Exploring Drug-Induced Alterations in Gene Expression in Mycobacterium Tuberculosis by Microarray Hybridization
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Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization Michael Wilson*†, Joseph DeRisi‡§, Hans-Henrik Kristensen¶ʈ, Paul Imboden¶**, Sangeeta Rane¶, Patrick O. Brown‡, and Gary K. Schoolnik*¶ *Department of Microbiology and Immunology, ‡Department of Biochemistry and the Howard Hughes Medical Institute, and ¶Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University, Stanford, CA 94305 Communicated by Barry R. Bloom, Harvard School of Public Health, Boston, MA, August 23, 1999 (received for review March 31, 1999) Tuberculosis is a chronic infectious disease that is transmitted by during its exposure to isoniazid (INH), a drug that blocks the cough-propelled droplets that carry the etiologic bacterium, My- mycolic acid biosynthetic pathway. cobacterium tuberculosis. Although currently available drugs kill We chose first to study the transcriptional response to INH most isolates of M. tuberculosis, strains resistant to each of these because it is given to more tuberculosis patients than any other have emerged, and multiply resistant strains are increasingly drug, and because among the arsenal of antituberculosis drugs, widespread. The growing problem of drug resistance combined INH is the agent to which resistance emerges most frequently (2, with a global incidence of seven million new cases per year 3). Additionally, our understanding of the intracellular conse- underscore the urgent need for new antituberculosis therapies. quences of INH activity on the biosynthesis of cell-wall lipids and The recent publication of the complete sequence of the M. tuber- the genes responsible for lipid metabolism has improved sub- culosis genome has made possible, for the first time, a compre- stantially in recent years. Therefore, a model system based on the hensive genomic approach to the biology of this organism and to effects of INH provides a conceptual framework for interpreting the drug discovery process. We used a DNA microarray containing the transcriptional responses that we would detect by the mi- 97% of the ORFs predicted from this sequence to monitor changes croarray method and allows us to compare these results with in M. tuberculosis gene expression in response to the antituber- published observations of genes and proteins that are known to culous drug isoniazid. Here we show that isoniazid induced several be INH-induced. In turn, because INH is lethal to sensitive genes that encode proteins physiologically relevant to the drug’s strains, the identification of enzymes that heretofore were not mode of action, including an operonic cluster of five genes encod- known to be within INH-inhibited pathways could lead to the ing type II fatty acid synthase enzymes and fbpC, which encodes selection of additional drug targets. The elucidation of the role trehalose dimycolyl transferase. Other genes, not apparently of these enzymes in the production of mycolic acids, as discussed within directly affected biosynthetic pathways, also were induced. below, also could lead to a deeper understanding of the biosyn- These genes, efpA, fadE23, fadE24, and ahpC, likely mediate thetic and regulatory processes involved in the production of this processes that are linked to the toxic consequences of the drug. characteristic class of lipids. Insights gained from this approach may define new drug targets INH selectively interrupts the synthesis of mycolic acids, the and suggest new methods for identifying compounds that inhibit major component of the waxy, outer lipid envelope of myco-  those targets. bacteria (4). Mycolic acids are branched, -hydroxy fatty acids composed of an intermediate-length (C24-C26), saturated alpha chain, and a longer (ϾC50) meromycolate chain that contains he transcription of genes encoding components of a mul- characteristic functional moieties. A convergence of elegant Ttienzyme pathway often is regulated in response to the genetic and biochemical evidence has shown that INH blocks a MICROBIOLOGY availability of cofactors, precursors, intermediates, or products type II fatty acid synthase (FAS-II) complex that is required for of the same pathway. Compounds, including drugs, that selec- full-length extension of the meromycolate chain (5–7). Although tively inhibit a pathway enzyme and thereby cause an accumu- the precise mechanism of INH-mediated killing remains unre- lation of precursors and a depletion of products can be expected solved, INH is thought to target three FAS-II complex proteins to selectively induce changes in the transcription of genes coding by binding to NADH in the pocket of the enoyl-acyl carrier for enzymes that comprise the affected