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Minor Modifications to the Phosphate Groups and the C3′ Acyl Chain Length of Lipid A in Two Bordetella pertussis Strains, BP338 and 18-323, Independently Affect Toll-like Receptor 4 Protein Activation*

  • Nita R. Shah
    Footnotes
    Affiliations
    Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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  • Sami AlBitar-Nehme
    Affiliations
    Equipe Structure et Activités des Endotoxines, UMR 8621 du Centre National de la Recherche Scientifique, Institut de Génétique et Microbiologie (IGM), Université de Paris-Sud, 91405 Orsay cedex, France
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  • Emma Kim
    Affiliations
    Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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  • Nico Marr
    Footnotes
    Affiliations
    Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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  • Alexey Novikov
    Affiliations
    Equipe Structure et Activités des Endotoxines, UMR 8621 du Centre National de la Recherche Scientifique, Institut de Génétique et Microbiologie (IGM), Université de Paris-Sud, 91405 Orsay cedex, France
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  • Martine Caroff
    Affiliations
    Equipe Structure et Activités des Endotoxines, UMR 8621 du Centre National de la Recherche Scientifique, Institut de Génétique et Microbiologie (IGM), Université de Paris-Sud, 91405 Orsay cedex, France
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  • Rachel C. Fernandez
    Correspondence
    To whom correspondence should be addressed. Tel.: 604-822-6824;
    Affiliations
    Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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  • Author Footnotes
    * This work was funded by an operating grant from the Canadian Institutes of Health Research (Grant MOP-102706) and a Programme International de Collaboration Scientifique (Franco-Canadien) grant from the CNRS to fund exchanges between the two countries.
    This article contains supplemental text, Table S1, and Figs. S1 and S2.
    1 The recipient of a Natural Sciences and Engineering Research Council of Canada scholarship.
    2 Present address: Dept. of Pediatrics, University of British Columbia and Child and Family Research Institute, Vancouver, British Columbia V5Z 4H4, Canada.
Open AccessPublished:March 06, 2013DOI:https://doi.org/10.1074/jbc.M112.434365
      Lipopolysaccharides (LPS) of Bordetella pertussis are important modulators of the immune system. Interaction of the lipid A region of LPS with the Toll-like receptor 4 (TLR4) complex causes dimerization of TLR4 and activation of downstream nuclear factor κB (NFκB), which can lead to inflammation. We have previously shown that two strains of B. pertussis, BP338 (a Tohama I-derivative) and 18-323, display two differences in lipid A structure. 1) BP338 can modify the 1- and 4′-phosphates by the addition of glucosamine (GlcN), whereas 18-323 cannot, and 2) the C3′ acyl chain in BP338 is 14 carbons long, but only 10 or 12 carbons long in 18-323. In addition, BP338 lipid A can activate TLR4 to a greater extent than 18-323 lipid A. Here we set out to determine the genetic reasons for the differences in these lipid A structures and the contribution of each structural difference to the ability of lipid A to activate TLR4. We show that three genes of the lipid A GlcN modification (Lgm) locus, lgmA, lgmB, and lgmC (previously locus tags BP0399–BP0397), are required for GlcN modification and a single amino acid difference in LpxA is responsible for the difference in C3′ acyl chain length. Furthermore, by introducing lipid A-modifying genes into 18-323 to generate isogenic strains with varying penta-acyl lipid A structures, we determined that both modifications increase TLR4 activation, although the GlcN modification plays a dominant role. These results shed light on how TLR4 may interact with penta-acyl lipid A species.

      Introduction

      Lipopolysaccharides (LPS) comprise the outer leaflet of the outer membrane in almost all Gram-negative bacteria and consist of two major regions: the hydrophilic polysaccharide and the hydrophobic lipid A region (
      • Trent M.S.
      • Stead C.M.
      • Tran A.X.
      • Hankins J.V.
      Diversity of endotoxin and its impact on pathogenesis.
      ). The polysaccharide region of LPS is composed of the polysaccharide core, which is found in most Gram-negative bacteria, and in some cases, a long, repeating O-antigen region. Bordetella pertussis LPS lacks a long O-antigen, and in its place is a short trisaccharide moiety linked to the core polysaccharide (
      • Caroff M.
      • Brisson J.
