Pseudomonas aeruginosa uses type III secretion system to kill biofilm-associated amoebae

Abstract

Bacteria and protozoa coexist in a wide range of biofilm communities of natural, technical and medical importance. Generally, this interaction is characterized by the extensive grazing activity of protozoa on bacterial prey populations. We hypothesized that the close spatial coexistence in biofilms should allow opportunistic pathogenic bacteria to utilize their eukaryote-targeting arsenal to attack and exploit protozoan host cells. Studying cocultures of the environmental pathogen Pseudomonas aeruginosa and the amoeba Acanthamoeba castellanii, we found that P. aeruginosa rapidly colonized and killed biofilm-associated amoebae by a quorum-sensing independent mechanism. Analysis of the amoeba-induced transcriptome indicated the involvement of the P. aeruginosa type III secretion system (T3SS) in this interaction. A comparison of mutants with specific defects in the T3SS demonstrated the use of the secretion apparatus and the effectors ExoU, ExoS and ExoT in the killing process, of which ExoU had the greatest impact. T3SS-mediated virulence towards A. castellanii was found to be controlled by the global regulators RpoN and RpoS and through modulation of cAMP and alginate biosynthesis. Our findings suggest that conserved virulence pathways and specifically the T3SS play a central role in bacteria-protozoa interactions in biofilms and may be instrumental for the environmental persistence and evolution of opportunistic bacterial pathogens.

Fig. 1

Lysis of Acanthamoeba castellanii settling on biofilms of Pseudomonas aeruginosa. Amoeba survival on (A) biofilms of P. aeruginosa PAO1 wild type versus QS-deficient rhlR/lasR mutant, and (B) biofilms of P. aeruginosa PA99 wild type versus T3SS-deficient PA99secr- mutant. Biofilms were pre-grown in flow cell systems for 3 days. Amoeba numbers include only structurally intact cells and are given as means ± standard deviation (n = 3).

Fig. 2

Colonization and killing of Acanthamoeba castellanii by Pseudomonas aeruginosa. (A) Colonization of A. castellanii by P. aeruginosa PA99 wild type and the T3SS-deficient mutant PA99secr- given as percentage of the total amoeba number after 24 hours. (B) Survival of A. castellanii when exposed to P. aeruginosa PA99 wild type and the T3SS-deficient mutant PA99secr- given as percentage of the total amoeba number after 24 hours. Error bars represent standard deviations (n = 3).

Fig. 3

Contribution of type III secreted effectors to the killing of A. castellanii. Amoeba lysis is given as relative reduction of initial cell numbers after 24 hours of co-incubation with P. aeruginosa PA99 wild type and the T3SS-deficient mutant PA99secr-, and mutants expressing only one of the three effectors, ExoS (PA99S), ExoT (PA99T), or ExoU (PA99U). Error bars represent standard deviations (n = 3).

Fig. 4

Contribution of global gene regulators to T3SS-mediated killing of A. castellanii. Amoeba survival is given as relative change of initial cell numbers after 24 hours of co-incubation with P. aeruginosa PAO1 wild type and mutants defective in rhlR/lasR, rpoS, vfr, mucA, rpoN. Error bars represent standard deviations (n = 3).