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Fortifying the barrier: the impact


of lipid A remodelling on bacterial
pathogenesis
Brittany D.Needham1 and M.Stephen Trent1,2
Abstract | Gram-negative bacteria decorate their outermost surface structure,
lipopolysaccharide, with elaborate chemical moieties, which effectively disguises them from
immune surveillance and protects them from the onslaught of host defences. Many of these
changes occur on the lipid A moiety of lipopolysaccharide, a component that is crucial for host
recognition of Gram-negative infection. In this Review, we describe the regulatory mechanisms
controlling lipid A modification and discuss the impact of modifications on pathogenesis,
bacterial physiology and bacterial interactions with the host immune system.

Microorganism-associated The bacterial cell envelope is a complex structure that often hostile surroundings7. During and after traffick-
molecular pattern protects the cell from the surrounding environment. ing to the cell surface, lipid A can undergo extensive
(MAMP). A component of a A defining feature of Gram-negative bacteria is the pres- remodelling, resulting in the wide variety of lipidA
commensal or pathogenic ence of an outer membrane, which is an asymmetrical structures that are observed across species (FIG.1d).
microorganism that is well
conserved and universally
bilayer with glycerophospholipids confined to the inner This is accomplished by diverse lipid A modification
recognized by the innate leaflet and lipopolysaccharide (LPS) anchored to the enzymes that remove or add acyl chains and phosphate
immune system. outer leaflet 1 (FIG.1a,b). Similarly to most cell envelope groups, as well as other enzymes that transfer various
Lipopolysaccharide is a typical components, LPS is made at the cytoplasmic face of the constituents onto the molecule, such as sugars, phos-
MAMP, and other examples
inner membrane and must be transported across the two phoryl containing groups and even an amino acid, all of
include peptidoglycan,
lipoproteins and flagella.
bilayers and the periplasm to become integrated in the which can influence bacterial interactions with the host.
Previously referred to as outer membrane1. The term lipid A modification is used throughout this
PAMPs (pathogen-associated LPS is composed of three domains: the lipid A Review to refer to biological modification of lipid A in
molecular patterns), hydrophobic anchor, the core oligosaccharide and the bacterial cell as well as artificial modification
the Oantigen1 (FIG.1b). Some organisms (for example, invitro. Modifications also occur on other moieties
Neisseriaspp.) produce lipooligosaccharide (LOS), in of LPS (BOX1), but these non-lipid A modifications
which the repeating Oantigen domain is absent and are not the focus of this Review.
is replaced by an extended core region2. Lipid A, the Lipid A alterations directly affect pathogenesis by
endotoxic portion of LPS and the site for many LPS changing outer-membrane permeability, promoting
modifications, is initially synthesized as a-1,6linked resistance to antimicrobial peptides and interfering with
disaccharide of glucosamine that is both phosphorylated the ability of the host to recognize LPS as a conserved
1
The Institute of Cellular
and fatty acylated (FIG.1c). In some organisms, such as microorganism-associated molecular pattern (MAMP)7.
and Molecular Biology, Escherichia coli K12, this structure represents the typical The diversity of LPS modification systems is quite
The University of Texas at form of lipid A in the outer membrane. Despite initial extraordinary, and the accumulated knowledge about
Austin. studies reporting that lipid A could be modified with these systems has provided deeper insight into bacte-
2
Section of Molecular
polar substituents (such as amino sugars3), it was nev- rial mechanisms that contribute to human disease and
Genetics and Microbiology,
The University of Texas at ertheless viewed as a static structure. This view changed immunity. In this Review, we describe the mechanisms
Austin, Austin, Texas 78712, in the late 1990s following the characterization of the that regulate lipid A remodelling, the host defence sys-
USA. lipidA biosynthesis pathway, which established a foun- tems that recognize this molecule and the strategies
Correspondence to M.S.T. dation for studies which discovered that lipidA is altered that bacteria have evolved to use lipid A modifications
e-mail:
strent@mail.utexas.edu
after synthesis46. In fact, Gram-negative organisms have for their own benefit. Finally, we discuss the interplay
doi:10.1038/nrmicro3047 evolved several LPS modification strategies that allow between lipid A modification systems and bacterial
Published online 10 June 2013 these organisms to adapt to their unpredictable and physiology.

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b
LPS

O antigen

Core
Lipid A
OM
a
Phospholipid OM protein

PG

Lipoprotein

IM

c d Cytoplasm
H2N
IM protein
Kdo O
EptB O P
HO OH HO O OH
HO HO
O O HO OH O O HO OH
HO HO
HO OH O O HO O O
OH
O OH O OH
O O
O O
P O O P O
HO O O O O O
OH O O OH O O
HO OH HO
O NH O H2N O NH O
O O
O O NH O O NH
O P ArnT HO O P O
O O O O
HO OH HO O P
O O HO O O HO
HO O
O HO HO O O
NH2
LpxO O
Lipid A EptA

O
O P O
HO O P
HO OH
LpxT

PagL
LpxR

Unmodied Kdo2lipid A Modied Kdo2lipid A PagP

Figure 1 | The cell envelope of Gram-negative bacteria. a|A cryoelectron tomography image of an Escherichiacoli
cell, showing the characteristic inner membrane (IM) and outer membrane (OM) (scale bar of Nature
200nm)Reviews
123 | Microbiology
. b|Schematic of
the Gram-negative cell envelope, showing the typical inner and outer bilayers that are separated by the periplasm, which
contains peptidoglycan (PG). The outer leaflet of the outer membrane contains lipopolysaccharide (LPS), which is
anchored to the membrane by the LPS lipid A domain1. The inner leaflet of the outer membrane and also the entire inner
membrane are composed of phospholipids only, and both bilayers can contain a range of different types of membrane
protein. c|The lipid A and inner core (Kdo (3deoxydmanno-octulosonic acid)) portion of LPS are shown. Unmodified
lipid A consists of a -1,6linked disaccharide of glucosamine that is both phosphorylated and fatty acylated7. This basic
structure can be extensively modified after synthesis. d|The LPS modifications that occur in Salmonella spp. The enzymes
responsible are controlled by either the PmrAB two-component system (red) or the PhoPQ two-component system (blue),
or have no known two-component regulatory system (green). The various possible modifications include the addition of
4amino4deoxylarabinose (aminoarabinose) moieties (by ArnT) and phosphoethanolamine moieties (by EptA and
EptB), as well as phosphorylation (by LpxT), deacylation (by PagL and LpxR7; resulting in loss of acyl chains, as indicated
by dashed lines), acylation (by PagP)7 and hydroxylation (by LpxO). Transcription of the gene encoding LpxO is modestly
induced by PhoPQ, but LpxO remains active in conditions in which PhoPQ is inactive, suggesting that the enzyme acts
independently of this two-component system124.

