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Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants.

Rossi O, Pesce I, Giannelli C, Aprea S, Caboni M, Citiulo F, Valentini S, Ferlenghi I, MacLennan CA, D'Oro U, Saul A, Gerke C - J. Biol. Chem. (2014)

Bottom Line: GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line.We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation.The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development.

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Affiliation: From the Novartis Vaccines Institute for Global Health and.

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MALDI-TOF spectra of lipid A preparations in reflectron ion-negative mode. Lipid A was extracted from GMMA from the following: A, Ss−p − OAg; B, Sf2a−p − OAg; C, Ss−p − OAg ΔmsbB; D, Sf2a−p − OAg ΔmsbB; E, Ss−p − OAg ΔhtrB; F, Sf2a−p + OAg ΔhtrB; H, Ss−p − OAg ΔhtrB (pACYChtrB); I, Sf2a−p − OAg ΔhtrB (pACYChtrB); J, Ss−p − OAg ΔhtrB grown at 12 °C; and K, Sf2a−p + OAg ΔhtrB. G, overlay of negative ion LIFT MALDI-TOF/TOF spectra in the low m/z range of the dominant species in lipid A from Ss−p − OAg ΔhtrB (E) and Sf2a−p − OAg ΔhtrB (F) after collision-induced dissociation. L, lipid A structures with molecular weights corresponding to the observed main peaks.
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Figure 2: MALDI-TOF spectra of lipid A preparations in reflectron ion-negative mode. Lipid A was extracted from GMMA from the following: A, Ss−p − OAg; B, Sf2a−p − OAg; C, Ss−p − OAg ΔmsbB; D, Sf2a−p − OAg ΔmsbB; E, Ss−p − OAg ΔhtrB; F, Sf2a−p + OAg ΔhtrB; H, Ss−p − OAg ΔhtrB (pACYChtrB); I, Sf2a−p − OAg ΔhtrB (pACYChtrB); J, Ss−p − OAg ΔhtrB grown at 12 °C; and K, Sf2a−p + OAg ΔhtrB. G, overlay of negative ion LIFT MALDI-TOF/TOF spectra in the low m/z range of the dominant species in lipid A from Ss−p − OAg ΔhtrB (E) and Sf2a−p − OAg ΔhtrB (F) after collision-induced dissociation. L, lipid A structures with molecular weights corresponding to the observed main peaks.

Mentions: The lipid A of LPS of the mutants was extracted and analyzed by MALDI-TOF. The spectra are reported in Fig. 2, and the structures of lipid A corresponding to the main peaks were assigned on the basis of mass and by comparison of results with similar mutants of E. coli (Fig. 2L) (40). The main peaks in the mass spectra obtained by MALDI-TOF from lipid A purified from GMMA from S. sonnei and S. flexneri 2a strains with wild-type (WT) LPS (Fig. 2, Ss−p − OAg (A) and Sf2a−p − OAg (B)) had an m/z corresponding to the theoretical mass of the hexa-acylated lipid A of 1,798 Da. The main peaks obtained by mass spectrometry from the Ss−p − OAg ΔmsbB GMMA (Fig. 2C) and Sf2a−p − OAg ΔmsbB (Fig. 2D) GMMA corresponded, in both strains, to a penta-acylated lipid A lacking a myristoyl chain (theoretical mass 1,588 Da, 210 m/z shift to WT lipid A due to the absence of a C14 fatty acid chain) consistent with msbB knock outs.


Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants.

Rossi O, Pesce I, Giannelli C, Aprea S, Caboni M, Citiulo F, Valentini S, Ferlenghi I, MacLennan CA, D'Oro U, Saul A, Gerke C - J. Biol. Chem. (2014)

MALDI-TOF spectra of lipid A preparations in reflectron ion-negative mode. Lipid A was extracted from GMMA from the following: A, Ss−p − OAg; B, Sf2a−p − OAg; C, Ss−p − OAg ΔmsbB; D, Sf2a−p − OAg ΔmsbB; E, Ss−p − OAg ΔhtrB; F, Sf2a−p + OAg ΔhtrB; H, Ss−p − OAg ΔhtrB (pACYChtrB); I, Sf2a−p − OAg ΔhtrB (pACYChtrB); J, Ss−p − OAg ΔhtrB grown at 12 °C; and K, Sf2a−p + OAg ΔhtrB. G, overlay of negative ion LIFT MALDI-TOF/TOF spectra in the low m/z range of the dominant species in lipid A from Ss−p − OAg ΔhtrB (E) and Sf2a−p − OAg ΔhtrB (F) after collision-induced dissociation. L, lipid A structures with molecular weights corresponding to the observed main peaks.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: MALDI-TOF spectra of lipid A preparations in reflectron ion-negative mode. Lipid A was extracted from GMMA from the following: A, Ss−p − OAg; B, Sf2a−p − OAg; C, Ss−p − OAg ΔmsbB; D, Sf2a−p − OAg ΔmsbB; E, Ss−p − OAg ΔhtrB; F, Sf2a−p + OAg ΔhtrB; H, Ss−p − OAg ΔhtrB (pACYChtrB); I, Sf2a−p − OAg ΔhtrB (pACYChtrB); J, Ss−p − OAg ΔhtrB grown at 12 °C; and K, Sf2a−p + OAg ΔhtrB. G, overlay of negative ion LIFT MALDI-TOF/TOF spectra in the low m/z range of the dominant species in lipid A from Ss−p − OAg ΔhtrB (E) and Sf2a−p − OAg ΔhtrB (F) after collision-induced dissociation. L, lipid A structures with molecular weights corresponding to the observed main peaks.
Mentions: The lipid A of LPS of the mutants was extracted and analyzed by MALDI-TOF. The spectra are reported in Fig. 2, and the structures of lipid A corresponding to the main peaks were assigned on the basis of mass and by comparison of results with similar mutants of E. coli (Fig. 2L) (40). The main peaks in the mass spectra obtained by MALDI-TOF from lipid A purified from GMMA from S. sonnei and S. flexneri 2a strains with wild-type (WT) LPS (Fig. 2, Ss−p − OAg (A) and Sf2a−p − OAg (B)) had an m/z corresponding to the theoretical mass of the hexa-acylated lipid A of 1,798 Da. The main peaks obtained by mass spectrometry from the Ss−p − OAg ΔmsbB GMMA (Fig. 2C) and Sf2a−p − OAg ΔmsbB (Fig. 2D) GMMA corresponded, in both strains, to a penta-acylated lipid A lacking a myristoyl chain (theoretical mass 1,588 Da, 210 m/z shift to WT lipid A due to the absence of a C14 fatty acid chain) consistent with msbB knock outs.

Bottom Line: GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line.We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation.The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development.

View Article: PubMed Central - PubMed

Affiliation: From the Novartis Vaccines Institute for Global Health and.

Show MeSH
Related in: MedlinePlus