Microbes and Infection 4 (2002) 325–331

www.elsevier.com/locate/micinf

Review

Outer membrane proteins: key players for bacterial adaptation

in host niches
Jun Lin, Shouxiong Huang, Qijing Zhang *
Food Animal Health Research Program, Department of Veterinary Preventive Medicine, The Ohio State University, 1680 Madison Avenue,
Wooster, OH 44691, USA

Abstract

Outer membrane proteins (OMPs) of Gram-negative bacteria have diverse functions and are directly involved in the interaction with
various environments encountered by pathogenic organisms. Thus, OMPs represent important virulence factors and play essential roles in
bacterial adaptation to host niches, which are usually hostile to invading pathogens. Understanding the structure and functions of bacterial
OMPs will facilitate the design of antimicrobial drugs and vaccines. In this paper, we will present a brief review on OMPs that contribute
to bacterial adaptive responses including iron uptake, antimicrobial peptide resistance, serum resistance, and drug/bile resistance. © 2002
Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Keywords: Outer membrane proteins; Pathogenesis; Adaptation; Resistance

1. Introduction 2. Iron uptake

The outer membrane of Gram-negative bacteria primarily
consists of phospholipids, lipopolysaccharide, and a group
of outer membrane proteins (OMPs) that account for ap-
proximately 50% of the outer membrane mass [1]. OMPs
include integral membrane proteins as well as lipoproteins
that are anchored to the outer membrane via N-terminally
attached lipid. Characterized by β-barrel structures, integral
OMPs are essential for maintaining the integrity and selec-
tive permeability of bacterial membranes [2]. In addition,
OMPs, whose production is often regulated by environmen-
tal cues, also play important roles in bacterial pathogenesis
by enhancing the adaptability of bacterial pathogens to
various environments. Several comprehensive reviews on
the structure and function of outer membranes have been
published [1–4]. This review will focus on those OMPs that
are involved in bacterial adaptive responses to frequently
encountered conditions upon infecting a host, such as
nutrient starvation (e.g. iron limitation), presence of potent
antimicrobial peptides (AMPs) in the circulating system and
mucosal surfaces, bactericidal activity of complements,
antibiotic treatments, and presence of detergent-like bile
salts in intestine.
* Corresponding author. Tel.: +1-330-263-3747; fax: +1-330-263-3677.
E-mail address: zhang.234@osu.edu (Q. Zhang).
© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
PII: S 1 2 8 6 - 4 5 7 9 ( 0 2 ) 0 1 5 4 5 - 9
Iron is the most abundant transition metal in living
organisms, with a critical role in many diverse biological
systems. All Gram-negative bacteria have an absolute re-
quirement for iron to survive. However, because of the low
solubility of ferric iron and the need to avoid its participa-
tion in generating toxic oxygen-derived free radicals, higher
organisms have evolved mechanisms for lowering the levels
of free iron to well below those required for the growth of
Gram-negative bacteria [5,6]. Most iron is located intracel-
lularly in eucaryotic cells as ferritin or as heme-compounds.
This source of iron is normally not available to invading
Gram-negative bacteria. The small amount of extracellular
iron that exists in body fluids is bound by high-affinity
iron-binding proteins, such as transferrin and lactoferrin in
serum and mucosal secretions [5,6]. Therefore, to obtain
sufficient iron for survival and multiplication, Gram-
negative bacteria have evolved sophisticated genetic sys-
tems for iron uptake. Two major strategies involving iron-
regulated OMPs are used by Gram-negative bacteria to
assimilate iron in iron-restricted niches: 1) expression of
specific outer membrane receptors (e.g. Tbp and Lbp in
Neisseria, HemR in Yersinia), some of which are TonB-
dependent, to directly bind host transferrin, lactoferrin, or
hemoprotein followed by the removal of iron from iron-
binding protein on the cell surface and internalization of
326 J. Lin et al. / Microbes and Infection 4 (2002) 325–331
iron into cells; and 2) utilization of TonB-dependent OMPs
(e.g. FhuA and FepA in Escherichia coli, whose three-
dimentional structures have been resolved [2,3]) that bind to
the iron–siderophore complex and promote iron–sidero-
phore across the membrane into cells. Siderophores
(500–1000 Da) are high-affinity iron chelators secreted by
bacteria and can competitively capture iron from host