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Box 1 | Non-lipid A modifications to lipopolysaccharide


downstream PhoPQ-regulated genes, but their direct
involvement in the modification of lipid A has not
Although most lipopolysaccharide (LPS) modifications that are known to affect yet been thoroughly investigated1417. The PhoPQ and
pathogenesis occur on the lipid A portion of the molecule, the remaining sugars of the PmrAB systems have been reviewed extensively else-
LPS molecule can also be modified. Extending from lipid A is the core oligosaccharide, where10,11, so a brief overview is provided here with a
which is subdivided into an inner and an outer core; the Oantigen is attached to the
focus on the more recent discoveries.
outer core (FIG.1b). Biosynthesis of these LPS domains varies more across Gram-negative
organisms than biosynthesis of lipid A, but within a genus, some components are
Functional PhoPQ systems are widespread among
relatively well conserved. The inner core typically consists of Kdo (3deoxydmanno- bacteria, although there is some interspecies vari-
octulosonic acid) and heptose sugars, whereas the outer core varies in sugar composition, ation in the activation signal used and the genes that
sugar arrangement and linkage to Oantigen. Oantigen, in addition to a tremendous are regulated10,18. Activation of the PhoPQ system in
variety in composition, can have strikingly different lengths, ranging from the complete Salmonellaenterica subsp. enterica serovar Typhimurium
absence of Oantigen to more than 100 repeating units of sugar backbones with by acidic pH, certain antimicrobial peptides, and the
branching chains1,109. These different variants of LPS, particularly the truncation of depletion of Mg 2+ and Ca2+ stimulates transcription
Oantigen, affect the ability of the bacterium to withstand harsh environments1,109,110. of pagP and pagL and subsequent upregulation of the
Various constituents, such as additional sugars, phosphate groups, phosphoethanol encoded proteins, which acylate and deacylate lipidA,
amine groups and phosphorylcholine groups, can modify the LPS core regions.
respectively 9,10,1921 (FIGS1d,2; TABLE1). Furthermore, both
Similarly, known Oantigen modifications include glycosylation111, acetylation112,
addition of phosphoryl constituents1 and ligation of acidic repeats such as colanic113
of these enzymes are subjected to post-translational reg-
and sialic114 acids. ulation (see below). Because the active sites of these two
In the context of cationic antimicrobial peptide (CAMP) resistance and Toll-like enzymes are found on the extracellular surface of the
receptor4 (TLR4) evasion, modifications to the LPS core seem to have more modest outer membrane and in close proximity to lipid A, they
roles in pathogenesis than do variations in Oantigen length and lipid A modifications, require tight control to ensure that lipid A modification
although core modifications can enhance resistance to some CAMPs and to is appropriately regulated.
components of the innate immune system, such as complement. A good example In S.Typhimurium, PhoPQ further influences lipid A
of how non-lipid A modifications can affect pathogenesis can be seen in Neisseria modification by activating the PmrAB system, although
gonorrhoeae. This bacterium protects itself from the complement system in human in E.coli and P.aeruginosa the two systems are not cou-
serum by modifying lipooligosaccharide (LOS; LPS in which Oantigen is absent and the
pled22. Direct activation of PmrAB in S.Typhimurium
core region is extended) through the addition of sialic acid and glucose, thus increasing
the affinity of LOS for the complement-inhibitory proteins factor H and complement
occurs upon sensing Fe3+, Al3+ and low pH, and leads
component4b (C4b)binding protein, respectively114,115. Furthermore, N.gonorrhoeae to the upregulation of genes such as arnT (also known
producing LOS with a terminal sugar of galactose has an increased association with as pmrK) and eptA (also known as lptA and pmrC),
dendritic cells compared to N.gonorrhoeae that produces LOS containing terminal which transfer 4amino4deoxylarabinose (ami-
Nacetylglucosamine. This association with dendritic cells leads to altered cytokine noarabinose) and phosphoethanolamine groups to
production and Tcell responses that could decrease immune responses against the lipid A, respectively 11 (FIGS. 1d,2a). Similarly to the
bacterium116. S. Typhimurium PmrAB system, the P. aeruginosa
PmrAB system is activated by the depletion of cations
(such as Mg 2+), but it is also activated by antimicrobial
Regulation of lipid A remodelling peptides23. In E.coli, S.Typhimurium, P.aeruginosa and
Lipid A modifications are often necessary only for a other bacteria, the activity of the enzymes controlled by
portion of the bacterial life cycle, such as host coloni- PhoPQ and PmrAB strengthen the integrity of the outer-
zation, and as a consequence the enzymes responsible membrane permeability barrier in the presence of anti-
for the modifications are subjected to both transcrip- microbial peptides and depleted cations, thus enhancing
tional and post-translational regulation. Many modifi- bacterial survival in the host 24,25.
cation enzymes are embedded in the outer membrane The ParRS and CprRS are independent two-component
in close proximity to lipid A, necessitating tight control systems that have been found only in P.aeruginosa and
for selective enzyme activity, whereas other enzymes respond to subinhibitory concentrations of antimicrobial
are constitutively active regardless of their localization. peptides12,13. Thus, these systems might have an important
Two-component systems, small RNAs (sRNAs), peptide role during infection. Although both systems upregulate
feedback loops and substrate availability are all involved pmrA, pmrB and the genes responsible for the addition
in directing the activity of these enzymes (FIG.2). of aminoarabinose to lipid A12,13, these systems are dif-
ferentially activated in response to certain antimicrobial
Transcriptional control. Two-component systems are peptides. For instance, the synthetic peptide CP28 acti-
typically composed of a sensor kinase and a response vates the CprRS system, whereas the ParRS system seems
regulator; the sensor kinase is autophosphorylated on to be more responsive to the peptide indolicidin13. The
stimulation and transfers its phosphate group to the two systems react similarly to other peptides, such as
response regulator, which then serves as a transcription polymyxin B and colistin, both of which are used to treat
factor. To date, various two-component systems have P.aeruginosa infections13. P.aeruginosa is a useful, but
been implicated in the regulation of lipid A modifica- complex, model pathogen for studying the regulation
Small RNAs tion enzymes, including the widespread PhoPQ and of lipid A modification because structurally divergent
Short, non-coding RNA PmrAB (also known as BasRS) systems, and the ParRS forms of lipid A are associated with different types of
molecules that can regulate
gene expression by interacting
and CprRS systems in Pseudomonas aeruginosa 813. infection, including lung infections that result in acute
with mRNA or can bind protein Other systems, such as EvgAS, RcsBC and BvgAS, have bronchiectasis, and chronic colonization of the cystic
targets to modify their activity. been linked to the activity of the PhoPQ system or to fibrosislung 26.

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a Transcriptional regulation b Post-translational regulation

Acylation Deacylation Acylation Deacylation


L-Ara4N
Outer membrane
PagP PagL PagP LpxR

Periplasm
L-Ara4N pEtN
P

PhoQ PmrB ArnT EptA LpxT


Inner membrane P P
P P
PhoP PhoP PmrA PmrA
MicA

P Modication
PmrA enzymes
Ribosome phoP

P Modication P
MgrR PhoP enzymes PmrA

PmrR
eptB MicF
LpxR
lpxR
Figure 2 | Transcriptional and post-translational regulation of lipid A modification enzymes. a|Transcriptional
Nature Reviews | Microbiology
control of lipidA modification enzymes includes gene regulation by two-component systems such as PhoPQ, leading
to acylation and deacylation of lipid A by upregulating transcription of the genes encoding the enzymes PagP and PagL,
respectively. The two-component system PmrAB leads to the addition of 4amino4deoxylarabinose (aminoarabinose;
l-Ara4N) and phosphoethanolamine (pEtN) to lipid A by upregulating transcription of the genes encoding the inner-
membrane enzymes ArnT and EptA, respectively, which modify lipid A as it is transported to the outer membrane10,11.
Expression of EptB (another phosphoethanolamine transferase) is repressed by the small RNA (sRNA) MgrR, which is
induced by PhoPQ30. Translation of the phoP mRNA is repressed by the sRNA MicA31, which leads to the loss of regulation
by the PhoPQ system. The sRNA MicF increases degradation of the lpxR mRNA, which encodes a lipid A deacylase.
b|Post-translational control of lipid A modification systems includes inhibition of the kinase LpxT (which phosphorylates
lipid A during transport to the outer membrane) by the small peptide PmrR, which is upregulated by the PmrAB system in
response to high levels of Fe3+ (REF.33). Post-translational regulation is also mediated by substrate availability. Membrane
perturbation can lead to the displacement of phospholipids from the inner leaflet to the outer leaflet of the outer
membrane, placing these donor substrates in close proximity to the acyltransferase PagP, and thus enhancing enzyme
activity. PagP cleaves the phospholipid substrate, restoring the composition of the outer membrane and increasing the
integrity of the permeability barrier by further acylating lipid A39. LpxR deacylates lipid A, but this activity is inhibited by
the aminoarabinose lipid A modification.

