Role of alginate O acetylation in resistance of mucoid Pseudomonas aeruginosa to opsonic phagocytosis

Abstract

Establishment and maintenance of chronic lung infections with mucoid Pseudomonas aeruginosa in patients with cystic fibrosis (CF) require that the bacteria avoid host defenses. Elaboration of the extracellular, O-acetylated mucoid exopolysaccharide, or alginate, is a major microbial factor in resistance to immune effectors. Here we show that O acetylation of alginate maximizes the resistance of mucoid P. aeruginosa to antibody-independent opsonic killing and is the molecular basis for the resistance of mucoid P. aeruginosa to normally nonopsonic but alginate-specific antibodies found in normal human sera and sera of infected CF patients. O acetylation of alginate appears to be critical for P. aeruginosa resistance to host immune effectors in CF patients.

FIG. 1

Susceptibility of variously acetylated mucoid P. aeruginosa strains to opsonic killing by human peripheral blood leukocytes (PBL) and complement at the indicated concentrations. Strain FRD1153 is an algJ mutant strain, unable to O acetylate alginate. Strain FRD1 is the parental strain of FRD1153. Plasmid pMF52 contains the algI, algJ, and algF genes under the control of the Ptrc promoter and restores the O-acetylation phenotype to FRD1153. Plasmid pMF54 is the cloning vector. Bars represent the mean CFU surviving, and error bars indicate the standard deviation. Asterisks indicate that at all complement concentrations tested, the percentage of bacteria surviving was significantly lower in both of the O-acetyl-deficient strains than in the O-acetyl-sufficient strains (P < 0.001, as determined by ANOVA and Fisher's PLSD test for pairwise comparisons).

FIG. 2

Acetate residues on MEP/alginate inhibit activation of the alternative pathway of complement. Exposure of 6.25% adsorbed normal human serum to 10⁷ CFU of each mucoid P. aeruginosa strain for 30 min followed by the removal of the bacteria and the evaluation of the residual complement-activating capacity of the serum showed that the fully O-acetylated strains, FRD1 and FRD1153(pMF52), activated <25% of the available complement, whereas the poorly O-acetylated strains, FRD1153 and FRD1153(pMF54), activated >98% of the available complement. Values of residual activity below 0% are due to experimental variation. Data are reported as mean and standard deviation. Asterisks indicate a significant difference between the value shown and that obtained with 100% residual activity (i.e., 100% red blood cell lysis; P <0.0001, as determined by ANOVA and Fisher's PLSD test).

FIG. 3

Opsonic killing of nonacetylated or acetylated mucoid P. aeruginosa strains in the presence of a nonopsonic monoclonal antibody (MAb), nonopsonic antibody to MEP in a normal human serum sample, and nonopsonic antibody in pooled sera from mice immunized with a high dose (50 μg) of MEP. Bars represent mean CFU killed, and error bars show the standard deviation. The percentage of the two nonacetylated strains killed by each of the three antibodies was significantly higher than the percentage of the acetylated strains killed by the corresponding antibodies (P < 0.001, as determined by ANOVA and Fisher's PLSD test for pairwise comparisons). C′, complement.

FIG. 4

Opsonic killing of nonacetylated or acetylated mucoid P. aeruginosa strains in the presence of opsonic monoclonal antibody (MAb 9/5/23) to MEP, opsonic antibody in serum from a person immunized with MEP, or opsonic antibody in sera pooled from mice immunized with a low dose (10 μg) of MEP. Bars represent the mean CFU killed, and error bars show the standard deviation. The percentage of the two nonacetylated strains killed by the opsonic MAb was significantly lower than the percentage killed by the other antibody preparations (P < 0.01, as determined by ANOVA and Fisher's PLSD test for pairwise comparisons). The immune mouse and human sera killed the nonacetylated strains because of the concomitant presence of nonopsonic antibody induced by immunization in mice (a 10-μg dose of MEP elicits both opsonic and nonopsonic antibodies) or occurring naturally in humans.