Construction of acetate auxotrophs of Neisseria meningitidis to study host-meningococcal endotoxin interactions

To facilitate studies of the molecular determinants of host-meningococcal lipooligosaccharide (endotoxin) interactions at patho-physiologically relevant endotoxin concentrations (i.e. < or =10 ng/ml), we have generated acetate auxotrophs NMBACE1 from encapsulated Neisseria meningitidis (serogroup B, strain NMB) and NMBACE2 from an isogenic bacterial mutant lacking the polysialic acid capsule. Growth of the auxotrophs in medium containing [(14)C]acetate yielded (14)C-lipooligosaccharides containing approximately 600 cpm/ng. Gel sieving resolved 14C-lipooligosaccharide-containing aggregates with an estimated molecular mass of > or =20 x 10(6) Da (peak A) and approximately 1 x 10(6) Da (peak B) from both strains. Lipooligosaccharides in peaks A and B had the same fatty acid composition and SDS-polyacrylamide gel electrophoresis profile. 14C-Labeled capsule copurified with (14)C-lipooligosaccharides in peak B from NMBACE1, whereas the other aggregates contained only 14C-lipooligosaccharide. For all aggregates, lipopolysaccharide-binding protein and soluble CD14-induced delivery of lipooligosaccharides to endothelial cells and cell activation correlated with disaggregation of lipooligosaccharides. These processes were inhibited by the presence of capsule but unaffected by the size of the aggregates. In contrast, endotoxin activation of cells containing membrane CD14 was unaffected by capsule but diminished when endotoxin was presented in larger aggregates. These findings demonstrate that the physical presentation of lipooligosaccharide, including possible interactions with capsule, affect the ability of meningococcal endotoxin to interact with and activate specific host targets.     

Specific high affinity interactions of monomeric endotoxin.protein complexes with Toll-like receptor 4 ectodomain

Potent Toll-like receptor 4 (TLR4) activation by endotoxin has been intensely studied, but the molecular requirements for endotoxin interaction with TLR4 are still incompletely defined. Ligand-receptor interactions involving endotoxin and TLR4 were characterized using monomeric endotoxin.protein complexes of high specific radioactivity. The binding of endotoxin.MD-2 to the TLR4 ectodomain (TLR4ECD) and transfer of endotoxin from CD14 to MD-2/TLR4ECD were demonstrated using HEK293T-conditioned medium containing TLR4ECD+/-MD-2. These interactions are specific, of high affinity (KD<300 pm), and consistent with the molecular requirements for potent cell activation by endotoxin. Both reactions result in the formation of a Mr approximately 190,000 complex composed of endotoxin, MD-2, and TLR4ECD. CD14 facilitates transfer of endotoxin to MD-2 (TLR4) but is not a stable component of the endotoxin.MD-2/TLR4 complex. The ability to assay specific high affinity interactions of monomeric endotoxin.protein complexes with TLR4ECD should allow better definition of the structural requirements for endotoxin-induced TLR4 activation.     

Endotoxin{middle dot}albumin complexes transfer endotoxin monomers to MD-2 resulting in activation of TLR4

Response to Gram-negative bacteria (GNB) is partially mediated by the recognition of GNB-derived endotoxin by host cells. Potent host response to endotoxin depends on the sequential interaction of endotoxin with lipopolysaccharide binding protein (LBP), CD14, MD-2 and TLR4. While CD14 facilitates the efficient transfer of endotoxin monomers to MD-2 and MD-2·TLR4, activation of MD-2·TLR4 can occur in the absence of CD14 through an unknown mechanism. Here, we show that incubation of purified endotoxin (E) aggregates (E(agg), M ( r )?≥?20 million) in PBS with?≥?0.1% albumin in the absence of divalent cations Ca(2+) and Mg(2+), yields E·albumin complexes (M ( r ) ～70,000). E·albumin transfers E monomers to sMD-2 or sMD-2·TLR4 ectodomain (TLR4(ecd)) with a 'K (d)' of ～4?nM and induces MD-2·TLR4-dependent, CD14-independent cell activation with a potency only 10-fold less than that of monomeric E·CD14 complexes. Our findings demonstrate, for the first time, a mechanistic basis for delivery of endotoxin monomers to MD-2 and for activation of TLR4 that is independent of CD14.     

