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microLife, 2022, 3,1–13
DOI: 10.1093/femsml/uqac011
Advance access publication date: 10 June 2022
Short Review
Lipid A heterogeneity and its role in the host
interactions with pathogenic and commensal bacteria
Sukumar Saha 1,2,*,ElderPupo 1, Afshin Zariri1, Peter van der Ley1
1Institute for Translational Vaccinology (Intravacc), Antonie van Leeuwenhoeklaan 9, 3721 MA Bilthoven, the Netherlands
2Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
∗Corresponding author: Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh. Tel: +880-1740847339;
E-mail: sukumar.saha@bau.edu.bd
Editor: Martin Loessner
Abstract
Lipopolysaccharide (LPS) is for most but not all Gram-negative bacteria an essential component of the outer leaet of the outer
membrane. LPS contributes to the integrity of the outer membrane, which acts as an effective permeability barrier to antimicrobial
agents and protects against complement-mediated lysis. In commensal and pathogenic bacteria LPS interacts with pattern recognition
receptors (e.g LBP, CD14, TLRs) of the innate immune system and thereby plays an important role in determining the immuneresponse
of the host. LPS molecules consist of a membrane-anchoring lipid A moiety and the surface-exposed core oligosaccharide and O-
antigen polysaccharide. While the basic lipid A structure is conserved among different bacterial species, there is still a huge variation
in its details, such as the number, position and chain length of the fatty acids and the decoration of the glucosamine disaccharide
with phosphate, phosphoethanolamine or amino sugars. New evidence has emerged over the last few decades on how this lipid A
heterogeneity confers distinct benets to some bacteria because it allows them to modulate host responses in response to changing
host environmental factors. Here we give an overview of what is known about the functional consequences of this lipid A structural
heterogeneity. In addition, we also summarize new approaches for lipid A extraction, purication and analysis which have enabled
analysis of its heterogeneity.
Keywords: lipid A, heterogeneity, outer membrane, barrier function, immune response, bacteria
Introduction
Lipopolysaccharide (LPS) is generally considered to be an essen-
tial component of the outer membrane of Gram-negative bacte-
ria and serves as a physical barrier, protecting the bacteria from
its surroundings. However, our understanding of the importance
of LPS in Gram-negative bacteria has changed considerably by
the discovery of mutant strains of Neisseria meningitidis,Moraxella
catarrhalis,andAcinetobacter baumannii which completely lack LPS.
This indicates that the essentiality of LPS in Gram-negative bac-
teria is more complex and varies considerably depending on the
species and strain background. While LPS may not be essential in
some organisms, several studies indicated that strains lacking LPS
are less virulent and more susceptible to antibiotics.The basic LPS
structure, including the membrane-anchoring lipid A moiety dif-
fers considerably among different bacterial species,and these dif-
ferences inuence not only the outer membrane barrier function
but also the immunological properties of LPS (see Fig. 1). While
hexa-acylated lipid A has the ability to induce the strongest proin-
ammatory reaction after binding with the TLR4/MD-2 receptor,
distinct lipid A structures of different bacteria can vary in their
immunogenic potential and TLR4-mediated signaling capacity.
Recent studies have begun to explore LPS alterations during in
vivo growth instead of lab-grown bacteria, which has been made
possible by advances in mass spectrometry techniques used for
analysis of complicated samples containing only small amounts
of LPS. In this way, much presently hidden structural variation
among strains has become apparent. It is not generally appreci-
ated (i) how heterogeneous the TLR4/MD-2 activating lipid A part
of LPS is, both within and between bacterial species, and (ii) how
this can profoundly affect virulence of pathogenic bacteria, or in-
teractions of commensal organisms with the immune system of
their host. In this review, we want to discuss lipid A heterogene-
ity in the context of interactions of pathogenic and commensal
bacteria with their hosts.
LPS deciency
For most Gram-negative bacteria LPS is an essential component
of the outer membrane. Studies with Escherichia coli which worked
out the lipid A biosynthesis pathway showed that only conditional
mutations are tolerated in the rst steps and complete knockouts
are not viable (Karow and Georgopoulos 1993). However, we re-
ported 15 years ago how Neisseria meningitidis can survive without
LPS when the lpxA gene is inactivated, which is required for the
rst step in the LPS biosynthesis pathway (Steeghs et al.1998).
Since that time, LPS deciency has also been reported for some
other bacterial species, i.e. Moraxella catarrhalis (lpxA),Yersinia ruck-
eri (lpxD) and Acinetobacter baumannii (lpxA, C.D) (Pengetal.2005,
Henry et al. 2012, Altinok et al.2016 ). Interestingly, in the case
of A.baumannii these LPS-decient mutants were rst isolated not
by directed inactivation of lpx genes but by selection for resis-
tance against colistin (Moffatt et al.2010). Subsequently, they were
also recovered as patient isolates after colistin treatment, show-
Received: March 18, 2022. Revised: May 17, 2022. Accepted: June 7, 2022
C
The Author(s) 2022. Published by Oxford University Press on behalf of FEMS. This is an Open Access article distributed under the terms of the Creative
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2|microLife, 2022, Vol. 3
Figure 1. (A) The basic structure of lipid A consists of two glucosamine units, in an β(1→6) linkage, with attached acyl chains and normally containing
one phosphate group on each glucosamine. The E. coli lipid A structure contains 6 acyl chains. Primary acyl chains are directly attached to the sugar
moieties and usually between 10 and 16 carbons in length, secondary acyl chains are esteried with the beta-hydroxyl groups of primary acyl chains.
