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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.

View Article: PubMed Central - PubMed

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

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A, electron microscopy of GMMA from different strains. GMMA were purified from Ss−p − OAg and Sf2a−p − OAg containing wild-type lipid A (Ss−p − OAgWT lip. A, Sf2a−p − OAgWT lip. A), Ss−p − OAg ΔmsbB, Sf2a−p − OAg ΔmsbB, Ss−p − OAg ΔhtrB, and Sf2a−p − OAg ΔhtrB, negatively stained, and viewed by electron microscopy (105,000-fold magnification) revealing the presence of well organized membrane particles with diameters ranging between 17 and 53 nm in each preparation. Bar length, 100 nm. B, SDS-PAGE. 10 μg (protein) of the GMMA shown in A and GMMA from Ss−p − OAg ΔhtrB (pACYChtrB) and Sf2a−p − OAg ΔhtrB (pACYChtrB) were separated by SDS-PAGE (12% polyacrylamide) and Coomassie stained. Four protein bands that were more abundant in GMMA from Sf2a−p − OAg ΔhtrB than in GMMA from other Sf2a−p − OAg strains were identified by peptide mass fingerprinting: 1, pyruvate dehydrogenase; 2, glutamine synthetase; 3, ketol-acid reductoisomerase; 4,d-3-phosphoglycerate dehydrogenase.
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Figure 1: A, electron microscopy of GMMA from different strains. GMMA were purified from Ss−p − OAg and Sf2a−p − OAg containing wild-type lipid A (Ss−p − OAgWT lip. A, Sf2a−p − OAgWT lip. A), Ss−p − OAg ΔmsbB, Sf2a−p − OAg ΔmsbB, Ss−p − OAg ΔhtrB, and Sf2a−p − OAg ΔhtrB, negatively stained, and viewed by electron microscopy (105,000-fold magnification) revealing the presence of well organized membrane particles with diameters ranging between 17 and 53 nm in each preparation. Bar length, 100 nm. B, SDS-PAGE. 10 μg (protein) of the GMMA shown in A and GMMA from Ss−p − OAg ΔhtrB (pACYChtrB) and Sf2a−p − OAg ΔhtrB (pACYChtrB) were separated by SDS-PAGE (12% polyacrylamide) and Coomassie stained. Four protein bands that were more abundant in GMMA from Sf2a−p − OAg ΔhtrB than in GMMA from other Sf2a−p − OAg strains were identified by peptide mass fingerprinting: 1, pyruvate dehydrogenase; 2, glutamine synthetase; 3, ketol-acid reductoisomerase; 4,d-3-phosphoglycerate dehydrogenase.

Mentions: GMMA from Shigella strains carrying different mutations showed similar morphology by electron microscopy (Fig. 1A) with average sizes of 30–32 nm in all six strains and a size distribution of 17–53 nm, measured with 30 GMMA per strain. A comparison of the GMMA sizes from all strains gave no significant difference (p = 0.90). To characterize whether the genetic lipid A modifications might alter the protein composition of GMMA, the protein pattern of GMMA from the different mutants was evaluated by SDS-PAGE (Fig. 1B). Although the overall pattern remained similar, four protein bands, identified as pyruvate dehydrogenase, glutamine synthetase, ketol-acid recutoisomerase, and d-3-phosphoglycerate dehydrogenase (Fig. 1B) by peptide mass fingerprinting, were found to be up-regulated in GMMA from Sf2a−p − OAg ΔhtrB. As these proteins are cytoplasmic proteins, no effect on the reactogenicity studies was expected.


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)

A, electron microscopy of GMMA from different strains. GMMA were purified from Ss−p − OAg and Sf2a−p − OAg containing wild-type lipid A (Ss−p − OAgWT lip. A, Sf2a−p − OAgWT lip. A), Ss−p − OAg ΔmsbB, Sf2a−p − OAg ΔmsbB, Ss−p − OAg ΔhtrB, and Sf2a−p − OAg ΔhtrB, negatively stained, and viewed by electron microscopy (105,000-fold magnification) revealing the presence of well organized membrane particles with diameters ranging between 17 and 53 nm in each preparation. Bar length, 100 nm. B, SDS-PAGE. 10 μg (protein) of the GMMA shown in A and GMMA from Ss−p − OAg ΔhtrB (pACYChtrB) and Sf2a−p − OAg ΔhtrB (pACYChtrB) were separated by SDS-PAGE (12% polyacrylamide) and Coomassie stained. Four protein bands that were more abundant in GMMA from Sf2a−p − OAg ΔhtrB than in GMMA from other Sf2a−p − OAg strains were identified by peptide mass fingerprinting: 1, pyruvate dehydrogenase; 2, glutamine synthetase; 3, ketol-acid reductoisomerase; 4,d-3-phosphoglycerate dehydrogenase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4155660&req=5

Figure 1: A, electron microscopy of GMMA from different strains. GMMA were purified from Ss−p − OAg and Sf2a−p − OAg containing wild-type lipid A (Ss−p − OAgWT lip. A, Sf2a−p − OAgWT lip. A), Ss−p − OAg ΔmsbB, Sf2a−p − OAg ΔmsbB, Ss−p − OAg ΔhtrB, and Sf2a−p − OAg ΔhtrB, negatively stained, and viewed by electron microscopy (105,000-fold magnification) revealing the presence of well organized membrane particles with diameters ranging between 17 and 53 nm in each preparation. Bar length, 100 nm. B, SDS-PAGE. 10 μg (protein) of the GMMA shown in A and GMMA from Ss−p − OAg ΔhtrB (pACYChtrB) and Sf2a−p − OAg ΔhtrB (pACYChtrB) were separated by SDS-PAGE (12% polyacrylamide) and Coomassie stained. Four protein bands that were more abundant in GMMA from Sf2a−p − OAg ΔhtrB than in GMMA from other Sf2a−p − OAg strains were identified by peptide mass fingerprinting: 1, pyruvate dehydrogenase; 2, glutamine synthetase; 3, ketol-acid reductoisomerase; 4,d-3-phosphoglycerate dehydrogenase.
Mentions: GMMA from Shigella strains carrying different mutations showed similar morphology by electron microscopy (Fig. 1A) with average sizes of 30–32 nm in all six strains and a size distribution of 17–53 nm, measured with 30 GMMA per strain. A comparison of the GMMA sizes from all strains gave no significant difference (p = 0.90). To characterize whether the genetic lipid A modifications might alter the protein composition of GMMA, the protein pattern of GMMA from the different mutants was evaluated by SDS-PAGE (Fig. 1B). Although the overall pattern remained similar, four protein bands, identified as pyruvate dehydrogenase, glutamine synthetase, ketol-acid recutoisomerase, and d-3-phosphoglycerate dehydrogenase (Fig. 1B) by peptide mass fingerprinting, were found to be up-regulated in GMMA from Sf2a−p − OAg ΔhtrB. As these proteins are cytoplasmic proteins, no effect on the reactogenicity studies was expected.

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