Flagellin glycosylation in Pseudomonas aeruginosa PAK requires the O-antigen biosynthesis enzyme WbpO

Pseudomonas aeruginosa PAK (serotype O6) produces a single polar, glycosylated flagellum composed of a-type flagellin. To determine whether or not flagellin glycosylation in this serotype requires O-antigen genes, flagellin was isolated from the wild type, three O-antigen-deficient mutants wbpL, wbpO, and wbpP, and a wbpO mutant complemented with a plasmid containing a wild-type copy of wbpO. Flagellin from the wbpO mutant was smaller (42 kDa) than that of the wild type (45 kDa), or other mutants strains, and exhibited an altered isoelectric point (pI 4.8) when compared with PAK flagellin (pI 4.6). These differences were because of the truncation of the glycan moiety in the wbpO-flagellin. Thus, flagellin glycosylation in P. aeruginosa PAK apparently requires a functional WbpO but not WbpP. Because WbpP was previously proposed to catalyze a metabolic step in the biosynthesis of B-band O-antigen that precedes the action of WbpO, these results prompted us to reevaluate the two-step pathway catalyzed by WbpO and WbpP. Results from WbpO-WbpP-coupled enzymatic assays showed that either WbpO or WbpP is capable of initiating the two-step pathway; however, the kinetic parameters favored the WbpO reaction to occur first, converting UDP-N-acetyl-D-glucosamine to UDP-N-acetyl-D-glucuronic acid prior to the conversion to UDP-N-acetyl-D-galacturonic acid by WbpP. This is the first report to show that a C4 epimerase could utilize UDP-N-acetylhexuronic acid as a substrate.     

Cloning and Characterization of Flagellin Genes and Identification of Flagellin Glycosylation from Thermophilic Bacillus Species

Flagellin glycosylation was identified in Bacillus sp. PS3 and Geobacillus stearothermophilus. In vivo complementation showed that these flagellin genes did not restore the motility of a Bacillus subtilis flagellin mutant, whereas the genes encoding non-glycosylated flagellin from Geobacillus kaustophilus and Bacillus sp. Kps3 restored motility. Moreover, four types of flagellins expressed in B. subtilis were not glycosylated. We speculate that glycosylation is required for flagellar filament assembly of these bacilli.     

TRIF Mediates Toll-like Receptor 5-induced Signaling in Intestinal Epithelial Cells

Toll-like receptors (TLRs) associate with adaptor molecules (MyD88, Mal/TIRAP, TRAM, and TRIF) to mediate signaling of host-microbial interaction. For instance, TLR4 utilizes the combination of both Mal/TIRAP-MyD88 (MyD88-dependent pathway) and TRAM-TRIF (MyD88-independent pathway). However, TLR5, the specific receptor for flagellin, is known to utilize only MyD88 to elicit inflammatory responses, and an involvement of other adaptor molecules has not been suggested in TLR5-dependent signaling. Here, we found that TRIF is involved in mediating TLR5-induced nuclear factor κB (NFκB) and mitogen-activated protein kinases (MAPKs), specifically JNK1/2 and ERK1/2, activation in intestinal epithelial cells. TLR5 activation by flagellin permits the physical interaction between TLR5 and TRIF in human colonic epithelial cells (NCM460), whereas TLR5 does not interact with TRAM upon flagellin stimulation. Both primary intestinal epithelial cells from TRIF-KO mice and TRIF-silenced NCM460 cells significantly reduced flagellin-induced NFκB (p105 and p65), JNK1/2, and ERK1/2 activation compared with control cells. However, p38 activation by flagellin was preserved in these TRIF-deficient cells. TRIF-KO intestinal epithelial cells exhibited substantially reduced inflammatory cytokine (keratinocyte-derived cytokine, macrophage inflammatory protein 3α, and IL-6) expression upon flagellin, whereas control cells from TRIF-WT mice showed robust cytokine expression by flagellin. Compare with TRIF-WT mice, TRIF-KO mice were resistant to in vivo intestinal inflammatory responses: flagellin-mediated exacerbation of colonic inflammation and dextran sulfate sodium-induced experimental colitis. We conclude that in addition to MyD88, TRIF mediates TLR5-dependent responses and, thereby regulates inflammatory responses elicited by flagellin/TLR5 engagement. Our findings suggest an important role of TRIF in regulating host-microbial communication via TLR5 in the gut epithelium.     

