Antifolate Activity of Epigallocatechin Gallate against <em>Stenotrophomonas maltophilia</em>

María Navarro-Martínez

Enma Navarro-Perán

Juan Cabezas-Herrera

Joaquín Ruiz-Gómez

Francisco García-Cánovas

José Rodríguez-López

Grupo de Investigación de Enzimología, Departamento de Bioquímica y Biología Molecular A, Facultad de Biología, Universidad de Murcia, E-30100 Espinardo,^{Servicio de Análisis Clínicos,}^{Servicio de Microbiología, Hospital Universitario Virgen de la Arrixaca, Murcia, Spain}³

^{Corresponding author. Mailing address: Grupo de Investigación de Enzimología, Departamento de Bioquímica y Biología Molecular A, Facultad de Biología, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain. Phone: 34-968-398284. Fax: 34-968-364147. E-mail:}se.mu@onutpen.

Received 2004 Oct 7; Revised 2004 Dec 7; Accepted 2005 Mar 6.

Abstract

The catechin epigallocatechin gallate, one of the main constituents of green tea, showed strong antibiotic activity against 18 isolates of Stenotrophomonas maltophilia (MIC range, 4 to 256 μg/ml). In elucidating its mechanism of action, we have shown that epigallocatechin gallate is an efficient inhibitor of S. maltophilia dihydrofolate reductase, a strategic enzyme that is considered an attractive target for the development of antibacterial agents. The inhibition of S. maltophilia dihydrofolate reductase by this tea compound was studied and compared with the mechanism of a nonclassical antifolate compound, trimethoprim. Investigation of dihydrofolate reductase was undertaken with both a trimethoprim-susceptible S. maltophilia isolate and an isolate with a high level of resistance. The enzymes were purified using ammonium sulfate precipitation, gel filtration, and methotrexate affinity chromatography. The two isolates showed similar levels of dihydrofolate reductase expression and similar substrate kinetics. However, the dihydrofolate reductase from the trimethoprim-resistant isolate demonstrated decreased susceptibility to inhibition by trimethoprim and epigallocatechin gallate. As with other antifolates, the action of epigallocatechin gallate was synergistic with that of sulfamethoxazole, a drug that blocks folic acid metabolism in bacteria, and the inhibition of bacterial growth was attenuated by including leucovorin in the growth medium. We conclude that the mechanism of action of epigallocatechin gallate on S. maltophilia is related to its antifolate activity.

Abstract

Stenotrophomonas maltophilia has emerged as an important nosocomial pathogen, especially for patients whose immune systems are compromised by debilitating diseases, and is associated with increasing case/fatality ratios. The major risk factors for S. maltophilia infection include long-term hospitalization, previous antimicrobial therapy, fungal infections, catheterization, and mechanical ventilation. S. maltophilia infection can cause bacteremia, endocarditis, pneumonia, mastoiditis, peritonitis, meningitis, or infections of the eyes, bones, joints, urinary tract, soft tissues, and wounds (4, 7, 10, 19, 21, 29, 30, 39). The management of infections caused by S. maltophilia is particularly difficult because of its inherent resistance to many currently available broad-spectrum antibiotics (5, 10, 11, 20, 22, 34).

The treatment of choice for S. maltophilia infection is trimethoprim-sulfamethoxazole (TMP-SMZ; cotrimoxazole), alone or in combination with ticarcillin-clavulanate (25, 32, 33). TMP-SMZ is bacteriostatic for most isolates; hence, high doses (12 to 15 mg/kg of body weight/day based on TMP) are usually recommended. Both drugs block folic acid metabolism in bacteria and are much more active together than either agent is alone. Sulfonamides are competitive inhibitors of the incorporation of p-aminobenzoic acid, while TMP is an inhibitor of the dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate:NADP^{oxidoreductase; EC 1.5.1.3) reaction. It is well known that DHFR catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF), which acts as a coenzyme for a number of 1-carbon transfer reactions, including those involved in nucleotide biosynthesis. Consequently, inhibition of DHFR leads to the disruption of DNA synthesis; this is the basis of the antibiotic action of DHFR inhibitors, the antifolates (}13). Although TMP is currently used for the treatment of S. maltophilia infections, the mechanism by which this compound inhibits S. maltophilia DHFR has not been well characterized. Therefore, in this study we purified the DHFR from this microorganism for the first time, and we present data on its inhibition by classical (methotrexate [MTX]) and nonclassical (TMP) antifolate compounds.

Recent studies have presented data on a number of biological activities of tea polyphenols, or catechins (14, 23, 26). It has been reported that tea catechins have antibacterial activity against various pathogenic bacteria (15, 16, 23, 37). There are three main varieties of tea, green, black, and oolong, which are all derived from the leaves of the Camellia sinensis plant. The difference between the teas results from their processing. Green tea is prepared from unfermented leaves, the leaves of oolong tea are partially fermented, and black tea is fully fermented. This difference in processing results in more of the polyphenols being destroyed in the black teas. Thus, green tea contains roughly 30% to 40% polyphenols, while black tea contains only 3% to 10%. Green tea, therefore, seems to have more of the beneficial effects mentioned above, but black teas still retain some of the benefits. Epigallocatechin gallate (EGCG) is the most abundant of these tea catechins (one 240-ml cup of brewed green tea contains up to 200 mg EGCG), and many health-related benefits, including antioxidant, antibiotic, and antiviral activities, have been attributed to this compound (26). Despite the great efforts made during the last 2 decades to understand the biological activity of tea, the exact mechanism(s) of action is not well defined. Therefore, deciphering the molecular mechanism by which green tea or EGCG exerts its antibacterial effects could be important because it may result in improved opportunities for the treatment of different bacterial infections. In attempting to explain the range of responses of S. maltophilia to tea phenols observed in our laboratory, we were struck by the structural similarity of EGCG to several inhibitors of DHFR, in particular, to the drugs MTX and TMP (Fig. (Fig.1).1). In order to probe the hypothesis that EGCG could act as an antifolate compound, we studied the inhibition of S. maltophilia DHFR by this tea compound and compared it with inhibition by TMP.

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FIG. 1.

Structural formulae of (−)-epigallocatechin gallate, TMP, and MTX.

Acknowledgments

This work was supported in part by grants from the EU INTAS program (project INTAS00-0727) to F.G.-C. and J.N.R.-L. and by grants from the Fondo de Investigación Sanitaria (FIS) (projects 01/3025 and 02/1567) to J.C.-H. M.D.N.-M. has a fellowship from Fundación Séneca, Murcia, Spain, and E.N.-P. has a fellowship from the Ministerio de Educación, Cultura y Deporte of Spain.

Acknowledgments

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