pathway, especially protein (ACP) reductase, InhA, and by forming a covalent, before a more generalized stress response ensues. The resulting ternary complex between the -ketoacyl-ACP synthase, KasA, gene expression profile would not only serve as a kind of and an acyl carrier protein, AcpM (6, 7). As a consequence of signature of the inhibitor used, but would, in the case of INH activity, mature mycolates are not produced and become inhibitors whose modes of action are unknown, incriminate the progressively depleted (4). At the same time, and in accordance affected pathway and perhaps the specific targeted enzyme with the pathway inhibition notion of INH action, an intracel- within the pathway. The realization of this idea requires that the expression of each gene in the genome be interrogated simul- taneously in the presence and absence of inhibitor. The increas- Abbreviations: INH, isoniazid; FAS, fatty acid synthase; RT, reverse transcription. ing availability of complete genome sequences provides one of †To whom reprint requests should be sent at present address: Affymax Research Institute, the two preconditions for this approach, and the development of 3410 Central Expressway, Santa Clara, CA 94501. E-mail: mike[email protected]. microarrays containing representatives of each of the genes §Present address: Department of Biochemistry and Biophysics, University of California, 513 provides the other. In the study described below, we have tested Parnassus Avenue, Box 0448, San Francisco, CA 94116. ʈ this method for pathway characterization. For this purpose, we Present address: Novo Nordisk A͞S, Enzyme Design, Novo Alle´, DK-2880 Bagsvaerd, Den- mark. fabricated a microarray containing DNA fragments correspond- **Present address: Department of Clinical Research, University of Berne, Tiefenaustr. 120, ing to nearly every predicted ORF of the recently completed and 3004 Berne, Switzerland. annotated Mycobacterium tuberculosis genomic sequence of The publication costs of this article were defrayed in part by page charge payment. This strain H37Rv (1). Here we describe the use of this array to article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. generate the transcriptional response profile of M. tuberculosis §1734 solely to indicate this fact. PNAS ͉ October 26, 1999 ͉ vol. 96 ͉ no. 22 ͉ 12833–12838 Downloaded by guest on September 28, 2021 Table 1. Genes induced by INH or ethionamide treatment of an INH-sensitive strain Fluorescent ratio† Classification ORF* Gene Proposed function INH ETH Ref.* FAS-II Rv2243 fabD Malonyl CoA-malony-ACP transacylase 2.8 3.1 Rv2244 acpM Acyl carrier protein 3.8 3.7 7 Rv2245 kasA -ketoacyl synthase 2.8 3.4 7 Rv2246 kasB -ketoacyl synthase 2.4 2.7 Rv2247 accD6 Acetyl-CoA carboxylase ( chain) 2.5 2.0 Mycolate transfer Rv0129c fbpC Trehalose dimycolyl transferase 2.1 2.2 19 Fatty acid -oxidation Rv3139 fadE24 Fatty-acyl-CoA dehydrogenase 2.3 2.4 Rv3140 fadE23 Fatty-acyl-CoA dehydrogenase 2.6 2.5 Detoxification Rv2428 ahpC Alkyl hydroperoxide reductase (subunit) 2.9 2.5 Transport Rv2846c efpA Efflux protein 2.5 2.1 Unknown Rv1592c 3.0 2.4 Rv1772 2.0 2.1 Rv0341 2.0 2.5 22 Rv0342 2.6 2.1 22 ETH, ethionamide. *ORF nomenclature is based on ref. 1. Citations for individual genes refer to reports of INH-induced expression of specific genes or their protein products. †Average fluorescent ratio (drug-treated͞untreated) resulting from the hybridization experiments of 2- to 6-hr drug exposures. lular pool of saturated fatty acids (C24-C26) accumulates that DNA fragment stocks. The most complete version contained presumably reflects the point at which meromycolate synthesis validated fragments representing 3,834 (97%) of the predicted is interrupted by INH (8, 9). The accumulation of these fatty acid 3,924 ORFs. The results of this array series are presented in pathway precursors is associated with the increased production Table 1, and the two less complex microarrays confirmed the of AcpM and KasA (7) and implies that the induction of their results of the larger version. The results presented in Fig. 1 are genes is the consequence of a regulatory feedback mechanism from a mini-array containing 203 different ORFs that were that senses the imbalance of mycolic acid biosynthetic interme- selected to contain a high proportion (29%) of genes encoding diates, which accumulate, and full-length mycolates, which are lipogenic or lipolytic enzymes. depleted. We reasoned that the INH-induced increase in KasA and AcpM, if mediated at the transcriptional level, should be Growth and Drug Treatment of M. tuberculosis Strains. The INHs, reflected by parallel changes in the abundance of the corre- ethionamides M. tuberculosis strain 1254 is a recent clinical sponding transcripts. Further, we predicted that INH also would isolate from San