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Departments of Medicine*,Microbiology‡ and GenomeSciences§, University ofWashington Medical School,Seattle, Washington 98195,USA.Correspondence to S.I.M.e-mail:millersi@u.washington.edudoi:10.1038/nrmicro1068

Animals and plants live in a ‘sea’ of microorganisms thatcolonize their surfaces. Most often, the interactionbetween host and bacteria is beneficial — for example,the commensal microbial flora can provide nutrientsand promote the full development of the human intesti-nal tract1. By contrast, many pathogenic microorgan-isms have evolved mechanisms to cross tissue barriers.Host recognition of microbial invasion promotes aninflammatory response through the secretion of antimi-crobial compounds and recruitment of phagocytic cellssuch as macrophages and neutrophils. These initialresponses to the presence of pathogens are called innateimmunity as they do not require any previous exposureto the pathogen in question. Instead, innate immunityrequires the recognition of evolutionarily conserved pat-terns associated with particular classes of pathogens2.Innate immune responses are also required for the initi-ation of acquired immunity, which relies on the clonalexpansion of individual antigen-specific lymphocytes3.Innate immune responses are found in all organisms,including those without adaptive immunity such asinsects, amoebas and plants, and they represent anancient system in the competition between single-celleukaryotes and prokaryotic microorganisms.

Innate immunity and infectious disease diversity?The host range of different pathogens varies tremen-dously. For example, Salmonella enterica serovar Typhi,which is the causative agent of typhoid fever, has

recently evolved to colonize humans and chimpanzees.By contrast, non-typhoidal Salmonella serovars have abroad host range and infect a wide variety of animals.The molecular determinants of such species specificityare generally unknown, although in some cases speci-ficity is determined by surface receptors that allowpenetration of the pathogen or a secreted toxin intohost tissues4–6.

Perhaps more perplexing than species specificity isthe variability in infectious disease outcome in individ-uals of a single species. In some human diseases, singlegene defects that result in the failure of a particularinnate immune receptor to be expressed cause a generalincreased susceptibility to infection7,8. In this review, wediscuss an alternative mechanism and argue that vari-ability in innate immune receptors and their bacterialligands could also explain why individual members of ahost population exhibit variable disease outcomes.

Two examples from studies of insect and plantpathogenesis help to illustrate these general principles.For example, Drosophila melanogaster relies exclusivelyon its innate immune system to combat bacterialinfections. Innate immune mechanisms include therecognition of bacterial and fungal pathogens andthe subsequent activation of signal-transduction cas-cades, which ultimately lead to the expression ofantimicrobial peptides that are directed against thesepathogens. A systematic test to study the susceptibilityof Drosophila to infectious disease and the relationship

LPS, TLR4 AND INFECTIOUSDISEASE DIVERSITYSamuel I. Miller*‡§, Robert K. Ernst* and Martin W. Bader§

Abstract | Innate immune receptors recognize microorganism-specific motifs. One suchreceptor–ligand complex is formed between the mammalian Toll-like receptor 4(TLR4)–MD2–CD14 complex and bacterial lipopolysaccharide (LPS). Recent researchindicates that there is significant phylogenetic and individual diversity in TLR4-mediatedresponses. In addition, the diversity of LPS structures and the differential recognition of thesestructures by TLR4 have been associated with several bacterial diseases. This review willexamine the hypothesis that the variability of bacterial ligands such as LPS and their innateimmune receptors is an important factor in determining the outcome of infectious disease.

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Increased interstrain variation has been observed in P. syringae pathovars, which supports the hypothesisthat innate immune pressure drives the diversity ofbacterial innate immune ligands14. This also results in arange of plant innate immune responses to variablebacterial molecules and might explain the differentdisease outcomes in individual plants and plantspecies. In this context, it is interesting to note that therecently described mammalian Nod proteins haveamino acid sequence similarity with plant R proteins,and might therefore form a common mechanism forthe intracellular signalling of bacterial infections15–17.

TLRs and innate immune recognitionSince Charles Janeway coined the term innate immunityand predicted the existence of pathogen-specific pattern-recognition receptors in the late 1980s, our under-standing of innate immunity has advanced immensely2.Pattern-recognition receptors recognize microorgan-ism-specific molecules that are essential and conserved(FIG. 1). The discovery of Toll, which is a Drosophilareceptor that is essential for the production of antimi-crobial peptides in response to fungal pathogens andwhich is also essential for fly development, led to thediscovery of a family of mammalian transmembranepattern-recognition Toll-like receptors (TLRs)18,19.TLRs recognize structural components unique tobacteria, fungi and viruses to signal and activateinflammatory responses. The ligands that are recog-nized by TLRs include lipopeptides (TLR2),

between this susceptibility and innate immunity hasrecently been carried out9. More than 100 differentDrosophila strains were tested for their susceptibility toinfection by the entomopathogen Serratia marcescens.These authors observed broad variability in susceptibil-ity in individual Drosophila strains. In support of theabove hypothesis, this varied susceptibility was linked topolymorphisms in 16 innate immune genes, most ofwhich are involved in the detection of conserved bacterialpatterns and downstream signalling pathways.

