Comparison of theexoSGene and Protein Expression in Soil and Clinical Isolates ofPseudomonas aeruginosa

Abstract

Exoenzyme S (ExoS) is translocated into eukaryotic cells by the type III secretory process and has been hypothesized to function in conjunction with other virulence factors in the pathogenesis of Pseudomonas aeruginosa. To gain further understanding of how ExoS might contribute to P. aeruginosa survival and virulence, ExoS expression and the structural gene sequence were determined in P. aeruginosa soil isolates and compared with ExoS of clinical isolates. Significantly higher levels of ExoS ADP-ribosyltransferase (ADPRT) activity were detected in culture supernatants of soil isolates compared to those of clinical isolates. The higher levels of ADPRT activity of soil isolates reflected both the increased production of ExoS and the production of ExoS having a higher specific activity. ExoS structural gene sequence comparisons found the gene to be highly conserved among soil and clinical isolates, with the greatest number of nonsynonymous substitutions occurring within the region of ExoS encoding GAP function. The lack of amino acid changes in the ADPRT region in association with a higher specific activity implies that other factors produced by P. aeruginosa or residues outside the ADPRT region are affecting ExoS ADPRT activity. The data are consistent with ExoS being integral to P. aeruginosa survival in the soil and suggest that, in the transition of P. aeruginosa from the soil to certain clinical settings, the loss of ExoS expression is favored.

Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patients compared with those of isolates from other origins

“…The prevalence of exoS was highest in CF pulmonary isolates (94 %; Table 3), in agreement with previous studies (Dacheux et al, 2000;Feltman et al, 2001). The prevalence of exoS was similar in isolates from plants (90 %; Table 3) and from soil isolates (Ferguson et al, 2001). Nevertheless, 80 % of all pulmonary isolates harboured exoS, indicating that this gene plays a major role in all pulmonary infections.…”

Single-Nucleotide-Polymorphism Mapping of the Pseudomonas aeruginosa Type III Secretion Toxins for Development of a Diagnostic Multiplex PCR System

“…The entire gene sequence was reconstructed from these data by using DNA analysis software. The sequences of strains previously published in GenBank were used for the evaluation of exoS (10,35). These included the following strains (accession number): 388 (L27629), PAK (AYO29248), CCU1 (AYO29240), CCU2 (AYO29241), CCU3 (AYO29252), CCU4 (AYO29243), CCU5 (AYO29246), CCU6 (AYO29250), CCU8 (AYO29249), CCU9 (AYO29239), DG1 (AYO29247), FRD1 (AYO29251), PA-U1 (AYO29245), PA-U3 (AYO29242), and ATCC 27853 (AYO29244).…”

Cell line differences in bacterially translocated ExoS ADP-ribosyltransferase substrate specificity

“…To analyse substrate modification in the cytosolic and membrane fractions, a 50 ml aliquot of each sample was mixed with 46 Laemmli sample buffer and an equivalent volume of each fraction was resolved by SDS-PAGE and immunoblotted for RalA, Rab proteins, or Rac1, as described above. ExoS ADPRT activity in remaining portions of cytosolic and membrane fractions was quantified, as described previously (Ferguson et al, 2001), and related to total protein concentrations in the respective sample determined using the Sigma Diagnostics Lowry-based protein assay. Data were analysed for statistical significance using one-way factorial ANOVA, as described above.…”