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. Author manuscript; available in PMC: 2016 Mar 17.
Published in final edited form as: Cell Host Microbe. 2015 Oct 14;18(4):456–462. doi: 10.1016/j.chom.2015.09.005

Mammalian lipopolysaccharide receptors incorporated into the retroviral envelope augment virus transmission

Jessica Wilks 1,*, Egil Lien 2, Amy N Jacobson 3, Michael A Fischbach 3, Nilofer Qureshi 4, Alexander V Chervonsky 5, Tatyana V Golovkina 1,†
PMCID: PMC4795803  NIHMSID: NIHMS767478  PMID: 26468748

Abstract

An orally transmitted retrovirus, Mouse Mammary Tumor Virus (MMTV), subverts the antiviral immune response by triggering IL-10 production via activation of Toll-like receptor 4 (TLR4). This process required lipopolysaccharide (LPS) produced by Gram-negative intestinal bacteria, but the mechanism by which the virus associated itself with LPS was unknown. Here, we find that the MMTV envelope contains the mammalian LPS-binding factors CD14, TLR4 and MD-2, which in conjunction with LPS binding protein (LBP), bind LPS to the virus and augment virus transmission. Furthermore, incorporation of a weak agonist LPS derived from bacteria of the Bacteroides genus (a prevalent LPS source in the normal gut) significantly enhances the ability of LPS to stimulate the TLR4 pathway. Thus, an orally transmitted retrovirus can capture, modify, and exploit mammalian receptors for bacterial ligands to ensure successful transmission.

Introduction

MMTV is a retrovirus that is transmitted as an endogenous, stably integrated provirus (Mtv) or as an exogenous virus (Goff, 2007). Exogenous MMTV is spread via the milk of infected females and is acquired by suckling pups. In the gut, the virus transcytoses through M cells in Peyer’s patches (Golovkina et al., 1999) and infects the underlying lymphoid cells, including T and B lymphocytes. Infected T and B cells traffic to the mammary gland and pass the virus to the mammary gland epithelia (Finke and Acha-Orbea, 2001). During lactation, the virus is secreted into the milk and is transmitted to the progeny.

Persistence of the virus requires commensal bacteria and functional TLR4, as the virus is eliminated in both TLR4-deficient specific pathogen free (SPF) (Jude et al., 2003) and wild-type (WT) germ-free (GF) mice (Kane et al., 2011). A series of genetic and biochemical experiments established that after infiltrating the host, the virus cloaks itself in LPS. The LPS-associated virus activates TLR4, which leads to IL-6-mediated production of the immunosuppressive cytokine IL-10 and blockage of the antiviral response (Jude et al., 2003; Kane et al., 2011). However, the nature of the virus-LPS interaction remained unknown. Here, we report that mammalian LPS receptors are integrated in the viral envelope and bind LPS thus, enabling the virus to activate the immune evasion pathway.

Results

To address whether MMTV directly binds LPS, a modified capture ELISA was used. Both anti-envelope (Env) glycoprotein antibodies (Abs) and biotinylated LPS detected plate-bound MMTV virions equally well (Figure S1), suggesting the virions directly interact with LPS.

Mammals have multiple LPS-binding proteins; some transfer LPS to other proteins, while others are directly involved in transmitting LPS-induced signals (Park and Lee, 2013). Specifically, the membrane-anchored protein CD14 binds LPS and transfers it to the MD-2-TLR4 complex (Viriyakosol et al., 2001). MD-2 directly binds LPS, and LPS-bound MD-2 triggers TLR4 dimerization and activation of downstream signaling (Park et al., 2009). Notably, in the absence of TLR4, MD-2 is not present on the cell surface. MMTV, an enveloped retrovirus, targets cells that express and display these LPS-binding proteins [(Lee et al., 2009; Miyake, 2006) and Figure S2]. Thus, we proposed that the virus acquires these proteins from the host and utilizes them to bind LPS. In fact, both TLR4 and CD14 were detected on purified MMTV virions (Figures 1A–1C), supporting this idea.

Figure 1. Mammalian LPS-binding receptors are present in MMTV virions.