E. coli lipid A, as an example, typically has four C14 hydroxy acyl chains attached to the sugars and one C14 and one C12 attached to the beta-hydroxy
groups. Lipid A is considered the most conserved domain of Gram-negative bacterial LPS but it still shows a great degree of diversity among bacterial
species. Differences are found in the number and modications of the phosphate residues, the number and length of the acyl chains and, though less
common, the chemistry of the disaccharide backbone. In E.coli, altering the phosphates, number and position of acyl chains of lipid A individually or in
combination can give a wide range of TLR4/MD-2 responses and cytokine production. Modication of lipid A can also provide resistance against
antimicrobial peptides by charge repulsion or decreasing of the uidity of the outer membrane, as well as alter the activation potential of the
inammasome. The presence and length of the secondary acyl chains (R1, R2, and R3) varies between bacteria and is linked to the ability of the lipid A
species to induce innate immune function. (B) Some examples showing the variation in lipid A structures from different bacteria. For N. meningitidis,
both wildtype and lpxL1 mutant structures are shown; for S.typhimurium, hexa- and hepta-acylated forms are shown.
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Saha et al. |3
ing that loss of LPS can have a selective advantage under specic
in vivo conditions (Cai et al.2012, Agodi et al.2014, Mavroidi et al.
2015). Also with N.meningitidis, an isolate from both blood and CSF
of a meningitis patient has been found that lacked LPS,in this case
due to a missense mutation in lpxH (Piet et al.2014). The specic
selective forces that led to its proliferation were unknown, but an-
tibiotic selection was not involved in this case. Possibly, evasion of
innate immunity mediated by TLR4 activation or specic immu-
nity mediated by anti-LPS antibodies led to the outgrowth of this
specic mutant strain.
A crucial question is why LPS is a crucial building block for the
outer membrane of some bacterial species while for others it is ap-
parently nonessential. Several studies have begun to address this
point. Differences in cell envelope stress response systems have
been suggested, or misassembly of major outer membrane pro-
teins. In some cases lipid A precursors may accumulate, and dif-
ferent bacteria may differ in their ability to tolerate this. An lptD
mutant of A.baumannii has a disruption in the LPS export path-
way and accumulates the precursor lipid IVa causing a growth
defect, while this is apparently not the case for an E.coli strain en-
gineered to express only this structure (Bojkovic et al.2016). In
the case of lpxH inactivation, a diacylated glucosamine interme-
diate may still be formed which is toxic for A.baumannii while this
is apparently not the case for N.meningitidis (Piet et al.2014). Even
closely related species such as N.meningitidis and N.gonorrhoeae dif-
fer in their tolerance for loss of LPS,as a lipid A biosynthesis block
could not be introduced in the latter despite several attempts by
us and others. For A.baumannii, comparative transcriptional pro-
ling has been done on strains without LPS, showing increased ex-
pression of lipoprotein and phospholipid transport genes (Henry
et al.2012). Further, screening of multiple strains showed that
only some could tolerate loss of LPS, and this was pinned down to
the absence of penicillin-binding protein 1A which is involved in
peptidoglycan biosynthesis (Boll et al.2016). Also, specic lipopro-
teins were overexpressed as potential compensatory mechanism.
Clarifying the molecular basis of such species-specic compen-
satory mechanisms is important for understanding basic aspects
of outer membrane biogenesis, but also has practical importance
as lipid A biosynthesis provides a promising target for new antibi-
otics. It is therefore crucial to know how widespread the ability of
bacteria is to survive without LPS.
Bacteria specically engineered for LPS deciency can have im-
portant biotechnological uses, as they lack a major activator of in-
nate immunity the presence of which is undesirable for many ap-
plications. In particular,LPS-decient strains can be used for mak-
ing less reactogenic inactivated whole-cell vaccines or live atten-
uated vaccines. However, complete loss of LPS may be disadvanta-
geous as compared to just attenuated LPS by mutations leading to
incomplete acylation, as the latter strains can be more robust and
still retain some adjuvant activity. Indeed, for N.meningitidis bac-
terial cells without LPS had strongly reduced immunogenicity, but
this was apparently not the case for M.catarrhalis and A.baumannii
(Garcia-Quintanilla et al.2014). This may be related to species dif-
ferences in compensatory mechanisms, as we found loss of outer
membrane lipoproteins in LPS-decient N.meningitidis, while in
A.baumannii their expression was reported to be increased instead
(Steeghs et al.2001, Henry et al.2012). As bacterial lipoproteins are
strong activators of TLR2, their increased or decreased expression
can also impact on overall innate immune activation.Other appli-
cations comprise the purication of proteins, polysaccharides or
other biomolecules without any endotoxin contamination. While
the isolation of completely LPS-decient E.coli has not been possi-
ble, an alternative is the use of strains engineered to make the
minimal structure compatible with viability, i.e. tetra-acylated
lipid A without KDO residues which lacks any detectable TLR4-
activating capacity (Golenbock et al.1991). However, it should be
remembered that TLR4 activation is highly species-specic, and
partial lipid A structures may still have agonistic activity in some
mammalian species. When comparing human, mouse, pig and
rabbit receptor activation by a panel of mutant LPS structures,
we found major species differences, with human TLR4/MD-2 the
most and rabbit TLR4/MD-2 the least discriminatory.
