Impending doom: antibiotic exposure and bacterial gene expression.

Abstract

The discovery of antibiotics ranks as one of the most significant medical achievements of the past century: Antibiotics have greatly attenuated the severity and lethality of bacterial infections. Because antibiotics have been so widely successful, they have been extensively used. This has resulted in strong selective pressure on bacteria that, in turn, has led to the rapid and widespread evolution of antibiotic resistance. Antibiotic resistance now poses a serious threat to the therapy of certain bacterial infections, and new approaches are needed for the development of the next generation of antibiotics. Functional genomics technologies to examine RNA and protein expression levels on a genomewide scale may prove to be important tools for drug discovery research. In this issue Gmuender et al. (2000) use a functional genomics approach to examine the changes in gene expression that occur in the human respiratory pathogen Haemophilus influenzae upon exposure to the antibiotics ciprofloxacin and novobiocin. Although both of these drugs inhibit bacterial DNA gyrase, they do so by different mechanisms. Novobiocin is a coumarin antibiotic that binds to the ATP binding site of the B subunit of gyrase and inhibits the supercoiling activity of the enzyme. Because transcription is known to be sensitive to the state of supercoiling, novobiocin may affect the transcription of many genes. In contrast, ciprofloxacin is a quinolone that binds to the A subunit of gyrase and interrupts the DNA cleavage and resealing activity of the enzyme. The failure to seal doublestrand breaks results in DNA damage and quinolones are known to induce DNA repair systems (Piddock et al. 1990). Thus, the two drugs may elicit different physiological responses despite binding to the same target. A distinguishing feature of this work is that Gmuender et al. (2000) examine gene expression at the level of transcription with microarrays and translation using two-dimensional polyacrylamide gel electrophoresis. The use of parallel cultures for the transcription and translation studies permits a direct comparison between differential RNA synthesis versus protein synthesis. The authors find that absolute levels of RNA and the corresponding protein exhibit a correlation coefficient of only 0.5. However, there is a correlation between the sign of the change of RNA and protein (i.e., if an RNA exhibits increased expression, the corresponding protein exhibits increased expression). Therefore, the observed levels of RNA and protein are qualitatively similar but the actual magnitude of the changes are significantly different. The clear conclusion from these results is that the microarray and 2D protein gel data cannot be interpreted quantitatively. Furthermore, extensive control experiments indicate that poor reproducibility of the 2D protein gels compared to microarray hybridization is a major limiting factor for comparing transcription and translation results. The effect of novobiocin and ciprofloxacin on H. influenzae gene expression was assessed at the minimum concentration of antibiotic known to inhibit bacterial growth (MIC) as well as at a tenfold higher concentration. Treatment with low concentrations of novobiocin resulted in only a few changes in gene expression, including increased expression of DNA gyrase subunit B and decreased expression of topoisomerase I, ribosome releasing factor as well as two hypothetical proteins. The changes in expression of DNA gyrase and topoisomerase I can be rationalized as a cellular response to the change in supercoiling due to inhibition of gyrase. It will be of interest to determine the function of the hypothetical proteins. Their grouping with gyrase and topoisomerase I suggests these hypothetical proteins may be involved in nucleic acid metabolism or DNA repair. If so, they may represent new targets for antibiotics that disrupt nucleic acid metabolism. At high concentrations of novobiocin, ∼140 genes exhibit significant changes in expression levels based on the microarray data. It is not obvious why, at the high concentration, such a large number of genes exhibit changes in expression. Perhaps the changes are secondary transcriptional effects due to the global changes in supercoiling. At both high and low concentrations, the length of time of exposure to novobiocin did not substantially affect the expression profile in that expression changes occurred at the earliest time points and remained relatively constant. Exposure of H. influenzae to a low concentration of ciprofloxacin also results in expression changes of only a small number of genes. However, these genes are dominated by DNA repair proteins involved in the SOS response such as the recA, uvrA and lexA genes. This result is not surprising in that the drug causes DNA damage. Also, similar to the case with novobiocin, the expression levels of a large number of genes were altered at high ciprofloxacin concentrations, which could be secondary effects E-MAIL timothyp@bcm.tmc.edu; FAX (713) 7987375. Article and publication are at www.genome.org/cgi/ doi/10.1101/gr.170901. Insight/Outlook