Figure 1

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(A) MMTV virions isolated from the milk of B6 wild-type (WT) and TLR4−/− mice were captured either by an anti-mouse TLR4 or anti-gp36Env Abs and detected with biotinylated anti-gp36Env Abs. Error bars represent the SE of three independent experiments. Significance was calculated using a two-sample t-test and asterisks indicate degree of significance (* = P<0.03). (B) Western blot of proteins from virions purified from the milk of B6 (WT) and CD14−/− females. The same density fraction isolated from uninfected milk (MMTV−) was used as a negative control. Anti-mouse CD14 Abs and anti-gp52Env Abs were used to detect CD14 and viral Env, respectively. Mouse CD14 recombinant protein was used as a positive control for anti-CD14 Abs. (C) Immunogold labeling of MMTV virions. Top panel, MMTV virions purified from B6 (WT) (a, b, c, d) or CD14−/− (e, f) mouse milk were stained with anti-gp52Env Abs followed by anti-mouse Ab coupled to gold particles (b, f) or anti-mouse Ab alone (a) or anti-CD14 Ab followed by anti-rat Ab coupled to gold particles (d, e) or anti-rat Abs alone (c). Bottom panel, Quantification of three independent experiments with over 50 virions examined per condition in each experiment. Error bars represent the SE. (D) MMTV virions isolated from the milk of mice lacking CD14, LBP, MD-2, TLR4 and from animals deficient in all four factors were captured with anti-gp36Env Abs bound to plastic. The ELISA was developed either with biotinylated LPS (E. Coli, serotype EH100) (top panel), or anti-gp36Env Abs (bottom panel). Error bars represent SE of three independent experiments. Significance was calculated using a two-sample t-test and asterisks indicate degree of significance (** =P<0.001).

To investigate the role of the mammalian LPS-binding machinery in viral acquisition of LPS, we used a genetic approach. WT females and females devoid of MD-2, TLR4, CD14, and another well-characterized LPS-binding factor, LBP (Park et al., 2009) were infected with MMTV via intraperitoneal injection as adults. The virions isolated from their milk were subjected to the LPS-binding assay. Whereas virus titers were comparable between all isolates (Fig. 1D, bottom panel), virions produced by both TLR4−/− and MD-2−/− females exhibited a significant reduction in LPS binding (Fig. 1D, top panel). This reduction was not observed with either the LBP or CD14 deficient virions. However, virions obtained from all LPS-binding factors-deficient mice completely lost their LPS-binding properties (Fig. 1D, top panel). Thus, even though the MD-2-TLR4 complex is the primary LPS binding factor, LBP and/or CD14 also contribute to LPS binding. In fact, the virus isolated from TLR4−/− mice was able to bind LPS (Fig. 4C).

Figure 4. MMTV augments the immunopotency of commensal LPS.

Figure 4

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(A) The immunostimulatory properties of LPS from B. theta and E. coli (serotype 055:B5) were compared by IL-6 ELISA after addition to B6 (WT) splenocytes. MD-2−, MD-2−/− splenocytes. Error bars represent SE of three independent experiments. Significance was calculated using a two-sample t-test and asterisks indicate degree of significance (** = P<0.001). (B) Binding of biotinylated B. theta and E. coli (serotype O26) LPS by SPF-MMTV virions captured to plastic with anti-gp36Env Abs was measured in serial dilutions. Error bars represent SE of three independent experiments. Significance was calculated using a two-way ANOVA. NS, non-significant. (C) B. theta or E. coli (serotype O26) LPS (at 40ng/ml of endotoxin) were incubated with or without GF-MMTV and spun down via 30% sucrose cushion. Endotoxin levels were measured in pelleted fractions using LAL assay. Fractions with no virus added had no detectable LPS (not shown). Virus-LPS pelleted fractions were added to B6 splenocytes at an endotoxin concentration of 71 pg/ml. The amount of secreted IL-6 was measured in tissue culture supernatants 16 h later. In separate cultures, virus-free B. theta and E. Coli LPS were added to B6 splenocytes at the same endotoxin concentration. Error bars represent SE of three independent experiments. Significance was calculated using a paired t-test and asterisks indicate degree of significance (*= P<0.03 and ** = P<0.001). (D) LPS species isolated from SPF-MMTV virions were added to B6 splenocytes as free form (‘viral’ LPS) or bound to SPF-MMTV virions at 35 pg/ml. The amount of secreted IL-6 was used as a read-out for TLR4 activation. Error bars represent SE of three independent experiments. Significance was calculated using a paired t-test and asterisks indicate degree of significance (* = P<0.03). (E) IL-6 secretion elicited by SPF-MMTV virions in B6 (WT), Caspase 1/4−/− (Caspase 1/4−), or MD-2−/− (MD-2−) splenocytes or by E. coli (serotype 055:B5) LPS from Sigma (S) or Enzo (E) was measured by ELISA. Endotoxin concentrations of 1 ng/ml in all samples were verified by the LAL assay. Error bars represent SE of three independent experiments. Significance was calculated using a paired t-test and asterisks indicate degree of significance (** = P<0.001).

