseudocolored composite image. The two channels were pseudocolored according to the fluorescence intensity, either red (INH treated) or green (INH untreated), and overlaid to give the images shown. Yellow shades are derived from the combination of red and green, indicating relatively equivalent expression levels. Predominantly red spots at positions B13–16 correspond to genes of the FAS-II gene cluster (Rv2244–7). The spots at coordinates (P2–3) are positive controls of DNA prelabeled with Cy3 (P2) or Cy5 (P3). Reference spots contained M. tuberculosis whole-genomic DNA (A1, P1, and P16) and ribosomal DNA (A4–10). (b) INH treatment of an INH-resistant, ethionamide-sensitive strain (4309A) revealed little change in relative mRNA levels. (c) Treatment of the same strain with ethionamide produced a response pattern that was similar to the pattern produced by treatment of the INH-sensitive strain with INH (a).

Figure 2

Temporal profile of INH-induced expression of selected genes. (a) Microarray-based determinations of selected gene responses resulting from 20- min, 40-min, 1-hr, 4-hr, and 8-hr treatments with INH. The results are depicted as ratios of the fluorescence intensities of labeled cDNAs from bacteria exposed to INH for the indicated times compared with the zero time point. Ratio values for inhA (■), fas (♦), kasA (○), fadE24 (▵), ahpC (+), and efpA (□) derived from microarray hybridization experiments are plotted with respect to the duration of INH exposure. (b) The change in abundance of selected transcripts in total RNA was estimated by RT-PCR samples prepared from organisms exposed to INH for 0, 1, 4, and 8 hr. Primers were designed to amplify internal regions of kasA (318 bp), efpA (521 bp), and ahpC (484 bp) and the internal reference genes inhA (432 bp) and fas (306 bp). RNA samples isolated from bacteria treated with INH for the indicated times (lanes 1–8) were reverse-transcribed and PCR-amplified with primers specific to kasA and inhA (Top), efpA and fas (Middle), and ahpC and fas (Bottom). The pairwise comparison of two genes for each experiment, using amplified fragments of similar size, allowed direct comparison of the corresponding transcripts. To assess RNA-specific amplification, a duplicate RNA sample for each time point was digested with RNase (lanes 1, 3, 5, and 7). Positive controls for the PCR step contained M. tuberculosis genomic DNA as template and were either treated (lane 11) or untreated (lane 12) with RNase. A PCR negative control reaction (lane 9), containing the appropriate primer pairs but no added template, was performed with each experiment.

Figure 3

Roles of INH-induced genes in the context of a proposed pathway for mycolic acid biosynthesis. (a) Mycolic acids of M. tuberculosis are elongated by the multienzyme, AcpM-dependent FAS-II complex (7, 23, 24). By analogy with the Escherichia coli FAS-II system (25), the enzymatic steps required for the addition of a two-carbon subunit, provided by malonyl-AcpM (highlighted in bold), are shown together with some of the proteins that contribute to the process. Activated INH (INH*) is thought to inhibit FAS-II by binding to NADH in the pocket of InhA and by forming a covalent, ternary complex between KasA and AcpM (6, 7). The operon-like cluster of genes encoding AccD6, FabD, KasA, KasB, and AcpM (red) all were induced by INH (Table 1), perhaps as a consequence of the depleted meromycolate pool. In contrast, neither fabG nor inhA, which are organized into a two-gene cluster at a separate position on the chromosome, was induced by INH (Fig. 2). (b) A proposed mycolate synthesis pathway shows the predicted roles of FAS-II, described above, and FAS-I, which elongates short-chain fatty-acyl-CoA esters (23, 26). The precise fatty acyl intermediates and ACP-charging enzymes that mediate the FAS-I to FAS-II transition have not been characterized. Meromycolate chains can be functionalized by several different moieties (27). For clarity, however, only the cyclopropyl-containing meromycolate and the enzymes that introduce the distal (Cma1) and proximal (Cma2) moieties are shown (28). The mycolyl transferase (FbpC) is one of several exported proteins predicted to catalyze the transesterification of mycolates to carbohydrate moieties in the cell wall (18). Microarray hybridization experiments showed that fbpC (red) also was induced by INH treatment.