The complexity of Gram-negative bacterial infectionof plants is also a result of innate immune and bacterialdiversity10,11. This diversity, which is a natural conse-quence of selection pressure on the mechanisms of recog-nition and response in both host and pathogen, has effec-tively resulted in a bacterial–plant ‘arms race’. Somebacterial pathogens can deliver diverse biologically activeproteins or effectors directly into plant cells through aninterspecies protein-transport system known as type IIIsecretion. For example, the genome of the plant pathogenPseudomonas syringae encodes up to 50 different type IIIeffectors that are involved in manipulating host cell func-tion12. Plants have developed multiple receptors that areencoded by R (resistance) genes, each of which specifi-cally recognizes one or a few type III effectors, eitherdirectly or through their biological activity13. The recogni-tion of bacterial effectors triggers a hypersensitiveresponse that prevents pathogen growth. Bacteria canescape recognition by the plant innate immune responseby increasing the diversity of type III secreted effectors.

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Figure 1 | The complexity of bacteria–host interactions. Pathogen-associated molecular patterns are recognized by the innateimmune systems that are present in multicellular organisms. Innate immune responses to these bacterial ligands vary not only from speciesto species, but also within one particular species and might determine diverse infectious disease outcomes. TLR, Toll-like receptor.

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cytosolic domain that is shared by all TLRs has highsimilarity to the cytosolic domain of the IL-1 cytokinereceptor and is essential for downstream signalling.Upon ligand binding, this domain associates with themyeloid differentiation factor 88 (MyD88) adaptorprotein, which is necessary for the recruitment of theprotein kinases interleukin-1 receptor-associated kinase 1(IRAK1) and IRAK6, and TNF-receptor-associatedfactor 6 (TRAF6) to the complex29. In addition to theirrole in mounting a pro-inflammatory response, TLRshave been shown to control activation of adaptiveimmune responses3. Recently, TLRs have also beenimplicated in intestinal development and protectionthrough recognition of commensal bacteria30.

Infectious disease outcomes and TLR diversityTLR diversity — both in recognition and in down-stream signalling pathways — has been identified notonly across species but also within individuals, and candetermine infectious disease outcome. One of the bestillustrations of this comes from the study of humanTLR4 gene polymorphisms31. Individuals carrying theAsp299Gly and/or Thr399Ile variant alleles, whichencode amino acids in the extracellular domain ofTLR4, are less responsive to inhaled Escherichia coliLPS, indicating that these polymorphisms result inreduced TLR4 signalling. Interestingly, in a single studycomparing individuals with clinical symptoms of septicshock to control individuals, only those with the less-responsive TLR4 alleles were observed in the septicshock group32. These individuals also had a greaterincidence of Gram-negative bacterial infection. Thesevariant alleles have also been associated with an increasedincidence of premature birth33, which could be relatedto prenatal Gram-negative bacteraemia and the inflam-matory bowel disease ulcerative colitis34. Individualscarrying these alleles also have a decreased risk ofatherogenesis, which is linked to the presence of theGram-negative bacterial pathogens Chlamydia andHelicobacter pylori35. The decreased risk of atherogenesiscould be a result of decreased responsiveness to pathogensand consequently lower concentrations of inflammatorycytokines,which can have atherogenic effects.

Additional evidence for a role for TLR4 polymor-phisms in disease susceptibility comes from studies bySmirnova et al. who sequenced the entire TLR4 gene of348 humans and 35 mouse strains36,37.According to theiranalysis, polymorphisms not only clustered in the extra-cellular domain, which is involved in the detection ofLPS, but were also found in the cytoplasmic domain,which is likely to modulate the magnitude of the responseto LPS. These studies indicated that amino acid polymor-phisms in TLR4 are rare and are probably a result ofselective pressure. The authors also implicated TLR4 insusceptibility to meningococcal sepsis38.

Polymorphisms in multiple genes encoding com-ponents of TLR downstream signalling pathwayscould also have important implications for infectiousdisease39. Data from a human volunteer study of theresponse of whole blood to LPS indicate that there isenormous and significant diversity in the cytokine

lipopolysaccharide (TLR4), flagellin (TLR5) andCpG DNA (TLR9)20–23 (FIG. 1). In addition, viral prod-ucts such as single-stranded and double-strandedRNA are recognized by TLR7 (human TLR8) andTLR3, respectively24–26. Recognition of these ligandscan occur both on the cell surface and inside the cell(for example, in late endosomes), depending on thesubcellular localization of each individual TLR27,28.

