Abstract
BackgroundWe 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.ResultsOptimum 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 (IC50) of l-serine was 630 μM for P. aeruginosa and 250 μM for E. coli, while IC50 of d-serine was much higher than that of l-serine; 76 mM in P. aeruginosa PGDH and 45 mM in E. coli PGDH.ConclusionsThese 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.
Highlights
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 show that in a PGDH assay using crude extracts that were isolated from overnight cultures of E. coli overexpressing the P. aeruginosa serA gene, l-serine directly inhibited the PGDH activity of the SerA protein
Cloning of P. aeruginosa serA gene To construct the plasmid used for the expression of glutathione S-transferase (GST)—P. aeruginosa PGDH fusion protein, BamHI–XhoI fragment carrying P. aeruginosa serA ORF was amplified by LA Taq DNA polymerase (TAKARA) under conditions recommended in the manufacturer’s protocol with 5-PA-serA-BamHI-ATG-356477 (5ʹ-GAGAGGATCCATGAGCAAGACCTCTCTCGA-3ʹ) and 3-PA-serAXho1-end-355248 primers (5ʹ-GAGACTCGAGTTAGAACAGCACGCGGCTAC-3ʹ); this insert corresponds to the nucleotides 355248 to 356477 in the PAO1 genome sequence
Summary
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 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. We 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 addition of l-serine was found to significantly reduce the phenotypes associated with the bacterial pathogenicity, including bacterial penetration through Caco-2 cell monolayers, bacterial swimming and swarming motility, bacterial adherence, and fly mortality [1]. We show that in a PGDH assay using crude extracts that were isolated from overnight cultures of E. coli overexpressing the P. aeruginosa serA gene, l-serine directly inhibited the PGDH activity of the SerA protein. To further confirm the direct inhibition of PGDH activity of P. aeruginosa SerA by l-serine, we purified the PGDH from P. aeruginosa using the glutathione S-transferase (GST) fusion protein system which is used for high-level expression and efficient purification of recombinant proteins
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