Activities of Pseudomonas aeruginosa effectors secreted by the Type III secretion system in vitro and during infection

Abstract

Pseudomonas aeruginosa utilizes a number of distinct pathways to secrete proteins that play various roles during infection. These include the type II secretion system, which is responsible for the secretion of the majority of exoproducts into the surrounding environment, including toxins and degradative enzymes. In contrast, the type III secretion system mediates the delivery of protein effectors directly into the cytoplasm of the host cell. Using tissue culture assays and a mouse acute-pneumonia model, we have determined the contribution of each of the type III effectors during infection. In strain PAK, ExoS is the major cytotoxin required for colonization and dissemination during infection. ExoT confers protection of tissue culture cells from type III-dependent lysis, while ExoY seemed to have little effect on cytotoxicity. ExoU is over 100-fold more cytotoxic than ExoS. The cytotoxicity of type II secretion was determined following deletion of the genes for the more toxic type III secretion system. The participation of these secretion systems during lifelong colonization of cystic fibrosis (CF) patients is unclear. By comparing clonal strains from the same patient isolated at the initial onset of P. aeruginosa infection and more than a decade later, after chronic colonization has been established, we show that initial strains are more cytotoxic than chronic strains that have evolved to reduce type III secretion. Constitutive expression of genes for the type III secretion system restored ExoS secretion but did not always reestablish cytotoxicity, suggesting that CF strains accumulate a number of mutations to reduce bacterial toxicity to the host.

FIG. 1.

Secretion profiles of P. aeruginosa strain PAK and isogenic derivatives induced for type III secretion. (A) Secreted proteins were separated by 12% PAGE and revealed by staining with Coomassie. The positions of ExoS, ExoT, and ExoY on the gel are indicated. WT, wild type. (B) Proteins were transferred onto PVDF membranes and detected with antibodies against ExoS (α-ExoS). In-frame deletion mutation of each exoenzyme gene resulted in the loss of that specific protein from the induced cultures.

FIG. 2.

Contribution of each exoenzyme to the cytotoxicity of CHO cells infected with PAK or mutant derivatives. CHO cells were infected with PAK at a MOI of 10, and supernatants were collected at 3 (white bars), 4 (gray bars), and 5 h (black bars) postinfection. Cleared supernatants were analyzed for LDH release as a measure of cell lysis. WT, wild type.

FIG. 3.

Survival of CHO cells after infection with PAK or mutant derivatives. CHO cells were infected with PAK at a MOI of 10 for 3 h. Cells were trypsinized, serially diluted, and seeded into media containing gentamicin. The foci that formed from CHO cells infected with various strains of PAK were enumerated. The relative plating efficiency was compared to that for uninfected cells, which was set to 100%. WT, wild type.

FIG. 4.

Colonization and dissemination of PAK in a mouse acute pneumonia model are attenuated in strains lacking exoS. Mice were inoculated intranasally with 5 × 10⁷ CFU of PAK or derivatives. At 16 h postinfection, the lungs (A), 300 mg of livers (B; gray bars), and spleens (B; white bars) were harvested and the number of CFU of P. aeruginosa was determined by serial dilution and plating on L agar. Error bars indicate standard deviations.

FIG. 5.

ExoU secretion enhances cytotoxicity of PAK and ΔSTY strains. (A) Secreted proteins from low-calcium-induced cultures were separated by SDS-PAGE. Total proteins were revealed by staining with Coomassie. Proteins were transferred onto PVDF membranes and detected with antibodies against ExoS or ExoU. WT, wild type. (B) Plating efficiency of CHO cells after 2 h of infection with PAK strains harboring exoU.

FIG. 6.

Colonization of PAK exoU strains in vivo requires the presence of exoS, exoT, and exoY. Mice were inoculated intranasally with 2 × 10⁶ CFU of PAK or derivatives. At 16 h postinfection, the lungs (A), 300 mg of livers (B; gray bars), and spleens (B; white bars) were harvested and the number of CFU of P. aeruginosa was determined by serial dilution and plating on L agar. Error bars indicate standard deviations. WT, wild type.

FIG. 7.

CF P. aeruginosa isolates lose the ability to secrete ExoS and induce cytotoxicity over the course of chronic colonization. (A) CHO cells were infected with PAK, PAKΔC (pscC deletion), PAKΔCΔX (pscC and xcp operon deletions), or CF isolates at an initial MOI of 10 for either 4.5 (white bars) or 7.5 h (black bars). Cleared supernatants were analyzed for LDH release as a measure for cell lysis. (B) Secreted proteins from low-calcium-induced cultures were separated by SDS-PAGE. Proteins were transferred onto PVDF membranes and detected with antibodies against ExoS (α-ExoS). Patients were designated A to G; early strains are labeled 1, and late strains are labeled 2.

FIG. 8.

Overexpression of exsA results in the secretion of ExoS or ExoU in vitro but does not correlate with cytotoxicity to CHO cells. Secreted proteins from low-calcium-induced cultures were separated by SDS-PAGE. Proteins were transferred onto PVDF membranes and detected with antibodies against ExoS (α-ExoS) or ExoU. CHO cells were infected with PAK, PAKΔC (pscC deletion), or CF isolates harboring pMMB-exsA at an initial MOI of 10 for either 3 (white bars), 4.5 (gray bars), or 6 h (black bars). Cleared supernatants were analyzed for LDH release as a measure of cell lysis. (A) Cultures induced with IPTG. WT, wild type. (B) Culture without IPTG addition.