Ligand binding to the extracellular domain of TLRsinitiates a complex signal-transduction cascade, whichultimately leads to activation of the transcription factornuclear factor-κB (NF-κB) and increased transcriptionof pro-inflammatory cytokines such as interleukin-6(IL-6) and tumour-necrosis factor (TNF) (FIG. 2). The

p65p50

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Figure 2 | Intracellular signalling by Toll-like receptors(TLRs). Stimulation of the extracellular domain of a TLRtriggers the intracellular association of myeloid differentiationfactor 88 (MyD88) with its cytosolic domain. Interleukin-1receptor-associated kinase 4 (IRAK4), IRAK1 and tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6) aresubsequently also recruited to the receptor complex. Througha series of other intermediate compounds (not shown), the IκBkinase (IKK) complex is phosphorylated, and in turnphosphorylates IκB, which allows nuclear factor (NF)-κB totranslocate to the nucleus.

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bilayer that consists of phospholipids on the inner leafletand the lipid anchor region of LPS, lipid A, on the outerleaflet43 (FIG. 3a). Lipid A, or endotoxin, is the only regionof LPS to be recognized by the innate immune system.Lipid A from the enteric commensal and occasionalpathogen E. coli is highly immune stimulatory, even atlow concentrations. Immune detection of lipid A is sosensitive and robust that a bloodstream infection cancause a severe complication called endotoxic shock — amajor clinical problem that leads to about 200,000deaths in the United States each year44.

Recognition of LPS occurs largely by the mammalianLPS receptor — the TLR4–MD2–CD14 complex —which is present on many cell types includingmacrophages and dendritic cells20,45–48 (FIG. 3b).Recognition of lipid A also requires an accessory protein— LPS-binding protein (LBP) — which convertsoligomeric micelles of LPS to a monomer for delivery toCD14, which is a glycosyl phosphatidylinositol (GPI)-anchored, high-affinity membrane protein that can alsocirculate in a soluble form. CD14 concentrates LPS forbinding to the TLR4–MD2 complex. How this complexrecognizes lipid A and signals across the plasma mem-brane is still not completely understood. The importanceof TLR4, CD14 and MD2 in LPS recognition is high-lighted by the unresponsive phenotype of mice carryingknockout mutations in any of these genes and the factthat, in humans, polymorphisms in these genes reducethe magnitude of LPS responses48–51. Additionally, inmice, a lack of these components leads to increased sus-ceptibility to infection by the Gram-negative pathogenSalmonella enterica serovar Typhimurium, illustratingthe general principle that functional innate immunerecognition is important for protection against bacterialinfections.

Human TLR4 and lipid ARecent research illustrates that humans, in contrast tomice, have evolved discriminatory mechanisms for dif-ferent lipid A structures, which could result in differentinfectious disease outcomes. Species comparisonsshow that the extracellular domain of TLR4 is less con-served than the cytoplasmic signalling domain.Furthermore, there is a hypervariable extracellulardomain in which the synonymous to non-synonymousbase ratios reflect evolutionary selection pressure todiscriminate between lipid A structures (FIG. 4a). It haslong been known that the mouse TLR4 complex ismore ‘promiscuous’ in recognizing lipid A than ishuman TLR4. For example, lipid

IVA, which is an inter-

mediate in the biosynthetic pathway of lipid A and isthe major component of Yersinia pestis lipid A, stimu-lates mouse TLR4 but is not recognized by humanTLR4, even at very high concentrations52,53. This andother differences in TLR recognition could explainwhy mice without other innate immune defects areresistant to a variety of human pathogens including Y. pestis, H. pylori and L. pneumophila. In addition, theanti-tumour drug Taxol, which has no structural simi-larity to lipid A, is recognized by the murine, but notby the human, TLR4 complex54.

response to LPS among different individuals, even inthe absence of TLR4 polymorphisms (M. Wurfel andT. Martin, personal communication). So, responsive-ness to LPS is distinct among individuals in the humanpopulation and seems to contribute to individual diseasesusceptibility.

Polymorphisms in individual TLR genes are notrestricted to TLR4. Susceptibility to leprosy, which iscaused by Mycobacterium leprae — a mycobacteriumthat does not contain LPS but does contain manyTLR2 ligands — has been linked to a mutation in theextracellular domain of TLR2 (REFS 40,41). Similarly, acommon mutation in TLR5 that reduces signalling inresponse to flagellin resulted in increased susceptibil-ity to infection by Legionella pneumophila, the agentof legionellosis42. In addition, human diversity indownstream signalling pathways has been describedin the case of IRAK4 and has been shown to be linkedto increased infections with pyogenic bacteria39.Taken together, these studies support the hypothesisthat differences in innate immune recognition by multi-ple receptors have an important role in the susceptibilityto infectious diseases.

The TLR4 complex and lipid AThe surface of Gram-negative bacteria is composed ofnumerous ligands for TLRs, including flagella, lipopro-teins, peptidoglycan and LPS. The Gram-negative bacte-rial envelope contains two membranes, in contrast tothe single membrane of Gram-positive bacteria. TheGram-negative outer membrane is an asymmetric

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Figure 3 | Chemical structure of lipopolysaccharide (LPS). a | LPS is composed of lipid A(endotoxin), core oligosaccharide and O-antigen. b | Components of the TLR4–MD2–CD14receptor complex. Different TLR4 regions are shown: leucine-rich repeats (LRR), a hypervariableregion (HYP) and the intracellular TIR domain.

