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, 193 (2), 399-410

MexT Regulates the Type III Secretion System Through MexS and PtrC in Pseudomonas Aeruginosa

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MexT Regulates the Type III Secretion System Through MexS and PtrC in Pseudomonas Aeruginosa

Yongxin Jin et al. J Bacteriol.

Abstract

The type III secretion system (T3SS) is the most important virulence factor in Pseudomonas aeruginosa, and its expression level varies in different isolates. We studied the molecular basis for such differences in two laboratory strains, PAK and PAO1. A chromosomal clone library from the high-T3SS-producer strain PAK was introduced into the low-producer strain PAO1, and we found that a mexS gene from PAK confers high T3SS expression in the PAO1 background. Further tests demonstrated that both mexS and its neighboring mexT gene are required for the repression of the T3SS in PAO1, while the PAK genome encodes a defective MexS, accounting for the derepression of the T3SS in PAK and the dominant negative effect when it is introduced into PAO1. MexS is a probable oxidoreductase whose expression is dependent on MexT, a LysR-type transcriptional regulator. Various genetic data support the idea that MexS modulates the transcriptional regulator function of MexT. In searching for the MexT-dependent repressor of the T3SS, a small gene product of PA2486 (ptrC) was found effective in suppressing the T3SS upon overexpression. However, deletion of ptrC in the PAO1 background did not result in derepression of the T3SS, indicating the presence of another repressor for the T3SS. Interestingly, overexpression of functional mexS alone was sufficient to repress T3SS even in the absence of MexT, suggesting that MexS is another mediator of MexT-dependent T3SS repression. Overexpression of mexS alone had no effect on the well-known MexT-dependent genes, including those encoding MexEF efflux pump, elastase, and pyocyanin, indicating alternative regulatory mechanisms. A model has been proposed for the MexS/MexT-mediated regulation of the T3SS, the MexEF efflux pump, and the production of elastase and pyocyanin.

