The porin and the permeating antibiotic: A
selective diffusion barrier in gram-
negative bacteria
Pages, J, James, C and Winterhalter, M
http://dx.doi.org/10.1038/nrmicro1994
Title The porin and the permeating antibiotic: A selective diffusion barrier in
gram-negative bacteria
Authors Pages, J, James, C and Winterhalter, M
Publication title Nature Reviews in Microbiology
Publisher Nature Publishing Group
Type Article
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Multidrug resistance (MDR) is frequ–ently reported
in clinical Gram-negative bacteria. This limits which
therapeutic options are available and is a major cause
of mortality when acquired as a nosocomial infection
1,2
.
Moreover, no truly novel active antibacterial compound
is currently in clinical trials. Thus, it is important to
decipher the molecular basis of the MDR mecha-
nisms
3–5
. MDR is prevalent in key Gram-negative clini-
cal pathogens, such as Escherichia coli, Salmonella spp.,
Klebsiella spp., Enterobacter spp., Campylobacter spp.,
Acinetobacter spp. and Pseudomonas spp. Three major
bacterial strategies have emerged for the development of
drug resistance: the membrane barrier limits the intra-
cellular access of an antibiotic; the enzymatic barrier
produces detoxifying enzymes that degrade or modify
the antibiotic; and the target protection barrier impairs
target recognition and thus antimicrobial activity
6
. These
mechanisms can act simultaneously in clinical isolates,
generating a high level of resistance. There are two dif-
ferent aspects to transport systems across the bacterial
membrane — influx and efflux. Here, we focus on the
influx of antibiotics, as the efflux has been extensively
discussed in recent reviews
5–8
.
The outer membrane is the first line of defence for
Gram-negative bacteria against toxic compounds
9
. This
barrier comprises a lipid bilayer that is impermeable to
large, charged molecules. Influx is largely controlled by
porins, which are water-filled open channels that span
the outer membrane and allow the passive penetration
of hydrophilic molecules
9–11
. Different types of porins
have been characterized in Gram-negative bacteria
and classified according to their activity (non-specific
or specific channel or selective pore), their functional
structure (monomeric or trimeric) and their regulation
and expression
9–14
.
E. coli produces three major trimeric porins —
OmpF, OmpC and PhoE — and pioneering studies
with these porins constitute the foundation of our cur-
rent knowledge of many other porins
9,10
. Thus, these
outer membrane proteins (OMPs) (and their homo-
logues in other Gram-negative bacteria) are termed
classical porins
9
. Despite their ‘non-specific’ nature,
the members of this family can be classified according
to a range of selective filters with respect to the charge
and size of the solutes and charges in key regions of the
porin channels: the OmpF and OmpC families show
a slight preference for cations, whereas PhoE selects
inorganic phosphate and anions
9–12
. Porins have been
purified and reconstituted in various experimental sys-
tems (for example, liposomes and planar membranes)
to analyse their physico-chemical parameters, such as
conductance, selectivity and voltage gating
10
.
*UMR-MD-1, Transporteurs
Membranaires,
Chimiorésistance et
Drug-Design, Facultés
de Médecine et de harmacie,
Université de la Méditerranée,
Marseille, 13385, France.
‡
School of Engineering and
Science, Jacobs University
Bremen, Bremen, 28759,
Germany.
Correspondence to J.-M.P.
e-mail: Jean-Marie.PAGES@
univmed.fr
doi:10.1038/nrmicro1994
Published online
10 November 2008
Nosocomial
Hospital-acquired infection.
Conductance
A measure of translocated
charges per unit time and
voltage gradient.
Selectivity
The translocation efficiency
of a channel for a particular
type of ion with respect to
another ion.
The porin and the permeating
antibiotic: a selective diffusion barrier
in Gram-negative bacteria
Jean-Marie Pagès*, Chloë E. James* and Mathias Winterhalter
‡
Abstract | Gram-negative bacteria are responsible for a large proportion of antibiotic-
resistant bacterial diseases. These bacteria have a complex cell envelope that comprises
an outer membrane and an inner membrane that delimit the periplasm. The outer membrane
contains various protein channels, called porins, which are involved in the influx of various
compounds, including several classes of antibiotics. Bacterial adaptation to reduce influx
through porins is an increasing problem worldwide that contributes, together with efflux
systems, to the emergence and dissemination of antibiotic resistance. An exciting challenge
is to decipher the genetic and molecular basis of membrane impermeability as a bacterial
resistance mechanism. This Review outlines the bacterial response towards antibiotic stress
on altered membrane permeability and discusses recent advances in molecular approaches
that are improving our knowledge of the physico-chemical parameters that govern the
translocation of antibiotics through porin channels.