Activation of the TLR4 pathway, specifically production of IL-6 and IL-10, is required for MMTV’s replication and successful transmission, and mice lacking TLR4, CD14, IL-6 or IL-10 eliminate the virus through successive generations (Jude et al., 2003; Kane et al., 2011). Therefore, the ability of MMTV virions lacking LPS-binding factors to activate TLR4 was tested. Virions lacking all LPS-binding factors on their membrane failed to activate the TLR4 pathway (Fig. 2A). In contrast, CD14-deficient virions induced signaling in WT cells (Fig. 2A). Interestingly, the presence of CD14 on either the viral particle or the host cell enabled LPS-bound virions to elicit TLR4 signaling. However, this activation was ablated when CD14 was absent altogether (Fig. 2A), indicating that CD14 must be present in the system to facilitate the transfer of the virus’s LPS cargo to the target cell.

Figure 2. LPS binding proteins carried by the virus are essential for immune activation and efficient transmission.

Figure 2

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(A) MMTV was isolated from the milk of the B6 (WT), MD-2−/− (MD-2−), CD14−/− (CD14−) and all LPS binding factors (All−) deficient females. Viral isolates with LPS content normalized to 1 ng/ml via the LAL assay were added to either WT or CD14−/− splenocytes. IL-6 in tissue culture supernatants was quantified by ELISA 16 h later. Error bars represent SE of three independent experiments. Significance was calculated using a two-sample t-test, and asterisks indicate degree of significance (** = P<0.001). LPS, 1 ng/ml of LPS from E. Coli, serotype 055:B5, (B) Top panel, B6 (WT) mice were fostered on either WT or MD-2−/− (MD-2−) viremic females for 48h. WT pups fostered on uninfected milk were used as a negative control. The deletion of SAg-cognate Vβ6+CD4+ T cells among CD4+ T cells was used as a read out for viral infection. X, animals confirmed as uninfected by qPCR. Bottom panel, Splenic DNA only from infected mice (not marked with X in top panel) was analyzed for integrated proviruses by qPCR. Data are presented as delta between the cycle threshhold (CT) obtained with MMTV-specific primers and IFN-gamma-specific primers. Significance was calculated using a two-sample t-test and asterisks indicate degree of significance (* = P<0.03, ** = P<0.001). (C) B6 mice were fostered on either BALB/cJ (WT) or BALB/cJ.LBP−/− (LBP−) viremic females for 48h. B6 pups fostered on uninfected BALB/cJ milk were used as a negative control. The deletion of SAg-cognate Vβ6+CD4+ T cells among CD4+ T cells was used to assess whether mice were infected. Error bars represent SE. Significance was calculated using a two-sample t-test and asterisks indicate degree of significance (*** = P<0.0001).

To determine whether the reduced ability to bind LPS would affect virus transmission in vivo, we followed the virus’s fate in mice infected with WT and MD-2− virions. MMTV encodes for a superantigen (v-SAg), which is essential for the virus life cycle (Ross, 2008). Presentation of v-SAg results in activation of SAg-cognate T cells and their subsequent deletion (Marrack et al., 1991). Deletion of SAg-cognate T cells is used as a read-out of successful infection, and the degree of T-cell deletion roughly correlates with the virus load (MacDearmid et al., 2006). Accordingly, WT neonates were fostered by either WT or MD-2−/− viremic females, and the presence of infectious virus was defined by the percentage of SAg cognate CD4+ T cells among all CD4+ T cells (Fig. 2B, top panel) and quantified by qPCR (Fig. 2B, bottom panel). Enveloped viruses, such as retroviruses, acquire their membrane as they egress from infected cells. Therefore, virions secreted by MD-2−/− females will acquire MD-2 upon one round of infection in WT mice, diminishing the phenotypic difference between WT and MD-2− viruses. To solve this problem, we exposed WT mice to infected foster mothers for only 2 days. Even though the viral titers secreted by WT and MD-2−/− females were comparable (Fig. 1D), the virus titer was reduced in mice infected with MD-2− virus (Fig. 2B). In addition, more mice fostered by MD-2−/− mothers remained uninfected (4/7) compared to mice fostered by WT mothers (2/14). Thus, MD-2− virions were significantly compromised in their ability to establish infection, definitively linking the virus’s LPS-binding capacity with successful transmission.