Glycosylation of Pseudomonas aeruginosa 1244 pilin: glycan substrate specificity

The structural similarity between the pilin glycan and the O-antigen of Pseudomonas aeruginosa 1244 suggested that they have a common metabolic origin. Mutants of this organism lacking functional wbpM or wbpL genes synthesized no O-antigen and produced only non-glycosylated pilin. Complementation with plasmids containing functional wbpM or wbpL genes fully restored the ability to produce both O-antigen and glycosylated pilin. Expression of a cosmid clone containing the O-antigen biosynthetic gene cluster from P. aeruginosa PA103 (LPS serotype O11) in P. aeruginosa 1244 (LPS serotype O7) resulted in the production of strain 1244 pili that contained both O7 and O11 antigens. The presence of the O11 repeating unit was confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Expression of the O-antigen biosynthesis cluster from Escherichia coli O157:H7 in strain 1244 resulted in the production of pilin that contained both the endogenous Pseudomonas as well as the Escherichia O157 O-antigens. A role for pilO in the glycosylation of pilin in P. aeruginosa is evident as the cloned pilAO operon produced glycosylated strain 1244 pilin in eight heterologous P. aeruginosa strains. Removal of the pilO gene resulted in the production of unmodified strain 1244 pilin. These results show that the pilin glycan of P. aeruginosa 1244 is a product of the O-antigen biosynthetic pathway. In addition, the structural diversity of the O-antigens used by the 1244 pilin glycosylation apparatus indicates that the glycan substrate specificity of this reaction is extremely low.     

Amino Acid Substitutions and Intragenic Duplications of Bacillus sp. PS3 Flagellin Cause Complementation of the Bacillus subtilis Flagellin Deletion Mutant

Bacillus sp. PS3 produces a glycosylated flagellin. In this study, a number of the glycosylated residues of the flagellin protein were found to be located in the central variable region of this protein. We also report that the motility defect of the Bacillus subtilis flagellin mutant was complemented by Bacillus sp. PS3 flagellin variants without glycosylation, which contained amino acid substitutions and intragenic duplications in the variable region of flagellin.     

Structural and genetic characterization of glycosylation of type a flagellin in Pseudomonas aeruginosa

Type a flagellins from two strains of Pseudomonas aeruginosa, strains PAK and JJ692, were found to be glycosylated with unique glycan structures. In both cases, two sites of O-linked glycosylation were identified on each monomer, and these sites were localized to the central, surface-exposed domain of the monomer in the assembled filament. The PAK flagellin was modified with a heterogeneous glycan comprising up to 11 monosaccharide units that were O linked through a rhamnose residue to the protein backbone. The flagellin of JJ692 was less complex and had a single rhamnose substitution at each site. The role of the glycosylation island gene cluster in the production of each of these glycosyl moieties was investigated. These studies revealed that the orfA and orfN genes were required for attachment of the heterologous glycan and the proximal rhamnose residue, respectively.     

Toll-like receptor 5 forms asymmetric dimers in the absence of flagellin

The structure of full-length human TLR5 determined by electron microscopy single-particle image reconstruction at 26 Å resolution shows that TLR5 forms an asymmetric homodimer via ectodomain interactions. The structure shows that like TLR9, TLR5 dimerizes in the absence of ligand. The asymmetry of the dimer suggests that TLR5 may recognize two flagellin molecules cooperatively to establish an optimal flagellin response threshold. A TLR5 homology model was generated and fitted into the electron microscopy structure. All seven predicted N-linked glycosylation sites are exposed on the molecular surface, away from the dimer interface. Glycosylation at the first five sites was confirmed by tandem mass spectrometry. Two aspartate residues proposed to interact with flagellin (Asp294 and Asp366) are sterically occluded by a glycan at position 342. In contrast, the central region of the ectodomains near the dimer interface is unobstructed by glycans. Ligand binding in this region would be consistent with the ligand binding sites of other TLRs.     

