Sensing Gram-Negative Bacterial Lipopolysaccharides: a Human Disease Determinant?^▿

Bacteria that produce hexaacyl LPS.

When mucosal pathogens that produce hexaacyl LPS cause damage to immunocompetent hosts, they generally induce either little or no inflammatory response (for example, secretory diarrhea) or local (sub)mucosal inflammation—the latter is clinically recognizable as pharyngitis, bronchitis, or cystitis (5) and as inflammatory diarrhea (enterocolitis). It is possible to view these local responses as representing innate immune success, since systemic dissemination rarely occurs. Here LPS is a virulence factor, since it elicits local inflammatory responses that may damage the host, and yet it also provides a signal that enables the host to prevent further invasion and systemic spread. It is interesting in this regard that Campylobacter jejuni produces hexaacyl LPS that has 16 carbon fatty acyl chains, is less stimulatory than E. coli LPS in various bioassays (84), and elicits less severe colonic inflammation than does Shigella flexneri or S. sonnei, species that produce hexaacyl LPS (6, 28). With colitis due to Campylobacter and most Shigella species (S. dysenteriae is an exception), bacteremia is unusual despite extensive mucosal injury. Nontypeable Haemophilus influenzae can cause bronchitis and can survive within adenoidal macrophages in asymptomatic children (38), and yet bacteremia is rarely noted. Salmonella enterica serovar Typhimurium elicits vigorous neutrophil recruitment to the gut mucosa; engineered mutants that produced pentaacyl LPS were less proinflammatory, but they were also found to be defective in elements of the type 3 secretion system that promotes pathogenicity (148). The latter example illustrates a well-known problem for students of microbial pathogenesis that is also a cautionary note for the present discussion: distinguishing the roles of individual bacterial molecules in disease processes is difficult when they are expressed or regulated in concert (137).

Successful innate immunity also prevents aerobic commensal mucosal bacteria such as Klebsiella spp. and (nonpathogenic) E. coli from causing disease in normal hosts. This may be a routine job for the MD-2-TLR4 recognition system—commensal bacteria probably traverse the mucosal epithelium frequently, via microabrasions, M cells, interdigitating dendritic cells, and other routes, and yet disease rarely ensues. By mobilizing inflammatory responses within the submucosa, cells that express MD-2-TLR4 would help contain and destroy these bacteria. Although other elements of innate and acquired immunity (antibodies, complement, antimicrobial peptides, NK cells, iron sequestration, etc.) also help prevent systemic dissemination by commensal microbes, even in concert these mechanisms may fail: in today's hospitals, the gram-negative bacteria isolated most often from patients with nosocomial severe sepsis and septic shock are E. coli and Klebsiella and Enterobacter species. Identifying virulence factors that favor the pathogenicity of these “extraintestinal” commensals-pathogens has been difficult (112, 129, 136), in part because the diseases they cause are often associated with epithelial barrier disruption or obstructed drainage conduits (146), because antimicrobial chemotherapy has selected resistant strains, or because their hosts are immunocompromised in some way (89). Bloodstream infection with these bacteria is typically low-grade and intermittent (154).

Stenotrophomonas maltophilia is a respiratory tract colonizer and pathogen in vulnerable hosts. It produces hexaacyl LPS and elicits airway inflammation, but it was poorly invasive in a mouse model (147). Although it can cause serious nosocomial pneumonia in immunocompromised patients, bacteremia occurs infrequently in nonneutropenic patients with S. maltophilia pneumonia (3). These observations raise the possibility that, even in some compromised hosts, S. maltophilia may be confined to the airways and lung by TLR4-based inflammation. The most abundant lipid A species in a nonpathogenic Acinetobacter strain was nonhexacylated (70), and yet LPS preparations obtained from clinical isolates of Acinetobacter baumannii were potently proinflammatory toward human cells (34). Structural information on A. baumannii LPS is awaited with interest; the clinical import of its host-LPS interaction may be similar to that suggested for Stenotrophomonas spp.