      • Martin A.
      • Karibian D.
      Structure of the Bordetella pertussis 1414 endotoxin.
      ); it is often referred to as lipooligosaccharide (LOS).
      The abbreviations used are: LOS
      lipooligosaccharide
      TLR4
      Toll-like receptor 4
      hTLR4
      human TLR4
      Lgm
      lipid A glucosamine modification
      l-Ara4N
      4-amino-4-deoxy-l-arabinose
      Tricine
      N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
      C55P
      undecaprenyl phosphate.
      The lipid A region of LPS plays an important role in modulating the host immune system via interaction with Toll-like receptor 4 (TLR4), which leads to downstream nuclear factor κB (NFκB) activation. Although there are many variations in lipid A structure among different species, and even among different strains of a particular bacterial species, the general structure of lipid A consists of a disaccharide backbone with a number of primary and secondary acyl chains. The canonical lipid A, that of Escherichia coli, has a (1′-6) diglucosamine backbone with the C1 and C4′ carbons modified with a phosphate group and has six acyl chains. However, even within a single bacterium, there is a heterogeneous population of lipid A species present in the outer membrane. For example, many E. coli strains are able to modify lipid A in a number of ways, such as altering the number and length of the acyl chains and adding accessory groups, such as 4-amino-4-deoxy-l-arabinose (l-Ara4N) via ArnT, onto the phosphates. Therefore, within the outer membrane of a single bacterium there are a variety of lipid A structures, with different modifications and acylation patterns (
      • Trent M.S.
      • Stead C.M.
      • Tran A.X.
      • Hankins J.V.
      Diversity of endotoxin and its impact on pathogenesis.
      ,
      • Caroff M.
      • Brisson J.
      • Martin A.
      • Karibian D.
      Structure of the Bordetella pertussis 1414 endotoxin.
      ,
      • Caroff M.
      • Karibian D.
      Structure of bacterial lipopolysaccharides.
      ,
      • Raetz C.R.
      • Reynolds C.M.
      • Trent M.S.
      • Bishop R.E.
      Lipid A modification systems in Gram-negative bacteria.
      ).
      Bacteria can use the modification of lipid A as a mechanism of resistance against cationic peptides or to affect the immune response mounted by the host (
      • Trent M.S.
      • Stead C.M.
      • Tran A.X.
      • Hankins J.V.
      Diversity of endotoxin and its impact on pathogenesis.
      ). For example, in E. coli and Salmonella, the addition of l-Ara4N to the phosphate groups increases resistance to cationic peptides (
      • Trent M.S.
      • Ribeiro A.A.
      • Lin S.
      • Cotter R.J.
      • Raetz C.R.
      An inner membrane enzyme in Salmonella and Escherichia coli that transfers 4-amino-4-deoxy-l-arabinose to lipid A: induction on polymyxin-resistant mutants and role of a novel lipid-linked donor.
      ). Other lipid A modifications affect TLR4 activation and complement resistance (
      • Trent M.S.
      • Stead C.M.
      • Tran A.X.
      • Hankins J.V.
      Diversity of endotoxin and its impact on pathogenesis.
      ).
      B. pertussis lipid A, like E. coli lipid A, consists of (1′-6) diglucosamine with 1- and 4′-phosphate groups, but unlike E. coli lipid A, it has only five acyl chains (
      • Caroff M.
      • Brisson J.
      • Martin A.
      • Karibian D.
      Structure of the Bordetella pertussis 1414 endotoxin.
      ). In addition, B. pertussis lipid A induces lower levels of TLR4 activation when compared with E. coli lipid A, and this difference is generally attributed to the penta-acyl nature of the lipid A, as opposed to the hexa-acyl structure of E. coli lipid A (
      • Trent M.S.
      • Stead C.M.
      • Tran A.X.
      • Hankins J.V.
      Diversity of endotoxin and its impact on pathogenesis.
      ,
      • Marr N.
      • Novikov A.
      • Hajjar A.M.
      • Caroff M.
      • Fernandez R.C.
      Variability in the lipooligosaccharide structure and endotoxicity among Bordetella pertussis strains.
      ). However, even among different B. pertussis strains, there is variation in the ability of LOS to activate TLR4 (
      • Marr N.