Figures

FIG. 1.
FIG. 1.
Expression and secretion of the T3SS in laboratory strains of P. aeruginosa PAO1 and PAK. (A) Overnight cultures were diluted to 2% in LB with (+) or without (−) 5 mM EGTA and grown at 37οC for 3 h. Proteins secreted into supernatants were concentrated 20-fold with trichloroacetic acid precipitation, loaded onto SDS-PAGE gels with equivalent bacterial cell numbers, and immunoblotted with antibody against ExoS. (B) β-Galactosidase activities in PAO1 and PAK carrying a PexsA-lacZ (pHW0032), PpopN-lacZ (pHW0243), or PpscN-lacZ (pHW0244) reporter gene. Overnight cultures were diluted to 2% in LB with 5 mM EGTA and grown at 37οC for 4 h before being assayed for β-galactosidase activities. Error bars indicate standard deviations of triplicate assays. Numbers on y axes of graphs are β-galactosidase activity units.
FIG. 2.
FIG. 2.
Expression and secretion of ExoS in selected transformants of PAO1 carrying the PAK chromosome DNA clone bank or empty vector pUCP19. (A) Overnight cultures carrying the exoT-lacZ reporter gene were diluted to 2% in LB with (+) or without (−) 5 mM EGTA plus appropriate antibiotic and grown at 37°C for 4 h before being subjected to β-galactosidase assay. Error bars indicate standard deviations of triplicate assays. Numbers on the y axis are β-galactosidase activity units. (B) Overnight cultures were diluted to 2% in LB with (+) or without (−) 5 mM EGTA and grown at 37°C for 3 h. Proteins secreted into supernatants were concentrated 20-fold with trichloroacetic acid precipitation, loaded onto SDS-PAGE gels with equivalent bacterial cell numbers, and immunoblotted with antibody against ExoS.
FIG. 3.
FIG. 3.
Expression and secretion of ExoS in PAO1 carrying various DNA fragments from PAK. (A) Overnight cultures carrying various constructs and the exoT-lacZ reporter gene were diluted to 2% in LB with (+) or without (−) 5 mM EGTA plus appropriate antibiotics and grown at 37°C for 4 h before being subjected to β-galactosidase assay. Error bars indicate standard deviations of triplicate assays. Numbers on the y axis are β-galactosidase activity units. (B) Overnight cultures were diluted to 2% in LB with 5 mM EGTA (+) or not (−) plus appropriate antibiotics and grown at 37°C for 3 h. Proteins secreted into supernatants were concentrated 20-fold with trichloroacetic acid precipitation, loaded onto SDS-PAGE gels with equivalent bacterial cell numbers, and immunoblotted with antibody against ExoS. (C) Schematic gene map for the indicated plasmid constructs.
FIG. 4.
FIG. 4.
T3SS secretion by mutant derivatives of PAK and PAO1. (A) Secretion of ExoS and ExoT by the indicated strains under type III-inducing and noninducing conditions. (B) Secretion of ExoS and ExoT in mexS mutants of PAO1 and PAK carrying mexS-complementing plasmid (pYAN0693) or empty vector (pUCP19). (C) Secretion of ExoS by mexT mutants of PAO1 and PAK carrying mexT-complementing plasmid or an empty vector (pUCP21). Overnight cultures were diluted to 2% in LB with (+) or without (−) 5 mM EGTA and grown at 37°C for 3 h.
FIG. 5.
FIG. 5.
Expression of mexE-lacZ fusion in various strain backgrounds. (A) Expression levels of mexE::lacZ fusion in PAO1, PAO1mexS::Ω, PAO1mexT::Gm, and PAK backgrounds. (B) Expression levels of mexE::lacZ fusion in PAO1 carrying indicated constructs. (C) Expression levels of mexE::lacZ fusion in PAO1mexT::Gm carrying mexT- and mexS-complementing plasmids or empty-vector control. Overnight cultures were diluted to 2% in LB with or without 5 mM EGTA and grown at 37°C for 4 h before being subjected to β-galactosidase activity assay. Error bars indicate standard deviations of triplicate assays.
FIG. 6.
FIG. 6.
Expression levels of mexS::lacZ and PA2486::lacZ fusions in PAO1 or PAO1mexT::Gm background. Overnight cultures were diluted to 2% in LB with or without 5 mM EGTA and grown at 37°C for 4 h before being subjected to β-galactosidase activity assay. Error bars indicate standard deviations of triplicate assays.
FIG. 7.
FIG. 7.
Test of protein-protein interactions between MexS and MexT using the Bacterial-Match two-hybrid system. pBT, bait vector; pTRG, target vector; pYAN0699, mexS from PAK cloned into pTRG; pYAN0700, mexS from PAO1 cloned into pTRG; pYAN0701, mexT from PAO1 cloned into pBT. The interaction between popN-TRG and pcr1-BT was used as a positive control.
FIG. 8.
FIG. 8.
Secretion of ExoS by the indicated strains. (A) ExoS secretion by PAO1mexT::Gm carrying different constructs derived from PAO1. Lane 1, PAO1 with pUCP20; lane 2, PAO1mexT::Gm with pUCP20; lane 3, PAO1mexT::Gm with mexT-complementing plasmid (pYX0908); lane 4, PAO1mexT::Gm with PA2486 overexpression plasmid (pYX1004); lane 5, PAO1mexT::Gm with PA2486 ATG→ATC overexpression plasmid (pYX1027); lane 6, PAO1mexT::Gm with PA2485 overexpression plasmid (pYX1030); lane 7, PAO1mexT::Gm with PA2487 overexpression plasmids (pYX1029). (B) ExoS secretion by PAO1mexT::Gm carrying mexS-complementing plasmid pYAN0693 (lane 1) or an empty vector (lane P). (C) ExoS secretion by PAO1mexS::Ω carrying mexT-complementing plasmid pYX0908. Lanes 1 to 3 are three parallel samples, and lane P is empty vector. Overnight cultures were diluted to 2% in LB with 5 mM EGTA plus appropriate antibiotics and grown at 37°C for 3 h. Proteins secreted into supernatants were concentrated with 15% trichloroacetic acid, loaded onto SDS-PAGE gels, and immunoblotted with antibody against ExoS.
FIG. 9.
FIG. 9.
Elastase and pyocyanin secretion assays. (A) Elastolytic activity of indicated strains on an elastin-agar plate incubated at 37°C for 40 h. (B) Pyocyanin production of indicated strains upon overnight growth at 37°C in LB medium.
FIG. 10.
FIG. 10.
Proposed model of MexS/MexT-mediated regulation of the type III secretion system (T3SS), the efflux pump (mexEF-oprN), and elastase and pyocyanin production. “X” denotes a LysR-family transcriptional regulator that is activated by MexS and represses the T3SS directly or indirectly through a downstream repressor.

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