A prominent exception to this pattern is Neisseria meningitidis, a mucosal colonizer that can cross the nasopharyngeal epithelium and invade the bloodstream without eliciting clinically apparent local inflammation. It then can infect vascular endothelial cells and perivascular tissues (51), grow to high density in the blood, and release large amounts of LPS-containing membrane blebs. How encapsulated, hexaacyl LPS-producing bacteria like N. meningitidis, Haemophilus influenzae type b, and E. coli K1 (in neonates) manage to invade and grow to high density in the blood is not known with certainty (128), but these colonizers-pathogens are clearly exceptions to the general pattern described above, and the contribution of the host-LPS interaction to disease pathogenesis is uncertain (see below). Producing hexaacyl LPS is necessary for N. gonorrhoeae to survive within mucosal epithelial cells (101), yet certain gonococcal strains can invade the bloodstream without inducing local inflammation (or, at least, symptoms), suggesting that they also can avoid recognition by submucosal phagocytes that express MD-2-TLR4. Sialylation of gonococcal LPS may contribute to this apparent camouflage, just as the saccharide composition of the N. meningitidis lipooligosaccharide seems to influence interactions with MD-2-TLR4 and several stages in meningococcal pathogenesis (100, 160).

Bacteria that produce nonhexaacyl LPS. (i) Systemic diseases in immunocompetent hosts.

In contrast to the mucosal commensals, colonizers, and pathogens, most systemic gram-negative pathogens do not produce hexaacyl lipid A. Many of them have habitats in soil, water, free-living amoebae, other vertebrates, or insects, and they often breech the body's epithelial defenses via nonmucosal routes (cuts, bites, inhalation, transport across conjunctival membranes) (91). Examples include Yersinia pestis, Francisella tularensis, Burkholderia pseudomallei, Legionella pneumophila, Bartonella species, and Leptospira species. Although each of these bacterial species has distinctive features that allow it to elicit injurious host responses, they share the ability to avoid recognition by MD-2-TLR4. Some may proliferate to high densities within the systemic compartment, reaching >1,000 CFU per ml of blood in severe cases (15, 114, 145, 153). Perhaps the most striking example is Y. pestis, which produces hexaacyl LPS when grown at 25°C (flea) and tetraacyl LPS at 37°C (mammalian host), an adaptation that likely accounts for its ability to cause high-grade bacteremia in mammals (83).

Alternative host sensory mechanisms have been identified for some of these bacteria. Mice can sense F. tularensis via TLR2/TLR6 (67, 75), for example. The tetraacyl LPS produced by Leptospira interrogans is sensed by TLR4 in rodents (143), its natural reservoir, but in humans the predominant Toll-like receptor for Leptospira LPS is TLR2 (92, 149). This observation highlights an interesting and important difference, first noted many years ago (47), between human and murine LPS recognition. Whereas human MD-2-TLR4 senses only hexaacyl lipid A, murine MD-2-TLR4 can recognize pentaacyl and even tetraacyl lipid A. This difference has been exploited in elegant studies of the molecular specificity of LPS recognition by MD-2 (reviewed in reference 81). Another example is Pseudomonas aeruginosa, which produces a pentaacylated LPS that is sensed by murine respiratory epithelium via TLR4 and yet does not activate human respiratory epithelial cells (62, 122, 158). Coxiella burnettii is able to induce cytokine production and granuloma formation in mice in a TLR4-dependent manner (54), and yet its tetraacyl LPS is a very weak agonist for human cells (135) and its polysaccharide chain may mask other Coxiella TLR ligands (117). In contrast, producing tetraacyl or pentaacyl LPS renders many other gram-negative bacteria less proinflammatory even in mice (24, 68, 83). Unfortunately, these species-dependent differences prevent confident generalization from many murine models to their putative counterparts in human disease.