The PhoPQ system has also been shown to mediate confer higher CAMP resistance30. In addition to regulat-
modification of lipid A through transcriptional acti- ing an sRNA, the PhoPQ system itself is controlled by
vation of non-coding sRNAs. Many sRNAs modulate another sRNA, MicA (FIG.2a), which inhibits translation
the expression of outer-membrane proteins 2729, but of PhoP by competitive binding to the ribosome-binding
Kdo recently the sRNA MgrR of E.coli was shown to regu- site of the phoP mRNA31. MicF represents another exam-
(3deoxydmanno-octulosonic late lipidA modification30 (FIG.2a). MgrR is a transcrip- ple of an sRNA that interacts with lipid A modification
acid). The sugar residue that
constitutes the inner core of
tional target of PhoP and is conserved in E.coli and enzymes. This sRNA binds to lpxR transcripts, which
lipopolysaccharide. This inner certain Citrobacter, Enterobacter and Klebsiellaspp.30. encode a lipid A deacylase (FIG.1c; TABLE1), and increases
core links the polysaccharide This sRNA regulates various genes, including the nega- degradation of the mRNA by exposing regions that are
chain to lipid A. tive regulation of eptB30, which encodes an enzyme susceptible to RNase E, a major contributor to RNA
that transfers phosphoethanolamine to the outer Kdo turnover in many bacteria32.
Cationic antimicrobial
peptide (3deoxydmanno-octulosonic acid) residue of LPS7
A type of positively charged, (FIG.1c). Although EptB activity results in a modest Post-translational control. In addition to transcriptional
amphipathic peptide that increase in resistance to the cationic antimicrobial peptide regulation, lipid A modification enzymes are subjected
associates with the negatively (CAMP) polymyxin B, it is unlikely that this is the main to post-translational control mechanisms. For example,
charged Gram-negative
membrane and is thought to
function of this enzyme because conditions that inhibit in S.Typhimurium, the PmrAB system orchestrates a
disrupt the membrane, leading EptB (through activation of the PhoPQ system) simulta- delayed negative feedback loop that can be activated
to cell lysis and death. neously induce the other PhoPQ-regulated genes, which by Fe3+ and allows initial uptake of the ion but sets in

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motion a shift in the cell surface charge that reduces their survival. The diversity of regulatory mechanisms
Fe3+ retrieval from the extracellular environment. This also illustrates the importance of the lipid A structure to
feedback loop is established through PmrAB-induced the membrane barrier.
expression of a short peptide, PmrR, which binds to
and inhibits the lipid A-modifying enzyme LpxT 33 Host defences that target lipid A
(FIG.2b; TABLE1). LpxT phosphorylates lipid A (FIG.1c) Considering the intimate contact that humans have
and thereby increases the overall net negative charge of with bacteria, the frequency of colonization with patho-
the outer membrane. However, when LpxT activity is genic Gram-negative bacteria is astonishingly low. This
inhibited, other modifying enzymes are capable of trans- is largely attributed to the formidable arsenal of host
ferring amine-containing constituents to lipidA34, and defences that eliminate invading pathogens by recog-
the outer membrane eventually becomes less negatively nizing and responding to highly conserved components
charged and so has a reduced affinity for Fe3+ (REF.33). of infectious agents, known as MAMPs41. Because LPS
This mechanism regulates lipid A-modifying enzymes is an essential component of the Gram-negative cell
such as LpxT and those encoded by genes that are tran- surface, it serves as an effective MAMP to trigger the
scriptionally induced by PmrAB (such as EptA and innate immune system1. The host offers a nutrient-rich
ArnT). It also prevents the intracellular accumulation but perilous environment for a bacterium. For example,
of toxic Fe3+ and dampens PmrA-dependent transcrip- intestinal colonization requires a bacterium to journey
tion in a delayed manner as a result of the lowered affin- through the acidic pH of the stomach and encounter
ity of PmrAB-activating Fe3+ for the membrane. Such toxic compounds such as bile and antimicrobials during
small peptide-mediated feedback control of the systems transit 42, and the bloodstream is swarming with LPS-
that regulate lipid A modification might be a com- binding proteins, antibodies, complement and immune
mon theme. In fact, other small peptides, such as SafA cells primed to detect LPS1,4346. Every point of entry for a
(in E.coli) and MgrB (in E.coli, S. Typhimurium and bacterium is well defended, but as many of the protective
Yersiniapestis), are inner-membrane peptides that inter- mechanisms rely on lipid A detection, the modification
act with the periplasmic domain of PhoQ and activate of lipid A affords the bacterium an opportunity to evade
or repress PhoQ activity, respectively 35,36. the immune system and establish an infection.
Other post-translational control mechanisms rely
on substrate availability. Although the acyltransferase Charge-dependent binding of lipid A. Charge-dependent
PagP is transcriptionally upregulated by PhoPQ in binding of various host molecules (such as CAMPs, plate-
organisms such as Salmonella spp. and E.coli, its acti- let factor 4 (PF4) and members of the complement sys-
vation is enhanced by environmental stress and intra- tem) to the bacterial cell surface is a major contributor to
cellular membrane stress37. For example, in E.coli, host protection. CAMPs are amphipathic molecules that
PagP remains dormant in the outer membrane under are present on mammalian mucosal surfaces, in bodily
standard growth conditions. However, treatment secretions (such as sweat and saliva) and in phagocytic
with membrane-perturbing agents, such as EDTA, cells. CAMP production is a highly conserved defence
results in displacement of the phospholipid donor mechanism of most organisms, but the peptides vary
substrate of PagP from the inner to the outer leaflet widely in terms of their composition and structure. The
of the outer membrane, placing it in close proxim- genes encoding CAMPs are among the most rapidly
ity to the PagP catalytic domain38. PagP cleaves the evolving mammalian genes47, and it has been hypoth-
phospholipid substrate, restoring the composition esized that this represents an example of coevolution,
of the outer membrane and increasing the integrity of the wherein the genes are under selective pressure to mutate
permeability barrier by further acylating lipid A39 (FIG.2b). as a consequence of the evolution of bacterial resistance to
The affinity and activity of lipid A modification the encoded proteins48. One of the primary mechanisms
enzymes are also modulated by the chemical composi- proposed for CAMP-mediated bactericidal activity
tion of lipid A. For example, in Yersinia enterocolitica, is association of the positively charged peptides with
LpxR deacylates lipid A at 37C, but at 21C LpxR activ- negatively charged lipid A, followed by CAMP inser-
ity is impeded. At 21C, pmrAB and the genes neces- tion into the bacterial membrane and disruption of the
Complement sary for aminoarabinose addition are induced, and the membrane potential, leading to cell death42,48.
An innate immune defence elevated amounts of aminoarabinose residues on lipidA PF4 is another positively charged host molecule that
mechanism involving many
proteins that function in
seem to suppress LpxR activity 40. In S.Typhimurium, is conserved across vertebrates and is released follow-
signalling cascades and also PagL is similarly repressed by aminoarabinose modi- ing platelet activation during infection. This molecule
form cell-lysing membrane fied lipid A, although this effect seems to be temperature binds the bacterial cell surface at the lipid A domain.
attack complexes. independent 21. Antibodies specific for PF4lipid A complexes are subse-
These various forms of post-translational regulation quently produced, leading to increased opsonization and
Opsonization
The tagging of pathogens by afford quick response times for modification of the outer phagocytosis of the bacterium43. Furthermore, comple-
molecules such as antibodies. defence barrier, as the enzymes are already present and ment proteins also bind lipid A and modulate the acti-
These molecules target the primed to function as soon as the appropriate signal is vation of the classical complement pathway to promote
foreign entity for destruction detected. Both transcriptional and post-translational bacterial clearance4446.
by immune system clearance
mechanisms such as
regulation work together, sometimes as functionally
phagocytosis and the redundant mechanisms, allowing Gram-negative bacte- Toll-like receptor4 signalling. Lipid A is recognized by
complement system. ria to adapt to diverse environments and thereby ensure the Toll-like receptor 4 (TLR4)MD2 (also known as