It is widely accepted that LBP solubilizes LPS aggregates in vivo, making this bacterial ligand accessible to CD14 and the MD-2/TLR4 receptor complex (Gutsmann et al., 2001; Hailman et al., 1994; Tobias et al., 1995). Even though virions secreted by LBP−/− females still bound LPS (Fig. 1D), this in vitro assay was performed with solubilized LPS. Thus, the potential physiological significance of LBP in the virus-LPS interaction may have been masked. To test the significance of LBP in virus transmission, we followed the fate of the virus secreted by LBP−/− females. WT mice fostered on LBP−/− mothers had significantly reduced virus titers compared to mice fostered on WT mothers, and 4/24 were uninfected (Fig. 2C). This strongly suggests that, like mammalian cells, virion-associated CD14 and MD-2/TLR4 require LBP to bind LPS monomers in vivo.

LPS consists of three chemically distinct components: O-antigen, core oligosaccharide, and lipid A (Raetz et al., 2007). There is significant structural heterogeneity in lipid A molecules, with acyl chain length number, acyl chain length, and phosphorylation state being the primary determinants of their immunostimulatory properties (Trent et al., 2006). Notably, the structure of LPS and its stimulatory properties varies drastically among Gram-negative bacteria, making the biological properties of virus-bound LPS a necessary avenue of investigation. To address whether MMTV binds purified lipid A from diverse bacterial species, LPS-free MMTV isolated from GF mice was incubated with lipid A from either E. coli [strong TLR4 agonist (Raetz and Whitfield, 2002)] or from Rhodobacter (R) sphaeroides [TLR4 antagonist (Kutuzova et al., 2001)]. MMTV bound both E. coli and R. sphaeroides lipid A to an equal degree (Fig. 3A). However, only virus-bound E. coli lipid A activated TLR4 signaling (Fig. 3A). Thus, MMTV does not discriminate between agonist and antagonist LPS/lipid A types for binding, but requires a TLR4 agonist to trigger TLR4 signaling.

Figure 3. MMTV binds the lipid A component of LPS from diverse species and enables agonist LPS to resist inhibition by an antagonist LPS.

Figure 3

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(A) LPS-free MMTV from GF MyD88/TLR4 double-deficient mice was incubated with 40 ng/ml of lipid A (E. coli, serotype R515) (E.c.) or R. sphaeroides (R.s.) and pelleted through 30% sucrose cushion. The amount of bound endotoxin was quantified by the LAL assay (left panel). The ability of virus-bound or free lipid A (normalized to 1 ng/ml via the LAL assay) to stimulate IL-6 production by B6 (WT) splenocytes was measured by ELISA (right panel). Neither E.c. or R.s. lipid A elicit IL-6 in MD-2−/− splenocytes. Error bars represent SE of three independent experiments. Significance was calculated using a two-sample t-test. NS - non-significant. (B) LPS from E. coli serotype 055:B5 (at 5, 50 and 100 ng/ml) or intact SPF-MMTV virions (at 5 ng/ml of endotoxin) was added to B6 splenocytes alone or together with increasing concentration of R.s. lipid A. IL-6 in tissue culture supernatants was quantified by ELISA 16 h later. X, is the factor by which R.s. lipid A concentration was increased relative to E.c. LPS. Error bars represent SE of three independent experiments. Significance was calculated using a paired t-test and asterisks indicate degree of significance (* = P<0.05)

MMTV-bound LPS is more immunopotent than virus-free LPS (Kane et al., 2011), indicating that binding of LPS to the viral membrane amplifies LPS-induced TLR4 activation. With this in mind and recognizing that R. sphaeroides lipid A antagonizes activation of TLR4 by agonist LPS (Kutuzova et al., 2001), we sought to determine whether virally-bound LPS was protected from this inhibition. Indeed, R. sphaeroides lipid A blocked TLR4 activation by free E. coli LPS, but virus-bound LPS resisted this inhibition (Fig. 3B). As competitive inhibition of free LPS by R. sphaeroides lipid A is concentration dependent (Manthey et al., 1993), the most parsimonious explanation for this result is that binding of LPS to the virion increases the effective local concentration, making MMTV-bound LPS more resistant to inhibition by R. sphaeroides lipid A.

