Effect of the global response regulator mora on the multi drug efflux pump MexCD oprj IN pseudomonas aeruginosa

CHAPTER 1
INTRODUCTION
P. aeruginosa is an opportunistic human pathogen which causes infections in individuals
immunocompromised as a result of burns or other severe trauma, underlying diseases,
including cancer, AIDS, diabetes and cystic fibrosis. Chronic lung infections caused by
P. aeruginosa is the main factor leading to the increased morbidity and premature
mortality seen in cystic fibrosis patients (1). The main reasons for persistence of P.
aeruginosa infections in hospital environment and in CF patients are attributed to its
ability to establish biofilms in lungs (CF patients), on implanted medical device or
damaged tissue and also to the emergence of multidrug resistant strains. Although
prolonged treatment with antibiotics is required to avoid a fast decline in the respiratory
functions of the infected patients, mutants resistant to multiple antimicrobials almost
constantly evolve and lead to failure of treatment (2). Hence, there is a great deal of
interest worldwide in understanding the basis of multidrug resistance so as to devise
suitable strategies to control multidrug resistant strains in hospitals and other
environments.
The pathogenesis of P. aeruginosa infections is multifactorial, as implicated by the
number and wide range of virulence determinants it possesses. These include, the
production and secretion of adhesions (biofilms), toxins (ExsS and ExoT via Type III
secretion system) and invasins (elastase, alkaline protease, hemolysins via Type II
secretion system), its motility, antiphagocytic surface properties, defense against immune
responses, genetic attributes (drug resistance) and ecological factors (2). One of the key

1


issues in understanding the complexity of P. aeruginosa pathogenicity is to uncover the
mechanisms that coordinately control some of these factors.
P. aeruginosa genome encodes proteins that are practically involved in all known
mechanisms of antimicrobial resistance and often these mechanisms work concurrently in
bestowing the multidrug resistant phenotype seen in this pathogen. Previously it was

believed that the limited permeability of the outer membrane of P. aeruginosa was the
main factor contributing to its multidrug resistance (3), but now it is clear that this
resistance is more due to the presence of specific antimicrobial efflux systems (4). There
are 428 such drug transporters present in P. aeruginosa at a density of 68% per Mbp of
genome which is among the highest occurrence in a single genome of any bacterial
species. Among these, clinically relevant antimicrobials are primarily accommodated by
the RND (Resistance Nodulation Division) family. Of these pumps, only MexAB-OprM
and MexXY-OprM (which are expressed constitutively in wild type cells and provide
intrinsic resistance) and MexCD-OprJ and MexEF-OprN (whose expression so far has
only been seen in acquired multidrug resistant strains) have been reported to provide
significant resistance to antibiotics when stably overproduced upon mutations. In this
study we are addressing the effects on the MexCD-OprJ and since it is an inducible pump
(its expression is induced by several chemicals and antibiotics used in hospitals),
studying the factors regulating/affecting expression of MexCD-OprJ is vital in controlling
the acquired resistance that develops in P. aeruginosa during antibiotic therapy.
We have previously identified a sensory regulator MorA that coordinately controls
motility and biofilm formation in P. aeruginosa and P. putida. The motility regulator
MorA controls the timing of flagella development and its loss led to changes in motility
2


and chemotaxis without affecting growth rate or cell size (5). As both motility and biofilm
formation are complex phenomenon, it suggested that MorA is likely to control multiple
targets directly or indirectly. Recent findings suggested that MorA regulates Type III
Secretion System in a transcriptional manner and it controls Type II Secretion in a posttranscriptional manner in P. aeruginosa (Ravichandran Ayshwarya’s PhD thesis). Hence,
system level analyses were conducted to unravel various pathways affected by MorA at
both transcriptional and post-transcriptional levels. A phosphoproteome analysis
indicated that MorA affects the phosphorylation state of several P. putida proteins
including signal transduction proteins, transcriptional regulators, flagellar associated
proteins and oxidative stress pathway proteins to name a few (6). MorA is predicted to be

involved in c-di-GMP signaling by virtue of its GGDEF (cyclase) and EAL
(phosphodiestrase) domains.

A global gene expression profiling demonstrated that over 80 genes were significantly
affected by the loss of morA, indicating its role as a high order or as a global regulator in
P. aeruginosa PAO1. Interestingly, the most affected genes were mexC, mexD and oprJ.
There was 4 to 20 fold increase in the RNA levels of the RND-type drug efflux pump
MexCD-OprJ operon cluster of MorA mutant strains, at early planktonic growth (Choy
Weng Keong’s PhD thesis). This finding was then first validated by quantitative real time
PCR (Xu Yanting’s thesis). Further validation was done by fusion of MexCD promoter to
reporter lacZ (Swee June’s report).