Mechanisms generating lipid A heterogeneity
Lipid A modications can affect many physiological processes
of bacteria, including structural integrity and permeability of
the outer membrane, susceptibility to antimicrobial peptides, im-
mune stimulation, formation of outer membrane vesicles and
pathogenesis. There are basically two mechanisms for generating
intrastrain lipid A heterogeneity: (i) regulation of gene expression
and enzyme activity, and (ii) mutations in genes responsible for
lipid A biosynthesis. LPS modications due to regulation of gene
expression and enzyme activity have been reviewed recently by
Simpson and Trent (Simpson and Trent 2019). Neisseria meningi-
tidis and Neisseria gonorrhoeae have been studied extensively for
phenotypic variation of LPS.These bacteria can change their sur-
face structures in response to their surroundings including the
host defense system, and the resulting antigenic variability con-
tributes to adaptation to their tissue microenvironment, distribu-
tion in the host and virulence. Clinical investigation has revealed a
large repertoire of different LPS structures among meningococcal
and gonococcal isolates, and mixed populations of organisms are
constantly generated due to the on-off switching of LPS biosyn-
thesis genes (Fransen et al.2010, Ladhani et al.2012, Rodenburg
et al. 2012, du Plessis et al.2014, Persa et al.2014,Pietetal.2014,
Fazio et al. 2015). Exchange of genetic material by transformation
and recombination also occurs frequently in N. meningitidis (Taha
et al.2002), further expanding the LPS gene repertoire.
Naturally occurring heterogeneity is also found in the lipid A
produced by N. meningitidis which leads to alteration of the acyla-
tion pattern as well as modulation of endotoxic activity (van der
Ley et al. 2001, Fransen et al. 2010, Brouwer et al.2011, Ladhani
et al.2012, Rodenburg et al.2012, du Plessis et al.2014, Persa et
al.2014, Fazio et al.2015). Pentaacylated and tetraacylated LPS
result from inactivation of the genes lpxL1 and lpxL2, respectively,
and both mutants activate human TLR4 much less efciently than
wild type bacteria (van der Ley et al.2001, Fransen et al.2010).
Meningococcal strains harbouring lpxL1 mutations occur natu-
rally, are more likely to cause a milder and more protracted dis-
ease in older children and young adults, and are often associated
with a particular genetic lineage (cc23). Such mutants may have
a selective advantage over wild type strains in their survival and
spreading due to their less efcient detection by the innate im-
mune system (Ladhani et al.2012, du Plessis et al.2014, Fazio et
al. 2015). Chronic meningococcemia is also associated with lpxL1
mutations (Brouwer et al.2011, Persa et al.2014). So structural
heterogeneity in Neisserial lipid A exists and this heterogeneity
correlates with the bioactivity of LPS.
Lipid A structure in vivo: new analytical
techniques
The structure of LPS from ex vivo samples collected from animal
or human bacterial infections is usually determined after exten-
sive cultivation of bacterial specimens in vitro. The basis for this
is that standard methods of structural analysis,including nuclear
magnetic resonance spectroscopy and mass spectrometry (MS),
commonly require much larger quantities (e.g., milligrams) of LPS
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4|microLife, 2022, Vol. 3
than those present in samples recovered from bacterial infec-
tions. Given that bacteria can modify LPS structure in response to
changes in their growth environment, this has the disadvantage
that the structure of LPS obtained after bacterial multiplication in
vitro may not fully reproduce that originally present in vivo.Con-
sequently, important effects of the in vivo biological environment
on LPS structure and their role in bacterial pathogenesis may be
neglected.
However, the analysis of LPS directly from ex vivo extracts is dif-
cult because these samples may typically contain low numbers
of bacteria (e.g., down to 105CFU/ml in signicant lower respi-
ratory tract and urinary infections (Khasriya et al.2013,Gadsby
et al.2015) of correspondingly low LPS content (e.g., in the order
of 102–103pg/ml) along with abundant host-derived material. To
approach this problem, micro-scale methods for extraction of low
levels of LPS together with mass spectrometry techniques of in-
creased sensitivity are being developed. In this context, two main
research strategies have been adopted comprising either direct
analysis of biological samples without in vitro culture or analy-
sis of small-scale cultures of bacterial isolates with minimal pas-
sage. The rationale for the latter strategy is that by keeping bacte-
rial passage in vitro at a minimum a less inaccurate picture of LPS
structure in vivo can be obtained. For instance, it has been shown
that disease-specic modication of the lipid A (e.g., addition of
palmitate to the lipid A of Pseudomonas aeruginosa in clinical cystic
brosis isolates) detected by MS analysis of the lipid A extracted
from minimally passaged bacteria can be lost after multiple bac-
terial passages in vitro (Ernst et al. 1999).Furthermore, small-scale
bacterial culture provides a means to amplify and isolate bacte-
ria from the biological matrix making samples less complex and
technically less challenging than the original biological extracts.
The structural analysis of LPS/lipid A from small-scale in vitro cul-
tures of clinical isolates, as small as a few bacterial colonies, has
been enabled by the introduction of sensitive MS-based micro-
methods including, among others, microwave-assisted enzymatic
lysis coupled to capillary electrophoresis (CE)-electrospray ioniza-
tion (ESI)-MS (Dzieciatkowska et al.2008), microwave-assisted en-
zymatic digestion and detergent-free mild hydrolysis in combi-
nation with matrix-assisted laser desorption ionization (MALDI)
time-of-ight (TOF) MS (Zhou et al. 2009) and ammonium hydrox-
ide/isobutyric acid micro-extraction followed by MALDI-TOF MS
(Hamidi et al.). Furthermore, it has been demonstrated that lipid
A mass spectra can also be obtained by directly analyzing intact
bacteria by MALDI-TOF MS in an optimized matrix solvent and
this method requires only as low as 104-105heat-inactivated bac-
teria for lipid A MS analyses (Larrouy-Maumus et al.2016).
