To specify critical factors responsible for Pseudomonas aeruginosa penetration through the Caco-2 cell epithelial barrier, we analyzed transposon insertion mutants that demonstrated a dramatic reduction in penetration activity relative to P. aeruginosa PAO1 strain. From these strains, mutations could be grouped into five classes, specifically flagellin-associated genes, pili-associated genes, heat-shock protein genes, genes related to the glycolytic pathway, and biosynthesis-related genes. Of these mutants, we here focused on the serA mutant, as the association between this gene and penetration activity is yet unknown. Inactivation of the serA gene caused significant repression of bacterial penetration through Caco-2 cell monolayers with decreased swimming and swarming motilities, bacterial adherence, and fly mortality rate, as well as repression of ExoS secretion; however, twitching motility was not affected. Furthermore, L-serine, which is known to inhibit the D-3-phosphoglycerate dehydrogenase activity of the SerA protein, caused significant reductions in penetration through Caco-2 cell monolayers, swarming and swimming motilities, bacterial adherence to Caco-2 cells, and virulence in flies in the wild-type P. aeruginosa PAO1 strain. Together, these results suggest that serA is associated with bacterial motility and adherence, which are mediated by flagella that play a key role in the penetration of P. aeruginosa through Caco-2 cell monolayers. Oral administration of L-serine to compromised hosts might have the potential to interfere with bacterial translocation and prevent septicemia caused by P. aeruginosa through inhibition of serA function.

Pseudomonas aeruginosa can penetrate through polarized epithelial cell monolayers produced by the human adenocarcinoma cell line Caco-2. We previously identified genes associated with bacterial translocation through Caco-2 cell monolayers by analysing transposon insertion mutants with dramatically reduced penetration activity relative to that of the wild-type P. aeruginosa PAO1 strain. In this study, we focused on the dnaK mutant because the association between this gene and penetration activity is unknown. Inactivation of dnaK caused significant repression of bacterial penetration through Caco-2 cell monolayers, with decreased swimming, swarming and twitching motilities; bacterial adherence; and fly mortality rate; as well as dramatic repression of type III effector secretion and production of elastase and exotoxin A. However, type IV pilus protein PilA expression was not affected. These results suggest that dnaK is associated with bacterial motility and adherence, which are mediated by flagella and pili, and with toxin secretion, which plays a key role in the penetration of P. aeruginosa through Caco-2 cell monolayers. Inactivation of P. aeruginosa dnaK function may interfere with bacterial translocation and prevent septicaemia caused by P. aeruginosa.

Background We previously demonstrated that the serA gene is associated with bacterial pathogenicity, including bacterial penetration through the Caco-2 cell monolayers, bacterial motility, bacterial adherence, and fly mortality. l -Serine is known to inhibit the d -3-phosphoglycerate dehydrogenase (PGDH) activity of the SerA protein, and it significantly reduced the bacterial pathogenicity as described above. We also demonstrated that in a PGDH assay using crude extracts isolated from overnight cultures of E . coli overexpressing the P . aeruginosa serA gene, l -serine inhibited the PGDH activity of the SerA protein. The basal PGDH activity of the negative control strain was high, presumably due to contamination of unknown proteins in the crude extracts. Therefore, to further confirm the direct inhibition of PGDH activity of P . aeruginosa SerA by l -serine, we purified and characterized the PGDH from P. aeruginosa and compared it with the previously characterized PGDHs from E. coli , and the human colon as controls. Results Optimum pH and ionic strength of the purified PGDHs were different depending on the three species; optimal activity of P. aeruginosa PGDH was at pH 7.5 with 50–100 mM Tris–HCl, E. coli PGDH was at pH 8.5 with 100–200 mM Tris–HCl, and human PGDH was at pH 9.0 with 100–200 mM Tris–HCl. The addition of l -serine reduced the activity of PGDH from P. aeruginosa and E. coli , but not the PGDH from human colon. The median inhibitory concentration (IC 50 ) of l -serine was 630 μM for P. aeruginosa and 250 μM for E. coli , while IC 50 of d -serine was much higher than that of l -serine; 76 mM in P. aeruginosa PGDH and 45 mM in E. coli PGDH. Conclusions These results suggest that l -serine significantly repressed P. aeruginosa pathogenicity through direct inhibition of the PGDH activity, but was not able to inhibit the human PGDH activity. Oral administration of l -serine to compromised hosts might interfere with bacterial translocation and prevent gut-derived sepsis caused by P. aeruginosa through inhibition of the function of the serA gene product.

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