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TLR4 has been shown to be responsible for differen-tial discrimination of naturally occurring P. aeruginosalipid A structures by mice and humans by the use of receptor chimaeras58. Human TLR4 responds to P. aeruginosa lipid A isolated from clinical isolates ofpatients with cystic fibrosis (CF) with a hexa-acylatedstructure, but not to environmental and other clinicalisolates with a penta-acylated structure. Mouse TLR4responds equally and robustly to both lipid A structures.This opportunistic pathogen colonizes the airways withresultant severe chronic disease in individuals with CF— who have a defect in the airway mucosa owing tomutation in the CF transmembrane receptor (CFTR)chloride transporter. Surprisingly, P. aeruginosa does notcolonize the airways of CFTR-knockout mice despitethese mice recapitulating other aspects of CF. Althoughother species differences, including pulmonary anatomyand transporter redundancy, could be involved in thesedifferences, the fact that the mouse TLR4 complex canrespond robustly to all Pseudomonas lipid A structuresmight facilitate the elimination of these organisms.

An 82-amino-acid hypervariable region in the extra-cellular domain of TLR4 was shown to be responsiblefor the ability of murine TLR4 to recognize lipid Astructures from environmental and non-CF clinical iso-lates of P. aeruginosa58. This is the region of TLR4 thatshows significant polymorphism in interspecies andwithin-species comparisons as a result of environmentalselection pressure (FIG. 4a). The relative lack of immunerecognition of environmental isolates by human TLR4might facilitate colonization of this pathogen in thecompromised CF airway of humans but not in mice,supporting the hypothesis that different immune recog-nition of the same microorganism by different speciescould determine disease outcome.

Structural variations in lipid AAlthough lipid A is an essential component of all Gram-negative bacterial membranes, it is a highly diversemolecule (FIG. 5). Lipid A diversity is observed both inthe number and length of fatty-acid side chains and in thepresence of terminal phosphate residues and associatedmodifications. This variability could have profoundimplications for disease, particularly in humans, owingto altered recognition by the TLR4 complex. Structure–function analysis of lipid A signalling indicates that thelength and number of acyl side chains are critical forTLR4 signalling in humans.A variety of studies indicatethat hexa-acylated E. coli lipid A with side chains of 12 to14 carbons in length is maximally stimulating in humancells, whereas altering the number or length of theattached fatty acids or altering the charge of lipid A canreduce the magnitude of the signal59–61 (FIG. 5a). Lipid Aisolated from H. pylori (FIG. 5e), L. pneumophila 62 and avariety of human pathogens that are important to bio-defence including Y. pestis and Francisella spp. havelipid A moieties that are poorly recognized by humanTLR4. These lipid A species typically consist of onlyfour or five acyl chains, some of which are 16–18 car-bons in length63,64. It seems likely that the potential forthese pathogens to cause severe disease in humans is

The creation of human–mouse chimeric receptorshas provided an insight into the molecular basis ofthe recognition of lipid A and its analogues. Forexample, recognition of Taxol and lipid

IVAby the

murine TLR4 complex is mediated by the MD2 com-ponent of the receptor, and a specific glutamineresidue in the carboxy-terminal region of the moleculehas been implicated as important for recognition55–57.In addition, comparison between the synonymous andnon-synonymous base ratios of MD2 indicates selec-tion pressure in the C-terminal region of the protein inall species examined, which is consistent with a role forthis part of the protein in differential recognition oflipid

IVa(FIG. 4b).

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Figure 4 | A hypervariable region of the TLR4 extracellular domain and the C-terminusof the accessory protein MD2 evolved across species. Results are shown from pair-wisealignments of human TLR4 (a) or MD2 (b) with those from other species. Alignments andconstruction of phylogenetic trees were performed using Multalin and DisplayFam,respectively (see the Online links box for further information). Mutation rates were calculatedusing DnaSP 3.51 (see the Online links box). Ka is the number of non-synonymoussubstitutions per non-synonymous site and Ks is the number of synonymous substitutionsper synonymous site. GenBank accession numbers for TLR4 sequences analysed: cat,AB060687; Chinese hamster, AF153676; cow, AB056444; gorilla, AH011592; horse,AY005808; human, U88880; mouse, AF110133; Olive baboon, AH008378; orangutan,AH011591; pygmy chimpanzee, AH008351; rabbit, AY101394; rat, NM019178. GenBankaccession numbers for MD2 sequences analysed: Chinese hamster, AF325501; cow,AF368418; horse, AY398685; human, AB018549; mouse, AB018550. nt, nucleotides.