iron-binding protein or iron-binding compounds. TonB and
TonB-dependent receptors are key players in iron uptake
under aerobic conditions. Several excellent reviews have
described the roles of iron-regulated OMPs in bacterial iron
uptake [7–11].
Since bacteria require iron for their growth and the levels
of free iron in vivo are well below microbial requirements,
possession of iron uptake systems and functional iron-
regulated OMPs are essential for bacterial survival and
virulence. There is a considerable body of experimental
studies demonstrating that iron-regulated OMPs are induced
under iron-restricted conditions in vivo. These examples
include Vibrio cholerae, Proteus, Klebsiella pneumoniae,
Pasteurella haemolytica, and Haemophilus influenzae
[12–16]. Specific antibodies to iron-regulated OMPs of
Gram-negative bacteria were found in sera of both animals
and humans [17–22], further indicating that iron-regulated
OMPs are induced during in vivo infection. In addition,
genetic knockout of iron-regulated OMP genes in Gram-
negative bacteria resulted in attenuated virulence compared
with wild-type. For example, inactivation of iron-regulated
OMPs in some Gram-negative bacteria, such as Pseudomo-
nas aeruginosa [23], Neisseria meningitidis [24], Vibrio
species [25,26], Salmonella enterica [27], Haemophilus
ducreyi [28], and Burkholderia cepacia [29], resulted in
reduced virulence in animal or human infection models.
Together, these findings indicate that iron-regulated OMPs
are important virulence factors.
3. Antimicrobial peptide resistance
Antimicrobial peptides are short, cationic, and bacteri-
cidal peptides that can be found in many animal species
ranging from insects to mammals (reviewed in [30–34]). As
a major component of host innate immunity, AMPs can
directly contact and disrupt the bacterial membrane by
permeating lipid bilayers, and ultimately lead to cell death
[34]. Approximately 400 AMPs have been reported in
insects, plants, animals, and humans [30]. Circulating ph-
agocytic leukocytes are the major source of several AMPs
including defensins. In addition, epithelial cells of the skin
and mucosal surfaces also synthesize and release AMPs,
which contribute to intrinsic mucosal immunity against
bacterial infections [31].
Bacterial pathogens have developed the means to curtail
the effect of AMPs. Direct degradation of AMPs and
modification of cell surface properties are two major strat-
egies used by Gram-negative bacteria to resist the bacteri-
cidal activity of AMPs. The former strategy is dependent on
the production of outer membrane-associated proteases,
which cleave AMPs outside of cells and enable bacteria to
evade killing by AMPs. For example, Stumpe et al. [35]
demonstrated that E. coli outer membrane protease OmpT
hydrolyzes AMP protamine before it enters growing cells of
E. coli. A PhoP-regulated outer membrane protease PgtE of
Salmonella typhimurium also contributes to resistance to
AMPs [36]. Sequence analysis indicated that PgtE has high
homology to E. coli OmpT (46% aa identity) and Yersinia
pestis Pla (72% aa identity). The PgtE deletional mutant
showed increased sensitivity to several cationic AMPs that
contain OmpT-cleavage site, suggesting that PgtE functions
in a similar manner to E. coli OmpT [36]. Direct evidence is
still lacking with regards the relationship between Y. pestis
Pla and AMP resistance. However, one study showed that
the subcutaneous LD50 of pla mutant was 4 to 6 logs
greater than that of wild type [37]. Considering the high
sequence similarities between Y. pestis Pla and S. typhimu-
rium PgtE, it is likely that protease-mediated resistance to
AMP may contribute, at least partially, to the attenuated
virulence of pla mutant.
Because AMPs act on bacterial membranes, Gram-
negative bacteria can also protect themselves from attack by
AMPs via modification of their cell surface properties to
prevent the binding of AMPs to the outer membrane or
decrease the permeability of the outer membrane [38–40].
For example, two-component regulatory systems in S. typh-
imurium including PhoP/PhoQ and PmrA/PmrB, promote
AMP resistance by activating transcription of genes that are
involved in the modification of lipid A, the bioactive
component of LPS [38,39]. The S. typhimurium
PhoP/PhoQ-activated gene pagP, which is essential for
addition of palmitate to Salmonella lipid A, encodes an
OMP with enzymatic activity involved in lipid A biosyn-
thesis [40,41]. Such lipid A modification changes the