      • Novikov A.
      • Hajjar A.M.
      • Caroff M.
      • Fernandez R.C.
      Variability in the lipooligosaccharide structure and endotoxicity among Bordetella pertussis strains.
      ). We have previously shown that LOS from B. pertussis strain BP338 (a derivative of Tohama I) induces greater TLR4 activation than strain 18-323, both of which are commonly used laboratory strains (
      • Marr N.
      • Novikov A.
      • Hajjar A.M.
      • Caroff M.
      • Fernandez R.C.
      Variability in the lipooligosaccharide structure and endotoxicity among Bordetella pertussis strains.
      ). Furthermore, we have shown that 18-323 LOS is antagonistic against E. coli LPS (
      • Marr N.
      • Novikov A.
      • Hajjar A.M.
      • Caroff M.
      • Fernandez R.C.
      Variability in the lipooligosaccharide structure and endotoxicity among Bordetella pertussis strains.
      ). Because lipid A is the region of LPS that interacts with the TLR4-MD2 complex, the variation in lipid A structure between BP338 and 18-323 is likely a contributing factor to the difference in TLR4 activation between these strains.
      Analysis of the lipid A structures of BP338 and 18-323 show two differences: 1) BP338 can modify the phosphates of lipid A with glucosamine (GlcN), whereas 18-323 cannot, and 2) the C3′ acyl chain length of BP338 lipid A is 14 carbons, whereas in 18-323, it is only 10 or 12 carbons in length (
      • Marr N.
      • Novikov A.
      • Hajjar A.M.
      • Caroff M.
      • Fernandez R.C.
      Variability in the lipooligosaccharide structure and endotoxicity among Bordetella pertussis strains.
      ). Previously, we have linked the gene BP0398, a homolog of ArnT, with GlcN modification of lipid A in BP338 and shown that a BP338 mutant that lacks this GlcN modification has decreased levels of TLR4 activation (
      • Marr N.
      • Tirsoaga A.
      • Blanot D.
      • Fernandez R.
      • Caroff M.
      Glucosamine found as a substituent of both phosphate groups in Bordetella lipid A backbones: role of a BvgAS-activated ArnT ortholog.
      ,
      • Marr N.
      • Hajjar A.M.
      • Shah N.R.
      • Novikov A.
      • Yam C.S.
      • Caroff M.
      • Fernandez R.C.
      Substitution of the Bordetella pertussis lipid A phosphate groups with glucosamine is required for robust NF-κB activation and release of proinflammatory cytokines in cells expressing human but not murine Toll-like receptor 4-MD-2-CD14.
      ). Previous work in E. coli suggests that a specific region of the enzyme LpxA, dubbed the “hydrocarbon ruler” region, plays a role in determining the length of the C3 and C3′ acyl chains in lipid A (
      • Williams A.H.
      • Raetz C.R.
      Structural basis for the acyl chain selectivity and mechanism of UDP-N-acetylglucosamine acyltransferase.
      ,
      • Wyckoff T.J.
      • Lin S.
      • Cotter R.J.
      • Dotson G.D.
      • Raetz C.R.
      Hydrocarbon rulers in UDP-N-acetylglucosamine acyltransferases.
      ). LpxA is the first enzyme in the Raetz lipid A biosynthesis pathway, and it is responsible for the addition of the C3 acyl chain onto the N-acetyl glucosamine (GlcNAc) precursor molecule (
      • Raetz C.R.
      • Guan Z.
      • Ingram B.O.
      • Six D.A.
      • Song F.
      • Wang X.
      • Zhao J.
      Discovery of new biosynthetic pathways: the lipid A story.
      ). In B. pertussis LOS, LpxA has been shown to play a role in the flexibility of the C3′ acyl chain length (
      • Sweet C.R.
      • Preston A.
      • Toland E.
      • Ramirez S.M.
      • Cotter R.J.
      • Maskell D.J.
      • Raetz C.R.
      Relaxed acyl chain specificity of Bordetella UDP-N-acetylglucosamine acyltransferases.
      ).