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that full resistance to Gram-negative bacterial infectionsrequires integration of information from a variety ofinnate immune receptors, although the abundance ofLPS and the data from mutant mice indicate that theTLR4 pathway might be the most important for Gram-negative bacteria.Variation in lipid A might be only onestrategy bacteria use to escape recognition by the innateimmune system. It is conceivable that the structures ofother bacterial ligands such as lipopeptides and fla-gellins also vary between species. The interactionbetween flagella and TLR5 could be important in thisregard65. Despite TLR5 recognition of a flagellar struc-tural motif that is conserved in many bacteria, singleamino acid changes have been shown to substantiallyalter TLR5 signalling. In this context, it is interestingto note that H. pylori flagellins have only low TLR5stimulatory activity, although the exact molecularmechanisms remain to be elucidated66.

attributable, at least in part, to their relative lack ofTLR4 signalling, as Tlr4-null mice are highly susceptibleto infection with Gram-negative microorganisms.Conversely, individuals with increased recognitionand/or response to these structures could be moreresistant or have an increased rate of survival, whichcould be reflected in an increased representation of thisphenotype in descendants of the Northern Europeanpopulation that survived the devastation of plague (Y. pestis) in the past 1,000 years. A caveat to thisassumption is that several reports suggest that lipid Astructures that are unable to signal through TLR4 areable to signal through TLR2, and of course other TLRligands could compensate for a relative lack of TLR4 sig-nalling62,64. So, caution is warranted in interpreting thedata on TLR responses in individuals or species thatcould have more robust signalling through other innateimmune receptor pathways. It is reasonable to conclude

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Figure 5 | The structural diversity of lipid A in Gram-negative microorganisms. Chemical structures of hexa-acylatedEscherichia coli (a) and Pseudomonas aeruginosa (b), hepta-acylated Salmonella enterica serovar Typhimurium (c), penta-acylatedPseudomonas aeruginosa (d), tetra-acylated Helicobacter pylori (e) and lipidIVa, a precursor of enteric lipid A or isolated from Yersiniapestis grown at 37°C (f). Colours indicate different fatty-acid carbon lengths.

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in part driven the diversity of CAMPs. Specific pro-teases and environmentally regulated lipid A alter-ations that promote resistance to CAMPs have agreater effect on susceptibility to α-helical peptidesand polymyxins, which are produced by invertebratesand Gram-positive bacteria, than to β-sheet pep-tides72,73, which are produced only by mammals andplants. It is also plausible that lipid A diversity is dri-ven by selective pressure from TLR4–MD2–CD14recognition. Regardless of the basis of selection, it isclear that selective pressure on both pathogen andhost results in marked variations in lipid A structuresand the host responses through TLR4, and this diver-sity is likely to have a role in the evolution of infectiousdiseases.

Lipid A modifications are induced both by sub-inhibitory concentrations of CAMPs (M.W.B.,unpublished observations) and by growth in mediumthat is limited for the divalent cations calcium andmagnesium74, the latter of which is required to stabilizethe bacterial outer membrane. Other environmentalconditions such as temperature and anaerobic condi-tions promote modification of lipid A in Y. pestis75 andP. aeruginosa, respectively (R.K.E., unpublished obser-vations). In S. typhimurium, a facultative intracellularpathogen that actively replicates in host macrophages,lipid A modifications are regulated by the environmentalsensor-kinase transcriptional regulatory systemPhoP–PhoQ67. PhoP–PhoQ is essential for S. typhi-murium virulence in mice and humans, survival withinmacrophages and resistance to CAMPs.

The regulation of lipid A structures in the salmonel-lae illustrates two important principles of microbialpathogenesis. First, microorganisms can sense hostenvironments (for example, through the PhoP–PhoQsystem), and second, microorganisms can remodeltheir surfaces to resist killing by innate immuneeffectors such as antimicrobial peptides. The PhoP–PhoQ system is fully activated after phagocytosis bymacrophages and signals to the bacterium its pres-ence in phagosomes76, which can contain a varietyof antimicrobial peptides and other surface-activecompounds, as well as being at acidic pH. Lipid Amodifications promote intracellular survival inmacrophages, probably by promoting resistance toantimicrobial peptides. This view is supported by arecent study77 that demonstrated that macrophagesdeficient in cathelicidin-related antimicrobial peptide(CRAMP) supported increased replication of a S. typhimurium phoP null mutant compared withCRAMP+/+ macrophages. So, it is likely that PhoP-dependent surface modifications that resist anti-microbial peptides are important for salmonellae toreplicate within macrophages. In addition to theirrole in promoting resistance to peptides, PhoP–PhoQ-regulated lipid A structures are poorly recognized bythe human TLR4 complex and are less stimulatorythan non-regulated lipid A74,78. This regulated alter-ation in TLR4 recognition might contribute to thepathogenesis of salmonellosis in humans in whichTLR4 is more selective in its recognition.