3


These preliminary results showed that MorA loss led to increased promoter mexCD
activity in a strain specific manner. Interestingly, this upregulation does not involve nfxB
which is the only known negative regulator of MexCD-OprJ operon.

Aim and Objectives:
Several findings suggest that cyclic-di-guanylate signaling might be involved in
coordinately regulating several pathogenicity related pathways at both transcriptional and
post-transcriptional levels. Hence, we build on this understanding by addressing the
effects on the multidrug efflux pump MexCD-OprJ and on the drug resistance phenotype
in P. aeruginosa by the response regulator MorA. To achieve this, the study had the
following specific objectives:

1. To study the time and strain dependent effects of MorA on the activity of promoter
of MexCD-OprJ.
2. To study the effects of MorA on the steady state RNA levels of mexCD-oprJ.

3. To study the effect of MorA on the drug resistance phenotype in P. aeruginosa
with respect to the MexCD-OprJ pump and how this is affected by constitutive
pump MexAB-OprM.

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CHAPTER 2
LITERATURE REVIEW
2.1 Bacteria used in our study – Pseudomonas aeruginosa
Pseudomonas aeruginosa is a member of the Gamma Proteobacteria class of bacteria. It
is a Gram-negative, aerobic rod belonging to the bacterial family Pseudomonadaceae.
There are eight groups in this family and P. aeruginosa is the type species of its group,
which contains twelve other members. P. aeruginosa strains have the ability to adapt to
and thrive in many ecological niches, particularly in soil and water, and also in plant and
animal tissues. It is capable of using more than 75 organic compounds as food sources;
this metabolic versatility contributes to its exceptional capability in colonizing ecological
niches where nutrients are limited.
P. aeruginosa strains are mono-flagellated and although the species is classified as an
aerobic bacterium, it can also be considered as a facultative anaerobe due to its ability to
proliferate under very low oxygen concentrations. It can grow especially well in moist
environments. Colony morphology exhibited by P. aeruginosa depends on the source
from which it is obtained. Natural isolates from soil/water produce small-rough colonies
while clinical isolates have a fried-egg or smooth mucoid appearance. The blue-green
appearance of these species is attributed to the production of two soluble pigments,
pyoverdin which is fluorescent and the blue colored pigment pyocyanin.
P. aeruginosa has a remarkable ability to form biofilms, which are dense bacterial
communities attached to a solid surface and surrounded by an exopolysaccharide matrix.
This protects it from adverse environmental factors and also contributes to antibiotic
5

resistance by forming a physical barrier to the entry of antimicrobial drug molecules
through the matrix. Molecular mechanisms that govern the switch from free-swimming
planktonic growth to the more resistant sessile biofilm phenotype are being studied
worldwide to improve treatment of the resistant bacteria. The process of biofilm
formation is complex and proceeds via many signaling pathways, which are regulated by
various signals like nutrient availability, temperature, osmolarity, pH, iron, and oxygen
(7). Pathogenesis of P. aeruginosa infections is multifactorial, as implicated by the

number and wide range of virulence determinants it possesses as shown in Table 2.1.
P. aeruginosa is an opportunistic pathogen, implying that it exploits some break in the
host defenses to initiate an infection. It is viewed as a highly adapted opportunistic
human pathogen, as P. aeruginosa strains do not normally infect uncompromised tissues.
Contrastingly, there are hardly any tissues that it cannot infect if the defenses are
compromised in any way .It can cause urinary tract infections, respiratory system
infections, dermatitis, soft tissue infections, bacteremia, bone and joint infections,
gastrointestinal infections and a variety of systemic infections, especially in
immunosupressed patients with severe burns and in cancer and AIDS patients. Infection
caused by P. aeruginosa occurs in three distinct stages: (1) bacterial attachment and
colonization; (2) local invasion; (3) disseminated systemic disease.
P. aeruginosa is the fourth most commonly-isolated nosocomial pathogen accounting for
10.1 percent of all hospital-acquired infections. Figure 2.1 illustrates the 133 clinical
isolates of P. aeruginosa that were collected from the infectious section unit of Loghman
Hospital, Iran from following sources; blood, urine, wounds, trachea, sputum, abscesses,
catheter, and body fluids during March 2007 to February 2008 (8). Hence, over the past
6


few decades, significant amount of research has been directed towards understanding the

factors implicated in its pathogenesis. The focus of this study is to investigate some of the
factors that coordinately regulate multidrug resistance with other virulence factors such
as motility and biofilm formation.
Table 2.1 Virulence Determinants of Pseudomonas aeruginosa
Virulence Determinant
1. Adhesins