An essential role for albumin in the interaction of endotoxin with lipopolysaccharide-binding protein and sCD14 and resultant cell activation

Experiments utilizing endotoxin aggregates, lipooligosaccharides (LOS) isolated from metabolically labeled Neisseria meningitidis serotype group B, demonstrate that albumin is an essential component of lipopolysaccharide binding protein- (LBP) and sCD14-dependent 1) disaggregation of LOS and 2) LOS activation of human umbilical vein endothelial cells (HUVEC). Aggregates of LOS (LOS(agg)) with an apparent M(r) >or= 2 x 10(7) were isolated by gel sieving on Sephacryl HR S500 in buffered balanced salts solution plus albumin. Incubation of LOS(agg) with LBP and sCD14 promoted LOS(agg) disaggregation in an albumin-dependent fashion to complexes that contain LOS and sCD14, but no LBP, with an apparent M(r) approximately 60,000 (LOS:sCD14) as determined by Sephacryl S200 chromatography. Isolation by gel filtration of LOS(agg):protein aggregates formed by the interaction of LOS(agg) with either LBP or sCD14 alone revealed that the sequence of LOS-protein interactions as well as the step(s) at which albumin is necessary for the production of bioactive LOS:sCD14 were specific. Efficient generation of LOS:sCD14 required 1) interaction of LOS(agg) with LBP before interaction with CD14 and 2) the presence of albumin during the interaction of LBP with LOS(agg). Activation of HUVEC by LOS(agg), as measured by IL-8 production, required both LBP and sCD14 and was thirty times more potent in the presence of albumin. In contrast, LOS:sCD14 did not require additional LBP, sCD14, or albumin to activate HUVEC but depended on the presence of albumin for optimal solubility/stability once formed. The albumin effect is apparently specific, because neither ovalbumin nor gelatin substituted for albumin in facilitating LBP:sCD14-dependent disaggregation of LOS(agg) or activation of endothelial cells. These results indicate that albumin is an essential facilitator of LBP/sCD14-induced LOS disaggregation that is required for activation of endothelial cells by LOS(agg).     

Molecular basis of reduced potency of underacylated endotoxins

Potent TLR4-dependent cell activation by gram-negative bacterial endotoxins depends on sequential endotoxin-protein and protein-protein interactions with LPS-binding protein, CD14, myeloid differentiation protein 2 (MD-2), and TLR4. Previous studies have suggested that reduced agonist potency of underacylated endotoxins (i.e., tetra- or penta- vs hexa-acylated) is determined by post-CD14 interactions. To better define the molecular basis of the differences in agonist potency of endotoxins differing in fatty acid acylation, we compared endotoxins (lipooligosaccharides (LOS)) from hexa-acylated wild-type (wt), penta-acylated mutant msbB meningococcal strains as well as tetra-acylated LOS generated by treatment of wt LOS with the deacylating enzyme, acyloxyacylhydrolase. To facilitate assay of endotoxin:protein and endotoxin:cell interactions, the endotoxins were purified after metabolic labeling with [3H]- or [14C]acetate. All LOS species tested formed monomeric complexes with MD-2 in an LPS-binding protein- and CD14-dependent manner with similar efficiency. However, msbB LOS:MD-2 and acyloxyacylhydrolase-treated LOS:MD-2 were at least 10-fold less potent in inducing TLR4-dependent cell activation than wt LOS:MD-2 and partially antagonized the action of wt LOS:MD-2. These findings suggest that underacylated endotoxins produce decreased TLR4-dependent cell activation by altering the interaction of the endotoxin:MD-2 complex with TLR4 in a way that reduces receptor activation. Differences in potency among these endotoxin species is determined not by different aggregate properties, but by different properties of monomeric endotoxin:MD-2 complexes.     