Regulating the structure of lipid AGram-negative bacteria have evolved mechanisms tomodify the structure of lipid A in different environments,including in host tissues (FIG. 6). These modifications canpromote resistance to host cationic antimicrobial pep-tides (CAMPs) and alter recognition by TLR4 (REF. 67).Environmentally regulated lipid A modifications, andthe enzymes responsible, were first characterized in S. typhimurium. Subsequently, they have also beenidentified in other human pathogens and commensals(TABLE 1). In the human pathogens Shigella spp. andthe salmonellae there is evidence that these environ-mentally regulated enzymes were acquired throughhorizontal gene transfer, indicating selection foraltered lipid A structures67. They are also found in theinsect pathogen Photorhabdus and the plant pathogenErwinia68,69, which suggests that regulation of lipid Ais an ancient mechanism under selection pressurefrom the innate immune system, and in particularCAMPs.

CAMPs are an integral component of innateimmunity with conserved amphipathic and cationicstructural features70,71. Common motifs include α-helical peptides, modified acylated peptides andcomplex β-sheet structures with disulphide bonds.Many different CAMPs are secreted by epithelial andimmune cells of various host organisms includinganimals, plants, Gram-positive bacteria and unicellulareukaryotes. There is evidence that bacterial resistancemechanisms, such as lipid A modifications, may have

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Figure 6 | The chemical structure of environmentallyregulated Salmonella enterica serovar Typhimurium lipid A modifications. PhoP-mediated modifications areshown in red and PmrA/PmrB-mediated modifications in blue.

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such as E. coli. PagL most likely functions to reducerecognition of lipid A when salmonellae colonize hosttissues78. Alterations in the lipid A component of theouter membrane do not occur in isolation and are partof a larger process of remodelling the bacterial surfacethat includes modifications to the carbohydrate com-ponent of lipid A and the protein component of thebacterial envelope.

Lipid A structures and human diseaseIn addition to the ability to regulate variations in theirlipid A structures, bacteria can also alter lipid A mole-cules by fixed mutation in response to long-term colo-nization of individual host environments. This hasbeen demonstrated for P. aeruginosa colonization of thelungs of patients with CF as clinical bacterial isolateshave a unique lipid A structure90 (FIG. 5b). The lipid Amolecules of such isolates are highly modified withaminoarabinose and fatty-acid side chains within thefirst few years of colonization. These lipid A moleculesare structurally different from those that are found inenvironmental isolates and isolates from other humandiseases including chronic pulmonary infections (FIG. 5d),which probably indicates a very early adaptation in theCF lung. The lipid A structures of approximately 36%of individuals with severe pulmonary disease have anadditional fatty acid, a hepta-acylated structure, whichis associated with resistance to β-lactam antibioticsand therefore may be selected for by antibiotic pres-sure (R.K.E., unpublished observations). Theseincreases in lipid A modification patterns in clinicalisolates are probably due to mutation, as these isolateshave constitutive modifications of their structures.

The airways of individuals with CF are colonizedshortly after birth with P. aeruginosa and these organ-isms evolve over decades, continuously promoting anacute inflammatory response that leads to airwaydestruction. It is likely that innate immune selectivepressure, such as that provided by antimicrobial pep-tides, promotes these constitutive lipid A modifications.In addition, lipid A from P. aeruginosa CF isolates, incontrast to isolates from other human infection sitesincluding chronic pulmonary and acute urinary, bloodand eye infections, markedly stimulate human TLR4signalling and might contribute to the inflammatoryresponse that is seen in patients with CF. This might bea model for other human chronic inflammatory dis-eases with a bacterial and innate immune componentsuch as inflammatory bowel disease and periodontaldisease caused by Porphyromonas gingivalis91, in whichselection of a bacterial clone with innate immune sig-nalling properties may be an essential component ofthe disease process.

Y. pestis lipid A. The study of lipid A structures andtheir regulation in Yersinia spp. is particularly instruc-tive for pathogen evolution as this genus includes threegenetically similar human pathogens — Yersiniapseudotuberculosis, Y. pestis and Yersinia enterocolitica.Y. pestis is the causative agent of plague, is transmittedby flea bites and is thought to have evolved from

The enzymes involved. PhoP–PhoQ-regulated structuralchanges in lipid A are illustrated in FIG. 5a and include theaddition of palmitate, aminoarabinose and phospho-ethanolamine to lipid A, as well as hydroxylation anddeacylation of specific fatty acids72,74,79–84.After inductionof the enzymes involved in these modifications byPhoP–PhoQ, the outer membrane is composed of amosaic of more than 20 individual lipid A structures.The molecular details of how these modifications arepromoted have recently been elucidated. Two geneclusters encoding eight proteins are necessary foraminoarabinose synthesis and addition, which usesUDP-glucose as a substrate79,85. Addition of theaminoarabinose moiety occurs at the bacterial innermembrane, where lipid A molecules are assembled fortheir export to the cell surface.Aminoarabinose is addedto the phosphate backbone of the lipid A molecule anddecreases the net negative charge, a change that pro-motes resistance to cationic antimicrobial peptides,especially polymyxins, and increases the virulence ofS. typhimurium for inbred susceptible mice by the oralroute79,84,86,87.