fluidity of the outer membrane and decreases the permeabil-
ity of the outer membrane enhancing the resistance of
S. typhimurium to α-helical cationic AMPs [40]. Whether
other Gram-negative bacteria use PagP-like enzymes to
mediate AMP resistance is largely unknown. However,
PagP-mediated lipid A palmitoylation is likely a general
mechanism for Gram-negative bacterial resistance to
a-helical cationic AMP because 1) other Gram-negative
bacteria, such as E. coli and Yersinia enterocolitica, are
shown to regulate the synthesis of palmitoylated lipid A in
response to low Mg2+ growth conditions in a manner similar
to the S. typhimurium PagP-mediated addition of palmitate
to lipid A [39]; and 2) homologs of PagP are encoded in
E. coli, Yersinia spp., and Bordetella spp. [41] and Le-
gionella pneumophila (Gene bank accession number:
AAK52070). Synergistic action of multiple resistance strat-
egies can greatly decrease the bactericidal activity of AMPs.
One such example is the study by Guina et al. [36], who
demonstrated that inactivation of both the protease gene
(pgtE) and the lipid A modification gene (pagP) in
 J. Lin et al. / Microbes and Infection 4 (2002) 325–331
S. typhimurium resulted in greater AMP sensitivity than that
in mutants containing a single mutation in either gene.
4. Serum resistance
Animal sera exhibit bactericidal activity primarily
through the action of complements. As an important factor
in protective immunity, complements play a critical role in
the resistance against bacterial infections [42]. The comple-
ment system is composed of approximately 20 interacting
soluble proteins that are constantly present in the blood and
extracellular fluids. After encountering an invading patho-
gen, the complement system can be activated through the
classical pathway (antibody-dependent) and/or the alterna-
tive pathway (antibody-independent), which ultimately re-
sult in the formation of pores in the membrane of the
invading bacterial pathogens. Soluble complement proteins
C4bp and factor H, which block the formation of C3
convertase and prevent complement activation, are key
regulators in the classical pathway and the alternative
pathway, respectively. To evade complement-mediated kill-
ing, Gram-negative bacteria have developed multiple strat-
egies to protect themselves from the bactericidal activity
(reviewed in references [43–46]). Three major components
(OMPs, LPS, and capsule) of Gram-negative bacteria are
involved in serum resistance. This section only focuses on
bacterial OMPs that contribute to serum resistance.
In general, OMPs of Gram-negative bacteria promote
bacterial resistance to complement-mediated killing by
preventing the activation of complement cascades and/or
blocking the formation of a lethal membrane attack complex
on the bacterial membrane. A well-characterized mechanism
is the binding of bacterial OMP to the main regulators of the
alternative pathway, factor H and factor H-like protein 1
(FHL-1) [47,48]. Expression of Por1A (a major OMP) in
Neisseria gonorrhoeae is associated with serum resistance
and virulence [49]. Mutation of por1A gene resulted in
serum sensitivity compared with the parent strain [50]. Ram
et al. [47] showed that Por1A binds to regulatory factor H
via loop 5 and thereafter increases the conversion of C3b to
iC3b, leading to decreased killing of N. gonorrhoeae by
complements. A similar mechanism was also observed in
another Gram-negative pathogen, Borrelia burgdorferi [48],
in which OspE was shown to bind strongly to factor H and
suppress ongoing complement activation. The C-terminal
short consensus repeat domains of OspE were characterized
as the specific binding site for factor H [48]. Besides OspE,
other OMPs of B. burgdorferi including OspA, OspC, or
OspD may be also involved in serum resistance, because
mutants lacking these OMPs are more susceptible to serum
killing than the wild-type strain [51,52]. Since there is no
specific binding of factor H to any of these OMPs [48],
other unknown mechanisms may account for the serum
resistance. Recently, Ram et al. [53] reported the binding of
C4bp, a key regulator of the classical pathway, to Por1A or
327
Por1B in N. gonorrhoeae. The N-terminal loop (loop1) of
Por1A is a specific C4bp binding domain while loop 5 and
loop 7 of Por1B together are required for C4bp binding [53].
Specific inhibition of C4bp binding to serum resistant PorA
or PorB strains resulted in complete killing of serum
resistant strains of N. gonorrhoeae in 10% of normal human
sera, further indicating the importance of OMPs in mediat-
