Point Mutations in the folP Gene Partly Explain Sulfonamide Resistance of Streptococcus mutans

Abstract

Cotrimoxazole inhibits dhfr and dhps and reportedly selects for drug resistance in pathogens. Here, Streptococcus mutans isolates were obtained from saliva of HIV/AIDS patients taking cotrimoxazole prophylaxis in Uganda. The isolates were tested for resistance to cotrimoxazole and their folP DNA (which encodes sulfonamide-targeted enzyme dhps) cloned in pUC19. A set of recombinant plasmids carrying different point mutations in cloned folP were separately transformed into folP-deficient Escherichia coli. Using sulfonamide-containing media, we assessed the growth of folP-deficient bacteria harbouring plasmids with differing folP point mutations. Interestingly, cloned folP with three mutations (A37V, N172D, R193Q) derived from Streptococcus mutans 8 conferred substantial resistance against sulfonamide to folP-deficient bacteria. Indeed, change of any of the three residues (A37V, N172D, and R193Q) in plasmid-encoded folP diminished the bacterial resistance to sulfonamide while removal of all three mutations abolished the resistance. In contrast, plasmids carrying four other mutations (A46V, E80K, Q122H, and S146G) in folP did not similarly confer any sulfonamide resistance to folP-knockout bacteria. Nevertheless, sulfonamide resistance (MIC = 50 μ M) of folP-knockout bacteria transformed with plasmid-encoded folP was much less than the resistance (MIC = 4 mM) expressed by chromosomally-encoded folP. Therefore, folP point mutations only partially explain bacterial resistance to sulfonamide.

Figure 1

Flow chart showing characterization of folP gene. Plasmids carrying folP gene were transformed in folP gene knockout bacteria to determine the effect of different mutations in plasmid encoded folP on bacterial resistance to sulfonamides.

Figure 2

Comparing sulfamethoxazole minimal inhibitory concentrations (MICs) for folP knockout E. coli cells transformed with pUC19 plasmid carrying differing chromosomal folP genes of streptococci. To determine the effect plasmid encoded mutant folP has on the sulfamethoxazole resistance of transformed C600ΔfolP E. coli bacteria, growths (in SMX containing media) were compared for folP-deficient E. coli transformed with pUC19 carrying triple-mutant folP (residues 37, 172, and 193: bar A), double-mutant folP (bars B, C, and D), single-mutant folP (residue 193: bar E), and wild-type folP (bar F) from S. mutans isolate 8. The sulfamethoxazole resistance of transformed folP deficient cells was notably increased by transformation with plasmid encoding triple-mutated folP (wildtype folP MIC = 20 μM, triple-mutant folP MIC = 50 μM). Note: the MIC (sulfamethoxazole) for chromosome-encoded folP in S. mutans isolate 8 was 4 mM (see Table 1). Controls comprising C600ΔfolP E. coli transformed with pUC19 encoding either mutant folP from S. mutans isolates 797 (bar G) and 135 (bar H) or wild-type folP from S. sobrinus isolate 7 (bar I) or S. downei isolate 477 (bar J) showed basal or less sulfamethoxazole resistance (MICs = 20–30 μM). *: S. mutans isolate 8 mutant folP; **: S. mutans isolate 797 mutant folP; ***: S. mutans isolate 135 mutant folP.