Table 1.
PCR primers used in this study.
Table 2.
Arbitrary PCR primers used in this study.
Fig 1.
Penetration activity of P. aeruginosa strains.
(A) The wild-type strain (WT), PAO1Tn::serA mutant (ΔserA), PAO1Tn::serA (pUCP19-serA) complementary strain (+serA), and PAO1Tn::flgE mutant (ΔflgE) were inoculated onto the apical surfaces of Caco-2 cell monolayers at an MOI of 100, and the number of bacteria in the basolateral medium was counted at 6 h after infection. The assay was performed in triplicate, and the results are expressed as the mean ± SD. E. coli DH5α was used as a negative control. *#: P < 0.05; *: vs WT; #: vs ΔserA (B) Influence of L-serine addition on penetration activity of the wild-type strain. The assay was performed in triplicate, and the results are expressed as mean ± SD. *: P < 0.05; *: vs WT.
Table 3.
Specification of genes responsible for penetration ability.
Fig 2.
(A) Western blot analysis to detect secretion of ExoS into the culture supernatant using type III secretion system-inducing conditions in the wild-type strain (WT), PAO1Tn::serA mutant (ΔserA), PAO1Tn::serA (pUCP19-serA) complementary strain (+serA), and WT in the presence of L-serine (30, 40, and 50 mM). A representative western blot image is shown. The arrow indicates the presence of ExoS with a deduced molecular weight of 48 kDa. (B) Expression ratio of ExoS based on western blot analyses of WT, ΔserA, +serA, and WT in the presence of 50 mM L-serine. Western blotting was repeated three times, and bands were quantified by ImageJ. Data is shown as the ratio of ExoS expression to that of the wild-type strain and is expressed as the mean ± SD. A significant difference was observed for ExoS expression between WT and ΔserA (*: P < 0.05).
Fig 3.
Staining of bacterial flagella.
Flagella staining of the wild-type strain (WT), PAO1Tn::serA mutant (ΔserA), PAO1Tn::serA (pUCP19-serA) complementary strain (+serA), and PAO1Tn::flgE mutant (ΔflgE) was performed as previously described [35].
Fig 4.
(A) Swimming motility of WT, ΔserA, +serA, and E. coli DH5α (as a negative control). A representative image from the swimming motility assay is shown. The major axis of swimming is the longest length of the swimming area. The assay was performed in triplicate, and the results are expressed as the mean ± SD. Significant differences were observed between WT and ΔserA (*: P < 0.05), between WT and +serA (#: P < 0.05), and between WT and DH5α (*: P < 0.05). (B) Swarming motility in the wild-type strain (WT), PAO1Tn::serA mutant (ΔserA), PAO1Tn::serA (pUCP19-serA) complementary strain (+serA), and E. coli DH5α (as a negative control). A representative image of the swarming motility assay is shown. The major axis of swarming is the longest length of the swarming area. The assay was performed in triplicate, and the results are expressed as the mean ± SD. Significant differences were observed between WT and ΔserA (*: P < 0.05), between WT and +serA (#: P < 0.05), and between WT and DH5α (*: P < 0.05). (C) Twitching motility in WT, ΔserA, +serA, and E. coli DH5α (as a negative control). A representative image of the twitching motility assay is shown. The major axis of twitching is the longest length of the twitching area. The assay was performed in triplicate, and the results are expressed as the mean ± SD. Significant difference was observed between WT and DH5α (*: P < 0.05).
Fig 5.
Influence of L-serine addition on swimming motility in the wild-type strain.
Swimming motility in the wild-type strain (WT), PAO1Tn::serA mutant (ΔserA), PAO1Tn::flgE mutant (ΔflgE) (as a negative control), and WT in the presence of L-serine (20, 30, 40, and 50 mM). A representative image from the swarming motility assay is shown. The major axis of swimming is the longest length of the swimming area. The assay was performed in triplicate, and the results are expressed as the mean ± SD. Significant differences were observed between WT and ΔserA (*: P < 0.05), between WT and ΔflgE (*: P < 0.05), between WT and WT in the presence of 20 mM L-serine (*: P < 0.05), between WT and WT in the presence of 30 mM L-serine (*: P < 0.05), between WT and WT in the presence of 40 mM L-serine (*: P < 0.05), and between WT and WT in the presence of 50 mM L-serine (*: P < 0.05).
