Pseudomonas aeruginosa gene products PilT and PilU are required for cytotoxicity in vitro and virulence in a mouse model of acute pneumonia

Abstract

Type IV pili of the opportunistic pathogen Pseudomonas aeruginosa mediate twitching motility and act as receptors for bacteriophage infection. They are also important bacterial adhesins, and nonpiliated mutants of P. aeruginosa have been shown to cause less epithelial cell damage in vitro and have decreased virulence in animal models. This finding raises the question as to whether the reduction in cytotoxicity and virulence of nonpiliated P. aeruginosa mutants are primarily due to defects in cell adhesion or loss of twitching motility, or both. This work describes the role of PilT and PilU, putative nucleotide-binding proteins involved in pili function, in mediating epithelial cell injury in vitro and virulence in vivo. Mutants of pilT and pilU retain surface pili but have lost twitching motility. In three different epithelial cell lines, pilT or pilU mutants of the strain PAK caused less cytotoxicity than the wild-type strain but more than isogenic, nonpiliated pilA or rpoN mutants. The pilT and pilU mutants also showed reduced association with these same epithelial cell lines compared both to the wild type, and surprisingly, to a pilA mutant. In a mouse model of acute pneumonia, the pilT and pilU mutants showed decreased colonization of the liver but not of the lung relative to the parental strain, though they exhibited no change in the ability to cause mortality. These results demonstrate that pilus function mediated by PilT and PilU is required for in vitro adherence and cytotoxicity toward epithelial cells and is important in virulence in vivo.

FIG. 1

P. aeruginosa pilT and pilU mutants have decreased ability to damage epithelial cells. Cytotoxicity of wild-type PAK, hyperpiliated pilT or pilU mutants, or nonpiliated pilA or rpoN mutants was assayed by incubation of the bacteria with the cell type indicated. Cell death was quantitated by LDH release and is expressed as a percentage of the total LDH released by lysis of cells not exposed to bacteria. Each assay was performed in triplicate, and error bars represent SEM. (A) Cytotoxicity of MDCK cells after incubation with bacteria for 9 h. ∗, P < 0.003 compared to PAK; #, P < 0.003 compared to rpoN. (B) Cytotoxicity observed on A549 cells after incubation with bacteria for 8 h. ∗, P < 0.001 compared to PAK. (C) Cytotoxicity observed on HeLa cells after incubation with bacteria for 5 h. ∗, P < 0.003 compared to PAK; #, P < 0.05 compared to rpoN. Statistical analysis was performed by using Student’s two-tailed t test with unequal variance.

FIG. 2

Association of P. aeruginosa with epithelial cells is reduced by inactivation of pilT or pilU. Wild-type PAK, hyperpiliated pilT or pilU mutants, or nonpiliated pilA or rpoN mutants were incubated with the indicated cell type, and cell-associated bacteria were quantitated after the amount of time indicated. Each assay was performed in triplicate and normalized to the number of CFU initially added to the assay. SEM is represented by the error bars. (A) Fraction of input bacteria associated with MDCK cells after 3 h. *, P < 0.04 compared to PAK; #, P < 0.04 compared to pilA. (B) Fraction of input bacteria associated with A549 cells after 3 h. *, P < 0.0001 compared to PAK; #, P < 0.003 compared to rpoN; ^, P < 0.05 compared to pilA. (C) Fraction of input bacteria associated with HeLa cells for 1 h. *, P < 0.006 compared to PAK; #, P < 0.003 compared to pilA. Statistical analysis was performed by using the Student’s two-tailed t test with unequal variance.

FIG. 3

Virulence of wild-type PAK, hyperpiliated pilT or pilU mutants, or nonpiliated pilA or rpoN mutants in a mouse model of acute pneumonia. Approximately 5 × 10⁷ CFU of each strain was used to intranasally infect 15 mice, each of which was monitored for viability over 7 days.