Modulation of Type III Secretion System in Pseudomonas aeruginosa: Involvement of the PA4857 Gene Product

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that causes serious acute or chronic infections in humans. Acute infections typically involve the type III secretion systems (T3SSs) and bacterial motility, whereas chronic infections are often associated with biofilm formation and the type VI secretion system. To identify new genes required for pathogenesis, a transposon mutagenesis library was constructed and the gene PA4857, named tspR, was found to modulate T3SS gene expression. Deletion of P. aeruginosa tspR reduced the virulence in a mouse acute lung infection model and diminished cytotoxicity. Suppression of T3SS gene expression in the tspR mutant resulted from compromised translation of the T3SS master regulator ExsA. TspR negatively regulated two small RNAs, RsmY and RsmZ, which control RsmA. Our data demonstrated that defects in T3SS expression and biofilm formation in retS mutant could be partially restored by overexpression of tspR. Taken together, our results demonstrated that the newly identified retS-tspR pathway is coordinated with the retS-gacS system, which regulates the genes associated with acute and chronic infections and controls the lifestyle choice of P. aeruginosa.

Keywords: Pseudomonas aeruginosa; bacterial virulence; regulatory mechanisms; tspR; type III secretion system.

FIGURE 1

TspR is required for the expression of T3SS genes. (A) The expression of exoS-lux was measured in the wild-type PAO1, tspR::Gm, and the complemented tspR::Gm strain (tspR::Gm/p-tspR), the tspR transposon strain (tspR::Tn), and the tspR::Tn complemented strain (tspR::Tn/p-tspR). The asterisks indicate the statistically different exoS expression compared to that in the wild-type strain as determined by Student’s t-test (^∗∗∗P < 0.001). (B) The expression and secretion of ExoS were examined in the indicated strains by Western-blotting. Strains were grown in LB with EGTA to an OD₆₀₀ = 0.6. Whole-cell extracts from the designated strains were separated by SDS-PAGE and immuno-blotted. (C) Effect of tspR mutation on swarming motility. Overnight cultures were spotted onto swarming plates (2 μl aliquots) and the plates were incubated at 37°C. The images captured after 16 h of growth. The experiments were repeated at least three times and similar results were observed. (D) TspR is required for biofilm formation. Quantification of crystal violet (CV) staining of biofilms was grown in microtiter plates for 14 h. ^∗∗P < 0.005 based on Student’s t-test. Photo of the tube binding assay was taken. EV, empty vector.

FIGURE 2

Mutation of tspR diminishes cytotoxicity and reduced the virulence of P. aeruginosa. (A) Cytotoxicity of the P. aeruginosa and its derivates. A549 cells were infected with the indicated strains at a MOI of 20. At 4 h post-infection, cells attached to the plate were measured with CV staining. Results represent mean ± SD, and data are representative of three independent experiments. (B) Microscopy images of A549 cells infected with the indicated strains. The control (CK) was an uninfected A549 culture. (C) The tspR mutation reduced the virulence of P. aeruginosa. C57BL6 mice were intranasally challenged with the wild-type strain, the tspR::Gm strain, and the complemented tspR::Gm strain at 5 × 10⁷ CFU in 50 μl of PBS, and moribund mice were killed to determine survival (Kaplan–Meier Curve with Log-Rank test, P = 0.0028, n = 6).

FIGURE 3

TspR affects expression of the exsCEBA operon at the transcriptional and post-transcriptional levels. (A) The activity of PexsC-lux was determined in the wild-type and the tspR::Gm strains. The results shown are the average of triplicate experiments. Error bars indicate the standard deviations. EV, empty vector. (B) exsA-FLAGs constructs. Ptac represents a tac promoter. (C) Western-blotting showing the expression levels of ExsA-FLAG under T3SS-inducing conditions in the exsA and tspR mutants.

FIGURE 4

RetS tunes the expression of tspR. (A) TspR had no effect on retS expression. The level of retS transcription was measured in the wild-type and tspR::Gm strains. The results shown are the mean ± SD, and data are representative from three independent experiments. (B) RetS positively regulates the activity of tspR. The activity of the tspR promoter-lux fusion (tspR-lux) in the wild-type, retS::Gm and retS::Gm complemented strains were measured. ^∗∗P < 0.005 between the wild-type and retS mutant by Student’s t-test. (C) The expression of exoS in the indicated strains (^∗∗∗P < 0.001). (D) Overexpression of tspR in the retS::Gm mutant restored the expression of ExoS to wild-type levels. Indicated strains were grown in LB with or without EGTA to an OD₆₀₀ = 0.6. Whole-cell extracts from the designated strains were separated by SDS-PAGE separation and subjected to Western-blotting. EV represents empty vector.

FIGURE 5

Two small RNAs, RsmY and RsmZ, are involved in tspR-mediated regulation. (A) The expression of rsmY-lux and rsmZ-lux was strongly induced in the tspR::Gm strain compared to that in the wild-type strain. ^∗∗P < 0.005 compared to the wild-type by Student’s t-test. (B) Mutantion of tspR in the rsmY::Gm or rsmZ::Gm strain reduced the levels of exoS. Results shown are the average of triplicate experiments. Error bars indicate SD. (C) The roles of RsmY and RsmZ in tspR-mediated T3SS gene expression. The indicated strains were grown in LB with or without EGTA to an OD₆₀₀ = 0.6. Whole-cell extracts from the designated strains were separated by SDS-PAGE and subjected to subsequent Western-blotting. EV represents empty vector.

FIGURE 6

TspR regulates biofilm formation through the RetS-RsmY/Z pathway. Quantification of CV staining of biofilms grown in microtiter plates for 14 h. ^∗P < 0.01 and ^∗∗P < 0.005 compared to the wild-type strain based on Student’s t-test. Photos of the tubes binding assay of the indicated strains were taken. EV, empty vector.

FIGURE 7

A schematic diagram showing that tspR is involved in the T3SS regulatory network in P. aeruginosa. The potential regulatory pathways and interactions including tspR were proposed based on our observations and those of previous studies. RetS and GacS-GacA are two major systems involved in T3SS regulation. In the present study, we identified a modulator, TspR, which tunes T3SS expression through two small sRNAs (RsmY and RsmZ) or the master regulator ExsA. In addition, the activity of tspR was shown to be controlled by RetS (blue lines). The solid arrows indicate positive regulation and the solid T-bars present negative regulation.