Factors involved
a) Pili (N-methyl-phenylalanine pilli
b) Polysaccharide capsule (glycocalyx)
c) Alginate slime (biofilm)

2. Invasins

a) Elastase
b) Alkaline protease
c) Hemolysins (phospholipase and
lecithinase)
d) Cytotoxin (leukocidin)
e) Siderophores and siderophore uptake
systems

3. Motility/chemotaxis

a) Flagella
b) Retractile pili

4. Toxins

a) Exoenzyme S

b) Exotoxin A
c) Lipopolysaccharide

5. Antiphagocytic surface
properties

a) Capsules, slime layers
b) LPS
c) Biofilm construction

6. Defense against serum
bactericidal reaction

a) Slime layers, capsules, biofilm
b) LPS
c) Protease enzymes

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7. Defense against immune
responses

a) Capsules, slime layers, biofilm
b) Protease enzymes

8. Genetic attributes

a) Genetic exchange by transduction
and conjugation

b) Inherent (natural) drug resistance
c) R factors and drug resistance
plasmids

9. Ecological criteria

a) Adaptability to minimal nutritional
requirements
b) Metabolic diversity
c) Widespread occurrence in a variety
of habitats

(Online text book of bacteriology by Kenneth Todar)

Abssess
2%

Cattether
2%

B.Fluids
3%

Blood
10%

Urine
32%

Sputum

12%
Wound
13%

Trachea
26%

Fig. 2.1 The frequency and source of P. aeruginosa isolates collected from 133 patients
during 11 months from the infectious section unit of Loghman Hospital, Iran (8).
2.2 Antibiotic resistance mechanisms in bacteria
Over the years, physicians have been administering antibiotic therapy successfully to
treat various bacterial infections. As new infections have been on the rise, an arsenal of
drugs have been developed and regularly launched into the market. Due to frequent usage
8


in hospitals, sometimes in an irrational manner, the pathogens have been long exposed to
various antimicrobial agents. As a survival strategy, these human pathogens have evolved
various modes of antibiotic resistance such as antibiotic inactivation, target modification,
efflux pumps and outer membrane permeability changes and target bypass. The manner
in which the various bacterial species acquire antibiotic resistance may vary but the
mechanisms can be broadly classified under biochemical and genetic aspects as
categorized in Figure 2.2.

Fig.2.2 Types of biochemical and genetic mechanisms of antibiotic resistance in bacteria
(2).

9

Antibiotic inactivation is one of the major mechanisms which involve production of
certain enzymes in bacteria which can destroy or modify activity of the drug. Many
enzymes inactivate the antibiotics by cleaving their hydrolytically susceptible bonds like
amides and esters. The main hydrolytic enzyme produced by many Gram-positive and
Gram-negative bacteria is β-lactamase that cleaves the β-lactam ring of the penicillin and
cephalosporin antibiotics (9). Other enzymes such as transferases inactivate antibiotics
such as aminoglycosides, chloramphenicol and macrolides by adding adenyl, phosphoryl
or acetyl groups to the periphery of the antibiotic molecule thereby preventing it from
binding to the target.

Target modification involves changes in the antibiotic target site in the bacteria which
prevents the antibiotic from binding to its target. Peptidoglycan structure, ribosome
structure, rRNA, protein synthesis and DNA synthesis in bacteria are all important targets
for the various classes of antibiotics. Since these structures and processes are vital for the
cell, the bacteria cannot modify it to a major extent such that it affects the normal
functioning of the organism. Nevertheless, it can accommodate for mutational changes in
the target site that do not affect its cellular function but reduce susceptibility to antibiotic
inhibition (10). A wide range of antibiotics interfere with protein synthesis
(aminoglycosides,

macrolides,

chloramphenicol,

fusidic

acid,

streptogramins,

oxazolidinones) and resistance to these is mainly mediated by ribosomal modification
and mutations in rRNA. Resistance to the macrolide, lincosamide and streptogramin B
group of antibiotics referred to as MLS(B) type resistance results from a post
transcriptional modification of the 23S rRNA component of the 50S ribosomal subunit

10


(66). Mutations in the 23S rRNA have been associated with resistance to macrolide and

oxazolidinones. Mutations in the 16S rRNA gene confer resistance to the
aminoglycosides (67). These ribosome mediated resistance mechanisms are important as
they account for almost one-third of the bacterial antibiotic resistance.