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Voltage gating
Effect observed for some
channels whereby a high
voltage gradient causes a
sudden closure of the ion
current. The molecular origins
remain unsolved.
β-lactam
A major family of antibiotic
molecules.
Antibiotherapy
A therapy that uses antibiotics
to treat infections.
Cephalosporins and
carbapenems
Two subclasses of the β-lactam
family.
General diffusion porins can be distinguished
from the specific and ligand-gated porins by their
poor substrate selectivity and their high probability of
presenting an open conformation in the absence of any
specific substrates
9,10
. Most porins that are involved in
antibiotic transport belong to the classical OmpF or
OmpC subfamilies. However, there are exceptions,
such as OprD of Pseudomonas aeruginosa and porins
from Acinetobacter baumannii and Neisseria spp.
For more examples, consult the Transport Protein
Database (see Further information). Many aspects of
porin activity and physiology have previously been
discussed in various reviews
9–15
.
β-lactams and fluoroquinolones are the prominent
groups in our current antibacterial arsenal
16
and their
respective activities are strongly affected by the influx
barrier in clinical isolates (TABLE 1). The porin channel
is the entry pathway for both β-lactams and fluoroqui-
nolones, which block the synthesis of peptidoglycan
and disrupt the activity of gyrase and topoisomerase,
respectively, and induce a bactericidal cascade
16,17
. In
this Review, we explore the recent clinical evidence for
distinct bacterial strategies of porin modification to limit
β-lactam uptake (FIG. 1): an exchange in the type of porin
expressed; a change in the level of porin expression; and
a mutation or modification that impairs the functional
properties of a porin channel. A possible emerging
mechanism, the synthesis of pore-blocking molecules,
is also discussed (BOX 1). The clinical prevalence of
these resistance strategies highlights the importance
of deciphering the antibiotic influx process. Therefore,
we also focus on recent state of the art techniques that
allow the quantification of antibiotic transport and the
understanding of molecular dialogue between the porin
channel and the antibiotic.
Role of porins in antibiotic resistance
Some Gram-negative bacteria, such as P. aeruginosa
and A. baumannii, possess an innate low susceptibility
to β-lactam molecules, a characteristic that is associated
with reduced outer-membrane permeability
9
. In P. aerugi-
nosa, this reduced permeability is due to the low number
of porins and their distinct physico-chemical properties
compared with the porins of the Enterobacteriaceae
9,18–20
.
In other Gram-negative species (such as Citrobacter,
Enterobacter, Escherichia and Klebsiella), β-lactam sus-
ceptibility is closely related to the presence of non-specific
porins that belong to the OmpC and OmpF groups
9,15
.
Several clinical studies have reported a modification of
the porin profile in antibiotic-resistant isolates: resistant
Enterobacteriaceae can exhibit a shift in the type of porin
they express, a reduction in the porin expression level or
the presence of a mutated porin (TABLE 1). These clinical
strains, isolated during patient antibiotherapy, exhibit a
characteristic decrease in cephalosporin and carbapenem
susceptibility. An altered porin phenotype is also com-
monly associated with the expression of degradative
enzymes, such as β-lactamases and cephalosporinases,
which efficiently confer a high level of β-lactam resist-
ance
14,21
. It is important to consider the clinical evidence
for the different strategies that are involved in reducing
influx across the outer membrane. By exploring each
mechanism in turn we illustrate a bacterial adaptive
response to antibiotherapy that leads to MDR.
Alterations in porin expression
Porin exchange. A study of Klebsiella pneumoniae strains
collected from different patients undergoing treatment
indicated that the isolates exhibited modified outer-
membrane permeability
22
. In most of these isolates,
OmpK35, which belongs to the OmpF porin group and
has a large channel size, was replaced with OmpK36,
which belongs to the OmpC porin group and possesses
a smaller channel size. This observation suggests that a
drastic modification of the porin balance occurs dur-
ing antibiotherapy. This is of particular interest owing
to the differential β-lactam susceptibility reported in
these K. pneumoniae porins. The level of susceptibility
to β-lactams, including cefepime, cefotetan, cefotaxime
and cefpirome, in strains expressing OmpK35 is 4–8
times higher than that conferred by OmpK36 (REF. 23).