Some gram-negative bacteria that cause systemic disease are acquired by ingestion but they either possess (Brucella abortus, S. enterica serovar Typhimurium), or may adapt to produce (S. enterica serovar Typhimurium, Yersinia enterocolitica), nonhexaacyl LPS. Equipped with mechanisms that facilitate invasion and an intracellular lifestyle, they make their way across the GI mucosa into the systemic compartment, often translocating in the terminal ileum without provoking clinically apparent inflammation in the bowel wall. They then establish residence within host cells, particularly in monocytes/macrophages in the liver, spleen, and lymph nodes. Although they may also circulate in the blood for many days, often within phagocytes, these intracellular pathogens infrequently cause severe sepsis or shock (144). Brucella LPS is heptaacylated, with unusual fatty acyl chains, and F. tularensis (which also can be acquired by ingestion) makes a complex array of lipid A molecules (116) that are poorly sensed by Toll receptors (50). Y. enterocolitica produces hexaacyl LPS at low temperatures but adapts to growth at 37°C by making almost exclusively tetraacyl (108) or pentaacyl (8) LPS; its most common clinical presentation is mesenteric adenitis. The Vi capsule seems to prevent recognition of S. enterica serovar Typhi by shielding the LPS and flagella from TLR4 and TLR5, respectively; very little neutrophilic infiltration of the mucosa has been found in patients with typhoid fever (105). Moraxella catarrhalis (heptaacyl LPS) and Bordetella pertussis (pentaacyl) can establish residence within adenoidal macrophages with little or no accompanying inflammatory reaction (53). In all of these host-pathogen relationships, the absence of inflammation-inducing (hexaacyl) LPS could play a permissive role in disease pathogenesis. A contribution to Salmonella pathogenesis is less certain: hyperacylation of lipid A to seven acyl chains (regulated by the phoP-phoQ system) was reported to favor Salmonella persistence in monocytes and reduce recognition by MD-2-TLR4 (44, 49), but the absence of the enzyme that adds the seventh acyl chain (pagP) did not reduce S. enterica serovar Typhimurium virulence in mice (7). Other adaptations may also be important for preventing host recognition of salmonellae, including a drop in flagellin production that reduces sensing via TLR5 (43).

The mucosal gram-negative aerobes and facultative anaerobes are greatly outnumbered by the gram-negative anaerobes that also inhabit the upper respiratory and GI tracts. These bacteria (those studied include Bacteroides, Porphyromonas, and Prevotella spp.) produce nonhexaacyl LPS, and they infrequently cause disease in normal hosts. One possible exception is Fusobacterium necrophorum, which can invade from the oropharynx to cause septic thrombophlebitis of the jugular vein (Lemierre's syndrome); its lipid A structure has not been reported. Tissue invasion by strict anaerobes may be limited most effectively by high oxygen tension (redox potential), although some of these bacteria may be able to induce inflammatory defenses via TLR2 (25). It is noteworthy that, at least in vitro, Bacteroides and Porphyromonas LPS can block the ability of hexaacyl LPS to stimulate cells via MD-2-TLR4 (20, 29). Whether this competition between hexaacyl and nonhexacyl LPS can modulate TLR4-based sensing in the (sub)mucosae of the respiratory tract or intestine is not known.

(ii) Systemic diseases in abnormal hosts.

This category could include two uncommon but often lethal pathogens, Capnocytophaga canimorsus and Vibrio vulnificus. Although the structure of C. canimorsus LPS has not been solved, its cells have low inflammatory potency in vitro and a nonhexaacyl LPS can be anticipated (39). Whereas many patients who have developed overwhelming bacteremia with this canine oral commensal have had inadequate splenic function or advanced liver disease, others have evidently been immunocompetent. Vibrio vulnificus bacteremia occurs most often in individuals whose clearance mechanisms are compromised by hepatic cirrhosis; increased iron availability may also be important. Although its lipid A structure has also not been published, it was at least 10-fold less potent than E. coli LPS in some bioassays (102). Both V. vulnificus and C. canimorsus can grow to high density in vivo, particularly in splenectomized individuals; it is not unusual to find them on microscopical examination of peripheral blood smears from infected patients.

(iii) Local diseases in abnormal hosts.

