Antimicrob Agents Chemother 47(3): 948-955

Proteomic Approach to Understanding Antibiotic Action

Institut für Mikrobiologie, Ernst-Moritz-Arndt-Universität, 17489 Greifswald,^{Bayer AG, Pharmaforschungszentrum, 42096 Wuppertal, Germany}²

^{Corresponding author. Mailing address: Bayer AG, Pharmaforschungszentrum, Postfach 10 17 09, 42096 Wuppertal, Germany. Phone: 49 (202) 368376. Fax: 49 (202) 364116. E-mail:}ed.ga-reyab@lh.iksnihcsibal.dlarah.

^{Present address: Pfizer Inc., Ann Arbor, MI 48105.}

Received 2002 Jul 23; Revised 2002 Oct 25; Accepted 2002 Nov 21.

Abstract

We have used proteomic technology to elucidate the complex cellular responses of Bacillus subtilis to antimicrobial compounds belonging to classical and emerging antibiotic classes. We established on two-dimensional gels a comprehensive database of cytoplasmic proteins with pIs covering a range of 4 to 7 that were synthesized during treatment with antibiotics or agents known to cause generalized cell damage. Although each antibiotic showed an individual protein expression profile, overlaps in the expression of marker proteins reflected similarities in molecular drug mechanisms, suggesting that novel compounds with unknown mechanisms of action may be classified. Indeed, one such substance, a structurally novel protein synthesis inhibitor (BAY 50-2369), could be classified as a peptidyltransferase inhibitor. These results suggest that this technique gives new insights into the bacterial response toward classical antibiotics and hints at modes of action of novel compounds. Such a method should prove useful in the process of antibiotic drug discovery.

Abstract

Infectious diseases are the leading cause of death worldwide, and rapid resistance development is a growing threat even in developed countries (33). In addition to improved hygiene and prudent use of existing antibiotics, the development of novel antibacterial classes is a key to keeping pace with the remarkable adaptability of the bacteria. The search for such novel substances involves understanding of the molecular mechanism of action of the inhibitor and the related bacterial response. Today two major strategies are used in antibiotic drug discovery: (i) evaluation of the structural variations among existing antibiotic classes in order to find compounds which hit the same targets by similar molecular mechanisms yet avoid cross-resistance and (ii) evaluation of novel antibiotic substances arising either from high-throughput target-based assays or from screening for antibacterial activity. If, through its antibacterial activity alone, a novel compound class arouses interest, its molecular target needs to be identified so that undesirable side effects on eukaryotic cells can be minimized (target identification). In addition, for structurally modified antibiotics or compounds derived from the target-based assays it is necessary to prove that interaction with the cellular target is indeed the direct cause for bacterial cell death (target validation).

In this study we used the proteomic approach to study the responses of bacteria to antibacterial compounds. We show that proteome analysis is useful for both target identification and target validation. Proteomics facilitates a broad view on the cell's physiological state and, in combination with radioactive pulse-labeling, allows us to study the cellular response to any changes in growth conditions (32). Recently, proteome analysis has developed to a state which permits investigation of large numbers of different samples. Thus, we started to build a database from two-dimensional (2D) protein analysis of bacterial responses to antibiotic treatment considering all important established and emerging antibiotic classes as well as some substances causing generalized cell damage. We chose the gram-positive model organism Bacillus subtilis in order to profit from its fully sequenced genome (23) and earlier proteome studies focusing on the description of protein signatures of environmental stimuli (2, 5, 7) which are accessible in the Sub2D database (J. Bernhardt and H. Werner, http://microbio2.biologie.uni-greifswald.de:8880/sub2d.htm).

Here we present data for 30 antimicrobial compounds, most of which have been well characterized in terms of their mechanisms of action. By comparison with known antibiotics, we were able to predict the mode of action of the structurally new antibacterial BAY 50-2369. Another example is nitrofurantoin, which has been used for decades in the treatment of urinary tract infections, although its precise mode of action is still unclear (15). Nitrofurantoin led to a protein pattern very similar to that of diamide, suggesting that nitrofurantoin causes nonnative disulfide bonds in the bacterial cell.

Our results show that, by mirroring the complex molecular reactions of bacteria, proteomics may enlarge our view of known antibiotics and help us find new drugs.

Acknowledgments

We thank Karin Binder for excellent support with 2D-PAGE and Doreen Kliewe, Thomas Kretschmann, Anita John, Dorian Schönfeld, Katy Raddatz, and Falko Hochgräfe for assisting in the identification of marker proteins. We thank Ben Newton for carefully reading and improving the manuscript.

This work was supported by a grant from Bayer AG, Wuppertal, Germany.

Acknowledgments

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