Fig 1.
LPS structure comparison of E. coli strains W3110, HW001, HW002, WBB06, HWB01 and HWB02.
Red numbers specify the glucosamine ring positions, and the black numbers indicate the predominant fatty acid chain lengths. The enzymes WaaC, WaaF, LpxM and LpxE are shown in res with arrows indicating their functional domains, and the groups removed by these enzymes are shown in blue. Expression of lpxE in W3110 forms the strain HW001 that synthesizes PS-MPLA, and deletion of lpxM in HW001 forms the strain HW002 that synthesizes PS-pentaacyl-MPLA. Inactivation of waaC and waaF in W3110, HW001 and HW002 resulted in strains WBB06, HWB01 and HWB02, respectively. WBB06, HWB01, and HWB02 synthesize Kdo2-lipid A, Kdo2-MPLA and Kdo2-pentaacyl-MPLA, respectively.
Table 1.
Bacterial strains and plasmids used in this study.
Fig 2.
Crude lipids were isolated from WBB06, HWB01 and HWB02 cells and analyzed by TLC and ESI/MS.
(A) TLC analysis. The lipid samples were dissolved in a solvent of chloroform and methanol (2:1, v/v) and developed in a solvent mixture of chloroform, methanol, acetic acid and water (25:15:4:4; v/v/v/v). (B) ESI/MS analysis.
Fig 3.
Comparison of the membrane permeability (A), surface hydrophobicity (B) and auto-aggregation (C) of E. coli strains W3110, HW001, HW002, WBB06, HWB01 and HWB02.
Error bars indicate the standard deviations from three parallel samples. *, p <0.05; **, p<0.01.
Table 2.
Comparison of antibiotic sensitivities of E. coli strains W3110, WBB06, HW001, HWB01, HW002 and HWB02.
Table 3.
CFU values (CFU/mL) of E. coli strains W3110, WBB06, HW001, HWB01, HW002 and HWB02 at OD600 of 0.02 before and after treatment with antibiotics of MIC as listed in Table 2.
Fig 4.
Comparison of TLR4 stimulation of whole cells of E. coli strains W3110, HW001, HW002, WBB06, HWB01 and HWB02.
The SEAP activity was assessed by reading the OD630 with a microplate reader. *, p <0.05; **, p<0.01.
Fig 5.
Immunogenicity of purified LPS, PS-MPLA, PS-pentaacyl-MPLA, Kdo2-lipid A, Kdo2-MPLA and Kdo2-pentaacyl-MPLA from W3110, HW001, HW002, WBB06, HWB01 and HWB02, respectively.
Levels of the MyD88 pathway cytokines IL-6 (A), IL-8 (D), TNF-α (B, E), and the TRIF pathway cytokine RANTES (C, F) were measured by ELISA. Error bars indicate the standard deviations from three parallel samples. *, p <0.05; **, p<0.01.