      To determine the contribution of the GlcN modification and C3′ acyl chain length of lipid A on TLR4 activation, we first set out to discover the genetic reasons for these structural differences between B. pertussis strains BP338 and 18-323. In the present study, we show that locus tags BP0399, BP0398, and BP0397 (which we are renaming lipid A GlcN modification A (lgmA), lgmB, and lgmC, respectively) are required for GlcN modification in BP338, and the absence of a complete Lgm locus in 18-323 is responsible for the inability of 18-323 to modify lipid A in this manner. We demonstrate that a single amino acid difference in the hydrocarbon ruler region of LpxA between these two strains is the reason for the difference in the lipid A C3′ acyl chain lengths. Then, to probe the effect these two modifications have on TLR4 activation, we generated strains with varying lipid A structures by adding the lipid A-modifying genes from BP338 into 18-323. Using in vitro assays, we show that both the GlcN modification and the longer C3′ acyl chain independently increase TLR4 activation. We also demonstrate that when both modifications are present, the effect of the GlcN on TLR4 activation supersedes that of the longer acyl chain. These results shed further light on how the TLR4 complex may interact with differently shaped lipid A species, especially penta-acyl lipid A.

      DISCUSSION

      Minute changes in the structure of lipid A can have great effects on how these lipid A species are able to interact with and cause dimerization of the TLR4 complex and downstream NFκB signaling (
      • Trent M.S.
      • Stead C.M.
      • Tran A.X.
      • Hankins J.V.
      Diversity of endotoxin and its impact on pathogenesis.
      ). Coats et al. (
      • Coats S.R.
      • Berezow A.B.
      • To T.T.
      • Jain S.
      • Bainbridge B.W.
      • Banani K.P.
      • Darveau R.P.
      The lipid A phosphate position determines differential host Toll-like receptor 4 responses to phylogenetically related symbiotic and pathogenic bacteria.
      ) suggest that in penta-acyl lipid A species, longer fatty acid chain lengths correlate with greater TLR4 activity. This hypothesis is based on the observation that penta-acyl E. coli LPS with acyl chain lengths of C12 and C14 is a weaker TLR4 agonist in comparison with Porphyromonas gingivalis penta-acyl LPS, which contains acyl chain lengths of C15, C16, and C17 (
      • Coats S.R.
      • Berezow A.B.
      • To T.T.
      • Jain S.
      • Bainbridge B.W.
      • Banani K.P.
      • Darveau R.P.
      The lipid A phosphate position determines differential host Toll-like receptor 4 responses to phylogenetically related symbiotic and pathogenic bacteria.
      ). In the present study, we have elucidated the genetic reasons for the structural differences between B. pertussis strain BP338 and 18-323 lipid A. lgmA, lgmB, and lgmC are responsible for the GlcN modification present on phosphate groups in BP338, but absent in 18-323, and the single amino acid difference at position 173 of B. pertussis LpxA is responsible for the presence of 14 carbon acyl chains at the C3′ position in BP338 and only 10 and 12 carbon acyl chains at this position in 18-323. Furthermore, we have shown that each difference in lipid A structure between BP338 and 18-323 effects TLR4 activation in penta-acyl B. pertussis lipid A.
      Previously, we had suggested that LgmA and LgmB (encoded by locus tags BP0399 and BP0398, respectively), which are homologs of glycosyl transferases ArnC and ArnT, respectively, were involved in the modification of B. pertussis lipid A with GlcN (
      • Marr N.
      • Tirsoaga A.
      • Blanot D.
      • Fernandez R.
      • Caroff M.
      Glucosamine found as a substituent of both phosphate groups in Bordetella lipid A backbones: role of a BvgAS-activated ArnT ortholog.
      ). Here, we show that lgmA and lgmB, along with the downstream gene lgmC (locus tag BP0397), are required for GlcN modification of lipid A, but not lgmD (locus tag BP0396), which has a start codon overlapping with the stop codon of lgmC. LgmC has a structural fold similarity to the YdjC-like superfamily of proteins (
      • Kelley L.A.
      • Sternberg M.J.
      Protein structure prediction on the Web: a case study using the Phyre server.
      ,
      • Imagawa T.
      • Iino H.
      • Kanagawa M.
      • Ebihara A.
      • Kuramitsu S.
      • Tsuge H.
      Crystal structure of the YdjC-family protein TTHB029 from Thermus thermophilus HB8: structural relationship with peptidoglycan N-acetylglucosamine deacetylase.