In addition to neutralizing the charge of the phos-phate groups, the greatest regulation of lipid A is in itsacylation. Salmonellae alter acylation through threeenzymes (two of which are not present in E. coli), andthe alterations are regulated by PhoP–PhoQ. Thereactions catalysed by PagP, PagL and LpxO lead tothe acylation, deacylation and hydroxylation of the lipidA molecule, respectively72,80,81. PagP and PagL both alterlipid A recognition by human TLR4 and are outermembrane enzymes that most likely function as serinehydrolases. PagP adds a C

16:0 fatty acid to lipid A72 by

transfer of a fatty acid from the inner leaflet to the outerleaflet of the outer membrane. This modification leadsto increased resistance to CAMPs, especially those withan α-helical structure. In addition to salmonellae, PagPis found in a subset of Gram-negative bacteria, furthersupporting its role during infection. L. pneumophila andBordetella spp. express PagP homologues, both of whichhave been demonstrated to be involved in virulence,specifically Legionella survival within macrophages88

and respiratory-tract colonization by Bordetella spp.89

PagL leads to deacylation of lipid A and, with the excep-tion of the salmonellae, is not present in enteric bacteria

Table 1 | Conservation of enzymes that modify lipid A

Species PhoP–PhoQ PmrF LpxO PagP PagL

Salmonella + + + + +

Shigella + + – + –

Yersinia + + – + –

Bordetella + + + + –

Pseudomonas + + + + +

Neisseria + – – – –

Erwinia + + – + –

Brucella + – + – –

(+) highly conserved, (–) not present. PhoP–PhoQ regulates expression of virulence genes in responseto environmental signals; PmrF transfers aminoarabinose to lipid A; LpxO transfers a hydroxyl group toa fatty acid group in lipid A; PagP transfers palmitate to lipid A; PagL deacylates lipid A.

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specific innate immune ligands, such as LPS, thatpromote detrimental inflammatory responses, themagnitudes of which are augmented by specific innateimmune phenotypes. Human phenotyping could alsopredict susceptibility by correlating responses to a par-ticular innate immune ligand with disease susceptibility.Such phenotyping and genotyping could lead to the pro-phylactic use of innate immune stimulators or blockers,with the aim of decreasing infection or response to thecorresponding microorganism. The analysis of innateimmune stimulators with specific properties could alsopromote the development of new therapeutic com-pounds that could reduce detrimental inflammatoryresponses.

Additionally, a better understanding of innateimmune diversity may lead to the development ofsafer and less expensive vaccines. Currently, manyapproved vaccines contain innate immune ligands asadjuvants, such as monophosphorylated lipid A fromSalmonella enterica serovar Minnesota93. A variety ofsynthetic lipid A analogues are also in developmentfor use as adjuvants, many of which have novel TLR4agonist or antagonist activities94–96. It is likely thatmany new lipid-A-type molecules and other innateimmune modifiers will be discovered in the future.These compounds can then be produced from recom-binant strains or synthesized as chemical analoguesand evaluated for their efficacy as adjuvants in vaccina-tion studies. Most vaccines, even those that are widelyused, are not 100% effective and have a low incidenceof serious side effects. To circumvent these problems,detailed analyses of human responses to innateimmune ligands may lead to the continuous improve-ment of adjuvants, which could be optimized for aparticular vaccine to promote efficacy by stimulatingspecific immunological responses. Synthetic stimula-tory molecules could also be used in prophylaxisagainst infectious diseases during epidemics or high-risk exposures. For instance, it has been shown thatstimulation with synthetic lipid A analogues withreduced inflammatory properties can protect miceagainst challenge with influenza virus97. During periodsof high exposure risk, prophylactic innate immunestimulators could be prescribed to reduce an individual’ssusceptibility to infection.

The discovery of the enzymes responsible for lipid Amodifications provides the opportunity to developantibacterial compounds that inhibit these enzymes andthereby promote CAMP-mediated killing and reduceinflammation. This application could be of use in thetreatment of chronic diseases such as CF in whichinhibition of specific lipid A modifications is expectedto reduce inflammation and augment susceptibility toinnate immunity. Inhibition of lipid A modificationscould also promote the utility of cationic antibiotics suchas polymyxin and aminoglycosides. This approach couldbe useful in treating a variety of other diseases such asnon-CF bronchiectasis and chronic obstructive pul-monary disease. The treatment of infections caused byintracellular pathogens that are more resistant to anti-biotic therapy due to the impermeability of the bacterial