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Table 1 | Modifications to the Kdolipid A domain of lipopolysaccharide


Enzyme* Enzyme Active-site Pathogenic organisms Activity Contributors to Effect of Refs
localization topology regulation modification
AlmG Inner Cytoplasmic V.cholerae Transfer of glycine to the Unknown CAMP resistance 75
membrane hydroxyl group of the
3-acyloxyacyl chain of
lipid A
ArnT Inner Periplasmic E.coli, P.mirabilis, Addition of PmrAB, as well as CAMP resistance 7,14,
(PmrK) membrane S.Typhimurium, S.flexneri, aminoarabinose to ParRS and CprRS 125128
P.aeruginosa, B.cepacia, lipidA in P.aeruginosa
Y.enterocolitica, Y.pestis,
Y.pseudotuberculosis,
K.pneumoniae, B.pertussis
and B.bronchiseptica
EptA Inner Periplasmic E.coli, V.cholerae, H.pylori, Addition of phospho PmrAB CAMP resistance 7,125,
(LptA and membrane S.Typhimurium, S.flexneri, ethanolamine to lipid A 129,130
PmrC) Neisseriagonorrhoeae and
Neisseria meningitidis
EptB Inner Periplasmic E.coli, S.Typhimurium and Transfer of phospho PhoPQ and the Modest CAMP 125,131,
membrane Y.pestis ethanolamine to Kdo sRNA MgrR resistance 132

EptC Inner Periplasmic C. jejuni Transfer of phospho Present under CAMP resistance 91,133
membrane ethanolamine to lipidA, normal laboratory and motility
the flagellar rod and conditions
other substituents
FlmF1 Inner Periplasmic F. tularensis Addition of glucose and Present under Possible role in 134
membrane mannose to lipid A normal laboratory CAMP resistance
conditions
FlmF2 Inner Periplasmic F. tularensis Addition of Present under Possible role in 134,135
membrane galactosamine to lipid A normal laboratory CAMP resistance
conditions
FlmK Inner Periplasmic F. tularensis Addition of glucose, Present under TLR4 evasion and 135
membrane mannose or normal laboratory a possible role in
galactosamine to lipid A conditions CAMP resistance
KdkA Inner Cytoplasmic H.influenzae, V.cholerae, Phosphorylation of Kdo Present under Possible effect on 125,136
membrane P.multocida, B.pertussis, normal laboratory toxin delivery
A.actinomycetemcomitans conditions
and S.putrefaciens
KdoH1 and Inner Cytoplasmic H.pylori, F. tularensis and Removal of outer Kdo Present under CAMP resistance 137139
KdoH2 membrane L.pneumophila region normal laboratory
(KdhA) conditions
KdoO Inner Cytoplasmic B.cepacia, B.ambifaria, Hydroxylation of Kdo Present under Unknown 140
membrane Y.pestis and normal laboratory
A.haemolyticus conditions
LmtA Inner Cytoplasmic L.interrogans Methylation of lipid A Present under Unknown 141
membrane normal laboratory
conditions
LpxD2 Inner Cytoplasmic F. tularensis Addition of shorter Low temperatures Increased 72
membrane (C16) primary acyl membrane
chains to lipidA at low fluidity
temperatures
LpxE Inner Periplasmic F. tularensis, H.pylori and Dephosphorylation of Present under CAMP resistance 7,125,
membrane P.gingivalis lipid A normal laboratory and TLR4 evasion 142
conditions
LpxF Inner Periplasmic F. tularensis, H.pylori, Dephosphorylation of Present under CAMP resistance 7,63,
membrane P.gingivalis and lipid A normal laboratory and TLR4 evasion 125,143,
L.interrogans conditions 144

LpxO Inner Cytoplasmic S.Typhimurium, Hydroxylation of lipid A Present under Coordination of 7,125,
membrane K.pneumoniae, acyl chains normal laboratory stress responses 145
P.aeruginosa, conditions
B.bronchiseptica and
L.pneumophila
LpxP Inner Cytoplasmic E.coli, S.Typhimurium, Alternative biosynthetic Low temperatures Membrane 7,146,
membrane Y.enterocolitica, Y.pestis, acyltransferase at low integrity and 147
and L.pneumophila temperatures fluidity

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Table 1 (continued) | Modifications to the Kdolipid A domain of lipopolysaccharide


Enzyme* Enzyme Active-site Pathogenic organisms Activity Contributors to Effect of Refs
localization topology regulation modification
LpxR Outer Extracellular E.coli O157:H7, Deacylation of lipid A The sRNA TLR4 evasion 7,125,
membrane H.pylori, V.cholerae, MicF and 148
S.Typhimurium, aminoarabinose
Y.enterocolitica, Y.pestis modifications to
and Y.pseudotuberculosis lipidA
LpxT Inner Periplasmic E.coli, S.Typhimurium and Phosphorylation of PmrAB and the Unknown 34
membrane Y.pestis lipid A and recycling small peptide
of undecaprenyl PmrR
pyrophosphate
PagL Outer Extracellular P.aeruginosa, B.cepacia, Deacylation of lipid A PhoPQ and Decreased TLR4 7,125,
membrane S.Typhimurium, aminoarabinose activation 128,149
B.pertussis and modifications to
B.bronchiseptica lipidA
PagP Outer Extracellular E.coli, S.Typhimurium, Acylation of lipid A PhoPQ and Selective CAMP 7,125,
membrane S.flexneri, P.aeruginosa, membrane resistance, 150152
Y.enterocolitica, Y.pestis, perturbation membrane
Y.pseudotuberculosis, integrity and
K.pneumoniae, B.pertussis, decreased TLR4
B.bronchiseptica, activation
B.parapertussis and
L.pneumophila
A.haemolyticus, Acinetobacter haemolyticus; A.actinomycetemcomitans, Actinobacillus actinomycetemcomitans; aminoarabinose, 4amino4deoxylarabinose;
B.bronchiseptica, Bordetella bronchiseptica; B.parapertussis, Bordetella parapertussis; B.pertussis, Bordetella pertussis; B.ambifaria, Burkholderia ambifaria;
B.cepacia, Burkholderia cepacia; CAMP, cationic antimicrobial peptide; C.jejuni, Campylobacter jejuni; E.coli, Escherichiacoli; F.tularensis, Francisella tularensis;
H.influenzae, Haemophilus influenzae; H.pylori, Helicobacter pylori; Kdo, 3deoxydmanno-octulosonic acid; K.pneumoniae, Klebsiella pneumoniae; L.pneumophila,
Legionella pneumophila; L.interrogans, Leptospira interrogans; N.gonorrhoeae, Neisseria gonorrhoeae; N.meningitidis, Neisseriameningitidis; P.multocida, Pasteurella
multocida; P.gingivalis, Porphyromonas gingivalis; P.mirabilis, Proteus mirabilis; P.aeruginosa, Pseudomonas aeruginosa; S.Typhimurium, Salmonella enterica subsp.
enterica serovar Typhimurium; S.putrefaciens, Shewanella putrefaciens; S.flexneri, Shigella flexneri; TLR4, Toll-like receptor4; V.cholerae, Vibrio cholerae;
Y.enterocolitica, Yersinia enterocolitica; Y.pestis, Yersinia pestis; Y.pseudotuberculosis, Yersinia pseudotuberculosis. *Alternative enzyme names in certain species are
given in brackets. Organisms containing homologues of these enzymes are excluded from the table if the respective modification has not been observed. The list of
enzymes in this table is likely to expand as the Kdolipid A region is further characterized in a range of species.

