piv does not impact Pseudomonas aeruginosa virulence in Galleria mellonella

Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen that can also infect mammals, invertebrates, and plants. Protease IV (PIV) is a secreted protease shown to be important in mammalian cornea, lung, and wound models of infection. It also contributes to P. aeruginosa virulence in many invertebrate models. Previous studies have shown that the expression of the gene encoding PIV is higher at 25°C than at 37°C. Thus, we hypothesized that piv would be more important for P. aeruginosa virulence at 25°C than at 37°C. To test this, we first demonstrated that more PIV is secreted by P. aeruginosa PAO1 cells grown at 25°C than at 37°C. We then determined the survival of larvae of the greater wax moth Galleria mellonella infected by PAO1 and an isogenic Δpiv mutant at both 25°C and 37°C. We found no significant difference in virulence between PAO1 and Δpiv at either 25°C or 37°C, although both strains were more virulent at 37°C than 25°C as measured by a decrease in median survival time. P. aeruginosa possesses an arsenal of virulence factors besides PIV, and thus loss of this single virulence factor may not result in attenuation in the highly susceptible G. mellonella larvae.IMPORTANCEPathogenesis of the important opportunistic pathogen Pseudomonas aeruginosa is often investigated using model organisms. Larvae of the greater wax moth, Galleria mellonella, are a popular non-mammalian model organism for P. aeruginosa infections that have been used to study highly attenuated mutants and characterize their defects in virulence. Our study shows that small differences in the virulence of P. aeruginosa, such as those caused by deleting the gene encoding a single virulence factor, may not be detectable in the G. mellonella model of infection. This is an important finding for researchers considering the choice of model organisms for virulence studies.

Keywords: Galleria; Pseudomonas aeruginosa; proteases; temperature regulation; virulence factors.

Fig 1

Mature PIV VSV-G protein levels in P. aeruginosa supernatants are thermoregulated. Supernatant harvested from PAO1 PIV VSV-G grown to the early stationary phase at 25°C and 37°C was concentrated, and total proteins were precipitated prior to analysis by immunoblotting with antibodies against the VSV-G tag (αVSV-G). A representative image of three biological replicates is shown. Mature PIV VSV-G found in supernatant is approximately 28 kD.

Fig 2

Schematic of G. mellonella larvae infection by P. aeruginosa strains at two temperatures. (A) Overnight cultures of PAO1 and Δpiv grown at 37°C were diluted to an OD₆₀₀ = 0.05 and cultured at 37°C and 25°C until reaching exponential phase at OD₆₀₀ approximately 0.5. G. mellonella larvae were infected with 100 CFU of each strain grown at each temperature and then incubated at the corresponding temperature to assess the role of piv in P. aeruginosa virulence at 37°C compared to 25°C. Created with BioRender.com. (B) PAO1 and Δpiv were grown overnight at 37°C and subcultured at 37°C and 25°C. The optical density (OD₆₀₀) of cultures was measured hourly for 8 h. Three biological replicates are shown with error bars representing standard deviation.

Fig 3

Deletion of piv does not impact the virulence of P. aeruginosa in G. mellonella. G. mellonella larvae were infected with ~100 CFU of PAO1 and Δpiv from exponential phase cultures grown at 37°C (A) or 25°C (B) and then housed at the corresponding temperature as depicted in Fig. 2. Larvae were also injected with an equal volume of PBS or not injected as negative controls. Thirty larvae were infected for each PAO1 and Δpiv group, 28 for the PBS group, and 25 for the no-infection group at each temperature. Kaplan-Meier survival curves represent the combined data from three independent experiments. Statistical significance was determined by the log-rank test. (C) Median survival for larvae infected with PAO1 and Δpiv at both 37°C and 25°C shown in (A) and (B). The mean of the three independent experiments with standard deviation is shown. Statistical significance was determined by two-way ANOVA with uncorrected Fisher’s LSD test: ns—not significant and ****P < 0.0001.