      ). NaxD, a member of this protein super family, was recently shown to function as a deacetylase as part of a pathway that modifies the lipid A 1-phosphate of Francisella novicida with galactosamine (
      • Llewellyn A.C.
      • Zhao J.
      • Song F.
      • Parvathareddy J.
      • Xu Q.
      • Napier B.A.
      • Laroui H.
      • Merlin D.
      • Bina J.E.
      • Cotter P.A.
      • Miller M.A.
      • Raetz C.R.
      • Weiss D.S.
      NaxD is a deacetylase required for lipid A modification and Francisella pathogenesis.
      ). Interestingly, the authors also showed that Bordetella bronchiseptica locus tag BB4247 (i.e. lgmC) is required for deacetylation of undecaprenyl phosphate (C55P)-GlcNAc and for the presence of GlcN-modified lipid A in total lipid extracts of B. bronchiseptica (
      • Llewellyn A.C.
      • Zhao J.
      • Song F.
      • Parvathareddy J.
      • Xu Q.
      • Napier B.A.
      • Laroui H.
      • Merlin D.
      • Bina J.E.
      • Cotter P.A.
      • Miller M.A.
      • Raetz C.R.
      • Weiss D.S.
      NaxD is a deacetylase required for lipid A modification and Francisella pathogenesis.
      ). Taken together, we hypothesize that LgmA functions to transfer GlcNAc to C55P, followed by deacetylation of this product by LgmC, and finally transfer of the GlcN from C55P-GlcN onto the phosphate of lipid A by LgmB. Because lgmD is closely linked with lgmC, LgmD may play a role in this pathway. If this is the case, in the absence of lgmD, as seen in the lgmD mutant, another B. pertussis protein would carry out the function of LgmD, thereby allowing the GlcN modification of lipid A.
      Differences between enzymes in the Raetz lipid A biosynthesis pathway (
      • Raetz C.R.
      • Guan Z.
      • Ingram B.O.
      • Six D.A.
      • Song F.
      • Wang X.
      • Zhao J.
      Discovery of new biosynthetic pathways: the lipid A story.
      ) can also be responsible for the variations observed in lipid A structures between different strains and species. LpxA, the first enzyme in this pathway, catalyzes the addition of an acyl chain onto the C3 carbon of GlcNAc, and when the di-GlcN backbone of lipid A is formed, this acyl chain can be in the C3 or the C3′ position (
      • Raetz C.R.
      • Guan Z.
      • Ingram B.O.
      • Six D.A.
      • Song F.
      • Wang X.
      • Zhao J.
      Discovery of new biosynthetic pathways: the lipid A story.
      ). Williams and Raetz (
      • Williams A.H.
      • Raetz C.R.
      Structural basis for the acyl chain selectivity and mechanism of UDP-N-acetylglucosamine acyltransferase.
      ) suggest that the length of the acyl chain at the C3 and C3′ positions of lipid A is controlled by the hydrocarbon ruler region of LpxA, that is, the amino acids located near the active site in proximity to the acyl chain of the substrate, such as Gly-173 and Gly-176 in E. coli LpxA. Previous work, where mutating Gly-173 of E. coli LpxA changed the acyl chain length specificity of the enzyme, supports this theory (
      • Wyckoff T.J.
      • Lin S.
      • Cotter R.J.
      • Dotson G.D.
      • Raetz C.R.
      Hydrocarbon rulers in UDP-N-acetylglucosamine acyltransferases.
      ). Our data also support this theory because amino acid 173 in B. pertussis LpxA is equivalent to Gly-176 in E. coli (Fig. 5), and if B. pertussis LpxA has a serine in this position (as seen in BP338), a C14OH acyl chain can be added at the C3′ position, whereas if a leucine is found in position 173 (as seen in 18-323), only C10OH and C12OH acyl chains are found. Therefore, we hypothesize that the larger leucine is obscuring the tip of the active site of LpxA, thus only allowing 10 or 12 carbon acyl chains to be added. An alignment of LpxA sequences from several Gram-negative species suggests a similar correlation between strains with shorter acyl chains at the C3 and/or C3′ positions (e.g. Pseudomonas aeruginosa and Rhodobacter sphaeroides) and the presence of a larger amino acid at positions equivalent to E. coli LpxA Gly-173, Gly-176, or both (Fig. 5). However, for P. gingivalis LpxA, this correlation, which is mostly based on sequence alignment, does not hold true and may be a reflection of the decreased sequence similarity of the P. gingivalis LpxA. Thus, although this analysis generally supports the role of both Gly-173 and Gly-176 of E. coli LpxA as a hydrocarbon ruler for the length of the acyl chain added to lipid A at the C3 and C3′ positions, there are likely more complexities involved in determining acyl chain length, especially in more distantly related LpxA species.