Recent studies have also started to explore lipid A alterations
during in vivo growth by directly characterizing biological sam-
ples without in vitro bacterial multiplication. For example, lipid A
micro-extraction by an ammonium hydroxide/isobutyric acid (El
Hamidi et al.2005) or TRI Reagent based method together with
MALDI-TOF MS have been used to probe lipid A structure directly
from organ tissues of mice infected with the human pathogen
Klebsiella pneumoniae (Llobet et al.2015). Lipid A mass spectra ob-
tained from lung (∼106CFU/gram of tissue) and spleen samples
showed that K. pneumoniae is able to alter its lipid A in vivo and
that this occurs in a tissue-dependent manner. Lipid A from lung
isolates were found to contain a 2-hydroxyacyl modication pro-
duced by the PhoPQ-regulated oxygenase LpxO, which was not
present in the lipid A recovered from spleen tissues. Remarkably,
minimal passage of bacteria from lung isolates in vitro led to the
loss of 2-hydroxyacyl modication of lipid A, reinforcing that envi-
ronmental conditions encountered by bacteria in vivo which affect
lipid A structure are not always replicated in vitro. Lipid A modi-
cation by the LpxO enzyme in vivo was found to facilitate innate
immune evasion by K. pneumoniae through decreasing the activa-
tion of inammatory responses and promoting resistance of bac-
teria to antimicrobial peptides (Llobet et al.2015).
A notable improvement in the sensitivity of lipid A structural
analysis has been achieved recently by the introduction of norhar-
mane (9H-pyrido[3,4-b]indole) as matrix for lipid A analysis by
MALDI-MS (Scott et al.2016). Replacement of the standard 2,5-
dihydroxybenzoic acid matrix with norharmane resulted in 10-
fold enhancement in the limit of detection of lipid A by MALDI-
MS enabling lipid A MS detection at the picogram level, which
approximates lipid A levels in clinical samples (Scott et al.2016).
Lipid A micro-extraction by ammonium hydroxide/isobutyric acid
(Hamidi et al.) in combination with Norharmane-based MALDI-
MS have been applied to the analysis of lipid A structure directly
from tissues extracted from mice (host) and ticks (vector) ex-
perimentally infected with Francisella novicida. These bacteria are
known to regulate lipid A structure in vitro depending on growth
temperature through the preferential addition of either a 3-OH
C18 acyl group at 37◦C or a shorter 3-OH C16 acyl group at 18◦C–
25◦C to lipid A by variants 1 and 2 of the lipid A-modifying N-
acyltransferase enzyme LpxD (Li et al.2012) respectively. Consis-
tent with these in vitro observations, MS analysis of lipid A directly
extracted from in vivo infected mouse spleens (∼106–107CFU per
spleen) and whole ticks (107CFU per tick) revealed that F. novicida
remodels its lipid A in vivo and incorporates a higher proportion
of a long 3-OH C18 acyl group in its lipid A when growing in the
higher body temperature of the mammalian host compared to the
tick vector (Scott et al.2016). Previously, incorporation of a long 3-
OH C18 acyl group in the lipid A by the enzyme LpxD1 has been
linked to increased antibiotic resistance and full expression of F.
novicida virulence in mice (Li et al.2012).
Lipid A structural heterogeneity and outer
membrane barrier function
Structures of lipid A vary widely among different bacterial species
(see Table 1). Sometimes a single bacterial species may contain
more than one lipid A structure (Raetz and Whiteld 2002). In
addition, specic environmental conditions can have a strong in-
uence on the lipid A composition. Under standard growth con-
ditions lipid A of E. coli is the biphosphorylated backbone disac-
charide β-d-GlcpN4P-(1→6)-α-d-GlcpN1P, which is hexaacylated
without any modications. However, exposure to unfavourable
environmental conditions including temperature, pH, excess of
metal ions, presence of antimicrobial peptides, or chelating agents
like EDTA can lead to profound changes in the lipid A composi-
tion (Raetz et al.2007, Klein et al.2009, Klein et al.2013). It in-
volves change in the number or composition of acyl chains,phos-
phate groups, or functional groups which are attached covalently
(Raetz et al.2007, Needham et al.2013). The effect of temperature
is clearly demonstrated in the case of Y. pestis:whenculturedat
21◦C–28◦C it expresses hexaacylated lipid A structures but when
the bacteria are grown at 37◦C it becomes tetraacylated (Kawa-
hara et al.2002, Rebeil et al.2004, Knirel et al.2005). In the case
of E.coli, growth at lower temperature leads to incorporation of a
longer mono-unsaturated secondary acyl chain by LpxP (Carty et
al.1999).
Salmonella spp overcome the action of cationic antimicrobial
peptides (CAMPs) by several modications of their lipid A, includ-
ing the addition of an additional palmitoyl chain (16:0), a 2-OH
group to one the secondary chains and Ara4N to the phosphates
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Saha et al. |5
Tab l e 1 . Lipopolysaccharides of Pathogenic and Commensal Bacteria
Lipopolysaccharides of Pathogenic Bacteria
Bacterial species
Intra or
extracellularaLipid A structure
TLR4 activation
potentialbReferences
Escherichia coli Intra- and
extracellular
Hexa-acylated Strong Beutler and Rietschel 2003, Raetz and
Whiteld 2002, Zähringer et al. 1994
Salmonella
typhimurium
Facultat ive
intracellular
Hexa and
Hepta-acylated
Guo et al. 1997, Guo et al. 1998
Salmonella minnesota Facultat ive
intracellular
Hexa-acylated, or
hepta-acylated
Janusch et al. 2002, Qureshi et al. 1985
Neisseria meningitidis Facultat ive
intracellular
Hexa- or penta-
acylated
Hexa-acylated
strains more
strongly activate
TLR4 than the
penta-acylated ones
Fransen et al. 2009, Kulshin et al. 1992
Neisseria gonorrhoeae Intra- and
extracellular
Hexa-acylated Post et al.2002
Klebsiella pneumoniae Mostly extracellular
but can survive
within the
macrophages
Hexa-and
hepta-acylated
Hepta-acylated form
weakly activates
TLR4
Kamaladevi and Balamurugan 2016, Silipo
et al.2002
Yersinia spp Facultat ive
intracellular
Tetra-acylated
(37◦C) and
hexa-acylated (26◦C)
Tetra-acylated form
weakly activates
TLR4
Kawahara et al.2002,Knireletal.2005,
Montminy et al. 2006, Rebeil et al. 2004,
Reinés et al. 2012
Proteus mirabilis Extracellular Hexa- or hepta-
acylated
McCoy Andrea et al. 2001, Zabłotni et al.