LY96) receptor, one of many pattern recognition receptors occurs after endocytosis of the TLR4MD2 receptor
(PRRs) of the mammalian innate immune system. This and is characterized by the production of interferon
receptor is present on a wide variety of cell types, includ- (IFN) and IFN-inducible proteins such as 10kDa
ing monocytes, lymphocytes and endothelial cells 1. IFNinduced protein (IP10; also known as CXCL10),
Binding of TLR4MD2 to lipid A triggers a signalling monocyte chemoattractant protein 1 (MCP1; also
cascade that leads to inflammation, cytokine produc- known as CCL2), RANTES (also known as CCL5)
tion and the eventual clearance of bacteria through and granulocyte colony-stimulating factor (G-CSF)55.
recruitment of effector cells, phagocytosis, cytotoxicity Although it is important for mounting an optimal
Pattern recognition
receptors
and activation of the complement system49 (FIG.3). This immune response to pathogens, the TRIF pathway does
Receptors of the innate inflammatory response can be severe, resulting in tissue not lead to severe inflammation. Unmodified LPS from
immune system. These damage, organ failure and death, especially in cases of E.coli induces signalling through both pathways, but
receptors bind microorganism- sepsis1. Unmodified E.coli lipid A, which contains six lipid A modifications can cause preferential recruitment
associated molecular patterns
acyl chains and two phosphate groups, is the strongest of one adaptor protein over the other 56 (BOX2). Invivo,
of infecting pathogens and
initiate signalling cascades known TLR4 ligand, and lipid A modifications can bacterial lipid A modifications are also known to affect
which lead to inflammation, weaken or abolish TLR4 signalling and change the nature the potency of TLR4 activation, as describedbelow 7.
cytokine release and activation of the downstream cytokine profile (see below)5053.
of the adaptive immune The detection of lipid A by TLR4 begins with LPS- Bacterial evasion strategies
response.
binding protein (LBP) and CD14 binding to LPS and the Modification of lipid A equips Gram-negative bacteria
Cytokine subsequent transfer of LPS to the TLR4MD2 complex. with an ability to evade immune recognition and sur-
A signalling protein involved in This complex can then signal through two major path- vive within a host. First, by changing the overall charge
the recruitment and regulation ways, which are named according to their adaptor of the bacterial surface through the addition of chemical
of cells that participate in the
proteins: myeloid differentiation primary response groups to lipid A, such as the addition of phospho
immune response.
protein 88 (MYD88) and TIR domain-containing adap- ethanolamine and aminoarabinose, resistance to innate
Sepsis tor inducing IFN (TRIF; also known as TICAM1)54 immune effectors (for example, CAMPS and complement
The severe and often fatal (FIG.3). Severe reactions to LPS are attributed to activation factors) increases. Second, changes in the structure of
inflammatory response of the of the MYD88 pathway, which induces the production of lipid A are important for bacterial pathogenesis because
body to the overwhelming
presence of infection (usually
pro-inflammatory cytokines such as tumour necrosis they directly affect recognition by the TLR4MD2 recep-
bacterial), characterized in part factor (TNF), interleukin6 (IL6) and IL12. The less tor, and the degree of lipid A acylation and phosphoryl
by organ failure. inflammatory TRIF (or MYD88independent) pathway ation is crucial for LPS recognition by TLR4MD2

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PmrAB-induced lipid A modifications increase resist-


LPS ance to CAMPS, but constitutive activation of PmrAB
has been shown to reduce the resistance of E.coli to the
LBP MD2 bile component deoxycholate58. Furthermore, PhoPQ-
mediated alterations have been shown to lower the
TLR4
CD14 Cell membrane resistance of S. Typhimurium to antibiotics in the pres-
ence of high Mg 2+ concentrations24. However, these costs
of lipid A modification seem to be outweighed by a
TIRAP
number of advantages for bacterial virulence, as exem-
plified by S. Typhimurium, Helicobacter pylori, Y.pestis,
MYD88 Francisella tularensis and Vibrio cholerae.

Salmonella enterica subsp. enterica serovar Typhi


Endosome murium. S. Typhimurium is an intracellular pathogen
that causes gastroenteritis in humans. As a long-standing
model organism for studying bacterial pathogenesis,
S.Typhimurium provides prime examples of complex
lipid A alterations7 (FIG.1d). The bacterium has a diverse
MYD88-dependent lifestyle and fine-tunes the composition of lipid A in
pathway response to the surrounding environment 7,59. During
infection, S. Typhimurium penetrates the epithelial
lining of the small intestine, invades lymphoid tissue
and infects host phagocytes60. The unmodified lipid A
synthesized by this organism is identical to that produced
TRAM by E.coli K12 (FIG.1c). However, survival is promoted
TRIF in the intestinal lumen and within host cells (where the
bacterium encounters CAMPs, low pH and possibly
MYD88-independent other unknown signals) by activation of the PhoPQ and
pathway
Cytosol PmrAB systems, leading to the addition of phospho
ethanolamine and aminoarabinose by EptA and ArnT,
respectively, and to acyl chain remodelling by PagP
and PagL19 (FIG.1d). Commensal and pathogenic E.coli
Pro-inammatory IFN-inducible
Nucleus cytokines genes
strains also encode these lipid A modification enzymes,
but little is known about their regulation invivo. In
S.Typhimurium, another enzyme, LpxO, hydroxylates
lipid A (FIG.1d) as part of a coordinated stress response61.
The combination of these modifications results in a
Figure 3 | Toll-like receptor4MD2 signalling. This simplified Toll-like receptor4 remodelled outer membrane with a reduced net nega-
Nature Reviews | Microbiology
(TLR4)MD2 signalling schematic illustrates the two responses the myeloid
tive charge and increased integrity, which enhances
differentiation primary response protein 88 (MYD88)dependent and TIR domain-
containing adaptor inducing IFN (TRIF) (or MYD88independent) pathways that virulence9,25.
can be differentially stimulated on binding of lipid A to the TLR4MD2 complex. This
binding occurs through the association of lipidA with lipidA-binding protein (LBP) and Helicobacter pylori. The human stomach is the sole
CD14, and leads to the production of cytokines and clearance of the pathogen49. The niche of H.pylori, and the organism is so well adapted
MYD88dependent pathway leads to the production of pro-inflammatory cytokines, to this environment that it colonizes roughly 50% of
whereas the less inflammatory TRIF pathway occurs after endocytosis of the TLR4MD2 the worlds population and can persist for decades
receptor and stimulates the expression of interferon (IFN)-inducible genes that are within a single host62. To survive chronically in the host
important for adjuvanticity but are less inflammatory than those cytokines induced by and remain undetected, H.pylori uses two constitu-
the MYDD88dependent pathway. tive lipidAmediated evasion strategies: repulsion of
CAMPs (which are present at high concentrations in
the gastric mucosa) and evasion of detection by TLR4
(REF.50). Bacteria can remove acyl chains and phosphate (FIG. 4a) . Similarly to most Gram-negative bacteria,
groups to evade detection by this PRR or to shift the type H.pylori synthesizes a hexa-acylated lipid A, but dis-
of cytokine response induced57. Finally, certain modi- plays a tetra-acylated molecule lacking phosphate groups
fications (such as those regulated by PhoPQ) alter the on the bacterial surface. This striking structural differ-
properties of the outer-membrane permeability barrier, ence between the originally synthesized lipidA and the
which provides resistance to harsh pH and antibiotics, surface-exposed molecule is due to the actions of sev-
among other stresses. eral enzymes, including dephosphorylation by LpxE and
There are benefits and costs associated with each LpxF, addition of phosphoethanolamine by EptA
modification: as bacteria adapt to protect themselves and deacylation by LpxR (TABLE1). These modifica-
against certain assaults, this might result in the loss of tions confer resistance to polymyxin B as well as other
protection against others. For instance, PhoPQ- and biologically relevant CAMPs63. The reduced acylation