The majority of Gram-negative commensals that populate the mouse and healthy human gut are of the Bacteroides genus (Human Microbiome Project, 2012), which synthesize relatively weak TLR4 agonists (Fig. 4A). Since MMTV must activate TLR4 to propagate within the host, we hypothesized that the virus must potentiate weak agonist LPS. First, it was determined that MMTV binds physiologically relevant LPS from the human commensal Bacteroides thetaiotaomicron (B. theta) to the same extent as E. coli LPS, one of the strongest TLR4 agonists (Fig. 4B). Second, GF-MMTV virions bound to B. theta LPS significantly strengthened the immunopotency of this commensal ligand similar to E. coli LPS (Fig. 4C). Moreover, this phenomenon was observed with LPS species purified directly from MMTV virions (Fig. 4D), suggesting that MMTV has the ability to intensify the immunostimulatory properties of agonistic commensal LPS found in the mouse gut.

Internalization of Gram-negative bacteria or LPS by innate immune cells activates a non-canonical inflammasome pathway (Kayagaki et al., 2013). This activation leads to the production and secretion of the inflammatory cytokine IL-1β, which is known to induce IL-6 (Cahill and Rogers, 2008). To determine whether the non-canonical inflammasome pathway was responsible for the increased immunopotency of virus-bound LPS, we examined virus-elicited IL-6 induction in WT and caspase 1/4-deficient mice, which lack both canonical and non-canonical pathways (Fig. 4E). Virus-bound LPS maintained its immunopotency in caspase1/4-deficient splenocytes, suggesting that inflammasome-dependent activation does not explain the enhanced immunopotency of virus-bound LPS.

Discussion

Although it has been previously noted that enveloped viruses acquire host proteins as they bud from the cell (Bubbers and Lilly, 1977; Cantin et al., 2005), few studies have linked the significance of this incorporation to virus replication, transmission, and pathogenesis. HIV-1 selectively incorporates the MHC class II glycoprotein within its membrane and uses it to accelerate entry into human T-lymphocytes (Bastiani et al., 1997; Cantin et al., 1997). HTLV-1 displays the complement regulatory proteins CD59 and CD55 on its surface to evade complement-mediated lysis (Spear et al., 1995). In addition, virion-associated MHC class I and class II molecules impact the ability of HIV virions to trigger cell death (Esser et al., 2001). In the case of both HIV-1 and HTLV-1, however, the effects of the host-derived membrane proteins on virus infectivity and pathogenesis were only demonstrated in vitro. In this study, we show that incorporation of mammalian LPS-binding proteins by an enveloped retrovirus enables the virus to bind LPS and evade the anti-retroviral immune response.

The most abundant commensal Gram-negative bacterial species within the human and mouse gastrointestinal tract produce LPS that are weak TLR4 agonists (Human Microbiome Project, 2012). However, when MMTV binds commensal LPS derived from B. theta, it strengthens this LPS immunopotency to such an extent that it exceeds immunopotency of E. coli LPS, a potent TLR4 agonist (Fig. 4C). How the virus modulates LPS potency remains to be elucidated and will require further investigation. Nevertheless, a few possibilities can be considered. First, LPS aggregates on the viral surface might strengthen TLR4 activation, as aggregated but not monomeric endotoxin activates TLR4 signaling at similar concentrations (Müller et al., 2003). Second, virion-bound LPS may have a greater effective concentration than free LPS, resulting in enhanced immunopotency. Third, delivery of LPS bound to the surface of the viral particle stabilizes or prolongs the ligand’s interaction with the cell membrane, facilitating dimerization of MD-2/TLR4 complexes. The latter events can potentially occur on the cell surface or inside the endocytic compartment where functional TLR4 is also located (Kagan et al., 2008; Tanimura et al., 2008).

The phenomenon of LPS exploitation is not restricted to MMTV, as other enteric viruses from completely different genera, such as poliovirus, also utilize LPS to enhance their environmental fitness and infectivity (Kuss et al., 2011; Robinson et al., 2014). Many non-enveloped enteric viruses target cells that express LPS-binding proteins (Iwasaki et al., 2002; Neal et al., 2006) and some were reported to encase themselves within the host cell membrane (Chen et al., 2015; Feng et al., 2013). Thus, it is possible that even naked viruses could potentially exploit host LPS receptors for their benefit. Further studies of this phenomenon may lead to new approaches for prevention and treatment of some viral infections.

