According to the innate immunity concept [1], animals defend themselves from microbes
by recognizing pathogen-associated molecular patterns. To detect many Gram-negative
bacteria, animals use the CD14–MD-2–TLR4 receptor mechanism to recognize the lipid
A moiety of the cell wall lipopolysaccharide (LPS). Lipid A is a glucosamine disaccharide
that carries phosphates at positions 1 and 4′ and usually has four primary (glucosamine-linked)
hydroxyacyl chains and one or more secondary acyl chains. Gram-negative bacteria produce
numerous variations on this basic structure, yet sensitive LPS recognition and pro-inflammatory
signaling by human TLR4 occur only when lipid A has both phosphates and is hexaacyl,
with two secondary acyl chains.
What might bacteria derive from producing this type of lipid A, and what do animals
gain from recognizing it? A survey of diverse lipid A structures found that the best-recognized
configuration is produced by most of the aerobic or facultatively anaerobic Gram-negative
bacteria that can live in the gastrointestinal and upper respiratory tracts. We hypothesize
that the CD14–MD-2–TLR4 mechanism evolved to recognize not just pathogens, but also
many of the commensals (normal flora) and colonizers that can inhabit the body's most
vulnerable surfaces. Producing this lipid A structure seems to favor bacterial persistence
on host mucosae, whereas recognizing it allows the host to kill invading bacteria
within subepithelial tissues and prevent dissemination. A conserved host lipase can
then limit the inflammatory response by removing a key feature of the lipid A signal,
the secondary acyl chains.
Acylation of Lipid A: Strengthening the Shield?
Gram-negative bacteria that inhabit water, soil, plants, or insects display impressive
diversity in their lipid A structures (see Table S1). Although the backbone is almost
always a bisphosphorylated disaccharide that has three or more primary fatty acyl
chains, the secondary acyl chains differ in their number, length, and degree of saturation.
In contrast, the lipid A structures produced by most of the aerobic and facultatively
anaerobic Gram-negative bacteria that live as human mucosal commensals, colonizers,
or pathogens [2] are monotonously similar: they have two phosphates, four primary
hydroxyacyl chains (3-hydroxymyrisate or 3-hydroxylaurate), and two saturated secondary
acyl chains (laurate, myristate, or both); we shall refer to this composition as “mucosal”
lipid A. Since these bacteria differ in many other ways, the fact that their lipid
As are so similar suggests that this structure may confer some advantage.
Mucosal secretions contain numerous cationic antimicrobial peptides (CAMPs) [3]. As
noted by Miller [4] and others, increased resistance to CAMPs and other host molecules
may explain why Gram-negative bacteria that colonize mucosae usually make LPS with
six or more acyl chains (Figure 1). Although we found no demonstration that this lipid
A structure enables commensal bacteria to thrive on mucosal surfaces, the evidence
that it does so for colonizers and pathogens is extensive. Having hexaacyl (rather
than pentaacyl) lipid A enables Bordetella and Haemophilus species to persist in the
respiratory tract [5–7] and Neisseria gonorrhoeae to survive within epithelial cells
[8]. Pseudomonas aeruginosa lives in water, produces a predominantly pentaacylated
LPS, and does not colonize the mucosae of normal humans. When P. aeruginosa colonize
the airways of children with cystic fibrosis, however, the bacteria often adapt by
producing hexaacylated (and even heptaacylated) lipid A [9]. Mucosal lipid A is also
found in intestinal pathogens: Shigella and Salmonella, pathogenic Escherichia coli
[10], Aeromonas species, Plesiomonas shigelloides, and Vibrio cholerae O1. In Salmonella
and some others, a PhoP/PhoQ-regulated transcriptional program promotes lipid A palmitoylation
(heptaacylation) along with other changes that increase resistance to CAMPs [4,11–13].
Other mucosal bacteria may also produce lipid A that is more hydrophobic than mucosal
lipid A, with longer secondary chains (Campylobacter jejuni) or more of them: heptaacyl
(Moraxella) or octaacyl (V. cholerae O139). Those that produce less hydrophobic lipid
A seem to be special cases: the pentaacyl LPS of Chlamydia species is found in spore-like
elementary bodies, and Helicobacter pylori, with tetraacyl LPS, is adapted to live
in the stomach.