Y. pseudotuberculosis approximately 1,500–20,000 yearsago92. Y. pseudotuberculosis and Y. enterocolitica areenteric organisms similar to non-typhoidal salmonel-lae that colonize the intestinal tract of a broad range ofanimals. Y. pestis evolved as a major pathogen owing toits ability to be transmitted to humans and other smallmammals by an insect vector. By contrast, infection bythe oral route with other Yersinia spp. results in milder,self-limiting disease in humans and a variety of animals.During its life cycle, Y. pestis is exposed to the innateimmune system of different hosts at different tempera-tures — humans and rodents at 37°C and fleas at21°C. Within insects, the bacteria are exposed to insectinnate immune pressure, which consists mostly ofCAMPs. Interestingly, Yersinia spp. regulate their lipid Astructures with temperature change, with Y. pestisexhibiting greater temperature-regulated diversity oflipid A structures, which is consistent with selection pres-sure within fleas. For example, at 21°C, Y. pestis lipid A ishighly acylated and more resistant to CAMPs, whereas at37°C, its lipid A is largely in lipid

IVAform, which is a

tetra-acylated lipid A precursor in enteric bacteria. Thistetra-acylated structure does not have a stimulatoryeffect on human innate immune signalling throughTLR4, so the ability of Y. pestis to synthesize a lipid Amolecule at 37°C that is not recognized by human TLR4could explain why this pathogen causes a much moresevere disease than Y. pseudotuberculosis. In support ofthis hypothesis, Y. pseudotuberculosis, which is nottransmitted by insect vectors but through thefaecal–oral route, exhibits the opposite temperatureregulation of lipid A – the molecules that are synthe-sized at the mammalian temperature of 37°C have ahigher fatty-acid content, are more stimulatory ofTLR4-dependent signalling than Y. pestis and bac-teria are more resistant to CAMPs. The regulation ofY. pestis lipid A structure and the differential stimulationof human TLR4 by these structures might explain inpart why insect-based transmission (TLR4 recogni-tion) is much less virulent than human-to-humanrespiratory transmission (TLR4 invisibility), whichresults in severe bacteraemia before septic shock.Therefore, alteration of pattern-recognition moleculesas a result of innate immune selective pressure mightrepresent a mechanism for a change in host range orvirulence.

Clinical implications of diversityAs human genotyping becomes more inexpensive andreliable, innate immune polymorphisms may be used asmarkers of susceptibility to infections and/or inflamma-tory diseases in the same way that human leukocyteantigen (HLA) genotyping has been used in transplan-tation and epidemiology for decades. Testing theresponses of individuals to specific bacterial innateimmune ligands might indicate their susceptibility to awide variety of infections and inflammatory conditionsthat probably involve a bacterial component, such asinflammatory bowel disease, CF, neurological disordersand arthritis. In such diseases, the unique mammalianenvironments might select bacterial populations with

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track the emergence, and predict the outcomes, ofinfectious diseases in specific populations and individu-als. This goal presents a significant challenge due to thediversity of microbial, plant and animal populations.Recent progress in defining the interactions betweeninnate immune receptor pathways and their bacterialligands supports the hypothesis that these interactionsare important factors in the diverse manifestations ofinfectious diseases.

outer membrane, such as L. pneumophila, Francisellatularensis and Burkholderia spp., could also be improvedby inhibition of lipid A modifications — a strategythat could work synergistically with the availableantimicrobial arsenal.

Important advances have been made in defining themolecular mechanisms underlying the interactionsbetween microorganisms and their hosts. The challengefor the twenty-first century is to use this information to

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AcknowledgementsWe would like to thank T. Freeman for calculating thesynonymous/non-synonymous content of TLR4 and MD2. We thankC. Wilson and members of the Miller lab for helpful discussions. Theauthors were supported by grants from the NIAID, and Cystic FibrosisFoundation grants to S.I.M., a NIH grant to R.K.E and an Emmy-Noether fellowship from Deutsche Forschungsgemeinschaft to M.W.B.S.I.M. and R.K.E. were funded by the NIAID Research Centre forExcellence in Biodefense and Emerging Infectious Diseases.

Competing interests statementThe authors declare no competing financial interests.

Online links

DATABASESThe following terms in this article are linked online to:Entrez: http://www.ncbi.nlm.nih.gov/Entrez/Drosophila melanogaster | Escherichia coli | Helicobacter pylori |IL-6 | IRAK1 | Legionella pneumophila | Mycobacterium leprae |NF-κB | PhoP | PhoQ | Porphyromonas gingivalis | Pseudomonassyringae | Salmonella enterica serovar Typhi | Salmonella entericaserovar Typhimurium | Serratia marcescens | TLR2 | TLR3 | TLR4 |TLR5 | TLR8 | TLR9 | TNF | TRAF6 | Yersinia enterocolitica |Yersinia pestis | Yersinia pseudotuberculosis

FURTHER INFORMATIONSamuel I. Miller’s laboratory:http://depts.washington.edu/micro/cvs/SamuelMiller.htmlDnaSP 3.51: http://www.ub.es/dnaspDisplayFam:http://prodes.toulouse.inra.fr/prodom/DisplayFam/DisplayFam.htmlMultalin: http://prodes.toulouse.inra.fr/multalin/multalin.htmlAccess to this links box is available online.

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