2018
Helicobacter pylori Facultat ive
intracellular
Tetra-acylated and
hexa-acylated
Hexa-acylated form
strongly activates
TLR4
Needham et al. 2013, Ogawa et al.2003,
Raetz et al. 2007
Campylovacter jejuni Facultat ive
intracellular
Hexa-acylated Weak Korneev et al. 2018
Moraxella sp Extracellular Hepta-acylated Masoud et al. 2011
Desulfovibrio
desulfuricans
Intra and
extracellular
Hexa- and
hepta-acylated
Wolny et al. 2011
Vibrio cholerae O1 Facultative
intracellular
Hexa-acylated Chatterjee and Chaudhuri 2003
Vibrio cholerae O139 Extracellular Octa-acylated Chatterjee and Chaudhuri 2003
Prevotella denticola Extracellular Penta-acylated Weak Larsen et al.2015
Prevotella intermedia Extracellular, some
strains are
intracellular
Penta-acylated Weak Hashimoto et al. 2003, Larsen et al. 2015
Porphyromonas
gingivalis
Intra- and
extracellular
Tetra- and penta
acylated
Penta-acylated form
strongly activates
TLR4
Colombo et al. 2007,Curtisetal.2011,
Kumada et al. 1995, Lee et al. 2018, Reife et
al. 2006
Francisella spp Facultati ve
intracellular
Tetra-acylated Weak Phillips et al. 2004, Que-Gewirth et al. 2004
Leptospira interrogans Intra and
extracellular
Hexa-acylated Boon Hinckley et al. 2005
Legionella
pneumophila
Facultat ive
intracellular
Hexa-acylated Weak Shevchuk et al. 2011
Bordetella pertussis Intra and
extracellular
Penta- acylated Weak El Hamidi et al. 2009
Bordetella
bronchiseptica
Intra- and
extracellular
Hexa-cylated Strong Marr et al. 2010
Chlamydia
trachomatis
Intracellular Penta-acylated Weak Yang et al.2019
Coxiella burnettii Intracellular Tetra-acylated Weak Toman et al. 2004
Capnocytophaga
canimorsus
Extracellular Penta-acylated Weak Ittig et al. 2012
Brucella spp Intracellular Hepta-acylated Weak Barquero-Calvo et al. 2007
Burkholderia mallei Facult ative
intracellular
Tet r a an d
Penta-acylated
Wea k Kor nee v et al . 2015
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6|microLife, 2022, Vol. 3
Tab l e 1 . Continued
Lipopolysaccharides of Pathogenic Bacteria
Bacterial species
Intra or
extracellularaLipid A structure
TLR4 activation
potentialbReferences
Burkholderia
cenocepacia
Facultat ive
intracellular
Penta-acylated Strong Korneev et al. 2015
Burkholderia dolosa Facultat ive
intracellular
Tetra-acylated Strong Lorenzo et al. 2013
Acinetobacter
baumannii
Facultat ive
intracellular
Hepta-acylated Strong Korneev et al.2015
Pseudomonas
aeruginosa
Facultat ive
intracellular
Tri- tetra- penta-
and Hexa-acylated
(depending on
environmental
conditions)
Weak Ernst et al.1999, Korneev et al.2015
Haemophilus
inuenzae
Facultat ive
intracellular
Hexa-acylated Weak White et al.1999
Shigella exneri Facultat ive
intracellular
Hexa-acylated d’Hauteville et al. 2002
Shigella sonnei Facult ative
intracellular
Hexa-acylated Bath et al. 1987
Serratia marcescens Facultat ive
intracellular
penta-acylated Strong Makimura et al. 2007
Stenotrophomonas sp Extracellular Hexa-acylated Naito et al. 2017
Aeromonas sp. Facult ative
intracellular
Hexa-acylated El-Aneed and Banoub 2005
Lipopolysaccharides of Commensal Bacteria
Bacterial species Intra or
extracellulara
Lipid A structure TLR4 activation
potentialb
References
Bacteroides spp Extracellular Penta- and
tetra-acylated
Weak d’Hennezel et al. 2017
Bacteroides fragilis Extracellular Penta-acylated Weak Berezow et al. 2009, Weintraub et al. 1989
Bacteroides dorei Extracellular Penta-acylated Weak Vatanen et al. 2016
Bacteroides
thetaiotaomicron
Extracellular Penta-acylated Weak Berezow et al. 2009
Acinetobacter spp Extracellular but
can survive inside
vacuoles
Hexa- and hepta-
acylated
Arroyo et al.2011, Beceiro et al. 2011,Boll
Joseph et al. 2015
Stenotrophomonas spp Extracellular Hexa-acylated Naito et al. 2017
Veillonella parvula Extracellular Not identied yet Weak Matera et al.1991, Matera et al. 2009
Delftia spp Extracellular Hexa-acylated Naito et al.2017
Prevotella
melaninogenica
Extracellular Penta-acylated Weak Council 2013
Providencia rettgeri Extracellular Hexa-acylated Munford 2008
Klebsiella oxytoca Extracellular Hexa-acylated Silipo et al. 2002
Burkholderia cepacia Facultat ive
intracellular
Penta-acylated Silipo et al.2005
Burkholderia
pseudomallei
Intracellular Penta-acylated Norris et al.2017
aIntracellular bacteria: Bacteria, which have the capability to enter and survive in the cells of the host organism. Extracellular bacteria: Bacteria, which can survive
and multiply outside the host cell. Facultative intracellular: They are basically extracellular but they can survive within the cells.