Fig 6.
Influence of L-serine addition on swarming motility in the wild-type strain.
Swarming motility of the wild-type strain (WT), PAO1Tn::serA mutant (ΔserA), PAO1Tn::flgE mutant (ΔflgE) (as a negative control), and WT in the presence of L-serine (20, 30, 40, and 50 mM). A representative image of the swarming motility assay is shown. The major axis of swarming is the longest length of the swarming area. The assay was performed in triplicate, and the results are expressed as the mean ± SD. Significant differences were observed between WT and ΔserA (*: P < 0.05), between WT and ΔflgE (*: P < 0.05), between WT and WT in the presence of 30 mM L-serine (*: P < 0.05), between WT and WT in the presence of 40 mM L-serine (*: P < 0.05), and between WT and WT in the presence of 50 mM L-serine (*: P < 0.05).
Fig 7.
Bacterial adherence to Caco-2 cells for the wild-type strain (WT), PAO1Tn::serA mutant (ΔserA), PAO1Tn::serA (pUCP19-serA) complementary strain (+serA), PAO1Tn::flgE mutant (ΔflgE), E. coli DH5α (as a negative control), and WT in the presence of L-serine (20, 30, 40, and 50 mM).
Bacterial adherence was determined based on the number of adhered bacteria per Caco-2 cell. The assay was performed in six replicates, and the results are expressed as the mean ± SD. Significant differences were observed between WT and ΔserA (*: P < 0.05), between WT and DH5α (*: P < 0.05), between WT and ΔflgE (*: P < 0.05), between WT and WT in the presence of 30 mM L-serine (*: P < 0.05), between WT and WT in the presence of 40 mM L-serine (*: P < 0.05), and between WT and WT in the presence of 50 mM L-serine (*: P < 0.05). Bacterial adherence of the +serA complementary strain was significantly restored as compared to that of ΔserA (#; P < 0.05).
Fig 8.
(A) SDS-PAGE analysis to verify overexpression of the P. aeruginosa serA and synthesis of SerA proteins in crude extract isolated from DH5α (ptac-85-serA) culture (SerA) and from DH5α (ptac-85) culture (mock). The red arrow indicates an overexpressed band at the expected size of 44 kDa for P. aeruginosa SerA. (B) Inhibitory effect of 50 mM L-serine on PGDH activity of SerA proteins (mU/mg) in crude extract isolated from DH5α (ptac-85-serA) culture (SerA) and from DH5α (ptac-85) culture (mock). The assay was performed in five replicates, and the results are expressed as the mean ± SD. Significant differences were observed between SerA and mock (*: P < 0.05), and between SerA and SerA in the presence of 50 mM L-serine (*: P < 0.05). There was no significant difference between mock and mock in the presence of 50 mM L-serine (ns: P > 0.9).
Fig 9.
Virulence of the wild-type strain (WT), PAO1Tn::serA mutant (ΔserA), PAO1Tn::serA (pUCP19-serA) complementary strain (+serA), E. coli DH5α (as a negative control), and WT in the presence of L-serine (10, 20, 30, 40, and 50 mM) was evaluated. Significant differences, based on the log-rank test, were observed between WT and DH5α (*: P < 0.05), between WT and ΔserA (*: P < 0.05), between WT and WT in the presence of 50 mM L-serine (*: P < 0.05), between WT and WT in the presence of 40 mM L-serine (*: P < 0.05), between WT and WT in the presence of 20 mM L-serine (*: P < 0.05), between WT and WT in the presence of 30 mM L-serine (*: P < 0.05), and between WT and WT in the presence of 10 mM L-serine (*: P < 0.05). Virulence of the +serA complementary strain was significantly restored as compared to that of ΔserA (#; P < 0.05).