Antifolate activity of epigallocatechin gallate against Stenotrophomonas maltophilia

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 microg/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.

FIG. 1.

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

FIG. 2.

Effect of EGCG on the viability of S. maltophilia isolate 1 in liquid medium (time-kill curve). S. maltophilia isolate 1 was cultured aerobically in cation-adjusted Mueller-Hinton broth at 35°C with reciprocation in the presence of EGCG at concentrations of 512 (+), 256 (•), 128 (, 64 (×), 32 (▵), 16 (□), and 0 (⧫) μg/ml. Culture samples (100 μl) were taken at the times indicated, and viability was measured by the plate colony count technique.

FIG. 3.

(A) Double-reciprocal plots of the reaction of DHFR from S. maltophilia isolate 1 (3 nM) with NADPH (100 μM) and DHF (variable substrate) in the presence of TMP at pH 7.4. TMP concentrations were 10 (•), 20 (○), 40 (▪), and 60 (◊) μM. Each point represents the mean ± standard deviation for five separate experiments. (B) Secondary plot for the apparent K_m^DHF, obtained from panel A, versus the concentration of TMP.

FIG. 4.

(A) Double-reciprocal plots of the reaction of DHFR from S. maltophilia isolate 1 (3 nM) with NADPH (100 μM) and DHF (variable substrate) in the presence of EGCG at pH 7.4. EGCG concentrations were 0 (•), 10 (○), 20 (▪), and 40 (◊) μM. Each point represents the mean ± standard deviation for five separate experiments. (B) Secondary plots for the apparent K_m^DHF, obtained from panel A, versus the concentration of EGCG.

FIG. 5.

Effects of TMP (black bar), SMZ (white bar), and EGCG (grey bar) on S. maltophilia growth after a 24-h incubation in the presence and absence of 0.4 mM leucovorin. The data are expressed assuming 100% growth for the untreated control. Bars represent the average growth for the 18 isolates, and the error bars represent the standard deviations of the data.