Fig 1.
The T3SS operon exsCEBA in P. aeruginosa was positively regulated by an adenosine tRNA methylthiotransferase PA3980 (MiaB).
(A) β-galactosidase activity of the PexsCEBA-lacZ transcriptional fusion in the wide-type PAO1 strain, ΔPA3980, and ΔPA3980 with in trans expression of PA3980. (B) β-galactosidase activity of the PexsCEBA-lacZ transcriptional fusion in the wide-type PAO1 strain, ΔPA3980, and ΔPA3980 with in trans expression of miaB from E. coli (miaB-E). (C) LC-MS measurement of the tRNA A37 N6-isopentenyladenosine (i6A) and 2-methylthio-N6-isopentenyladenosine (ms2i6A) in the wide-type PAO1 strain and ΔPA3980 mutant. ns, not significant; ***, P < 0.001 compared to the wild-type PAO1 strain based on Student’s t test.
Fig 2.
MiaB positively regulated T3SS gene expression and was necessary for the virulence in P. aeruginosa.
(A) Five clustered operons involved in the T3SS in P. aeruginosa. Orange: needle complex; Blue: translocation apparatus; Pink: regulation. (B) Relative expression of T3SS genes in the wild-type PAO1 and ΔmiaB mutant measured by RT-qPCR. (C) Productions of the cytosol ExsA protein and the secreted ExoS and PcrV proteins determined by western blot in the strains of wide-type PAO1, ΔmiaB, and ΔmiaB with in trans expression of miaB. ECP: extracellular protein, ICP: intracellular protein. RNA polymerase (RNAP) was used as an internal control for ICP. ECPs were normalized with the cell density. (D) Cytotoxicity was evaluated by monitoring LDH release from the A549 cells infected by the strains of wide-type PAO1, ΔmiaB, and ΔmiaB with in trans expression of miaB. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to the wild-type PAO1 strain based on Student’s t test.
Fig 3.
MiaB modulated T3SS gene expression through the master regulator ExsA.
(A) β-galactosidase activity of the PexsCEBA-lacZ transcriptional fusion in the wide-type PAO1 strain, ΔmiaB, ΔexsA mutants and mutants with in trans expression of exsA or miaB. (B) Productions of ExsA, ExoS and PcrV proteins measured by western blot in the wide-type PAO1 strain, ΔmiaB, ΔexsA mutants and mutants with in trans expression of exsA or miaB. ns, not significant; **, P < 0.01; ***, P < 0.001 compared to the indicated group based on Student’s t test.
Fig 4.
LadS was involved in the MiaB-mediated regulation of T3SS gene expression.
(A) Relative fluorescence intensity of the pLadS-gfp transcriptional fusion measured in the wild-type PAO1 strain and the ΔmiaB mutant. (B) Relative fluorescence intensity of the pRetS-gfp transcriptional fusion measured in the wild-type PAO1 strain and the ΔmiaB mutant. (C) Relative expression of the ladS gene in the wild-type PAO1 and ΔmiaB mutant measured by RT-qPCR. (D) Relative fluorescence intensity of the pLadS-gfp transcriptional fusion measured in the wild-type PAO1 strain and the ΔmiaB mutant when cell growth at the OD600 of 0.5, 1.0, 1.5 and 2.0. (E) β-galactosidase activity of the PexsCEBA-lacZ transcriptional fusion in the wide-type PAO1 strain, ΔmiaB, ΔladS and the ΔmiaBΔladS mutants as well as the ΔmiaBΔladS mutant with in trans expression of miaB and ladS. (F) Production of the ExoS protein measured by western blot in the wide-type PAO1 strain, ΔmiaB, ΔladS and the ΔmiaBΔladS mutants as well as the ΔmiaBΔladS mutant with in trans expression of miaB and ladS. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; compared to the wild-type PAO1 strain or indicated group based on Student’s t test.
Fig 5.
gacA, rsmY, rsmZ were repressed by MiaB to activate T3SS gene expression.
(A) Relative expression of gacA, rsmY and rsmZ measured by RT-qPCR in the strains of wide-type PAO1, ΔmiaB, and ΔmiaB with in trans expression of miaB. (B) Relative expression of gacA measured by RT-qPCR in the strains of wide-type PAO1, ΔmiaB, ΔladS and ΔmiaBΔladS. (C) Relative expression of rsmY and rsmZ measured by RT-qPCR in the strains of wide-type PAO1, ΔmiaB, ΔgacA and ΔmiaBΔgacA. (D) Production of the ExoS protein measured by western blot in the wide-type PAO1 strain, ΔmiaB, ΔgacA, ΔrsmY, ΔrsmZ and double or triple deletion mutants. ns, not significant; **, P < 0.01; ***, P < 0.001 compared to the wild-type PAO1 strain based on Student’s t test.
Fig 6.
MiaB was positively regulated by the transcription regulator Vfr.
(A) Expression of vfr and miaB in the strains of ΔmiaB and Δvfr relative to that in the wild-type PAO1 strain was measured by RT-qPCR, respectively. (B) β-galactosidase activity of the PexsCEBA-lacZ transcriptional fusion in the wide-type PAO1 strain, Δvfr, and Δvfr with in trans expression of miaB. (C) Productions of the intracellular ExsA and extracellular ExoS proteins measured by western blot in the wide-type PAO1 strain, Δvfr, and Δvfr with in trans expression of miaB. (D) β-galactosidase activity of the PexsCEBA-lacZ transcriptional fusion in the wide-type PAO1, ΔmiaB, Δvfr, and ΔmiaBΔvfr strains. (E) Productions of the intracellular ExsA and extracellular ExoS proteins measured by western blot in the wide-type PAO1, ΔmiaB, Δvfr, and ΔmiaBΔvfr strains. ns, not significant; ***, P < 0.001 compared to the wild-type PAO1 strain or the indicated group based on Student’s t test.
Fig 7.
Disruption of the spermidine transporter by deleting spuE led to repressed expression of miaB and T3SS genes.
(A) Expression of spuE and miaB in the strains of ΔmiaB and Δvfr relative to that in the wild-type PAO1 strain was measured by RT-qPCR, respectively. (B) β-galactosidase activity of the PexsCEBA-lacZ transcriptional fusion in the wide-type PAO1 strain, ΔspuE, and ΔspuE with in trans expression of miaB. (C) Productions of the intracellular ExsA and extracellular ExoS proteins measured by western blot in the wide-type PAO1 strain, ΔspuE, and ΔspuE with in trans expression of miaB. (D) β-galactosidase activity of the PexsCEBA-lacZ transcriptional fusion in the wide-type PAO1, ΔmiaB, ΔspuE, and ΔmiaBΔspuE strains. (E) Productions of the intracellular ExsA and extracellular ExoS proteins measured by western blot in the wide-type PAO1, ΔmiaB, ΔspuE, and ΔmiaBΔspuE strains. ns, not significant; **, P < 0.01; ***, P < 0.001 compared to the wild-type PAO1 strain or the indicated group based on Student’s t test.
Fig 8.
A diagram showing the MiaB-mediated regulation of T3SS in P. aeruginosa.
The adenosine tRNA methylthiotransferase MiaB was upregulated by the cAMP-dependent regulator Vfr and the spermidine transporter-dependent pathway. MiaB independently repressed the expression of ladS, gacA, rsmY and rsmZ in the LadS-Gac/Rsm signaling pathway, which released RsmA to activate the transcription of exsCEBA and T3SS gene expression through the intrinsic regulator ExsA. Solid lines indicate direct regulations while dashed lines indicate probable indirect regulations. Arrows and T-shaped symbols indicate upregulation (activation) and repression, respectively.