Experimental Procedures

Mice

All animals were bred and maintained at the animal facility of The University of Chicago. SPF MMTV-infected and uninfected C3H/HeN were maintained in our colony. TLR4−/− C57BL10/ScNJ (Poltorak et al., 1998), B6.CD14−/− (Moore et al., 2000), B6.IL-1R1−/− (Glaccum et al., 1997), BALB/cJ.LBP−/− (Jack et al., 1997), B6.Caspase 1/4−/− (Kuida et al., 1995), BALB/cJ and B6 mice were obtained from The Jackson Laboratory. B6.MD-2−/− mice (Nagai et al., 2002) were crossed to B6 mice for 4 generations before the MD-2−/− was fixed at homozygosity. BALB/cJ.LBP−/− were crossed to B6 mice for 6 generations and then crossed to B6.CD14/MD-2/TLR4 triple-deficient mice and intercrossed to generate B6.LBP/CD14/MD-2/TLR4-quadruple deficient mice. GF C3H/HeN.MyD88/TLR4 double-deficient mice have been published (Kane et al., 2011). The studies described here have been reviewed and approved by the Animal Care and Use Committee at The University of Chicago.

Detection of TLR4 on MMTV virions

MMTV(LA) viral variant (Golovkina et al., 1997) was used in these studies. A capture ELISA was developed to detect TLR4 in virions. Briefly, anti-mouse TLR4 Abs (Biolegend, San Diego, CA) and anti-gp36Env Ab (Purdy et al., 2003) were bound to a 96-well plate overnight. After blocking, purified virions were added to the capture Abs and the reactions were developed with biotinylated anti-gp36Env Abs.

Detection of CD14 in purified virions

MMTV virions isolated from the milk of infected B6, B6.CD14−/− mice, as well as ‘virus density’ fractions isolated from the milk of uninfected B6 females were subjected to western blot analysis using anti-mouse CD14 (Santa Cruz Biotechnology, Dallas, TX) and anti-gp52Env Abs (Purdy et al., 2003).

Transmission electron microscopy (TEM)

Purified milk-borne virions were placed on glow discharge carbon coated gold grids and incubated with either mouse anti-gp52Env or rat anti-CD14 Abs followed by anti-mouse or anti-rat Abs coupled to gold-conjugated Abs, respectively. Samples were fixed with glutaraldehyde and stained with Uranyl Acetate and examined under 300KV at FEI Tecnai F30.

MMTV-LPS capture ELISA

Anti-gp36Env Abs were bound to 96-well plates and incubated with purified SPF-MMTV virions. ELISA was developed with either biotinylated LPS or biotinylated anti-gp36Env Abs.

MMTV infection

To compare transmission of MD-2+ and MD-2− virions, as well as LBP− and LPB+ virions, newborn B6 were allowed to suckle on infected WT, MD-2−/−, or LBP−/− females. Deletion of SAg-cognate T cells was used as a read-out for MMTV infection, and qPCR was used to quantify viral titers.

LPS binding to GF-MMTV virions

Binding of TLR4/MyD88 double-deficient GF-MMTV virions to E. coli lipid A, R. sphaeroides lipid A, B. thetaiotaomicron LPS, or E. coli LPS was performed as published (Kane et al., 2011). Endotoxin concentration was quantified via LAL assay.

Supplementary Material

Suplemental material

Acknowledgments

The authors are thankful to Dr. Betty R. Theriault, Alan Vest, and Kristin Kolar for their help in monitoring the gnotobiotic animals. Helen Beilinson and Felicity Deiss for help in genotyping mice. The Golovkina laboratory, Chervonsky laboratory, and Howard Shuman for helpful discussion. This work was supported by the NIH/NIAID grant AI090084 to T.V.G., by T32 AI007090 to J.W., by NIH grant AI075305 and support of the Research Council of Norway to E.L., by NIH grant DP2 OD007290 to M.A.F., and by the NSF grant 1144247 to A.N.J., by P30 DK42086 grant to the DDRCC (University of Chicago), by P30 CA014599 grant to The University of Chicago, by the Sandler Family Foundation, the Gordon and Betty Moore Foundation, and P30 CA082103, and DE-AC02-05CH11231 to University of California, San Francisco.

Footnotes

Author Contributions

J.W. produced all LPS binding factors-deficient mice and performed most of the experiments; T.V.G. performed electron microscopy and western blot analysis; A.N.J and M.A.F. purified B. theta LPS; N.Q. and E.L. purified R. sphaeroides lipid A; L.E. and A.V.C. contributed to experimental design; J.W. and T.V.G. wrote the manuscript. T.V.G. conceived the project, analyzed the results; All authors discussed the results and commented on the manuscript.

None of the authors of this manuscript have a financial interest related to this work.

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