Community surveillance enhances Pseudomonas aeruginosa virulence during polymicrobial infection

Abstract

Most infections result from colonization by more than one microbe. Within such polymicrobial infections, microbes often display synergistic interactions that result in increased disease severity. Although many clinical studies have documented the occurrence of synergy in polymicrobial infections, little is known about the underlying molecular mechanisms. A prominent pathogen in many polymicrobial infections is Pseudomonas aeruginosa, a Gram-negative bacterium that displays enhanced virulence during coculture with Gram-positive bacteria. In this study we discovered that during coinfection, P. aeruginosa uses peptidoglycan shed by Gram-positive bacteria as a cue to stimulate production of multiple extracellular factors that possess lytic activity against prokaryotic and eukaryotic cells. Consequently, P. aeruginosa displays enhanced virulence in a Drosophila model of infection when cocultured with Gram-positive bacteria. Inactivation of a gene (PA0601) required for peptidoglycan sensing mitigated this phenotype. Using Drosophila and murine models of infection, we also show that peptidoglycan sensing results in P. aeruginosa-mediated reduction in the Gram-positive flora in the infection site. Our data suggest that P. aeruginosa has evolved a mechanism to survey the microbial community and respond to Gram-positive produced peptidoglycan through production of antimicrobials and toxins that not only modify the composition of the community but also enhance host killing. Additionally, our results suggest that therapeutic strategies targeting Gram-positive bacteria might be a viable approach for reducing the severity of P. aeruginosa polymicrobial infections.

Fig. 1.

PA0601 is required for GlcNAc and peptidoglycan sensing in P. aeruginosa. (A) Pyocyanin, (B) elastase, and (C) PQS levels produced by WT P. aeruginosa (PA14), the P. aeruginosa PA0601 transposon mutant (PA0601⁻), and the genetically complemented PA0601 mutant (PA0601⁻ complement) in the presence of no inducer (succinate), GlcNAc, or peptidoglycan. PA14 and PA0601⁻ contain the empty complementation plasmid pEX1.8, whereas the PA0601⁻ complement contains pAK601 (pEX1.8 expressing PA0601). *P < 0.05 by Student t test compared with the no-inducer (succinate) control. Error bars represent SD, n = 3.

Fig. 2.

GlcNAc/peptidoglycan sensing enhances P. aeruginosa virulence in polymicrobial infections. Kaplan-Meier survival curves of (A) antibiotic-untreated Drosophila after infection with WT P. aeruginosa (PA14), the PA0601 mutant (PA0601⁻), and the genetically complemented PA0601 mutant (PA0601⁻ complement); (B) antibiotic-treated flies infected with P. aeruginosa WT or the PA0601 mutant; and (C) antibiotic-treated flies infected with WT P. aeruginosa or the PA0601 mutant in the presence and absence of peptidoglycan. Curves are representative of a minimum of two biological replicates, n = 60 for each replicate. *P < 0.0001 by the log–rank test for comparison of percentage survival after infection with PA14 compared with infection with PA0601⁻.

Fig. 3.

(A) Decreased levels of the pqsA transcript in antibiotic-treated Drosophila. Reverse transcriptase PCR was used to examine pqsA transcript levels during Drosophila infection under three conditions: (i) no antibiotic treatment of flies and no peptidoglycan added; (ii) antibiotic-treated flies with no peptidoglycan added; (iii) antibiotic-treated flies with peptidoglycan added. gDNA is the genomic DNA positive control, and −RT is the negative control using RNA as the PCR template. The constitutively expressed clpX gene was used as a control. A representative ethidium bromide stained agarose gel is shown. (B) P. aeruginosa colonization reduces the native Gram-positive flora of Drosophila. Gram-positive bacteria were enumerated by viable counts on phenylethyl alcohol agar. Shown are cfu per fly from uninfected flies (control), and flies infected with either WT P. aeruginosa (PA14) or the PA0601 mutant (PA0601⁻). *P < 0.01 by Student t test compared with PA14-infected flies. Error bars represent SD, n = 3.

Fig. 4.

Competitive indices (CI) of WT P. aeruginosa (PA14) and the PA0601 mutant (PA0601⁻) in a murine wound infection model during coculture with S. aureus. Competitive index is defined as the output ratio of P. aeruginosa:S. aureus after 4 d within the chronic wound divided by the input ratio of P. aeruginosa:S. aureus used to initiate the infection at day 1. Each symbol (● or ■) represents values obtained from infection of an individual mouse. P < 0.01 by Mann-Whitney U test for PA14 CI compared with PA0601⁻ CI, n = 12. The number of cfu/g of wound tissue for WT P. aeruginosa (average 5 × 10⁸, SD 3 × 10⁸) and the PA0601 mutant (average 2 × 10⁸, SD 0.8 × 10⁸) in coculture infections after 4 d were not significantly different, P = 0.125 via Mann-Whitney U test n = 12.