As noted above, bacteria that have natural habitats in water, plants, or soil can cause disease in special hosts. Environmental isolates of Pseudomonas aeruginosa produce pentaacyl LPS, but this bacterium can adapt to the tracheobronchial tree of children with cystic fibrosis by making hexaacyl or even heptaacyl LPS (33); the extent to which this adaptation influences inflammatory responses in the airways is not known. Tissue P. aeruginosa isolates have also had pentaacyl LPS (32). Some local tissue infections caused by P. aeruginosa (for example, invasive external otitis) can be associated with minimal inflammatory reactions, as would be anticipated if the bacteria produced pentaacyl LPS and lacked other potent proinflammatory molecules. In some settings the inflammatory response to P. aeruginosa can be more vigorous; although the basis for this difference is not well understood, many strains express a type 3 secretion system that can deliver toxic proteins to host cells (58) and flagellated strains may be sensed by TLR2 and TLR5 (2). Burkholderia cepacia is a plant pathogen that can also colonize the airways of humans with cystic fibrosis. It has nine genomic species that differ in their ability to cause a life-threatening pneumonia (“cepacia syndrome”) and in the lipid A structures of their LPSs. Earlier investigations found that B. cepacia LPS was as stimulatory to human monocytes as E. coli LPS (159), and the LPS from one genomovar I strain was reported to have pentaacyl lipid A (120). More recently, De Soyza et al. found that the proinflammatory potency of strains from different genomovars was mimicked by that of their isolated LPSs; a representative ET-12 (pneumonia-associated) B. cenocepacia strain made hexaacyl LPS, whereas LPS from a less proinflammatory strain was pentaacyl (27).

Summary.

For most bacteria that produce hexaacyl LPS, LPS recognition via MD-2-TLR4 seems to contribute to disease within and beneath the mucosae of the GI, respiratory, and urinary tracts. One may infer that this defense also helps prevent dissemination into and within the systemic compartment (also see references 5 and 81). For bacteria that do not produce hexaacyl LPS, failure to activate host defenses via MD-2-TLR4 may allow or favor growth and dissemination in vivo.

Most of the experimental evidence for these generalizations has come from studies of bacterial mutants in animal models of disease. Since LPS-altering mutations can be associated with changes in other (possibly unrecognized) bacterial virulence determinants (124, 148, 156), reaching definitive conclusions regarding the role of LPS recognition per se may not be possible. Nonetheless, production of hexaacyl lipid A was associated with the ability of both Shigella flexneri and Salmonella enterica serovar Typhimurium to elicit gut-damaging inflammatory responses—in other words, to be pathogenic (28, 148). Moreover, S. enterica serovar Typhimurium bacteria mutated to produce pentaacyl LPS grew to high densities in vivo without causing death (68). In contrast, engineering a pathogenic strain of Yersinia pestis to produce hexaacyl instead of tetraacyl LPS rendered this otherwise lethal microbe avirulent—here the presence of hexaacyl LPS made Y. pestis recognizable by MD-2-TLR4 and thus very sensitive to killing by innate defenses, even when bacteria were injected subcutaneously or intravenously (83). This striking result is the most convincing evidence to date that the absence of hexaacyl LPS permits expression of other bacterial virulence determinants in vivo. Mutations in murine TLR4 also may not provide definitive tests of the LPS-MD-2-TLR4 interaction, but producing a functional TLR4 decreased susceptibility to bacteremia and/or systemic injury caused by several gram-negative bacteria that produce hexaacyl LPS (22, 23, 81, 93, 152). Conversely, the avirulent Y. pestis mutant that produced hexaacyl LPS was completely virulent in Tlr4^−/− mice (83).

Limitations.

Although LPS sensing may play an important role in shaping many human diseases, many other innate mechanisms also contribute to defense against gram-negative bacteria. They include mobilizing inflammatory responses through recognition of other bacterial molecules, natural and acquired antibodies, complement activation, antimicrobial peptides, and many aspects of the acute phase response (90). Acquired immunity is also obviously critical for preventing recurrent disease.