Innate Immune Detection of Flagellin Positively and Negatively Regulates Salmonella Infection

Salmonella enterica serovar Typhimurium is a flagellated bacterium and one of the leading causes of gastroenteritis in humans. Bacterial flagellin is required for motility and also a prime target of the innate immune system. Innate immune recognition of flagellin is mediated by at least two independent pathways, TLR5 and Naip5-Naip6/NlrC4/Caspase-1. The functional significance of each of the two independent flagellin recognition systems for host defense against wild type Salmonella infection is complex, and innate immune detection of flagellin contributes to both protection and susceptibility. We hypothesized that efficient modulation of flagellin expression in vivo permits Salmonella to evade innate immune detection and limit the functional role of flagellin-specific host innate defenses. To test this hypothesis, we used Salmonella deficient in the anti-sigma factor flgM, which overproduce flagella and are attenuated in vivo. In this study we demonstrate that flagellin recognition by the innate immune system is responsible for the attenuation of flgM⁻ S. Typhimurium, and dissect the contribution of each flagellin recognition pathway to bacterial clearance and inflammation. We demonstrate that caspase-1 controls mucosal and systemic infection of flgM⁻ S. Typhimurium, and also limits intestinal inflammation and injury. In contrast, TLR5 paradoxically promotes bacterial colonization in the cecum and systemic infection, but attenuates intestinal inflammation. Our results indicate that Salmonella evasion of caspase-1 dependent flagellin recognition is critical for establishing infection and that evasion of TLR5 and caspase-1 dependent flagellin recognition helps Salmonella induce intestinal inflammation and establish a niche in the inflamed gut.     

Functional Characterization of Flagellin Glycosylation in Campylobacter jejuni 81-176

The major flagellin of Campylobacter jejuni strain 81-176, FlaA, has been shown to be glycosylated at 19 serine or threonine sites, and this glycosylation is required for flagellar filament formation. Some enzymatic components of the glycosylation machinery of C. jejuni 81-176 are localized to the poles of the cell in an FlhF-independent manner. Flagellin glycosylation could be detected in flagellar mutants at multiple levels of the regulatory hierarchy, indicating that glycosylation occurs independently of the flagellar regulon. Mutants were constructed in which each of the 19 serine or threonines that are glycosylated in FlaA was converted to an alanine. Eleven of the 19 mutants displayed no observable phenotype, but the remaining 8 mutants had two distinct phenotypes. Five mutants (mutations S417A, S436A, S440A, S457A, and T481A) were fully motile but defective in autoagglutination (AAG). Three other mutants (mutations S425A, S454A, and S460A) were reduced in motility and synthesized truncated flagellar filaments. The data implicate certain glycans in mediating filament-filament interactions resulting in AAG and other glycans appear to be critical for structural subunit-subunit interactions within the filament.Flagellins from many polarly flagellated bacteria are glycosylated (reviewed in reference 22). The best-characterized examples are the flagellins from Campylobacter spp. that are decorated with as many as 19 O-linked glycans that can contribute ∼10% to the weight of flagellin (38). The genes encoding the enzymes for biosynthesis of the glycans found on Campylobacter flagellins and the respective glycosyltransferases are located adjacent to the flagellin structural genes in one of the more hypervariable regions of the Campylobacter genome (3, 16, 28, 37). Most strains appear to carry the genes for synthesis of two distinct nine-carbon sugars that decorate flagellin: pseudaminic acid (PseAc) and an acetamidino form of legionaminic acid (LegAm) (23). In contrast, Campylobacter jejuni strain 81-176 contains only the pathway for synthesis of PseAc (9) and derivatives of PseAc that include an acetylated form (PseAcOAc), an acetamidino form (PseAm), and a form of PseAm with a glutamic acid moiety attached (PseAmOGln) (25, 34, 38). The flagellins of C. jejuni strain NCTC 11168 have recently been shown to be glycosylated with PseAc and LegAm, as well as two novel derivatives of PseAc, a di-O-methylglyceric acid and a related acetamidino form (24). Thus, although all of the flagellar glycans appear to be based on either PseAc and/or LegAm, there are variations among strains that contribute to serospecificity and reflect the heterogeneity of the flagellin glycosylation loci (23, 24).The function of the glycosyl modifications to flagellar structure and to the biology of campylobacters is not fully understood. Although most polarly flagellated bacteria appear to glycosylate flagellin, mutation of the genes involved in glycosylation does not generally result in loss of motility (22). However, flagella from C. jejuni, Campylobacter coli, and Helicobacter pylori, all members of the epsilon division of Proteobacteria, are unable to assemble a filament in the absence of a functional glycosylation system (7, 33). Also, changes in the glycans on campylobacter flagellins have been shown to affect autoagglutination (AAG) and microcolony formation on intestinal epithelial cells in vitro (5, 9). Thus, a mutant of C. jejuni 81-176 that was unable to synthesize PseAm assembled a flagellar filament, but the sites on the flagellin subunits that were normally glycosylated with PseAm were instead glycosylated with PseAc. This mutant was reduced in AAG, adherence, and invasion of INT407 cells and was also attenuated in a ferret diarrheal disease model (9). C. coli VC167 has both PseAc and LegAm pathways. Mutants that were defective in either pathway could still assemble flagellar filaments composed of subunits that were modified with the alternate sugar, but these mutants showed defects in AAG (7). A VC167 double mutant, defective in both PseAc and LegAm synthesis, was nonflagellated (7). Collectively, these data suggest that some glycosylation is required for either secretion of flagellin or for interactions between subunits within the filament.Flagellar biogenesis in C. jejuni is a complex process that is highly controlled by the alternate sigma factors σ²⁸ and σ⁵⁴, a two-component regulatory system composed of the sensor kinase FlgS and the σ⁵⁴-response regulator FlgR, and the flagellar export apparatus (15, 39). Both flgR and flgS genes undergo slip strand mismatch repair in C. jejuni strain 81-176, resulting in an on/off-phase variation of flagellar expression (13, 14). The major flagellin gene, flaA, and some other late flagellar genes are regulated by σ²⁸; the genes encoding the minor flagellin, flaB, and the hook and rod structures are regulated by σ⁵⁴. Here, we examine several aspects of glycosylation to flagellar function in C. jejuni 81-176. We demonstrate that some components of the flagellar glycosylation machinery are localized to the poles of the cell, but independently of the signal recognition particle-like flagellar protein, FlhF, and that flagellin glycosylation occurs independently of the flagellar regulon. We also show that the glycans on some amino acids appear to play a structural role in subunit interactions in the filament, while others affect interactions with adjacent filaments that result in AAG.     