The clinical isolates collected after antibiotic treatment
exhibited an altered porin phenotype, with a simultane-
ous overexpression of an AcrAB efflux pump for extru-
sion of incoming antibiotic molecules. Together, these
modifications severely decrease the intracellular drug
concentration
22
.
Several studies have observed that there is a rela-
tionship between the balance of porin expression
and β-lactam susceptibility in clinical K. pneumoniae
isolates
24–30
. A similar phenomenon has been reported
in a patient infected with Salmonella enterica subsp.
enterica serovar Typhimurium. All isolates collected
Table 1 | Porin modification in Gram-negative bacteria
Bacteria Characterized
porins
3D structure
of porins
Porin alteration
in clinical isolates
Enterobacter cloacae,
Enterobacter aerogenes
Omp36*, Omp35
‡
None Omp36
§
, Omp35
§
,
Omp 36
||
Escherichia coli OmpC*, OmpF*,
OmpN
¶
, PhoE
OmpC
31
,
OmpF
30
, PhoE
30
OmpC
§
, OmpF
§
,
OmpC
||
Klebsiella pneumoniae OmpK36*,
OmpK35
‡
, OmpK37
¶
OmpK36
32
OmpK35
§
,
OmpK36
§
Morganella morganii Major porin
(36 kDa)
None Major porin
Neisseria gonorrhoeae Major porin None Major porin (PorA
§||
,
PorB
§||
)
Pseudomonas
aeruginosa
Porins, OprD OprD
33
Porins
§
, OprD
§
,
OprD
||
Salmonella enterica
subsp. enterica
serovars Typhimurium
and Enteritidis
OmpC*, OmpF
‡
,
OmpD
None Major porins
(OmpC
§
, OmpF
§
,
OmpD
§
)
Serratia marcescens Omp1*, Omp2
‡
None Major porins
(Omp1
§
, Omp2
§
)
Shigella dysenteriae None None Major porins
(OmpC
§
, OmpF
§
)
*OmpC family.
‡
OmpF family.
§
Identification of porin loss in resistant isolate.
||
Identification of
porin mutation in resistant isolate.
¶
Quiescent porin family.
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before the start of antibiotherapy were susceptible to
cephalosporins (cephalexin, cefazolin and cefoxitin)
31
.
However, only days after the start of cephalexin treat-
ment, a cephalosporin-resistant strain was isolated from
a wound drain sample. This direct clonal descendent of
the original pre-therapy strain exhibited complete resist-
ance to all cephalosporins. No significant increase in
β-lactamase activity was observed. The OmpC–OmpF
balance is strongly regulated by different genetic control
systems, such as EnvZ–OmpR and RNA anti-sense regu-
lators (MicF and MicC)
6,9–10,12,13
. In vitro, osmoregulation
of porin synthesis in the susceptible parental strain was
normal: only OmpC-type porins were expressed in high
osmolarity medium and both OmpC- and OmpF-type
porins were detected in low osmolarity medium. By
contrast, the resistant isolate expressed only OmpF-type
porins in low ionic strength conditions, and the synthe-
sis of OmpC- and OmpF-type porins was fully repressed
at high osmolarity, mimicking the conditions that are
present in vivo
31
.
These observations pinpoint a key step in bacterial
adaptation: expression of the OmpC porin with its
restrictive channel is generally favoured in vivo, mainly
owing to conditions of high osmolarity in patients. This
regulation naturally limits the entry of large, charged
molecules. The different diffusion rates of cepha-
losporins through OmpC and OmpF has been shown
using liposome swelling assays
32
. Continued exposure
to sub-inhibitory antibiotherapy selects for step by step
porin-expression modifications, resulting in further
reduced influx at each stage
14
. Complete impermeability
to β-lactams is achieved through total loss of the OmpC
porin in resistant isolates and can represent an ‘extreme
step’ in the porin adaptive response. This sacrifice can
result in severe loss of bacterial fitness owing to restricted
entry of nutrients, but can enable survival in the face
of intensive and continuous antibiotherapy
6,33
. A rapid
change in the balance of porin expression in response
to antibiotic treatment confers a noticeable advantage to
the pathogen compared with the commensal microflora
that is susceptible to β-lactams.
A possible option for Enterobacteriaceae to maintain
fitness following the loss of OmpC involves a further
possible porin exchange to exploit a quiescent porin
34
.
