Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung

Abstract

Pseudomonas aeruginosa uses a type III secretion system to promote development of severe disease, particularly in patients with impaired immune defenses. While the biochemical and enzymatic functions of ExoU, ExoS, and ExoT, three effector proteins secreted by this system, are well defined, the relative roles of each protein in the pathogenesis of acute infections is not clearly understood. Since ExoU and ExoS are usually not secreted by the same strain, it has been difficult to directly compare the effects of these proteins during infection. In the work described here, several isogenic mutants of a bacterial strain that naturally secretes ExoU, ExoS, and ExoT were generated to carefully evaluate the relative contribution of each effector protein to pathogenesis in a mouse model of acute pneumonia. Measurements of mortality, bacterial persistence in the lung, and dissemination indicated that secretion of ExoU had the greatest impact on virulence while secretion of ExoS had an intermediate effect and ExoT had a minor effect. It is of note that these results conclusively show for the first time that ExoS is a virulence factor. Infection with isogenic mutants secreting wild-type ExoS, ExoS defective in GTPase-activating protein (GAP) activity, or ExoS defective in ADP-ribosyltransferase activity demonstrated that the virulence of ExoS was largely dependent on its ADP-ribosyltransferase activity. The GAP activity of this protein had only a minor effect in vivo. The relative virulence associated with each of these type III effector proteins may have important prognostic implications for patients infected with P. aeruginosa.

FIG. 1.

Type III effector proteins secreted by wild-type and isogenic mutant forms of strain PA99. Each strain was grown in low-calcium medium to induce type III secretion. Immunoblot analysis with a mixture of antibodies to ExoS, ExoT, ExoU, PopB, and PopD was then performed on the culture supernatants from each strain. The locations of bands representing ExoU, ExoT, ExoS, PopB, and PopD are shown to the left. The band that migrates between ExoS and PopB is a product of ExoU breakdown (data not shown).

FIG. 2.

Virulence of strains expressing individual type III secreted effector proteins in a mouse model of acute pneumonia. Bars represent the bacterial dose needed to cause mortality in 50% of infected mice. Shorter bars indicate increased virulence. Values were calculated on the basis of at least 10 mice per LD₅₀. Data are shown as the average log₁₀ number of CFU per mouse.

FIG. 3.

Persistence of strains expressing individual type III secreted effector proteins in the lung through 18 h postinfection. Each symbol represents the bacterial load in the lung of an individual animal after 18 h. Bars represent the median value. §, Significant difference from PA99null (P < 0.05).

FIG. 4.

Virulence of strains expressing wild-type or mutated ExoS in a mouse model of acute pneumonia. Lines represent the number of animals surviving in each experimental group over time (n = 10 per strain, pooled from two separate experiments). §, Significant difference from PA99null+S (P < 0.0001).

FIG. 5.

Persistence of strains expressing wild-type or mutated ExoS in the lung through 18 h postinfection. Each symbol represents the bacterial load in the lung of an individual animal after 18 h. Bars represent the median value. §, Significant difference from PA99null+S (P < 0.0001).