Pilin glycosylation in Neisseria meningitidis occurs by a similar pathway to wzy-dependent O-antigen biosynthesis in Escherichia coli

Pili (type IV fimbriae) of Neisseria meningitidis are glycosylated by the addition of O-linked sugars. Recent work has shown that PglF, a protein with homology to O-antigen 'flippases', is required for the biosynthesis of the pilin-linked glycan and suggests pilin glycosylation occurs in a manner analogous to the wzy-dependent addition of O-antigen to the core-LPS. O-Antigen ligases are crucial in this pathway for the transfer of undecraprenol-linked sugars to the LPS-core in Gram-negative bacteria. An O-antigen ligase homologue, pglL, was identified in N. meningitidis. PglL mutants showed no change in LPS phenotypes but did show loss of pilin glycosylation, confirming PglL is essential for pilin O-linked glycosylation in N. meningitidis.     

Genetic organization of chromosomal S-layer glycan biosynthesis loci of Bacillaceae

S-layer glycoproteins are cell surface glycoconjugates that have been identified in archaea and in bacteria. Usually, S-layer glycoproteins assemble into regular, crystalline arrays covering the entire bacterium. Our research focuses on thermophilic Bacillaceae, which are considered a suitable model system for studying bacterial glycosylation. During the past decade, investigations of S-layer glycoproteins dealt with the elucidation of the highly variable glycan structures by a combination of chemical degradation methods and nuclear magnetic resonance spectroscopy. It was only recently that the molecular characterization of the genes governing the formation of the S-layer glycoprotein glycan chains has been initiated. The S-layer glycosylation (slg) gene clusters of four of the 11 known S-layer glycan structures from members of the Bacillaceae have now been studied. The clusters are ～16 to ～25 kb in size and transcribed as polycistronic units. They include nucleotide sugar pathway genes that are arranged as operons, sugar transferase genes, glycan processing genes, and transporter genes. So far, the biochemical functions only of the genes required for nucleotide sugar biosynthesis have been demonstrated experimentally. The presence of insertion sequences and the decrease of the G+C content at the slg locus suggest that the investigated organisms have acquired their specific S-layer glycosylation potential by lateral gene transfer. In addition, S-layer protein glycosylation requires the participation of housekeeping genes that map outside the cluster. The gene encoding the respective S-layer target protein is transcribed monocistronically and independently of the slg cluster genes. Its chromosomal location is not necessarily in close vicinity to the slg gene cluster. Published in 2004.     