Endotoxin-binding proteins modulate the susceptibility of bacterial endotoxin to deacylation by acyloxyacyl hydrolase

Acyloxyacyl hydrolase (AOAH) is an eukaryotic lipase that partially deacylates and detoxifies Gram-negative bacterial lipopolysaccharides and lipooligosaccharides (LPSs or LOSs, endotoxin) within intact cells and inflammatory fluids. In cell lysates or as purified enzyme, in contrast, detergent is required for AOAH to act on LPS or LOS (Erwin, A. L., and Munford, R. S. (1990) J. Biol. Chem. 265, 16444-16449 and Katz, S. S., Weinrauch, Y., Munford, R. S., Elsbach, P., and Weiss, J. (1999) J. Biol. Chem. 274, 36579-36584). We speculated that the sequential interactions of endotoxin (E) with endotoxin-binding proteins (lipopolysaccharide-binding protein (LBP), CD14, and MD-2) might produce changes in endotoxin presentation that would allow AOAH greater access to its substrate, lipid A. To test this hypothesis, we measured the activity of purified AOAH against isolated, metabolically labeled meningococcal LOS and Escherichia coli LPS that were presented either as aggregates (LOSagg or LPSagg)+/-LBP or as monomeric protein (sCD14 or MD-2)-endotoxin complexes. Up to 100-fold differences in the efficiency of endotoxin deacylation by AOAH were observed, with the following rank order of susceptibility to AOAH: E:sCD14>or=endotoxin aggregates (Eagg):LBP (molar ratio of E/LBP 100:1)>Eagg, Eagg:LBP (E/LBP approximately 1, mol/mol), or E:MD-2. AOAH treatment of LOS-sCD14 produced partially deacylated LOS still complexed with sCD14. The underacylated LOS complexed to sCD14 transferred to MD-2 and thus formed a complex capable of preventing TLR4 activation. These findings strongly suggest that LBP- and CD14-dependent extraction and transfer of endotoxin monomers are accompanied by increased exposure of fatty acyl chains within lipid A and that the acyl chains are then sequestered when LOS binds MD-2. The susceptibility of the monomeric endotoxin-CD14 complex to AOAH may help constrain endotoxin-induced TLR4 activation when endotoxin and membrane CD14 are present in excess of MD-2/TLR-4.     

Effects of Vitamin A on In Vitro Maturation of Pre-Pubertal Mouse Spermatogonial Stem Cells

Testicular tissue cryopreservation is the only potential option for fertility preservation in pre-pubertal boys exposed to gonadotoxic treatment. Completion of spermatogenesis after in vitro maturation is one of the future uses of harvested testicular tissue. The purpose of the current study was to evaluate the effects of vitamin A on in vitro maturation of fresh and frozen-thawed mouse pre-pubertal spermatogonial stem cells in an organ culture system. Pre-pubertal CD1 mouse fresh testes were cultured for 7 (D7), 9 (D9) and 11 (D11) days using an organ culture system. Basal medium was supplemented with different concentrations of retinol (Re) or retinoic acid (RA) alone or in combination. Seminiferous tubule morphology (tubule diameter, intra-tubular cell type), intra-tubular cell death and proliferation (PCNA antibody) and testosterone level were assessed at D7, D9 and D11. Pre-pubertal mouse testicular tissue were frozen after a soaking temperature performed at -7°C, -8°C or -9°C and after thawing, were cultured for 9 days, using the culture medium preserving the best fresh tissue functionality. Retinoic acid at 10^-6M and retinol at 3.3.10^-7M, as well as retinol 10^-6M are favourable for seminiferous tubule growth, maintenance of intra-tubular cell proliferation and germ cell differentiation of fresh pre-pubertal mouse spermatogonia. Structural and functional integrity of frozen-thawed testicular tissue appeared to be well-preserved after soaking temperature at -8°C, after 9 days of organotypic culture using 10^-6M retinol. RA and Re can control in vitro germ cell proliferation and differentiation. Re at a concentration of 10^-6M maintains intra-tubular cell proliferation and the ability of spermatogonia to initiate spermatogenesis in fresh and frozen pre-pubertal mouse testicular tissue using a soaking temperature at -8°C. Our data suggested a possible human application for in vitro maturation of cryopreserved pre-pubertal testicular tissue.     