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Box 2 | Lipid A as a tool for immune system modulation


transition between hosts coincides with a switch in the
composition of lipid A68 (FIG.4b). Inside the flea, Y.pes
By chemically or biologically modifying the phosphate groups and acyl chains of lipidA, tis grows at a temperature of between 21C and 27C
the therapeutic potential of this lipopolysaccharide (LPS) component can be harnessed and synthesizes a hexa-acylated lipid A similar to the
while limiting the inflammatory effects of the molecule50,54,64. In fact, a chemically highly inflammatory E.coli lipid A that strongly stimu-
detoxified mixture of monophosphorylated lipid A species (MPL) derived from
lates TLR4 (REF.69). In humans, Y.pestis encounters a
Salmonella enterica subsp. enterica serovar Minnesota (the predominant species of
which is 3Odeacyl4-monophosphoryl lipid A; see the figure) recently became the
temperature of 37C and replaces the agonist form of
first US Food and Drug Agency (FDA)-approved vaccine adjuvant in more than 70years, lipidA with a tetra-acylated form that is a TLR4 antago-
bringing the number of FDA-approved Toll-like receptor (TLR) agonists up to three54,117. nist. The tetra-acylated form of lipid A is believed to
We predict that TLR ligands are likely to be the future of vaccines and could benefit allow the pathogen to proliferate undetected in the
other areas as well, such as cancer research, gene therapy and bacterial production of bloodstream during the early stages of infection70,71.
pharmaceuticals. Heterologous expression of the E.coli acyltransferase
Lipid A derivatives like MPL have been well studied for their agonistic properties in LpxL in Y.pestis restores hexa-acylated lipid A and
terms of immune stimulation54. However, MPL is the only lipid A that has been tested in strong TLR4stimulatory properties70. Strains that
human cancer vaccine trials118, and almost all studies have focused on cervical cancer, can produce only hexa-acylated lipid A are also aviru-
as MPL is used as the vaccine adjuvant against the oncogenic human papilloma virus
lent in mice, suggesting that deacylation of lipid A is
(HPV)117. Considering the evidence that TLR4 signalling can be biased to produce
certain types of cytokine responses56, further study of other modified lipid A structures,
required for TLR4 evasion and is crucial for Y.pestis
as well as chemically synthesized lipid A variants119, could prove advantageous for the pathogenesis70.
development of vaccine adjuvants. To facilitate such work, an Escherichiacoli library Similarly to Y.pestis, the lipid A of F.tularensis is modi-
was recently engineered to synthesize lipid A variants with a spectrum of endotoxicity120. fied according to temperature, affecting membrane integ-
These lipid A variants have the potential to be used as components of whole cells, rity and pathogenesis in the environmental vectors of this
LPS or purified lipid A and might be OH
pathogen, such as protozoa and arthropods (1826C),
used as vaccine adjuvants in the O
4 6 O 6 and mammalian hosts (37C). This bacterium has two
future. P 5 O 4
HO O
HO
5 O homologues of LpxD, an essential lipid A biosynthesis acyl
Whole-cell vaccine strains of some OH O
O
3 2
NH 1 HO 2 1 transferase that incorporates longer acyl chains at high
organisms, such as Salmonella enterica 3 NH
O OH temperature (37C) than at lower temperatures (25C and
subsp. enterica serovar Typhimurium, O
have been engineered to synthesize
O 18C)72. A mutant strain that is unable to produce lipid A
O O
altered lipid A structures in order to O with longer acyl chains is avirulent in mice and is also more
O
reduce the toxicity of the vaccine. O susceptible to antibiotics and CAMPs owing to increased
Heterologous expression of an antigen membrane permeability 72. These two examples of lipidA
from Streptococcus pneumoniae in modifications emphasize the fact that appropriate adapta-
such strains provides protection tion in response to temperature is important for altering
against both S.Typhimurium and the fluidity of the outer membrane, and that this alteration
S. pneumoniae51,52. Similarly, is needed to maximize Y.pestis and F.tularensis virulence.
outer-membrane vesicles from
Neisseria meningitidis producing
Vibrio cholerae. V.cholerae, the causative agent of the
modified lipid A are now prime
candidates for vaccination against
severe diarrhoeal disease cholera, commonly survives
N.meningitidis121, and also against on marine crustaceans such as copepods and can be
other organisms for which antigens acquired by humans through ingestion of copepods, lead-
can be heterologously expressed in ing to intestinal disease73. The V.cholerae O1 El Tor bio-
N.meningitidis and targeted to the type is currently causing a seventh worldwide pandemic
vesicle lumen or surface122. 3-O-deacyl-4-monophosphoryl lipid A affecting an estimated 35 million people, but the previ-
ous six pandemics were caused by the classical biotype
Nature Reviews | Microbiology
V.cholerae O1. These two biotypes can be differenti-
and phosphorylation of lipidA also lead to decreased ated by their sensitivity to polymyxin B74, as V.cholerae
stimulation of TLR4 and the downstream signalling cas- O1 El Tor is 100fold more resistant to polymyxin B
cade50,64,65. When these modification systems are inacti- than the classical V.cholerae O1 strains. This resistance
vated through mutation, H.pylori displays hexa-acylated, is conferred by an unusual lipid A modification. A three-
Biotype bis-phosphorylated lipid A (FIG.4a), which is a strong gene operon in V.cholerae (almEFG) encodes enzymes
A subtype of a bacterial
species that can be
stimulator of TLR4 (REF.63). The constitutive lipidA that transfer a glycine or diglycine residue to a 3linked
distinguished from other modifications to the acyl chains and phosphate groups acyl chain of lipid A75 (FIG.4c). This is the first described
subtypes by biological are adaptations that allow this bacterium to persist in the amino acid modification of lipid A, and it has revealed a
characteristics such as motility, harsh gastric environment amidst the several antibacterial unique link between Gram-negative and Gram-positive
resistance to cationic
components of the innate immune response. cell wall decoration75,76, as Gram-positive bacteria can
antimicrobial peptides and
antibiotics, and the production transfer the amino acid alanine to wall teichoic acids and
of virulence factors. Yersinia pestis and Francisella tularensis. Y.pestis is wall lipoteichoic acids and thus reduce the net negative
notorious for its role in human disease throughout his- charge of the cell wall76. The amino acid modification of
Pandemic tory, causing the Black Death plague that killed approxi- lipidA in V.cholerae has little effect on TLR4 activation,
The widespread occurrence
of a human infectious disease
mately one-third of the European population in the suggesting that it has evolved for the distinct purpose
that is spread over a large fourteenth century 66. The bacterium has a complex life of promoting survival in the presence of CAMPs in the
geographical region. cycle, colonizing both the flea and human host 67. This marine and human host environment 75.