      In this study, we have shown that slight variations in the structure of B. pertussis penta-acyl lipid A result in differences in TLR4 activation, suggesting that these structural modifications of lipid A affect how it interacts with the TLR4-MD2 complex. The solved crystal structure of dimerized TLR4-MD2 in complex with E. coli lipid A or lipid IVa (a tetra-acyl precursor of lipid A that acts as a TLR4 antagonist) can give us clues as to which amino acids may be important in this interaction (
      • Park B.S.
      • Song D.H.
      • Kim H.M.
      • Choi B.S.
      • Lee H.
      • Lee J.O.
      The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex.
      ,
      • Ohto U.
      • Fukase K.
      • Miyake K.
      • Satow Y.
      Crystal structures of human MD-2 and its complex with antiendotoxic lipid IVa.
      ). In the case of E. coli lipid A, Park et al. (
      • Park B.S.
      • Song D.H.
      • Kim H.M.
      • Choi B.S.
      • Lee H.
      • Lee J.O.
      The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex.
      ) show that lipid IVa, which harbors only four acyl chains, sits deeper in the MD2 pocket when compared with hexa-acyl lipid A, and therefore, suggest that the phosphates of lipid IVa cannot interact with key positive amino acids that the phosphates of lipid A interact with to cause TLR4 dimerization. In the case of B. pertussis lipid A, which has only five acyl chains, we propose, in the absence of the GlcN modification on the phosphate groups and with shorter C3′ acyl chains, as seen in 18-323, that the phosphate groups of lipid A are not able to interact with the key positively charged residues in TLR4 to cause dimerization and activation. However, when the C3′ acyl chain length is increased, this may position the lipid A molecule slightly more out of the MD2 pocket and slightly closer to the positively charged residues in TLR4, thus significantly increasing TLR4 dimerization and activation. Alternatively, when the phosphates are modified with positively charged GlcN, we hypothesize the lipid A must now interact with negatively charged residues in TLR4 to cause dimerization. Because B. pertussis lipid A modified with only GlcN has greater TLR4 activation when compared with nonmodified lipid A, we propose that the penta-acyl lipid A is able to interact with negatively charged residues in TLR4 to promote dimerization. Adding both GlcN and longer acyl chain modifications to 18-323 lipid A also increases TLR4 activation when compared with both wild type 18-323 and 18-323 with only the longer acyl chain modification, but this level is statistically equal to that of 18-323 with only the GlcN modification. This suggests that the slight increase in the C3′ acyl chain length in the GlcN-modified lipid A does not change the position within the TLR4 complex enough to significantly affect the interaction between the negatively charged residues in TLR4 and the GlcNs.
      The structure of the TLR4-MD-2 complex bound to E. coli hexa-acyl lipid A has shed light on the interactions between LPS and TLR4 that lead to TLR4 dimerization and activation, especially the key interactions between the negatively charged 1- and 4′-phosphate groups of LPS and positively charged amino acid residues within TLR4 (
      • Park B.S.
      • Song D.H.
      • Kim H.M.
      • Choi B.S.
      • Lee H.
      • Lee J.O.
      The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex.
      ). However, many Gram-negative species have penta-acyl lipid A, and the structure of TLR4-MD2 bound to penta-acyl lipid A has yet to be determined. A greater understanding of how different lipid A structures interact with the TLR4-MD2 complex and the effect of these structural differences on TLR4 activation will reveal new insights on how the immune system recognizes different bacteria. This may lead to the generation of tools to specifically modify the TLR4 activation potential of bacterial strains, and therefore, the ability of these strains to modulate the immune response.

      Acknowledgments

      We thank J. Felberg for contributions to the project.

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