Single-nucleotide-polymorphism mapping of the Pseudomonas aeruginosa type III secretion toxins for development of a diagnostic multiplex PCR system

Abstract

We mapped the coding single nucleotide polymorphisms in four toxin genes-exoS, exoT, exoU, and exoY-of the Pseudomonas aeruginosa type III secretion system among several clinical isolates. We then used this information to design a multiplex PCR assay based on the simultaneous amplification of fragments of these genes. Eight strains of known genotype were used to test our multiplex PCR method, which showed 100% sensitivity and specificity in this small sample size. This assay appears to be promising for the rapid and accurate genotyping of the presence of these genes in clinical strains of P. aeruginosa.

FIG. 1.

Genotyping of exoS, exoT, exoU, and exoY in clinical and laboratory P. aeruginosa isolates by multiplex PCR. Agarose gel electrophoresis shows bands representing amplified DNA fragments of each gene. All isolates were genotype positive for exoT, but only strain 19660 was genotype negative for exoY. Strains positive for exoU were genotype negative for exoS. M.W.M., molecular weight marker; standard, standard DNA fragments representing each gene; bps, base pairs.

FIG. 2.

Position and frequency of exoS DNA and amino acid sequence variations (n = 13) and position of PCR primers, GAP, and ADP-ribosyltransferase functional domains. The x axis represents the nucleotide or amino acid position; the y axis represents the percentage of P. aeruginosa isolates that differed in sequence from PAO1 at each position. Arg-146 is essential for Rho GAP activity, whereas Glu-381 is essential for ADP-ribosyltransferase activity. E-son in the ADP-ribosyltransferase domain is the binding site of a 14-3-3 protein (factor for activating ExoS [FAS]). The exoS sequence of various isolates reported by Ferguson et al. (10) was used in this analysis.

FIG. 3.

Position and frequency of exoT DNA and amino acid sequence variations (n = 8) and the position of PCR primers and signal sequence, GAP, and ADP-ribosyltransferase domains. The x axis represents the nucleotide or amino acid position; the y axis represents the percentage of P. aeruginosa isolates that differed in sequence from PAO1 at each position.

FIG. 4.

Position and frequency of exoU DNA and amino acid sequence variations (n = 6) and position of PCR primers. The x axis represents the nucleotide or amino acid position; the y axis represents the percentage of P. aeruginosa isolates that differed in sequence from PA103 at each position.

FIG. 5.

Position and frequency of exoY DNA and amino acid sequence variations (n = 9) and position of PCR primers and conserved regions between P. aeruginosa, B. pertussis, and B. anthracis adenylate cyclases. The x axis represents the nucleotide or amino acid position; the y axis represents the percentage of P. aeruginosa isolates that differed in sequence from PAO1 at each position. Two lysine residues (Lys-81 and Lys-88) in conserved region I and two arginine residues (Arg-212 and Arg-214) in conserved region II are essential for the adenylate cyclase activity of ExoY.