ing gonococcal serum resistance.
Many other OMPs are implicated in serum resistance
despite the fact that the resistance mechanisms are un-
known. OmpX of E. coli belongs to a group of integral
OMPs that not only play important roles in adhesion and
invasion but also promote bacterial resistance to the bacte-
ricidal activity of complements [54–56]. The OmpX ho-
mologs in Gram-negative bacteria include Ail of Y. entero-
colitica [55], Rck and PagC of S. typhimurium [55], OmpX
of Enterobacter cloacae [57], and OmpK17 of K. pneumo-
niae [58]. The recent elucidation of the crystal structure of
OmpX of E. coli revealed a unique structural feature of
OmpX, the protruding β-sheet formed by loop 2 and loop 3
[56]. These structural and functional studies have revealed
that the external loops of the proteins in the OmpX family
are responsible for serum resistance and other virulence
properties. In addition, other OMPs, such as OmpA of
E. coli [59], TraT and YadA of Y. enterocolitica [60,61], Brk
of Bordetella pertussis [62], and DsrA of H. ducreyi [63],
were also involved in serum resistance, although the under-
lying mechanisms responsible for the resistance are still
unknown.
5. Multi-drug resistance and bile resistance
In parallel to the availability of multiple antibiotics for
medical uses, bacterial organisms have evolved a variety of
mechanisms for drug resistance (reviewed in references
[64,65]), which has greatly compromised the effectiveness
of antibiotic treatments. Although specific mechanisms are
associated with resistance to individual antibiotics, the
multi-drug efflux pumps function as general and intrinsic
drug resistance systems in Gram-negative bacteria and are
responsible for bacterial resistance to a variety of harmful
molecules including antibiotics (reviewed in references
[66–69]). Bacterial resistance to bile salts, a group of
bactericidal detergents present in the intestinal tract of
animals, is also attributable to multi-drug efflux pumps.
Multi-drug efflux pumps from different species of Gram-
negative bacteria usually share a common structural theme,
including an inner membrane transporter, a periplasmic
fusion protein, and an outer membrane channel protein [66].
Functioning together, the three components form a multi-
purpose efflux system that allows efflux of a variety of
substrates across the two membranes directly into the
surrounding medium. As an essential component in the
tri-partite efflux pump, the OMP component can interact
with different transporter complexes and exhibits functional
328 J. Lin et al. / Microbes and Infection 4 (2002) 325–331
diversity [70,71]. For example, the well-characterized OMP
TolC of E. coli can function with at least four distinct
cytoplasmic membrane transport systems and plays an
essential role in export of different substrates ranging from
structurally unrelated antimicrobials, heavy metals, and
detergents to large toxins [70,71]. The importance of OMP
in efflux systems and antibiotic resistance is supported by
the finding that inactivation of the OMP gene in an efflux
system could result in decreased efflux ability and increased
susceptibility to a wide range of antibiotics and other
antimicrobials. An isogenic tolC mutant exhibited hypersen-
sitivity to various hydrophobic inhibitors due to the mal-
functioning of the AcrAB multi-drug efflux system and its
inability to exclude antimicrobial agents [72]. Genetic
knockout of tolC homolog gene in Salmonella enteritidis
resulted in an avirulent phenotype, suggesting that tolC-
related proteins are important for pathogenicity [73]. Dis-
ruption of the oprM gene, which encodes an OMP of 50 kDa
involved in the MexAB and MexXY efflux systems of
P. aeruginosa [74,75], increased the susceptibility of this
organism to many antibiotics [76–78]. Notably, disruption
of oprM often resulted in more drug susceptibility than
mexA or mexB mutations [76–78], which further highlights
the critical role of the OMP components in efflux systems
and multi-drug resistance.
Overexpression of efflux-related OMPs can result in
increased drug resistance. For example, mutation in mexR,
which encodes a repressor of the mexAB-oprM multi-drug
efflux operon of P. aeruginosa, resulted in the overexpres-
sion of the 50-kDa OprM and increased multidrug resis-
tance in P. aeruginosa [74,75,79]. In addition, overexpres-
sion of OprJ, an OMP component in the MexCD efflux
system, was also correlated with enhanced multidrug resis-
tance in P. aeruginosa [80]. Recently, Ziha-Zarifi et al. [81]