bTLR4 activation potential is given relative to that of hexa-acylated and bis-phosphorylated LPS of E. coli.
(Guo et al.1997). In a similar way, Klebsiella pneumoniae resists the
host innate immune system by addition of Ara4N to the lipid
A phosphates and by switching from a potently agonistic hexa-
acylated to a weak hepta-acylated lipid A (Kamaladevi and Bal-
amurugan 2016). Some gastrointestinal pathogens, such as Heli-
cobacter pylori and Campylobacter jejuni have a different pattern of
lipid A components. Lipid A of most clinical isolates of H. pylori
lacks 4-phosphate while an ethanolamine phosphate is attached
to the reducing GlcN of the lipid A, which is only tetra-acylated
with longer chains of 16-18 carbon atoms. Much more diversity
is observed in the lipid A of C. jejuni where the disaccharide back-
bone of the most common forms has both glucosamine (GlcN) and
diacylglycerol (DAG). The phosphate groups are substituted with
bis-phosphorylated ethanolamine or with 3 or 4 additional phos-
phate units and some strains express from tetra-to hexa-acylated
lipid A forms (Moran et al.1997, Stephenson et al. 2013,Korneev
et al. 2018). Extreme structural diversity is reported among differ-
ent strains of Vibrio cholera biotype E1 Tor which expresses hexa-
acylated lipid A modied with either glycine or diglycine units
attached at the 3-position (C12:0) with a 3-OH groups (Hank-
ins et al.2011, Henderson et al. 2017). Glycine or diglycine mod-
ication decrease the negative charge of the bacterial surface
which provides resistance to cationic antimicrobial peptides such
as polymyxin. On the other hand, The pandemic V.cholerae bio-
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Saha et al. |7
type Classical strain is devoid of these glycine/diglycine modi-
cations and is polymyxin-sensitive (Henderson et al.2017). An in-
teresting form of lipid A micro-diversity among strains of the hu-
man gut bacterium Desulfovibrio desulfuricans has been reported
by (Zhang-Sun et al.2015). Desulfovibrio desulfuricans shares the
di-phospho-di-glucosamine lipid A with Enterobacteriaceae, with
also the same fatty acid distribution. However, some strains of D.
desulfuricans isolated from the same host have a different lipid A
structure with different pro-inammatory properties.
High levels of lipid A heterogeneity are also present in the gen-
era of the Bacteroidetes phylum, consisting of Bacteroides,Alistipes,
Parabacteroides,andPrevotella (Wexler and Goodman 2017). Bac-
teroides LPS expresses a different lipid A structure compared to
the prototypical Proteobacterial lipid A, since different Bacteroides
species only possess penta- and tetra acylated species, contain-
ing branched fatty acids (15–17 carbon atoms in length), and the
sugar backbone is substituted with only one phosphate group
(Weintraub et al.1989, Berezow et al. 2009, Vatanen et al.2016).
This lipid A modication contributes to reduced activation of the
TLR4/MD-2 LPS receptor (Rietschel et al.1998, Phillips et al.2004,
Que-Gewirth et al. 2004, Kanistanon et al.2008, Munford 2008,
Coats et al.2009, Cullen and Trent 2010, Coats et al.2011, Cullen
et al.2011). Broad conservation of the LpxF enzyme across com-
mensal Bacteroidetes is responsible for removing a single lipid
A phosphate residue which provides resistance to antimicrobial
peptides and increases bacterial resilience in the intestine (Cullen
et al.2015).
Reports are not available on the LPS from gut Prevotella species
but the lipid A and R-LPS structures have been characterized from
two Prevotella species (P. denticola and P. intermedia) found in the
human oral cavity (Hashimoto et al.2003, Di Lorenzo et al.2016).
Their lipid A moieties are penta-acylated and decorated by phos-
phoethanolamine, and have low inammatory capacity similar to
Bacteroides (Larsen et al.2015). Tetra- and penta acylated lipid
A structures of Porphyromonas gingivalis LPS have been shown to
activate the TLR4-mediated NF-κB signaling pathway differen-
tially and modulate the expression of proinammatory cytokines
(Herath et al.2013). Over-acylated and bi-phosphorylated LPS
have been identied in P. gingivalis isolates from patients with
chronic periodontitis (Herath et al.2013,Strachanetal.2019).
Zhang-Sun and his co-workers(Zhang-Sun et al.2019) very re-
cently characterized the LPSs of three Ralsonia species: Ralstonia
eutropa, R. mannitolilytica,andR. pickettii. They showed that lipid A
of R. pickettii is penta-acylated and was of low inammatory ca-
pacity compared to the other two species having hexa-acylated
lipid A. Production of outer membrane vesicles (OMVs) was in-
creased by the under-acylated LPS containing R. pickettii and these
can enter into the blood more easily than do the bacteria. Dea-
cylation of lipid A also lead to increased production of OMVs in
Salmonella enterica serovar Typhimurium (Elhenawy et al.2016).