Many other disease-associated Gram-negative bacteria have nonmucosal habitats. Their
lipid A moieties differ from the typical mucosal structure by having shorter or longer
acyl chains, unsaturated acyl chains, only four or five chains, or only one phosphate
(see Table S1): Legionella (water habitat, often in free-living amoebae), Burkholderia
pseudomallei (soil and water), Yersinia pestis (small rodents, lice), Coxiella burnetti
(intracellular, livestock), Leptospira (water, animal urine), and Francisella tularensis
(ticks, rabbits, other small animals). These pathogens usually enter vertebrate tissues
via insect bites or cuts, within inhaled droplets, or across the conjunctivae. Brucellae
(livestock), which inhabit macrophages yet are typically acquired via ingestion, also
produce a nonmucosal LPS [14].
How Animals Sense Mucosal Gram-Negative Bacteria: Shield as Signal
Whereas bacterial peptide resistance and outer membrane impermeability seem to vary
directly with the number of acyl chains, the inflammation-inducing CD14–MD-2–TLR4
sensory mechanism best recognizes lipid A that has the hexaacyl mucosal lipid A structure
[15–20] (Figure 1). In support, Hajjar et al. [21] reported that a discrete extracellular
region of human TLR4 enables recognition of hexaacyl, but not pentaacyl, P. aeruginosa
LPS. Further discrimination is performed by MD-2 [22]. The same recognition pattern
has been found for all mammals tested except rodents [23].
Evidence that lipid A structure influences the recognition of intact bacteria by host
cells came from mutating enzymes that attach secondary acyl chains to the backbone.
Somerville et al. [24] found an E. coli mutant that was unable to attach the secondary
myristate at position 3′ and could not stimulate human endothelial cells. Having a
hexaacyl LPS also enhances other responses to intact bacteria, including the induction
of tumor necrosis factor by Salmonella in vivo [25], the initiation of intestine wall
inflammation by S. flexneri [26], and the production of IL-8 by bladder epithelial
cells infected with E. coli [27]. These and other studies convincingly showed that
LPS is the major structure sensed by most host defense cells when they interact with
bacteria that produce mucosal lipid A [28].
Although the TLR4-based mechanism for sensing LPS has been highly conserved [29],
how it benefits the host is only partly understood. Gastrointestinal epithelial cells
evidently do not express TLR4 on their lumenal surfaces under normal in vivo conditions,
so it is unlikely that they sense LPS or Gram-negative bacteria in the fecal stream
[30]. On the other hand, Rakoff-Nahoum et al. [31] recently found that subepithelial
TLR4-dependent sensing protects damaged gut from injury by commensal bacteria. Perhaps
the key function of this system is to sense bacteria as they enter submucosae, thus
mobilizing defenses that confine bacterial invasion, and the inflammatory response
to it, to the local site [32,33]. Shigella invasion through the colonic epithelium
prompts intense local inflammation, for example; the fact that Shigella possess hexaacyl
LPS may help the host confine infection to the intestine (bacteremia rarely occurs)
[26,34]. TLR4-dependent responses to other enteric pathogens may also damage the intestinal
wall, yet these bacteria also do not often spread to the bloodstream [35]. Similar
local responses may help restrict disease caused by most Haemophilus and Bordetella
species to the respiratory tract, most strains of N. gonorrhoeae to the urethral mucosa,
and E. coli to the bladder [27]. In contrast, producing heptaacylated LPS may help
Salmonellae [11] avoid recognition within the intestinal submucosa and grow in extraintestinal
tissues.
Most mucosal Gram-negative bacteria that enter the bloodstream are rapidly killed.
In many instances, LPS sensing is required for effective elimination [32,36]. Few
Gram-negative bacteria grow to high density in the blood of immunocompetent humans
[37]; of these, Y. pestis [38] and B. pseudomallei [39] produce lipid A structures
that are poorly sensed by TLR4 [40–42]. As suggested by others [29,40,42], these bacteria
may be effective human pathogens, at least in part, because the TLR4 mechanism does
not recognize them. This notion may also apply to other bacteria that lack the mucosal
lipid A structure and are weak TLR4 agonists: F. tularensis [43,44], L. pneumophila
[45], C. burnetti [46], H. pylori [47], Brucellae [48,49], and Leptospira [50] species.
