Secretion of Pseudomonas aeruginosa type III cytotoxins is dependent on pseudomonas quinolone signal concentration

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that can, like other bacterial species, exist in antimicrobial resistant sessile biofilms and as free-swimming, planktonic cells. Specific virulence factors are typically associated with each lifestyle and several two component response regulators have been shown to reciprocally regulate transition between biofilm-associated chronic, and free-swimming acute infections. Quorum sensing (QS) signal molecules belonging to the las and rhl systems are known to regulate virulence gene expression by P. aeruginosa. However the impact of a recently described family of novel quorum sensing signals produced by the Pseudomonas Quinolone Signal (PQS) biosynthetic pathway, on the transition between these modes of infection is less clear. Using clonal isolates from a patient developing ventilator-associated pneumonia, we demonstrated that clinical observations were mirrored by an in vitro temporal shift in isolate phenotype from a non-secreting, to a Type III cytotoxin secreting (TTSS) phenotype and further, that this phenotypic change was PQS-dependent. While intracellular type III cytotoxin levels were unaffected by PQS concentration, cytotoxin secretion was dependent on this signal molecule. Elevated PQS concentrations were associated with inhibition of cytotoxin secretion coincident with expression of virulence factors such as elastase and pyoverdin. In contrast, low concentrations or the inability to biosynthesize PQS resulted in a reversal of this phenotype. These data suggest that expression of specific P. aeruginosa virulence factors appears to be reciprocally regulated and that an additional level of PQS-dependent post-translational control, specifically governing type III cytotoxin secretion, exists in this species.

Fig. 1

Fig. 1A. Serial ETA’s collected over a 3 week period from a patient developing VAP exhibit evidence of a transition from chronic (green tinged appearance) to acute (bloody appearance) mode of infection. B. P. aeruginosa isolates demonstrate a shift in fluorescent metabolite production upon exposure to UV light. C. Serial isolates are clonal by standard PFGE analysis.

Fig. 2

Fig. 2A. Elastase production is approximately 2-fold higher in strains 1 and 2 compared to strains 3, 4 and 5. B. Pyoverdin production is also greater in the initial two isolates compared to the latter 3. C. Biofilm formation by all five strains. D. Analysis of intracellular (IC) and secreted total protein fractions (S) of each strain demonstrates that while all strains produce ExoU and ExoT, only the latter 3 isolates secrete these cytotoxins. E. Cytotoxicity assays profiling epithelial cell toxicity of all 5 strains demonstrate that the latter 3 isolates are significantly more cytotoxic compared to strains isolated earlier in the infectious process.

Fig. 3

Fig. 3A. Substantially higher concentrations of extracellular organic compounds (including quinolone signals) in cell-free extract are produced by strains 1 and 2, compared to strains 3, 4 and 5. B. Q-PCR analysis confirms that strains 1 and 2 express approximately 12-fold higher pqsA copy number compared to strains 3, 4 and 5.

Fig. 4

Fig. 4A. Inhibition of AHQ biosynthesis by farnesol addition induces a type III secretion phenotype in all five isolates. B. Strains cultured without exogenous extract exhibit cytotoxin secretion by the latter 3 strains (Control), those exposed to PQS-containing extract (PQS⁺) obtained from clinical strain 1 (this study) or the laboratory strain PAO1 exhibit no evidence of cytotoxin secretion, while strains cultured in the presence of extract from PAO1ΔPqsH exhibit a secretion profile identical to the control cultures, confirming the role of PQS in type III cytotoxin secretion.