described the in vivo emergence of muti-drug resistant
mutants of P. aeruginosa. Immunoblotting of OMP of 11
bacterial pairs (isolated before and after drug therapy)
demonstrated the overexpression of OprM in all the post-
therapy isolates. Ten out of the 11 post-therapy isolates had
mutations (insertion, deletion, or point mutation) in repres-
sor gene mexR. These results indicate that mutations in a
regulatory gene (such as mexR) can lead to overexpression
of the drug efflux systems and increased resistance to
antibiotics. For the foodborne pathogen Campylobacter
jejuni, a 55-kDa OMP was overexpressed in two mutants
which were resistant to pefloxacin or cefotaxime [82]. Both
mutants showed cross-resistance to other structurally unre-
lated antibiotics and showed a lower intracellular accumu-
lation level of antibiotics than the parent strain, indicating
that this OMP was involved in multi-drug resistance via a
multi-drug efflux system with broad specificity. However,
detailed analysis of this protein and further identification of
its related efflux system has not been reported since then.
Recently, our lab has characterized a multifunctional efflux
pump in C. jejuni (unpublished data in this lab). This
putative efflux pump shares high homology with the Mex-
AB–OprM efflux system in P. aeruginosa. Transposon in-
sertional mutation of the inner membrane efflux transporter
of the C. jejuni efflux system abolished the production of the
transporter protein and its associated outer membrane pro-
tein, leading to a significant increase in susceptibility to
structurally unrelated antibiotics, heavy metals, and bile
salts (unpublished data in this lab).
Bile is produced in the liver, stored in the gallbladder,
and released into the small intestine for digestion of food
and fats. Bile contains groups of detergent-like bile salts
which can kill bacterial cells by destroying the lipid bilayer
of the membrane [83]. Many Gram-negative bacteria, espe-
cially those enteric pathogens, are highly resistant to the
bactericidal activity of bile salts. As reviewed recently by
Gunn [84], multiple mechanisms, some of which are regu-
lated by two-component regulatory systems, contribute to
bacterial resistance to bile. Here, we will briefly discuss the
roles of OMPs in bile resistance.
The outer membrane of Gram-negative bacteria plays a
key role in bile resistance, which requires the expression
and function of multiple OMPs. In general, these OMPs
either affect the membrane permeability (e.g. porins) to bile
salts, or are involved in the efflux of these antimicrobial
detergents. Porins are the most abundant OMPs and exist in
the bacterial outer membrane as stable trimers, which are
highly resistant to bile salts. Different porin molecules form
various sizes of pores with dissimilar charge properties, and
therefore have varied permeability to bile salts. By regulat-
ing the production of a specific porin, bacteria can modulate
their membrane permeability and decrease the sensitivity to
bile salts. For example, the E. coli outer membrane porins
OmpF and OmpC were involved in bile resistance, with
OmpC playing a more significant role than OmpF, possibly
due to the smaller pore size of OmpC than that of OmpF
[85]. Similarly, Provenzano and Klose [86] reported that
ToxR-dependent modulation of the outer membrane porins
OmpU and OmpT production is critical for V. cholerae bile
resistance, with OmpU playing a more significant role than
OmpT in bile resistance. OmpU, similar to E. coli OmpC
which has a small pore size, is more cationic selective than
OmpT and is less permeable to negatively charged bile salts
[86]. However, porins do not block a significant portion of
bile salts that exist in lipophilic, uncharged forms and can
directly cross through the outer membrane. Those bile salts
entering into bacterial cells are eliminated from the cells by
another major mechanism involving multi-drug efflux
pumps.
Efflux of bile salts from the bacterial cytoplasm directly
out of the cell is a well-characterized mechanism of bile
resistance, and is mediated by the multi-drug efflux systems
discussed in the previous section. For example, the TolC
(outer membrane channel protein)-dependent AcrAB and
EmrAB efflux pumps are important players in excluding
bile salts out of E. coli cells [72,85]. The multi-drug efflux
system MtrCD–MtrE mediates resistance of N. gonor-
rhhoeae to structurally diverse hydrophobic agents includ
 J. Lin et al. / Microbes and Infection 4 (2002) 325–331 329

