Pathogenicity islands PAPI-1 and PAPI-2 contribute individually and synergistically to the virulence of Pseudomonas aeruginosa strain PA14

Abstract

Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and severe chronic lung infections in cystic fibrosis patients. The reference strains PA14 and PAO1 have been studied extensively, revealing that PA14 is more virulent than PAO1 in diverse infection models. Among other factors, this may be due to two pathogenicity islands, PAPI-1 and PAPI-2, both present in PA14 but not in PAO1. We compared the global contributions to virulence of PAPI-1 and PAPI-2, rather than that of individual island-borne genes, using murine models of acute pneumonia and bacteremia. Three isogenic island-minus mutants (PAPI-1-minus, PAPI-2-minus, and PAPI-1-minus, PAPI-2-minus mutants) were compared with the wild-type parent strain PA14 and with PAO1. Our results showed that both islands contributed significantly to the virulence of PA14 in acute pneumonia and bacteremia models. However, in contrast to the results for the bacteremia model, where each island was found to contribute individually, loss of the 108-kb PAPI-1 island alone was insufficient to measurably attenuate the mutant in the acute pneumonia model. Nevertheless, the double mutant was substantially more attenuated, and exhibited a lesser degree of virulence, than even PAO1 in the acute pneumonia model. In particular, its ability to disseminate from the lungs to the bloodstream was markedly inhibited. We conclude that both PAPI-1 and PAPI-2 contribute directly and synergistically in a major way to the virulence of PA14, and we suggest that analysis of island-minus strains may be a more appropriate way than individual gene knockouts to assess the contributions to virulence of large, horizontally acquired segments of DNA.

FIG. 1.

(A) Survival graph for the acute murine pneumonia model. Mice were monitored for 96 h. Ten mice were used for each strain. Data were generated from two independent experiments for each strain, except for the ΔexoU and ΔPAPI-1ΔexoU strains, for which data were generated from a single experiment only. Horizontal lines represent the percentage of mice surviving postinfection for each strain. (B) Acute murine pneumonia symptom scores at 18 h postinfection. Ten mice were used for each strain. Each data point represents a single mouse. All data were generated from two independent experiments for each strain. The asterisk indicates a significant difference from wild-type PA14 (P < 0.05). The bars represent mean symptom scores ± standard deviations.

FIG. 2.

P. aeruginosa bacterial burdens in the nasopharynx, lungs, and blood at 18 h postinfection in the acute murine pneumonia model. Ten mice were used for each strain. All data were generated from two independent experiments for each strain. Asterisks and number symbols indicate significant differences (P < 0.05) from wild-type PA14 and PA14ΔPAPI-2, respectively. The error bars represent the standard errors of the means.

FIG. 3.

(A) Comparison of the invasive ability of wild-type P. aeruginosa PA14 with those of isogenic mutants lacking PAPI-1 and/or PAPI-2 using an A459 human lung carcinoma cell-based assay. The percentages of internalized bacteria protected from amikacin killing that were recovered after lysis of the remaining intact A459 cells relative to the original inocula are shown. Strains that are highly cytotoxic for A459 cells would result in an apparent reduction in invasive ability. Bars represent means and standard deviations. The asterisks indicate no significant difference between the strains compared. (B) Comparison of the cytotoxicity of P. aeruginosa strain PA14 with those of its isogenic mutants lacking PAPI-1 and/or PAPI-2 against A459 human lung carcinoma cells. The percentage of cytotoxicity was calculated relative to that of a maximum-LDH-release control. Bars represent means and standard deviations. Asterisks indicate significant differences from PA14 (P < 0.05).

FIG. 4.

Survival graph for Galleria mellonella killing assay. A total of 30 larvae were used for each strain. Horizontal lines represent the percentage of G. mellonella larvae surviving after inoculation with each bacterial strain at the indicated time point.

FIG. 5.

(A) P. aeruginosa bacterial burdens in blood over a 24-h period following intravenous infection with 2 × 10⁶ CFU. A total of five mice were used for each strain, and the same five mice were used for each time point. Each data point represents the mean CFU/ml of blood. All data were generated from a single experiment for each strain. The standard errors of the means are indicated. No data were available for the 24-h time point for mice infected with PA14, because severe signs of disease required culling of these mice at 6 h postinfection. (B, C, and D) Intravenous infection symptom scores at 2, 6, and 24 h postinfection, respectively. A total of five mice were used for each strain. No symptom score data were available at 24 h for PA14. Each symbol represents a single mouse. The bars represent mean symptom scores ± standard deviations. Asterisks mark strains exhibiting significant differences from PA14 (P < 0.05).

FIG. 6.

Numbers of neutrophils (A), lymphocytes (B), macrophages (C), and monocytes (D) in lung tissue obtained 18 h postinfection in the murine acute pneumonia model. Four mice were used for each strain. The measurements refer to the number of target cells per milliliter of lung homogenate. Asterisks indicate significant differences from wild-type PA14 (P < 0.05). The standard errors of the means are shown.