Staphylococcus aureus Inhibits IL-8 Responses Induced by Pseudomonas aeruginosa in Airway Epithelial Cells

Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) are major respiratory pathogens and can concurrently colonize the airways of patients with chronic obstructive diseases, such as cystic fibrosis (CF). Airway epithelial cell signalling is critical to the activation of innate immune responses. In the setting of polymicrobial colonization or infection of the respiratory tract, how epithelial cells integrate different bacterial stimuli remains unknown. Our study examined the inflammatory responses to PA and SA co-stimulations. Immortalised airway epithelial cells (Beas-2B) exposed to bacteria-free filtrates from PA (PAF) induced a robust production of the neutrophil chemoattractant IL-8 while bacteria-free filtrates from SA (SAF) had a minimal effect. Surprisingly, co-stimulation with PAF+SAF demonstrated that SAF strongly inhibited the PAF-driven IL-8 production, showing that SAF has potent anti-inflammatory effects. Similarly SAF decreased IL-8 production induced by the TLR1/TLR2 ligand Pam₃CysSK₄ but not the TLR4 ligand LPS nor TLR5 ligand flagellin in Beas-2B cells. Moreover, SAF greatly dampened TLR1/TLR2-mediated activation of the NF-κB pathway, but not the p38 MAPK pathway. We observed this SAF-dependent anti-inflammatory activity in several SA clinical strains, as well as in the CF epithelial cell line CFBE41o-. These findings show a novel direct anti-inflammatory effect of SA on airway epithelial cells, highlighting its potential to modulate inflammatory responses in the setting of polymicrobial infections.     

The Adjuvant Activity of Alphavirus Replicons Is Enhanced by Incorporating the Microbial Molecule Flagellin into the Replicon

Ligands of pattern recognition receptors (PRRs) including Toll-like receptors (TLRs) stimulate innate and adaptive immune responses and are considered as potent adjuvants. Combinations of ligands might act in synergy to induce stronger and broader immune responses compared to stand-alone ligands. Alphaviruses stimulate endosomal TLRs 3, 7 and 8 as well as the cytoplasmic PRR MDA-5, resulting in induction of a strong type I interferon (IFN) response. Bacterial flagellin stimulates TLR5 and when delivered intracellularly the cytosolic PRR NLRC4, leading to secretion of proinflammatory cytokines. Both alphaviruses and flagellin have independently been shown to act as adjuvants for antigen-specific antibody responses. Here, we hypothesized that alphavirus and flagellin would act in synergy when combined. We therefore cloned the Salmonella Typhimurium flagellin (FliC) gene into an alphavirus replicon and assessed its adjuvant activity on the antibody response against co-administered antigen. In mice immunized with recombinant alphavirus, antibody responses were greatly enhanced compared to soluble FliC or control alphavirus. Both IgG1 and IgG2a/c responses were increased, indicating an enhancement of both Th1 and Th2 type responses. The adjuvant activity of FliC-expressing alphavirus was diminished but not abolished in the absence of TLR5 or type I IFN signaling, suggesting the contribution of several signaling pathways and some synergistic and redundant activity of its components. Thus, we have created a recombinant adjuvant that stimulates multiple signaling pathways of innate immunity resulting in a strong and broad antibody response.     

Flagellar glycosylation in Burkholderia pseudomallei and Burkholderia thailandensis

Glycosylation of proteins is known to impart novel physical properties and biological roles to proteins from both eukaryotes and prokaryotes. In this study, gel-based glycoproteomics were used to identify glycoproteins of the potential biothreat agent Burkholderia pseudomallei and the closely related but nonpathogenic B. thailandensis. Top-down and bottom-up mass spectrometry (MS) analyses identified that the flagellin proteins of both species were posttranslationally modified by novel glycans. Analysis of proteins from two strains of each species demonstrated that B. pseudomallei flagellin proteins were modified with a glycan with a mass of 291 Da, while B. thailandensis flagellin protein was modified with related glycans with a mass of 300 or 342 Da. Structural characterization of the B. thailandensis carbohydrate moiety suggests that it is an acetylated hexuronic acid. In addition, we have identified through mutagenesis a gene from the lipopolysaccharide (LPS) O-antigen biosynthetic cluster which is involved in flagellar glycosylation, and inactivation of this gene eliminates flagellar glycosylation and motility in B. pseudomallei. This is the first report to conclusively demonstrate the presence of a carbohydrate covalently linked to a protein in B. pseudomallei and B. thailandensis, and it suggests new avenues to explore in order to examine the marked differences in virulence between these two species.     

A Non-Synonymous Coding Variant (L616F) in the TLR5 Gene Is Potentially Associated with Crohn's Disease and Influences Responses to Bacterial Flagellin

Background and Objectives

Although numerous studies have implicated TLR5, or its ligands, bacterial flagellins, in the pathogenesis of Crohn''s disease (CD), genome-wide association studies (GWAS) have not reported associations with the TLR5 gene. We aimed to examine potential CD-associated TLR5 variants and assess whether they modified inflammatory responses to bacterial flagellins.