The LPS analysis makes several assumptions not previously discussed. First, the structures produced by bacteria growing in culture are assumed to resemble those produced in vivo. Second, since many bacteria produce more than one lipid A structure, the most abundant one is assumed to dominate host recognition. Third, bioassays performed using isolated cells or experimental animals (often rodents) are assumed to reflect accurately how humans and other animals sense these structures. A fourth assumption, that the literature survey for LPS structures was complete and accurate, is compromised by the disparate nature of the sources and the author's unintended yet indisputable selection biases. Perhaps the most important of these limitations is that of the structural heterogeneity of the LPSs. Not only do many gram-negative bacteria produce more than one lipid A structure, but the relative abundances of the different structures can vary with environmental conditions that include the media and temperature used to grow the bacteria in culture. Such influences may account for reported differences in the acylation of S. flexneri lipid A, for example (18, 28). Improved methods are needed to analyze the lipid A structures that exist in vivo in different disease and nondisease states.

Another illustration emphasizes this heterogeneity. For many years, LPS has been thought to be transferred from bacterial membranes by LPS-binding protein to soluble or membrane-bound CD14, which in turn delivers it to MD-2-TLR4. In fact, there is strong evidence that LPS-binding protein and CD14 are most important for sensing LPS molecules that have long polysaccharide chains (smooth or S-form LPS) (41, 56, 131). Interestingly, CD14 can deliver S-form LPS to cells in a way that selectively activates the MyD88-independent intracellular pathway downstream of TLR4 (63). Early studies found that the lipid A moiety of S-form LPS is hypoacylated (64). If confirmed, this heterogeneity would further complicate the interpretation of the lipid A structures found in bacteria that produce both S-form and short chain (R-form) LPS molecules.

Finally, although many investigators have found that animal cells recognize hexaacyl LPS more sensitively than they detect other gram-negative bacterial components (24, 56, 83), few head-to-head comparisons of LPS with other bacterial components have been performed to ascertain their relative potencies as TLR ligands for different cell types (158).

Possible implications: innate immunity.

What does the LPS-TLR4 example suggest about innate recognition of other bacteria? Some of the other PRRs are also quite specific. NOD1 and NOD2 sense distinct substructures of peptidoglycan, TLR2/TLR6, and TLR2/TLR1 heterodimers can distinguish lipopeptides that have two and three acyl chains, respectively, and TLR9 recognizes definable oligonucleotide sequences (42). Since these microbial molecular “patterns” have been much more highly conserved than has the structure of lipid A, it is likely that these TLRs sense a much greater diversity of microbes than does MD-2-TLR4 (80). As might be expected, it is these sensors that seem to figure most prominently in diseases caused by gram-negative bacteria that make nonhexaacyl LPS.

Disease susceptibility.

Functional mutations in MD-2 or TLR4 could be expected to influence diseases caused by bacteria that make hexaacyl LPS. In keeping with this prediction, TLR4 polymorphisms were found more frequently in patients with meningococcal disease than in controls (123). On the other hand, the large number of studies with negative or marginally positive results published to date suggests that it may be difficult to find reproducible associations between TLR4 polymorphisms and disease susceptibility or severity. The association of the TLR4 Asp299Gly allele with protection from cerebral malaria suggests that nonbacterial diseases may have influenced the evolution of the gene in diverse human populations (35), although a confounding role for endotoxemia has not been excluded (139). It is also possible that the associations sought so far have not included mucosal infectious diseases, such as shigellosis, in which LPS recognition by MD-2-TLR4 seems to play an important role. Since LPS binds MD-2 (20, 45, 94), disease-linked polymorphisms in MD-2 also deserve more attention (48). Polymorphisms in other host sensors are more likely to be associated with increased susceptibility to microbes, such as Legionella pneumophila (52) and Y. pestis, that make nonhexaacyl LPS.

Pathogenesis.

Whereas LPS has long been regarded as necessary for gram-negative bacterial virulence, its contribution to the outcome of infectious diseases has seemed quite variable. Considering how and where animals sense LPS highlights the two faces of the host-parasite encounter: LPS recognition by an animal host can trigger inflammatory reactions that are both damaging and protective.

Many experts have regarded fulminant meningococcemia as a paradigm for serious systemic gram-negative bacterial diseases (46). As noted above, fulminant disease caused by N. meningitidis is actually a special case. It is not simply the most dramatic extreme of a continuous spectrum of sepsis severity, so information gained from patients with meningococcemia is not necessarily useful for understanding the pathophysiology of other kinds of sepsis (71).