When considering these examples of low-activity penta-
acylated lipid A, it is interesting to note that a bioinformatic anal-
ysis of available bacterial whole genome sequences showed that
while the lpxL gene was found in most gram-negative bacteria, the
lpxM gene was exclusively present in Gammaproteobacteria (Brix
et al.2015). Since LpxM is required for adding the sixth acyl chain,
most bacteria may thus make only the less inammatory penta-
acylated form. Therefore, the typical hexa-acylated lipid A from
E.coli may not be representative, and low-activity LPS forms may
be much more common than generally assumed.
The permeability properties of the outer membrane barrier
have a major impact on the susceptibility or resistance to an-
tibiotics. Mutations altering the lipid or protein composition of
the outer membrane can lead to the development of drug resis-
tant bacteria (Pagès et al.2008). LPS plays a major role in the
outer membrane permeability and modications in the lipid A
structure can affect antimicrobial susceptibility of the organisms
(Vaara 1992,Delcour2009). LPS decient mutants of E. coli hav-
ing deletions in any of the genes required for the inner part of
core-oligosaccharide are more susceptible towards cationic AMPs
(Ebbensgaard et al.2018) such as melittin, indolicidin, cecropin
and colistin. LPS-decient colistin resistant A. baumannii clinical
isolates have been reported previously (Moffatt et al.2010, Moffatt
et al.2013)E. coli and Salmonella typhimurium are protected by their
LPS from the antimicrobial activity of medium and long chain
fatty acids (Freese et al.1973). Fatty acid resistance of meningo-
cocci largely depends on the specic composition of the LPS core
oligosaccharide as well as hexa-acylation of the lipid A (Murray et
al.2001, Fisseha et al.2005, Schielke et al. 2010).
The gut microbiome, lipid A structure and the
immune response
The gut microbiome, the largest ecosystem in the human body,
performs a variety of important functions including immune
modulation. Their capsular polysaccharides, cell envelope con-
stituents and metabolites are the key components which inu-
ence the immune system of the host (Arnolds and Lozupone 2016,
Kespohl et al.2017). It is now well established that microbes are in
constant contact with the immune system on every surface of the
human body and provide training to the immune system in early
life (Olszak et al.2012). The host-microbiome interactions in the
intestine induce antimicrobial responses from the epithelia in-
cluding the secretion of antibacterial factors such as α-defensins,
angiogenins and RegIIIc, protecting against pathogenic microbes
and subsequent abnormal immune responses (Hooper et al.2003,
Cash et al.2006). Gut microbiota play an important role in neu-
trophil migration and function (Ogawa et al.2003) and also con-
tribute to the differentiation of T cell populations into Th1, Th2,
andTh17(Kespohletal.2017) or into regulatory T cells (Francino
2014). The cytokines secreted from Th17 cells have signicant im-
pact on intestinal homeostasis and inammation (Sonnenberg et
al. 2011, Rossi and Bot 2013). Short-chain atty acids such as bu-
tyrate produced by Clostridia spp cause increased production of
regulatory T cells in the intestine, and supplementation of these
bacteria confers resistance to colitis in mice (Atarashi et al.2011).
A derivative of indole, indoxyl 3-sulphate, produced by the gut
microbiota from tryptophan modulates immune functions which
may confer protection from graft versus host disease (Ghimire et
al.2018). The gut epithelium is equipped with pattern recognition
receptors (PRRs) which can directly sense commensal bacteria as
well as pathogens in the intestine, leading to activation of proin-
ammatory pathways and innate immunity of the host (Macpher-
son et al.2005). The PRRs recognize a variety of common bacterial
structures such as LPS of the Gram-negative and lipoteichoic acid
of the gram-positive cell wall.
As described above, the detailed lipid A structures differ con-
siderably among distinct bacterial species and determine the im-
munological activity of LPS (Brandenburg et al.1993,Seydeletal.
2000). For example, an LpxF-decient mutant B. thetaiotaomicron
strain was unable to stably colonize the murine gut, thus conrm-
ing the need for lipid A dephosphorylation in colonization of the
intestine (Cullen et al. 2015). This new nding shows that vari-
ation in the lipid A of species such as Bacteroides inuences the
commensal composition in the host gut. Another study by Cullen
and colleagues showed that modication of surface structures
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8|microLife, 2022, Vol. 3
with phosphoethanolamine (PEA) by the transferase EptC is key
for promoting commensalism of the bacterium Campylobacter je-
juni in its avian host (Cullen et al.2013). EptC adds PEA to the lipid
A, but also modies the agellar rod protein and several N-linked
glycans (Cullen et al.2013). Thus, the exact cause of the reduced
colonization by the EptC-decient mutant strain is difcult to pin-
point. The addition of PEA to the lipid A increased its ability to
activate TLR4/MD-2, but at the same time provided resistance to
cationic antimicrobial peptides, which indicates that in this case
resisting antimicrobial peptides is more important than reducing
the pro-inammatory response.
