Children with Cystic Fibrosis Produce an Immune Response against Exoenzyme S, a Type III Cytotoxin ofPseudomonas aeruginosa

To the Editor —Cystic fibrosis (CF) is a common lethal autosomal genetic disorder among white persons, affecting ∼1 in 3500 live births. The lungs of patients with CF frequently are colonized by ⩾1 bacterial pathogen, typically Staphylococcus aureus and Pseudomonas aeruginosa. Chronic colonization of the lungs of patients with CF with mucoid strains of P. aeruginosa is associated with progressive lung destruction and increased mortality [1]. P. aeruginosa produces both secreted and cell-surface virulence determinants. Secreted virulence factors can provide nutrients for growth, enhance invasive potential, or directly damage host tissue. Type III cytotoxins are delivered directly by the bacterium into the eukaryotic cell cytoplasm. In both acute lung infection and in cultured cells, type III cytotoxins act at the site of infection to subvert the local immune response [2]. P. aeruginosa produces several type III secreted cytotoxins, including exoenzyme S (ExoS). ExoS is a bifunctional cytotoxin that inhibits 2 independent eukaryotic signal-transduction pathways [3]. Type III cytotoxins enter the cell through a transport channel formed by 2 bacterial proteins, PopB and PopD, in a process involving another type III protein, PcrV. Immunization with PcrV is an effective intervention in acute pneumonia caused by P. aeruginosa [4]. The presence of an antibody response has been used to implicate the expression of several virulence factors of P. aeruginosa in children with CF, including exotoxin A, lipopolysaccharide, and outer-surface proteins [5, 6]. In a recent study in the Journal Burns et al. [7] reported a longitudinal assessment of P. aeruginosa in young children with CF, which concluded that most patients had evidence of infection by age 3 years. These results suggest that P. aeruginosa infection occurs early in CF but may be intermittent or undetectable by culture [7] and that combined serologic testing and culturing can effectively detect infection. Here, we present evidence that some children with CF produce antibodies to ExoS and other type III antigens, which suggests that they are infected with acutely cytotoxic strains of P. aeruginosa Infants with mutations in the CF transmembrane regulator (CFTR) were identified by genotyping. Serum samples and cough cultures were collected from these infants during routine evaluations, as defined elsewhere [8]. Serum antibodies against the cytotoxin ExoS and components of the type III system, PopB and PcrV, were detected by Western blot, using purified recombinant PcrV and a P. aeruginosa cell extract, which contained ExoS and PopB. In a random screening of serum samples (diluted 1:1000) obtained from 12–24-month-old infants with CF, 7 of 13 serum samples were observed to contain antibodies against ExoS and PopB, and 5 of these 7 positive serum samples had a weak response to PcrV. A longitudinal study of 3 infants with positive serum samples was performed, and findings were compared with the growth of P. aeruginosa from oropharyngeal cultures obtained during vigorous coughing (figure 1). In serum samples from 1 infant, increasing titers of antibodies against ExoS and PopB were found before a positive oropharyngeal culture for P. aeruginosa was obtained, whereas, in serum samples from the other 2 infants, the appearance of antibody against ExoS and PopB coincided with positive oropharyngeal cultures. In each longitudinal study, the titer of antibodies against PcrV was lower than the titer to ExoS or PopB Longitudinal analysis of serum antibody titers to type III antigens of Pseudomonas aeruginosa in infants with cystic fibrosis (CF). Culture supernatants of P. aeruginosa PA103 were subjected to SDS-PAGE (12% gels). Proteins were transferred to nitrocellulose for reaction with serum samples from 3 infants with CF, taken at the indicated ages, and were diluted 1:500 (A and B) or 1:1000 (C). Western blot was used to detect immunoreactivity, using goat anti–human IgG horseradish peroxidase as the secondary antibody. The antibody titer is expressed as a fraction of the immune response against PcrV from serum of an adult with CF (obtained under National Heart, Lung, and Blood Institute protocol 98-H-62). Antibody reactivity is a representative titer determination from ⩾2 independent analyses, in which ratios to the positive control serum were similar. +, Detection of a positive oropharyngeal culture of P. aeruginosa; −, detection of a negative oropharyngeal culture of P. aeruginosa. ExoS, exoenzyme S; SC, oropharyngeal culture There were no differences with respect to gender, CFTR genotype profile, or gastrointestinal disease status among the 7 children whose serum samples tested positive and the 6 children whose serum samples tested negative for type III reactive antibodies between ages 12 and 24 months. Of the 6 children whose serum samples tested negative for type III reactive antibodies, only 1 had a transiently positive P. aeruginosa culture result between ages 12 and 24 months, whereas all 7 children whose serum samples tested positive for type III reactive antibodies had early positive P. aeruginosa culture results (P=.005, Fisher’s exact test) Type III cytotoxins have been implicated in P. aeruginosa disease associated with epithelial cell and lung injury [9] and with expression of ExoS, which is shown to accelerate injury in cultured epithelial cells. This pilot study presents the first determination that some children with CF are infected by P. aeruginosa that express the type III cytotoxin ExoS. These results imply that there is a role for type III cytotoxins during initial infection and may present a new diagnostic strategy that uses the immune response to ExoS and new vaccine strategies against the type III antigen PcrV to delay or prevent infection by P. aeruginosa in the lungs of children with CF. PcrV is thought to be a protective antigen [4], and the weak response of this antigen in children with CF, relative to other type III antigens, may be the basis for chronic infection by P. aeruginosa