This novel porin subfamily (structurally related to
the OmpC and OmpF subfamilies), termed OmpN-
type porins, comprises E. coli OmpN, K. pneumoniae
OmpK37 and Salmonella enterica subsp. enterica serovar
Typhi (S. typhi) OmpS2. It has been demonstrated that
when OmpK37 is expressed in a porin-null K. pneumo-
niae strain, a strong decrease in β-lactam susceptibility
Box 1 | Effects of porin blockers
Polyamines are polycationic molecules that modulate the activity of various ion channels
83
. Among these, spermidine,
cadaverine and putrescine are produced by bacteria
83
. So far, few data are available concerning the in vitro effects of
polyamines on the diffusion of antibiotics through the porins of Escherichia coli and Enterobacter cloacae. Spermine has
been reported to inhibit OmpF channel properties
83–86
, to protect E. coli from colicin action and to decrease the diffusion
of norfloxacin and cefepime through OmpF
87
. In addition, cadaverine has been shown to reduce ampicillin and
cephaloridine susceptibility in E. coli by promoting an inhibition of ionic flux through cationic porins
85,88
. Thus, inhibition
of porin transport by excreted cadaverine might represent a mechanism that provides bacterial cells with the ability to
survive acid stress and nitrosative stress
89–91
. In the presence of antibiotics that use porins to cross the membrane barrier,
cadaverine might play the part of pore modulator and reduce the penetration rate of these antibacterial agents. The
conditions found in the intestinal tract favour the synthesis of polyamines and can be exploited by the enterobacterial
pathogens that colonize this site. This adaptive response might function to block the transport of toxic compounds, such
as bile acids and β‑lactam antibiotics.
Nature Reviews | Microbiology
Outer
membrane
Inner
membrane
Periplasm
Normal synthesis
of wild-type porin
Decreased synthesis
of wild-type porin
Normal synthesis of
restricted-channel porin
Normal synthesis
of mutated porin
Normal synthesis of
wild-type porin with
a channel blocker
Figure 1 | Multidrug resistance mechanisms associated with porin modification.
Shows the various resistance mechanisms that are associated with porin modification.
The β-lactam molecules and porin trimers are represented by blue circles and pink
cylinders, respectively. The thickness of the straight arrows reflects the level of
β-lactam penetration through porin channels. The curved arrows illustrate the uptake
failure that occurs with: a change (decrease) in the level of porin expression; an
exchange in the type of porin that is expressed (restricted-channel porin); and
mutation or modification that impairs the functional properties of a porin channel
(mutated porin). The effect of pore-blocking molecules (black circles) is shown at the
bottom of the figure.
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Bacteraemia
A medical condition in which
bacteria enter the bloodstream.
is observed compared with the susceptibility that is
observed when OmpK35 or OmpK36 is synthesized
at the same level
35
. This has so far only been illustrated
in vitro and, although the conditions required for native
expression of these quiescent porins are not yet known,
it seems that the structural organization of the internal
loop 3 of OmpN can constitute a selective filter for
charged molecules. Expression of these porins would
allow normal nutrient acquisition, but the presence of
a bulky tyrosine residue, located inside the pore eyelet,
could restrict the channel size and impair the penetration
of large β-lactams, such as cefotaxime or cefoxitime
35
.
Decreased porin expression. A study reported the effect
of imipenem on Enterobacter aerogenes strains collected
from four patients during the course of imipenem ther-
apy
36
(2–9 weeks). The emergence of resistant variants
occurred rapidly, within 5 days of the start of treatment.
Molecular epidemiological analysis indicated that the
resistant variants evolved from an original susceptible
E. aerogenes strain that belonged to the prevalent clone
37
.
In addition, restoration of imipenem susceptibility was
observed in isolates that were recovered a few days after the
treatment ended. This suggests that an efficient regulation
mechanism is involved in porin expression
36
. An asso-
ciation was reported between the presence of the major
Omp36 porin (OmpC homologue) and the β-lactam sus-
ceptibility of the various isolates that were collected during
this study. The absence of Omp36 always correlated with
β-lactam (cephalosporin and imipenem) resistance and
imipenem susceptibility
36
. These data clearly show that
in vivo antibiotic treatment can select for the emergence
of a resistant phenotype that is associated with porin loss
from an original susceptible isolate. The simultaneous
detection of an efflux pump in these resistant isolates
suggests that a complex process regulates both influx
and efflux
36,38
. In addition, up to 6% of highly β-lactam-
resistant E. aerogenes isolates collected over a 1-year
period lacked porins, indicating the importance of porin
regulation that is associated with β-lactamase production
in the emergence of β-lactam-resistant strains
39
.