It is also noteworthy that none of the major gram-negative bacterial biothreat agents produces hexaacyl LPS. Yersinia pestis and Francisella tularensis make tetraacyl LPS when grown at 37°C (104), and Burkholderia pseudomallei LPS (inferring from its weak potency [78] and the reported structures of other Burkholderia species) is probably pentaacyl. Producing an LPS that is poorly detected by MD-2-TLR4 may be essential for these microbes to cause disease (83).

Diagnosis.

The most widely employed test for the presence of endotoxin in fluids is based on LPS sensing by the clotting system of the horseshoe crab. This assay is not specific for hexaacyl LPS (132, 133). LPS recognition by the horseshoe crab likely evolved as a defense from aquatic bacteria; the assay's ability to recognize widely diverse lipid A structures enhances its ability to detect environmental contamination of biologicals, but it also may account for some of the problems observed when the test has been used to detect endotoxemia and gram-negative bacteremia in human patients (the correlations between disease severity, gram-negative bacteremia and endotoxemia have generally been poor) (57). A newer test for endotoxemia is based on an antibody that also does not discriminate hexaacyl from nonhexaacyl LPS (111).

Recent studies have found clear-cut differences in the mRNA profiles of blood leukocytes from children with gram-positive infections (by S. pneumoniae and S. aureus, TLR2 agonists) and gram-negative infections (by E. coli, a TLR4 agonist) (107). Future improvements may equip clinicians to distinguish inflammatory responses to gram-negative bacteria that produce hexaacyl LPS from responses elicited by bacteria that do not (30). It is noteworthy in this connection that TLR4 is the only TLR that activates both the MyD88 and TRIF (MyD88-independent) intracellular signaling pathways, although different hexaacyl LPSs may activate the two paths to different degrees (63, 161). Perhaps this property will assist molecular diagnosis. Future clinical trials may also benefit from categorizing gram-negative diseases according to the likely host-LPS interaction, since therapies that interfere with the LPS-TLR4 interaction should not be useful for diseases caused by bacteria that make nonhexaacyl LPS (12).

Therapy.

Two TLR4-targeting drugs are currently in clinical trials, and monoclonal antibodies that prevent LPS sensing by MD-2-TLR4 are in development (26). Eritoran is a tetraacyl synthetic lipid A analog that competes with LPS for binding MD-2 (69). It potently blocks human responses to intravenously injected LPS (74, 85). TAK-242 is a cyclohexene derivative that inhibits TLR4-mediated responses to LPS (59). Adjunctive therapy with these drugs should be most effective when the offending bacteria produce hexaacyl LPS and the drug can reach them in infected tissue spaces. Lipid A antagonists might hasten recovery from shigellosis or other mucosal gram-negative diseases, and yet it is also possible that, by diminishing the local inflammatory response, they could favor systemic dissemination. Also providing effective bactericidal therapy will thus be essential. Similarly, combining TLR4 inhibition with bactericidal chemotherapy might improve the outcome of tissue infections, such as pneumonia or pyelonephritis, due to bacteria that produce hexaacyl LPS (Stenotrophomonas spp., probably Acinetobacter spp., and any member of the Enterobacteriaceae family).

Experiments performed using mice have found that enzymatic deacylation of lipid A is required to prevent prolonged reactions to bacteria that produce hexaacyl LPS (73, 118). The deacylating enzyme, acyloxyacyl hydrolase, is made by phagocytes, including the Kupffer cells of the liver (118), and by renal cortical epithelial cells that secrete it into the urine (36). It is possible that increasing the amount or activity of this enzyme could hasten the recovery of patients with diseases caused by gram-negative bacteria that produce hexaacyl LPS.

Inhibiting the bacterial myristoyl or lauryl transferases, and thus preventing the addition of the secondary acyl chains to the lipid A backbone, should in theory prevent or diminish LPS recognition by MD-2-TLR4. This could reduce the local inflammatory response induced by mucosal pathogens such as H. influenzae or M. catarrhalis, but it also might enable invasive bacteria to evade TLR4-based host defenses. Inhibition of the early constitutive steps in lipid A biosynthesis (104) should be less likely to have unwanted consequences, since LPS hypoacylation may significantly weaken the barrier function of the bacterial cell wall.

