Novel roles for two-component regulatory systems in cytotoxicity and virulence-related properties in Pseudomonas aeruginosa

Abstract

The rapid adaptation of the opportunistic bacterial pathogen Pseudomonas aeruginosa to various growth modes and environmental conditions is controlled in part through diverse two-component regulatory systems. Some of these systems are well studied, but the majority are poorly characterized, even though it is likely that several of these systems contribute to virulence. Here, we screened all available strain PA14 mutants in 50 sensor kinases, 50 response regulators and 5 hybrid sensor/regulators, for contributions to cytotoxicity against cultured human bronchial epithelial cells, as assessed by the release of cytosolic lactate dehydrogenase. This enabled the identification of 8 response regulators and 3 sensor kinases that caused substantial decreases in cytotoxicity, and 5 response regulators and 8 sensor kinases that significantly increased cytotoxicity by 15-58% or more. These regulators were additionally involved in motility, adherence, type 3 secretion, production of cytotoxins, and the development of biofilms. Here we investigated in more detail the roles of FleSR, PilSR and WspR. Not all cognate pairs contributed to cytotoxicity (e.g. PhoPQ, PilSR) in the same way and some differences could be detected between the same mutants in PAO1 and PA14 strain backgrounds (e.g. FleSR, PhoPQ). This study highlights the potential importance of these regulators and their downstream targets on pathogenesis and demonstrates that cytotoxicity can be regulated by several systems and that their contributions are partly dependent on strain background.

Keywords: FleSR; PilSR; Pseudomonas aeruginosa; WspR; adherence; biofilm; cytotoxicity; motility; two-component regulatory systems.

Figure 1.. Flagella regulatory mutants demonstrated deficiencies in swarming (A) and swimming (B, C) motility, biofilm formation (D) and cytotoxicity (E). Data shown is with strains PA14 and PAO1. Swarming was performed on BM2-glucose containing casamino acids and 0.5% agar; as the two strains exhibit different swarming behaviours the data is shown as a representative photo for PA14 (dendritic movement) and a bar graph for PAO1 (circular movement). Swimming was measured as the ability of bacteria to move through LB-containing 0.25% agar. A non-flagellated fliC mutant was used as a control (B). A fleQ mutant demonstrated aberrant swimming motility (C). Intriguingly, mutations in fleR and fleS had a more pronounced effect on swarming (panels A and B) and cytotoxicity (panel E) in PA14 than in PAO1. Student's t-test, ***p < 0.001.

Figure 2.. Effect of mutants in regulators of type IV pili on twitching, biofilm formation and swarming. Various regulators showed alterations in some but not all of these pilus-related phenotypes. (A) Twitching motility along the agar-plastic interface of LB plates containing 1% agar was reduced in pilG, pilH and pilR regulator mutants but not in pilS. The non-piliated pilB was used as a control. (B) Pilus-related regulatory mutants demonstrated alterations in their abilities to produce static biofilm. Overnight cultures were diluted into LB and grown for 24 hours in polystyrene 96-well plates. The cultures were then removed, the wells washed, and the remaining biofilm stained with crystal violet. The amount of crystal violet bound corresponded directly to the amount of biofilm formed. (C) Swarming motility across the surface of semi-solid 0.5% agar. Only the non-piliated pilB mutant demonstrated a reduction in swarming. Student's t-test, mean average with standard error. *p < 0.05, **p < 0.01, NS not significant.

Figure 3.. The effect of the Wsp chemosensory system on biofilm formation (A) and twitching motility (B). A lesser (stimulatory) effect was observed for swarming motility (C). Available Cup fimbriae mutants did not show any significant abnormalities in their cytotoxic abilities towards cultured HBE cells (D). Student's t-test, ***p < 0.001.