Thiolas et al.
40
found a correlation between a suc-
cessive antibiotherapy, imipenem followed by colistin,
and the isolation of E. aerogenes strains with sequential
adaptive modifications. A decrease in porin produc-
tion was detected in the resistant isolates that were
collected during imipenem therapy, and a lipopolysac-
charide alteration associated with porin recovery was
reported in the isolates that were obtained during col-
istin therapy
40
. A similar scenario was reported in the
case of a patient who presented with bacteraemia due
to Enterobacter cloacae and was treated with imipenem
and amikacin over 3 weeks
41
. Two weeks after the ces-
sation of antibiotherapy, a resistant strain was isolated.
Characterization of the susceptible and resistant isolates
indicated that transcription of the major E. cloacae
porins was severely decreased and that the expression
of an inhibitor-sensitive efflux system was increased in
the resistant isolate
41
. This observation suggests that a
genetic regulation cascade through the Mar operon is
involved in porin expression. This regulon, which was
described in Enterobacteriaceae, comprises a repressor
(marR) that binds to an operator (marO) upstream of an
activator
5–7
(marA). De-repression of marA in response
to several chemical and antibiotic stresses triggers a cas-
cade of events that results in global control of membrane
permeability by the downregulation of porin synthesis
and overexpression of efflux pump components
5–8,15,42
.
In K. pneumoniae, several isolates have an insertion
sequence (such as IS5 and IS26) in the OmpK36 and
OmpK35 genes
43–45
. This insertion, which abolishes
porin expression, effects an efficient bacterial response
to β-lactam stress.
Mutations in porins. X-ray crystallography has resolved
the intricate structural details of several porins (reviewed
in REFS 10,11). The internal loop 3 forms a constriction
at about half the height of the β-barrel. This ‘eyelet’ gov-
erns channel size and ion selectivity
10,11
. In the eyelet,
several positively charged amino-acid residues (the
arginine cluster) on one side of the lumen face negatively
charged residues on the opposite side, creating a strong
electrostatic field that influences translocation through
the porin (BOX 2). The conserved internal loop 3 (REF. 46)
therefore constitutes a crucial region of the enterobac-
terial porin channel and has a major influence on the
influx of antibiotics. Genetic mutations in this loop
can alter the levels of susceptibility to antibiotics that
translocate porin channels. In this section, we focus
on naturally occurring porin mutations that have been
detected in clinical isolates.
Low et al.
47
performed a molecular study of several
E. coli strains that were collected during long-term anti-
biotherapy. Over a period of 2 years, a complex treat-
ment regimen, comprising the successive use of several
antibiotic classes (including fluoroquinolones, cepha-
losporins and carbapenems), was used. Seven isolates
from blood samples or liver abscesses were collected at
different stages of the treatment. These isolates exhibited
progressively increased levels of antibiotic resistance, and
all harboured the same two mutations (D18E and S274F)
in the OmpC porin, which might influence antibiotic
Box 2 | Porin structure and activity
The crystal structures of porins from Rhodobacter capsulatus and Escherichia coli
indicate the existence of a conserved 16‑strand anti‑parallel β‑barrel structure for each
monomer that contains a long internal loop, which is bent inside the pore
11
. OmpF of
E. coli is the best studied porin, both functionally and structurally. Its crystal structure
66
enabled better understanding of channel function properties, such as solute‑exclusion
limit and biological activity
9–11
. Using the three‑dimensional (3D) structure, several
OmpF mutants have been constructed to examine the role of specific residues during
assembly and function. In the pore constriction area (eyelet region), various
mutagenesis and electrophysiological studies have focused on the positive cluster
(Lys16, Arg42, Arg82 and Arg132) and the negative face (Asp113, Glu117 and Asp121),
which are important for the electrostatic field that governs the diffusion of charged
molecules
9,10
. The recent 3D structure of OmpC
67
, the major porin that is detected in
clinical Gram‑negative bacteria, indicates that the general organization of the channel
is well conserved between OmpF and OmpC, except that the respective pore lining is
altered at the extracellular entrance
66,67
(located just before the constriction region).
The comparison between OmpF, OmpC activity and the solved structure of the major
Klebsiella pneumoniae porin
92
, OmpK36, defines the two groups of major enterobacterial
porins, OmpC‑ and OmpF‑class porins (TABLE 1).
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