NATURE REVIEWS | MICROBIOLOGY VOLUME 11 | JULY 2013 | 475

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a Chronic persistance Wild type Helicobacter pylori Mutant


OH OH
O
O P O
HO O HO O O
O O OH O O
H. pylori HO O
HO
NH O NH O
O O
O O NH O O NH
O P O P
O O O
HO O HO OH
O HO O O HO
NH2
O HO O HO

CAMP resistant CAMP sensitive


TLR4 evasive TLR4 agonist
Gastric epithelium

b Initial evasion 37 C Yersinia pestis 2127 C


OH OH
O O
P O P O
HO O O HO O O
OH O O OH O O
HO HO
O NH O O NH O
O O
O O NH O O NH
Temperature- O P O P
O O O O
dependent switch O HO
HO OH
O O HO
HO OH

HO O HO

Flea infected
with Y. pestis
2127 C

37 C
TLR4 antagonist TLR4 agonist

c CAMP resistance Classical O1 biotype Vibrio cholerae El Tor O1 biotype


OH OH
O O
P O P O
HO O O HO O O
OH O O OH O O
HO HO
O NH O O NH O
O O
O O NH O O NH
O P O P
O O
HO OH Glycine O O
HO OH
O O HO O O HO
O
O HO N2H O HO
O

Copepod host H O
N
colonized N2H
with V. cholerae O

Diglycine

CAMP sensitive CAMP resistant

Host modification of lipid A to promote tolerance to commensals


Nature Reviews but| Microbiology
to mount a
Host tolerance of commensal bacteria. The host envi- defence against pathogens. In general, enteric bacteria
ronment at mucosal surfaces is rich in LPS, as this produce highly stimulatory, hexa-acylated lipidA 77,
compound is produced in abundance by commensal although decorating the cell surface with such an effi-
Gram-negative bacteria. This means that the immune cient immunostimulant seems counterintuitive as a
response at these surfaces needs to be modulated strategy for commensal bacteria. In the gastrointestinal

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Figure 4 | Lipid A modification strategies that promote survival in the capacity of the host to react to a secondary exposure.
host. Helicobacterpylori, Yersiniapestis and Vibriocholerae have evolved different By contrast, wild-type mice are able to recover and
mechanisms of lipid A modification to aid colonization of their respective niches inside respond normally to LPS within 510days80. AOAH
the host. Differences in structure between the pairs of lipidA molecules are highlighted activity increases during LPS challenge in rabbits and
in red. a|The human stomach is the sole niche of H.pylori, and the bacterium has
mice, suggesting that this enzyme has a primary role in
adapted to this environment by constitutively modifying lipid A to a form that resists
cationic antimicrobial peptides (CAMPs) and evades the lipid A receptor, Toll-like
LPS detoxification79,80.
receptor4 (TLR4). Surface expressed H. pylori lipid A consists of a tetra-acylated form Host alkaline phosphatases have also been shown to
that lacks the 4-phosphate group and is substituted at the C1 position with a modify LPS through the removal of phosphate groups.
phosphoethanolamine63. Lipid A modification mutants present a hexa-acylated, Whether these phosphatases act on both lipid A and the
bis-phosphorylated species that is a TLR4 agonist. b|When residing in the flea vector, sugar chains of LPS requires further study, although sev-
Y.pestis produces an endotoxic hexa-acylated lipid A that is a strong immunostimulant eral studies have shown that alkaline phosphatases inter-
in humans. Following transmission to the human host, the bacterium senses a shift in act with LPS invivo8183. Similarly to deacylation, removal
temperature (from 2127C to 37C) and synthesizes tetra-acylated lipid A, which of the lipid A phosphate groups results in reduced TLR4
escapes detection by TLR4, and immune stimulation is thereby curtailed70. c|V.cholerae stimulation and reduced LPS potency 50. Recently, a
normally inhabits freshwater, estuarine and oceanic environments and often colonizes
direct link between LPS and one of these phosphatases
marine organisms such as copepods, which are important for cholera transmission.
Copepod colonization provides an environment that probably induces lipid A
(intestinal alkaline phosphatase (Iap; also known as
modifications which are crucial for marine and host survival, a prospect that is currently Alpi1)) was identified in zebrafish. This particular phos-
under investigation. The V.cholerae O1 ElTor biotype modifies lipid A with a glycine or phatase probably cleaves phosphate groups from the
diglycine residue, providing resistance to CAMPs, whereas the classical V.cholerae O1 lipidA domain of LPS, and it controls the inflammatory
biotype remains susceptible75. response to gut microorganisms. Zebrafish with muta-
tions in iap are sensitive to LPS, whereas in wild-type
zebrafish, LPS induces TLR4dependent signalling that
upregulates the transcription of iap, effectively lowering
lumen, tolerance of commensals is partly attributed to LPS toxicity 83.
the limited expression of TLR4 on the apical surface of
epithelial cells, which means that TLR4 expression in Lipopolysaccharide scavenging. Many other host mol-
the subepithelial layers is important to restrict infec- ecules have been identified that associate with LPS
tion following damage to the outer layer of cells. On (usually at the lipid A domain) and assist in reducing
the other hand, Paneth cells in the small intestine have endotoxin concentration in the body and trafficking LPS
recently been shown to produce multiple antimicro- for disposal. These molecules have been shown to reduce
bial proteins in order to control inflammation and LPS toxicity by direct binding to LPS or by competing
infection caused by Gram-negative bacteria, and this with it for association with essential cofactors of TLR4
response is triggered directly in a TLR4dependent (CD14 and LBP)84. For example, pre-incubation of the
manner by the presence of bacteria and purified antimicrobial peptide LL37 with LPS results in reduced
LPS78. Paneth cells seem perfectly situated to respond cytokine induction in exposed THP1 cells compared
appropriately to commensal bacteria, producing an with exposure to LPS alone, suggesting that LL37 is an
antimicrobial moat that keeps bacterial numbers bal- LPS scavenger 85. The abundant plasma protein 2glyco
anced to avoid excessive inflammation but permit protein I (2GPI) forms complexes with LPS through
enough commensal colonization to impede invasion an LPS-binding domain that is well conserved across
by pathogens78. vertebrates86. Binding of 2GPI to LPS causes a confor-
mational change in 2GPI that leads to the association
Host detoxification of lipid A. When the innate immune of these complexes with monocytes and subsequent
response has been triggered by Gram-negative bacteria clearance of LPS87. LBP is important for binding LPS
that have successfully breached the mucosal and epi- and recruiting it to CD14 and TLR4 to induce immune
thelial barriers, LPS must be inactivated or removed signalling, but it has also been implicated in a mecha-
Wall teichoic acids from the bloodstream to reset the immune response nism of neutralizing LPS by transferring the endotoxin
Long anionic glycopolymers for subsequent LPS exposure. Various host response to high- and low-density lipoprotein and to chylomicrons,
that are covalently linked to mechanisms are involved in clearance of and toler- enhancing clearance of LPS following uptake by the
the peptidoglycan of ance to LPS from commensal and pathogenic bacteria. liver 88,89. The direct binding of host factors to lipidA,
Gram-positive bacteria and
extend beyond the cell wall.
Covalent modification of lipid A by host enzymes is as well as the effects of lipid A modifications on the
one mechanism to inactivate lipid A. The mamma- above mechanisms, have not yet been investigated, but
Wall lipoteichoic acids lian lipase acyloxyacyl hydrolase (AOAH) is produced further studies could reveal additional evasion strate-
Teichoic acids that are by macrophages, dendritic cells and neutrophils and gies that are used by Gram-negative bacteria to promote
anchored to the plasma
cleaves all secondary linked acyl chains of lipid A pathogenesis.
membrane of Gram-positive
bacteria and extend into the (that is, the Olinked acyl chains that are attached to
peptidoglycan layer. one of the four primary acyl chains linked directly to Effects on other cellular processes
the sugar backbone)79. This renders the LPS molecule Recent studies have revealed links between lipid A
Chylomicrons less inflammatory owing to impaired interactions with modifications and diverse processes within the bacterial
Small micelles that are
composed of lipids,
TLR4 (REFS50,64). Exposure of AOAH-deficient mice to cell. These associations support the notion that lipidA
lipoproteins and proteins, and LPS results in prolonged tolerance or unresponsiveness modifications are crucial for bacterial pathogenesis and
function to transport lipids. to LPS; this effect can last for months, thus limiting the survival.