Methods and Principal Results

A two-stage study was carried out. In stage 1, we genotyped tagging single-nucleotide polymorphisms (tag-SNPs) in the TLR5 gene in a sample of CD cases (<20 years of age, N = 566) and controls (N = 536). Single SNP and haplotype analysis was carried out. In Stage 2, we assessed the functional significance of potential CD-associated variant(s) vis-à-vis effects on the inflammatory response to bacterial flagellin using HEK293T cells. We observed marginal association between a non-synonymous coding SNP rs5744174 (p = 0.05) and CD. Associations between SNP rs851139 that is in high linkage disequilibrium (LD) with SNP rs5744174 were also suggested (p = 0.07). Haplotype analysis revealed that a 3 marker haplotype was significantly associated with CD (p = 0.01). Functional studies showed that the risk allele (616F) (corresponding to the C allele of SNP rs5744174) conferred significantly greater production of CCL20 in response to a range of flagellin doses than the comparator allele (616L).

Conclusions

Our findings suggest that a non-synonymous coding variation in the TLR5 gene may confer modest susceptibility for CD.     

Flagellin/TLR5 responses in epithelia reveal intertwined activation of inflammatory and apoptotic pathways

Flagellin, the primary structural component of bacterial flagella, is recognized by Toll-like receptor 5 (TLR5) present on the basolateral surface of intestinal epithelial cells. Utilizing biochemical assays of proinflammatory signaling pathways and mRNA expression profiling, we found that purified flagellin could recapitulate the human epithelial cell proinflammatory responses activated by flagellated pathogenic bacteria. Flagellin-induced proinflammatory activation showed similar kinetics and gene specificity as that induced by the classical endogenous proinflammatory cytokine TNF-alpha, although both responses were more rapid than that elicited by viable flagellated bacteria. Flagellin, like TNF-alpha, activated a number of antiapoptotic mediators, and pretreatment of epithelial cells with this bacterial protein could protect cells from subsequent bacterially mediated apoptotic challenge. However, when NF-kappaB-mediated or phosphatidylinositol 3-kinase/Akt proinflammatory signaling was blocked, flagellin could induce programmed cell death. Consistently, we demonstrate that flagellin and viable flagellate Salmonella induces both the extrinsic and intrinsic caspase activation pathways, with the extrinsic pathway (caspase 8) activated by purified flagellin in a TLR5-dependant fashion. We conclude that interaction of flagellin with epithelial cells induces caspase activation in parallel with proinflammatory responses. Such intertwining of proinflammatory and apoptotic signaling mediated by bacterial products suggests roles for host programmed cell death in the pathogenesis of enteric infections.     

Activation of Human Toll-like Receptor 4 (TLR4)·Myeloid Differentiation Factor 2 (MD-2) by Hypoacylated Lipopolysaccharide from a Clinical Isolate of Burkholderia cenocepacia

Lung infection by Burkholderia species, in particular Burkholderia cenocepacia, accelerates tissue damage and increases post-lung transplant mortality in cystic fibrosis patients. Host-microbe interplay largely depends on interactions between pathogen-specific molecules and innate immune receptors such as Toll-like receptor 4 (TLR4), which recognizes the lipid A moiety of the bacterial lipopolysaccharide (LPS). The human TLR4·myeloid differentiation factor 2 (MD-2) LPS receptor complex is strongly activated by hexa-acylated lipid A and poorly activated by underacylated lipid A. Here, we report that B. cenocepacia LPS strongly activates human TLR4·MD-2 despite its lipid A having only five acyl chains. Furthermore, we show that aminoarabinose residues in lipid A contribute to TLR4-lipid A interactions, and experiments in a mouse model of LPS-induced endotoxic shock confirmed the proinflammatory potential of B. cenocepacia penta-acylated lipid A. Molecular modeling combined with mutagenesis of TLR4-MD-2 interactive surfaces suggests that longer acyl chains and the aminoarabinose residues in the B. cenocepacia lipid A allow exposure of the fifth acyl chain on the surface of MD-2 enabling interactions with TLR4 and its dimerization. Our results provide a molecular model for activation of the human TLR4·MD-2 complex by penta-acylated lipid A explaining the ability of hypoacylated B. cenocepacia LPS to promote proinflammatory responses associated with the severe pathogenicity of this opportunistic bacterium.