Therapy-induced “endotoxic” shock was first noted in patients infected with B. melitensis or S. enterica serovar Typhi (109, 125). Since brucellae do not produce hexaacyl LPS (the S. enterica serovar Typhi lipid A structure has not been reported), the basis for shock is unlikely to have been treatment-induced endotoxin release. No clinical tests of this hypothesis have been reported from studies of patients with brucellosis or typhoid fever, but inflammatory reactions due to antibiotic-induced endotoxin release have been observed when treating local tissue infections due to bacteria that produce hexaacyl LPS (4, 103). In contrast, antibiotic treatment of patients with Burkholderia pseudomallei bacteremia increased endotoxin levels in plasma with no change in signs or symptoms (121).

Prevention.

Dutch workers found that the absence of one of its secondary acyl chains greatly reduced proinflammatory responses to meningococcal LPS while retaining its ability to act as an adjuvant in mice (140). Of the other lipid A-derived adjuvants, the monophosphoryl lipid A (MPLA) from Salmonella minnesota LPS has been studied most intensively (98, 138). Its most active congener, which has six acyl chains, potently induces human macrophages to produce chemokines and cytokines via a TLR4-dependent mechanism. Synthetic analogs based on the MPLA structure are also good adjuvants and immunomodulators, at least in mice (98). The basis for their lack of toxicity is not known, since they and LPS have induced very similar responses in some cell types (130). One recent report noted that MPLA failed to activate caspase-1, which catalyzes IL-1β precursor processing, so that IL-1 secretion was impaired (95). Another found that MPLA predominantly activates cell responses via a MyD88-independent pathway that is associated with less production of IL-1 and gamma interferon (77).

Despite producing a full complement of virulence factors, the Y. pestis strain engineered to make hexaacyl LPS at 37°C was so avirulent that it could be used as a vaccine in mice (83). This strategy might be used with other bacteria that make nonhexaacyl LPS, including other biothreat agents, provided that hexaacyl LPS-containing vaccines are not too proinflammatory for injection into humans.

Questions for future research.

In addition to the important unknowns mentioned in the paragraphs above, there are many unanswered questions. (i) How do cells that express MD-2-TLR4 sense native LPS? If the LPS acyl chains must fit into a pocket in MD-2, as recently reported for tetraacyl lipid A (69, 94), how does hexaacyl LPS bind to MD-2 and induce TLR4 activation (dimerization)? What extracts LPS from the bacterial membrane in a way that allows it to bind MD-2? Is this a job for LPS-binding protein (142), and, if so, does it occur both on the cell surface and within host cell phagosomes (11)? (ii) What is the basis for “mutualism” between aerobic, hexaacyl LPS-producing gram-negative bacteria and the mammalian upper respiratory and gastrointestinal mucosae? Do these bacteria derive some benefit from being sensed by host TLR4? Does producing a hexaacyl LPS contribute to this mutualism? (iii) How do gram-negative aerobic bacteria that lack hexaacyl lipid A survive in the gut? This question is especially interesting with regard to the pathogens that translocate across the gastrointestinal mucosa: might brucellae be protected within macrophages from an infected animal host or Vibrio vulnificus by oyster cells? If not, what are the cell wall molecules in these bacteria that prevent killing by antimicrobial peptides or bile salts? (iv) How do some bacteria that make hexaacyl LPS, most notably the pathogenic Neisseria spp., evade submucosal defenses and cross into the bloodstream without eliciting a (protective) local inflammatory response? In particular, what roles do capsule, LPS sialylation, and pili play in this process (65)? (v) What are the most important innate defenses toward bacteria that lack hexaacyl LPS? How can these defenses be enhanced without provoking harmful systemic inflammation? (vi) How does MD-2-TLR4 discriminate between lipid A and the host carbohydrate and protein ligands that have been reported to activate cells via TLR4 (134)? What are the downstream intracellular and systemic consequences of recognizing these different agonists? (vii) Can TLR4 stimulation or inhibition be used to improve outcome from diseases caused by bacteria that make hexaacyl LPS?