Amino acid repetitions in the dihydropteroate synthase of Streptococcus pneumoniae lead to sulfonamide resistance with limited effects on substrate K(m)

Abstract

Sulfonamide resistance in Streptococcus pneumoniae is due to changes in the chromosomal folP (sulA) gene coding for dihydropteroate synthase (DHPS). The first reported laboratory-selected sulfonamide-resistant S. pneumoniae isolate had a 6-bp repetition, the sul-d mutation, leading to a repetition of the amino acids Ile(66) and Glu(67) in the gene product DHPS. More recently, clinical isolates showing this and other repetitions have been reported. WA-5, a clinical isolate from Washington State, contains a 6-bp repetition in the folP gene, identical to the sul-d mutation. The repetition was deleted by site-directed mutagenesis. Enzyme kinetic measurements showed that the deletion was associated with a 35-fold difference in K(i) for sulfathiazole but changed the K(m) for p-aminobenzoic acid only 2.5-fold and did not significantly change the K(m) for 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine pyrophosphate. The enzyme characteristics of the deletion variant were identical to those of DHPS from a sulfonamide-susceptible strain. DHPS from clinical isolates with repetitions of Ser(61) had very similar enzyme characteristics to the DHPS from WA-5. The results confirm that the repetitions are sufficient for development of a resistant enzyme and suggest that the fitness cost to the organism of developing resistance may be very low.

FIG. 1

The nucleotide sequences of the folP genes from all Washington State isolates are compared. Only nucleotides that differ from those of CP1015 are shown. Isolate WA-133 had a sequence identical to that of WA-152. Isolates WA-54, WA-127, WA-970195, and WA-263 had sequences identical to that of WA-45.