Table 1
Major mechanisms of OMP-mediated bacterial adaptive responses to host environment
Adaptive responses Mechanisms Examples of OMPs References
Iron uptake Expression of OMPs to directly bind host transferrin, lactoferrin, or hemoprotein Tbp, Lbp, HemR [7,9,10]
Synthesis of high-affinity iron–siderophores and expression of OMPs to bind the FhuA, FepA [8,11]
siderophore complexes
Antimicrobial peptide Direct degradation of antimicrobial peptides through production of outer membrane OmpT, PgtE [35,36]
resistance associated proteases
Modification of bacterial surface through production of OMPs with enzymatic PagP [40,41]
activities
Serum resistance Prevent the activation of complement cascades by binding to factor H or C4bp, Por1A, Por1B OspE [47,48,53]
down-regulators of complement activation
Unknown OmpX [54–56]
Multidrug resistance Key roles in multi-drug efflux systems TolC, OprM [72,74,75]
Bile resistance Modulate membrane permeability by regulating the productions of specific porins OmpC, OmpU [85,86]
Key roles in multi-drug efflux systems TolC [72,85]
.

ing bile salts [87]. Insertional inactivation of the inner Acknowledgements

membrane efflux transporter gene mtrD rendered gonococci
hypersusceptible to bile salt cholic acid. Inactivation of a
putative multi-drug resistance pump from V. cholerae also
resulted in increased susceptibility to bile salt deoxycholate
when compared with the parent strain [88]. We have
recently demonstrated that a multi-drug efflux pump in
C. jejuni contributes greatly to bile resistance. A transposon
mutant with the inactivated efflux system resulted in a more
than 1000-fold decrease in MIC of bile salts (unpublished
data in this lab). Because C. jejuni is an enteric pathogen,
we speculate that this multi-functional efflux system may
enhance the survival of C. jejuni under the harsh conditions
(including bile salts) encountered in the gastrointestinal
tract.
6. Conclusion
It can be concluded that OMPs play important roles in the
pathobiology of Gram-negative bacterial pathogens. Some
OMP-mediated adaptive responses to host environments
and the associated mechanisms are summarized in Table 1.
Although significant advances have been made regarding
the structure and function of OMPs, the number of OMPs
that have been characterized represents only a small portion
of the total OMPs revealed by bacterial genome sequences.
The biological roles of the majority of bacterial OMPs are
still unknown and remain to be determined in future studies.
With the aid of the newly developed technologies, such as
functional genomics and DNA microarrays, characterization
of bacterial OMPs can now be performed at a previously
unprecedented large scale. These new systems in conjunc-
tion with other strategies, such as signature-tagged mu-
tagenesis, subtractive and differential hybridization, in vivo
expression technology, and differential fluorescence induc-
tion, etc., will reveal more OMPs that are essential for
bacterial virulence and adaptation during in vivo infection.
These identified OMPs will be promising targets for the
design of antimicrobial drugs and vaccines.
Work in our laboratory was supported by a grant from the
Research Enhancement Competitive Grants Program of
OARDC at the Ohio State University. We would like to
thank Srinand Sreevatsan, Linda Michel, and Orhan Sahin
for helpful critiques of this manuscript.
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