Isolation of monomeric and dimeric secreted MD-2. Endotoxin.sCD14 and Toll-like receptor 4 ectodomain selectively react with the monomeric form of secreted MD-2

Potent cell activation by endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). MD-2 plays an essential role by bridging endotoxin (E) recognition initiated by lipopolysaccharide-binding protein and CD14 to TLR4 activation by presenting endotoxin as a monomeric E.MD-2 complex that directly and potently activates TLR4. Secreted MD-2 (sMD-2) exists as a mixture of monomers and multimers. Published data suggest that only MD-2 monomer can interact with endotoxin and TLR4 and support cell activation, but the apparent instability of MD-2 has thwarted efforts to more fully separate and characterize the individual species of sMD-2. We have taken advantage of the much greater stability of sMD-2 in insect culture medium to fully separate sMD-2 monomer from dimer by gel sieving chromatography. At low nanomolar concentrations, the sMD-2 monomer, but not dimer, reacted with a monomeric complex of E.sCD14 to form monomeric E.MD-2 and activate HEK293/TLR4 cells. The monomer, but not dimer, also reacted with the ectodomain of TLR4 with an affinity comparable with the picomolar affinity of E.MD-2. These findings demonstrate directly that the monomeric form of sMD-2 is the active species both for reaction with E.CD14 and TLR4, as needed for potent endotoxin-induced TLR4 activation.     

Novel roles in human MD-2 of phenylalanines 121 and 126 and tyrosine 131 in activation of Toll-like receptor 4 by endotoxin

Potent mammalian cell activation by Gram-negative bacterial endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). TLR4 activation requires simultaneous binding of MD-2 to endotoxin (E) and the ectodomain of TLR4. We now describe mutants of recombinant human MD-2 that bind TLR4 and react with E.CD14 but do not support cellular responsiveness to endotoxin. The mutants F121A/K122A MD-2 and Y131A/K132A MD-2 react with E.CD14 only when co-expressed with TLR4. Single mutants K122A and K132A each react with E.CD14 +/- TLR4 and promote TLR4-dependent cell activation by endotoxin suggesting that Phe(121) and Tyr(131) are needed for TLR4-independent transfer of endotoxin from CD14 to MD-2 and also needed for TLR4 activation by bound E.MD-2. The mutant F126A MD-2 reacts as well as wild-type MD-2 with E.CD14 +/- TLR4. E.MD-2(F126A) binds TLR4 with high affinity (K(d) approximately 200 pm) but does not activate TLR4 and instead acts as a potent TLR4 antagonist, inhibiting activation of HEK/TLR4 cells by wild-type E.MD-2. These findings reveal roles of Phe(121) and Tyr(131) in TLR4-independent interactions of human MD-2 with E.CD14 and, together with Phe(126), in activation of TLR4 by bound E.MD-2. These findings strongly suggest that the structural properties of E.MD-2, not E alone, determine agonist or antagonist effects on TLR4.     

Transfer of monomeric endotoxin from MD-2 to CD14: characterization and functional consequences

Potent Toll-like receptor 4 (TLR4)-dependent cell activation by endotoxin depends on sequential transfer of monomers of endotoxin from an aggregated form to CD14 via the lipopolysaccharide-binding protein and then to MD-2. We now show that monomeric endotoxin can be transferred in reverse from MD-2 to CD14 but not to lipopolysaccharide-binding protein. Reverse transfer requires an approximately 1000-fold molar excess of CD14 to endotoxin-MD-2. Transfer of endotoxin from MD-2 to extracellular soluble CD14 reduces activation of cells expressing TLR4 without MD-2. However, transfer of endotoxin from MD-2 to membrane CD14 (mCD14) makes cells expressing MD-2.TLR4 sensitive to activation by the endotoxin-MD-2 complex. An endotoxin-mutant (F126A) MD-2 complex that does not activate cells expressing TLR4 alone potently activates cells expressing mCD14, MD-2, and TLR4 by transferring endotoxin to mCD14, which then transfers endotoxin to endogenous wild-type MD-2.TLR4. These findings describe a novel pathway of endotoxin transfer that provides an additional layer of regulation of cell activation by endotoxin.