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Multitarget lipid A modificationenzymes.InCampylo called Pseudomonas quinolone signal (PQS), which is


bacter jejuni, the enzyme EptC modifies both lipid A and packaged into vesicles and induces vesicle formation
a structural protein required for the assembly of flagella. in P.aeruginosa as well as other bacteria100. The exact
EptC is a homologue of E.coli EptA, and both enzymes mechanism of OMV formation is not understood; how-
transfer a phosphoethanolamine residue to the 1 and ever, PQS was found to interact with the 4phosphate
4positions of lipid A (TABLE1), masking both phosphate group and acyl chains of lipid A, indicating that modifi-
groups and promoting CAMP resistance. Interestingly, cation of lipid A could influence the efficiency of vesicle
in C.jejuni, EptC also transfers an essential phospho shedding 101.
ethanolamine to FlgG (a flagellar rod protein90), and The structure of lipid A and the Kdo residues of LPS
EptC mutants lack wild-type motility and produce fewer also affect the delivery of toxins. For example, two similar
flagella, which are required for virulence. This promiscuous toxins cholera toxin (CT), produced by V.cholerae,
C.jejuni enzyme also modifies carbohydrates of the LOS and heat-labile enterotoxin (LT), produced by entero-
core and glycosylated proteins91. toxigenic E.coli (ETEC) are both secreted, are struc-
turally similar and bind the same host cell receptor.
Recycling of biosynthetic intermediates. During the assem- However, CT causes a more severe diarrhoeal disease
bly of bacterial polymers (for example, peptidoglycan, the than LT102. Although the topic is controversial103, the dif-
capsule and LPS), a wide array of glycoconjugates must ference in toxin severity seems to be due to the partial
be trafficked across the inner membrane92. These glycan sequestration of LT, as it binds to Kdolipid A on the
intermediates are assembled on a universal carrier lipid bacterial cell surface after secretion. By contrast, the Kdo
known as undecaprenyl phosphate (C55-P)93. The C55-P sugar attached to lipid A in V.cholerae is phosphorylated
transports the glycan precursors via a pyrophosphate by Kdo kinase (KdkA) (TABLE1), and this modifica-
linkage (C55-PPprecursor), and when the precursor is tion inhibits CT association with the outer membrane,
removed, the carrier lipid is released as a pyrophosphate thereby allowing robust toxin secretion104.
and requires dephosphorylation in order to be recycled93.
Within the periplasm, LpxT transfers a secondary phos- Conclusions and future prospects
phate group onto lipid A at the 1 position (FIG.1c) and The wide variety of lipid A modifications equips Gram-
uses C55-PP as a donor 94. This finding represented a negative bacteria for survival in their respective niches
novel and unexpected link between the modification in the host and other environments. Other functions of
of lipidA and the recycling of C55-PP. Furthermore, the lipid A are highlighted by the interplay of lipid A modi-
connection between these systems implies that C55-PP fications with a diverse set of cellular processes, and both
could serve as a high-energy phosphate donor for various of these facets of lipid A biology emphasize the impor-
periplasmic components, although these potential targets tance of a continued effort to understand the regulation,
are largely unidentified. benefits and host response to lipid A in its numerous
modifiedforms.
Activation of outer-membrane proteins. As a membrane However, many questions remain unanswered.
component, LPS is more than just a platform for activi- The continued development of animal models is para-
ties at the cell surface95. It also has a role in the activa- mount to more closely mimic the host infection site.
tion of omptins, a class of proteases that is widespread Considerable progress has been made in this regard by
among Gram-negative enteric bacteria. The activity of the recent generation of a transgenic mouse expressing the
one member of this class, OmpT, has been evaluated in human TLR4 receptor 105. Furthermore, there is much
the presence of hexa-acylated and tri-acylated lipidA to learn about how lipid A and its modifications
forms, and the protease was found to be active only in affect the outcome of polymicrobial infections, with
the presence of hexa-acylated lipid A96. Interestingly, recent studies suggesting that LPS has an important
another omptin named plasminogen activator (Pla), role within such communities. For instance, purified
which is central to Y.pestis pathogenesis, is more active LPS from intestinal commensal bacteria has been
in Y.pestis grown at 37C and producing tetra-acylated found to promote replication and transmission of
Flagellar rod lipid A than in cells grown at 20C and producing viruses106,107. Pathogenesis of orally acquired polio
The central, structural hexa-acylated lipidA97. virus, which replicates in the intestine before spread-
component of bacterial flagella ing to cause a severe systemic infection, is supported
that spans the periplasm.
Outer-membrane vesicles and toxin delivery. Lipid A by the intestinal microbiota and purified LPS107. Mouse
Flagella modifications also affect the delivery of proteins and mammary tumour virus (MMTV) manipulates the
Whip- or tail-like appendages toxins that are associated with the outer membrane innate immune response by binding LPS and eliciting
that are synthesized by many and outer-membrane vesicles (OMVs). In organisms such TLR4dependent production of IL10, a cytokine that
bacteria and are important for
as Porphyromonas gingivalis and P.aeruginosa, negatively is required for MMTV persistence106. Although these
motility.
charged LPS is enriched in OMVs98,99. Furthermore, viruses bind LPS and this enhances infectivity invitro,
Outer-membrane vesicles in P.gingivalis, the lipid A found in OMVs is more how lipid A modifications affect these phenomena is
Small, spherical outer- deacylated than that present in whole-cell membranes, currently unknown.
membrane blebs that are suggesting that lipid A modification facilitates vesicle Improved methods for studying lipid A modifica-
released from Gram-negative
bacterial cells and contain
formation or the sorting of outer-membrane proteins tions invivo could also help elucidate any potential
membrane and periplasmic that are packaged into vesicles98. In P.aeruginosa, OMV links between modified lipid A and diseases involving
components. formation is stimulated by a small signalling molecule TLR4, such as inflammatory bowel disease, diabetes,

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REVIEWS

rheumatoid arthritis and atherosclerosis108. Important enzymes are distributed and organized within the
insights into human TLR4 polymorphisms might now membrane is also unclear. For example, it is not known
be possible using the humanized mouse model105, and whether lipid rafts exist in bacteria and whether some
it should also be possible to examine how different or all of the modification enzymes are enriched at these
variants of TLR4 affect susceptibility to infection by sites. Lipid A molecules with certain modifications
organisms with modified lipid A. This could potentially could also be specifically localized in the membrane.
lead to more personalized medical treatment through Such an organization could have a major influence on
the development of a new range of modified TLR4 the activation of key outer-membrane proteins or on
agonists that could serve as customized immunomodu- OMV formation and protein sorting.
latory agents to induce or repress particular cytokine With further study, inhibitors of modification
responses. enzymes could be developed for use as antibiotics or in
It remains a mystery why lipid A is essential in vir- combination with currently used antibiotics. Although
tually all Gram-negative organisms. Furthermore, why immense progress has been made in our understand-
lipid A biosynthesis is well conserved only to have com- ing of lipid A modification systems in Gram-negative
plex (and varying) modifications occur later, sometimes bacteria and their importance for pathogenesis, there is
constitutively, is not understood. How the modification much work yet to bedone.

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Mol. Microbiol. 55, 425440 (2005). hydrolase in the inner membrane of Francisella M. Stephen Trents homepage:
125. Trent,M.S., Stead,C.M., Tran,A.X. & Hankins,J.V. novicida. Mol. Microbiol. 78, 820836 (2010). http://www.icmb.utexas.edu/directory/details.asp?id=3806
Diversity of endotoxin and its impact on pathogenesis. 140. Chung,H.S. & Raetz,C.R. Dioxygenases in ALL LINKS ARE ACTIVE IN THE ONLINE PDF
J.Endotoxin Res. 12, 205223 (2006). Burkholderia ambifaria and Yersinia pestis that

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