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