According to this hypothesis, engineering these bacteria to produce mucosal lipid
A should alter their ability to cause disease.
The most obvious exceptions are the pathogenic Neisseriae. Both N. meningitidis and
N. gonorrhoeae produce a mucosal lipid A and colonize mucosal surfaces. How they invade
the bloodstream is not known [51], but they usually seem to do so without triggering
local inflammation. They illustrate the important point that the lipid A–TLR4 interaction
is but one element of the confrontation between bacterial pathogen and animal host.
Destroying the Signal: Acyloxyacyl Hydrolysis
Whereas Dictyostelium discoideum produces several lipid A–deacylating enzymes, only
one has been found in mammals. Acyloxyacyl hydrolase (AOAH) removes only the secondary
chains from lipid A; it cleaves saturated, short secondary chains, as are found in
the mucosal lipid A structure, more rapidly than it removes long unsaturated ones
[52,53]. A phylogenetic analysis revealed high conservation for both the AOAH large
subunit, which has the bacterial GDSL lipase motif [54], and the small subunit, a
member of the saposin-like protein family [55] and the likely LPS recognition motif
(Figure 2). Indeed, AOAH has evidently been more highly conserved than has TLR4 [29]
In vertebrates, AOAH is produced by neutrophils, dendritic cells, renal cortical epithelial
cells, and monocyte-macrophages. AOAH treatment greatly reduces LPS sensing via TLR4
[52], and LPS may remain stimulatory for weeks in mice that cannot deacylate it [56].
AOAH thus can limit inflammatory responses to bacteria that produce mucosal lipid
A. Deacylation occurs slowly, reaching completion after the early recognition phase
of antibacterial innate immunity has occurred.
Other Signals
The host response to LPS also has noninflammatory, immunostimulatory elements. Lipid
A analogs that lack the optimal configuration for inducing inflammation may be excellent
adjuvants, enhancing acquired immune responses in ways that mimic those induced by
LPS itself [57,58]. The mucosal lipid A motif triggers inflammation (and toxicity),
whereas adjuvanticity may also follow TLR4-based recognition of lipid A molecules
that have only one phosphate and secondary chains of various lengths, numbers, and/or
configurations. These structure–function relationships have been exploited to produce
analogs that are either LPS antagonists or nontoxic adjuvants.
LPS recognition by CD14–MD-2–TLR4 has received intensive study because it initiates
the inflammatory response to so many disease-associated Gram-negative bacteria. Less
is known about how animals sense their far more abundant flora of strictly anaerobic
Gram-negative bacteria, although doing so may be important for establishing beneficial
mutualism between bacteria and host [59,60]. Like the tetraacylated LPS of Porphyromonas
gingivalis, the pentaacylated monophosphoryl LPS of Bacteroides fragilis seems to
be sensed principally by TLR2 [61–63] and can inhibit recognition of mucosal LPS by
TLR4 [64,65].
Conclusions
An immune system that only recognizes pathogens would leave animals vulnerable to
the commensal and colonizing microbes that enter subepithelial tissues at sites of
microtrauma throughout life [31]. An innate defense that detects and responds to mucosal
commensals as well as pathogens is obviously not impenetrable, however; even commensals
may induce damaging responses when host defenses are impaired by trauma, cuts, or
tubes that provide conduits across epithelia, immunosuppression, or an inherited immune
defect [29,66]. An even greater gap in host defense may be exposed when a Gram-negative
pathogen evades TLR4 recognition by producing a nonmucosal lipid A.
If the synthesis proposed here is correct, it would not be surprising to learn that
other elements of innate immunity also sense commensal microbes. Animals may also
have conserved enzymatic mechanisms for extinguishing microbial signals that are sensed
via other receptors [67,68].
Supporting Information
Table S1
Lipid A Structures of Various Gram-Negative Bacterial LPSs
(208 KB DOC)
Click here for additional data file.
Table S2
GenBank and Ensemble Accession Numbers Used
(25 KB DOC)
Click here for additional data file.
Accession Numbers
The SwissProt (http://www.ebi.ac.uk/swissprot) primary accession numbers discussed
in this paper are AOAH (P28039), CD14 (P08571), MD-2 (Q9Y6Y9), and TLR4 (000206).