The role of LPS in the gut system has been studied in some
pathological conditions such as inammatory bowel disease and
enterocolitis. The LPS concentration in the blood of such patients
was estimated to be more than 40%–60% higher as compared to
healthy individuals (Pastor Rojo et al.2007). Inammatory bal-
ance, cell death, and intestinal permeability depend on the shifts
in the microbiota during infection, disease and trauma through
compositional changes in the resident LPS population. Serotype
specic LPS responses may be responsible for the pathogenesis
of IBD and other chronic inammatory diseases in the intestine
(Stephens and von der Weid 2019). It is plausible that the in-
tact intestinal epithelium prevents the entry of too much LPS
into the systemic circulation in healthy individuals while in dis-
eased states such barrier functions are disrupted, allowing ab-
sorption of much higher levels of LPS into the circulation lead-
ing to endotoxemia (Pastor Rojo et al. 2007). But also under non-
pathological conditions, small amounts of LPS can be absorbed
from the intestinal tract and can be detected at low concentra-
tions (1–200 pg/mL) in the blood plasma of healthy individuals
(Laugerette et al. 2011). The impact of such low LPS concentra-
tions in the circulation remains undened. It is well established
that low-activity lipid A from some bacteria can interfere with
appropriate TLR4/MD-2 signaling via competitive inhibition (Cur-
tis et al.2011, Tan et al.2015). Interestingly, d’Hennezel and his
co-workers(d’Hennezel et al. 2017) reported that members of Bac-
teroidetes group are the major contributors of LPS in human gut
and their LPS is non-stimulatory and inhibits TLR4-dependent cy-
tokine production. Puried capsular polysaccharide from B. frag-
ilis suppresses the production of IL-17 and reduces inamma-
tion in the colon by modulating CD4 lymphocytes to produce
IL-10 (Mazmanian et al.2008). These results underline the im-
portant role of gut commensal bacteria in the management of
inammation.
A growing body of evidence suggests that the gut microbiome
may be a key factor in inuencing predisposition to autoimmunity,
inammatory disorders and allergic diseases ( Eppinga et al. 2014,
Fyhrquist et al.2014, Scher et al.2015,Forbesetal.2016,Zákos-
telská et al.2016). On this note, the early childhood exposure in
priming and establishing immune responses seems to be key. This
was demonstrated when oral inoculation of Clostridium in early
life resulted in resistance to induced gut inammation in adult
mice (Atarashi et al.2011). More recently, a study followed the gut
microbiota in the rst three years of life of infants from Northern
Europe (Finland and Estonia) where early-onset of autoimmune
diseases are common and Russia where it is less prevalent, and
found low amounts of Bacteroides species in Russian infants while
they were dominant in the Finnish and Estonian infants (Vatanen
et al.2016). This translated to exposure to an underacylated lipid
A structure, which in turn inhibits the innate immune signaling
and prevents the induction of endotoxin tolerance (Vatanen et al.
2016). This induction of immune tolerance by exposure to highly
potent LPS from E. coli showed a decrease in the incidence of dia-
betes in mice, whereas the same could not be observed with mice
treated with underacyated LPS from B. dorei (Vatanen et al.2016).
Altogether, these studies highlight that it is not merely the pres-
ence of commensal species or the amount of LPS present in the
gut, but also the specic lipid A structure they make which has
a major role in the development of inammatory diseases, au-
toimmunity and allergies. In addition the exposure to highly po-
tent lipid A structures in early years of life is important for the
induction of regulatory T cells and immune tolerance, thereby
preventing the onset of these immunological disorders later
in life.
Immune mechanisms involved in intestinal commensalism
and the role of LPS related to gut health and disease need to
be investigated further, considering the vast number of different
gram-negative bacterial species in the gut. Untill now, isolation of
LPS from gut microbiota involved conventional methods and the
data generated are mostly derived from bacteria grown in vitro.
As described above, their lipid A structures may differ consider-
ably when they are present in the human gut. So it is essential to
develop research methods including innovative mass spectrome-
try analyses of highly complex in vivo samples to investigate to
role of gut microbial LPS under natural conditions and preferably
in both diseased and healthy situations in order to validate the
present concepts (Cani 2018).
Biotechnological applications
The great range of possible lipid A structural modications can be
utilized for vaccine development. The innate immunity activation
capacity of LPS makes it a potent adjuvant, either as a component
of LPS-containing vaccines based on whole bacterial cells or outer
membrane vesicles (OMVs), or when added as puried component
to subunit vaccines. Fine-tuning the biological activity of LPS can
be done by utilizing the natural heterogeneity of lipid A and select-
ing those with an optimal balance between immunostimulatory
and toxic properties. In addition, the great natural variety of lipid
A biosynthetic and modifying enzymes can be put to use for the
generation of novel variants with improved adjuvant properties. A
combinatorial lipid A bioengineering approach has been used to
generate extensive panels of such LPS derivatives in Escherichia coli
(Needham et al.2013)andNeisseria meningitidis (Zariri et al. 2016).
In Bordetella pertussis, heterologous expression of lipid A biosyn-
thesis enzymes enabled the depletion of endotoxic activity of vac-
cines based on whole cells or OMVs (Geurtsen et al.2006, Asensio
et al.2011,Arenasetal.2020). Complete genetic detoxication of
LPS in E.coli led to the development of endotoxin-free production
strains for recombinant proteins (Mamat et al.2015). For studies
of the role of the microbiome in health and disease, and possi-
ble therapeutic modications to its composition, it will be impor-
tant to take the lipid A structures of the relevant bacteria into
account, for instance through MS structural analysis. Advances
in new analytical techniques (Pupo et al.2021) will make this in-
creasingly possible, but have until now not been used to their full
potential.
Acknowledgements
We would like to extend our thanks to Dr. Md. Abdul Hannan,
Department of Biochemistry and Molecular Biology, Bangladesh
Agricultural University, Mymensingh, Bangladesh, for helping us
in preparing the gure for this manuscript.
Conict of interest statement. The authors declare no conicts of in-
terest.
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Saha et al. |9
Funding
This work was nancially supported by a Marie Curie fellowship
of the European Commission (project number 796009).
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