Efflux pumps located in the membrane facilitate the export of antibiotics out of the cell
thereby maintaining low intracellular antibiotic levels. They impact all classes of
antibiotics in particular macrolides, tetracycline and fluoroquinolones because these
interfere with protein and DNA synthesis and therefore must be intracellular to exert their
effect. Both global and local regulators are involved in regulating efflux gene expression
in various bacteria. Efflux pumps are described in the next section in detail as they are the
focus of this study.
In terms of genetic aspects, antibiotic resistance emerges from mutations in cellular genes
or by the acquisition of foreign resistance genes or by a combination of both mechanisms.
Mutations in various chromosomal loci arise due to spontaneous mutations,
hypermutators and adaptive mutagenesis. On the other hand, foreign antibiotic resistance
elements can be acquired primarily through horizontal gene transfer mediated by
conjugation, transformation, or transduction.
2.3 Types of Multidrug Efflux Pumps in Bacteria
Multidrug efflux pumps are transporters known to extrude structurally different organic
compounds. Drug transporters or efflux pumps are the major determinants of resistance

to antibacterials in virtually all cell types, ranging from prokaryotic to eukaryotic cells.
11


Their ability to extrude a wide spectrum of structurally unrelated drugs/compounds
makes them one of the major factors contributing to multidrug resistance (MDR) in
pathogenic strains. In the case of P. aeruginosa, the genome is quiet packed with
transporters of different types (Transport Classification Database: www.tcdb.org).
Bacterial multidrug efflux transporters are generally classified into five super-families,
primarily based on the amino acid sequence homology. These include the major
facilitator superfamily (MFS), the ATP-binding cassette (ABC) family, the resistancenodulation-division (RND) family, the small multidrug resistance (SMR) protein family
and, very recently, the multidrug and toxic compound extrusion (MATE) family as
represented in Figure 2.3 (11).

Fig. 2.3 Members of the five characterized super-families of multidrug efflux pumps in
bacteria (11,12). MFS - Major Facilitator Superfamily ; SMR - Small Multidrug
Resistance family ; MATE - Multidrug And Toxic compounds Efflux family ; RND Resistance/Nodulation/Cell Division family and ABC - ATP-binding cassette
superfamily.

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The MFS family represents one of the largest groups of secondary active transporters
with well characterized multidrug pumps like Bmr and Blt of Bacillus subtilis (13),
MdfA of E. coli (14), LmrP of Lactobacillus lactis (15), NorA and QacA of S. aureus(16).
As monomers, they can function to export drugs only into the periplasm while in Gram negative bacteria they associate with MFPs and OM channels to export out the substrate
across the two membranes (17). Functioning of MFS, includes solute uniport,
solute/cation symport, solute/cation antiport and solute/solute antiport with inwardly
and/or outwardly directed polarity. Apart from its role in drug efflux, MFS permeases are
also involved in the transport of simple sugars, oligosaccharides, inositols, amino acids,

nucleosides, organophosphate esters and a large variety of organic and inorganic anions
and cations (18).
The SMR family includes proton driven drug efflux pumps as represented by EmrE in E.
Coli (19) and it pumps the substrates into the periplasmic space. This family comprises of
more than 250 annotated members, which are classified into three groups – small
multidrug pumps, the paired SMR proteins and suppressors of groEL mutant proteins.
Apart from substrate specificity towards cationic dyes, QACs and disinfectants it can also
accommodate clinically relevant antibiotics like aminoglycosides, amikacin and
vancomycin (20).
The MATE family consists of sodium ion-driven drug efflux pumps such as NorM from
Vibrio parahaemolyticus. They provide resistance to multiple cationic toxic agents
including fluoroquinolones (17). The range of substrates that it pumps out is much
narrower compared to RND family and only approximately 20 transporters have been
characterized to date (21).
13


The ABC transporters are a very large family, members of which collectively export a
wide array of substrates and as the name suggests they are driven by ATP hydrolysis.
Main examples include P-glycoprotein and LmrA from Lactococcus lactis. They are
conserved from bacteria to humans with about 48 ABC transporters present in humans
and 80 in the gram-negative bacterium E. coli. In bacteria, they function in the efflux of
surface components of the bacterial cell, proteins involved in bacterial pathogenesis,
peptide antibiotics, heme, drugs and siderophores (22).
Mainly, it is the drug exporters of the RND family that are primarily responsible in
providing the clinically relevant antibiotic resistance in the Gram-negative bacteria like
P. aeruginosa.
2.4 Structure, Mechanism and Regulation of the Bacterial Multidrug Efflux Pumps
Bacterial drug efflux pumps are categorized into five families, i.e., ABC superfamily,
MFS, MATE family, SMR family and RND superfamily as explained in section 2.3. A

significant amount of research has been done in the structural and biochemical
elucidation of these pumps. Crystal structures are available for many MFS transporters
such as the lactose/H+ permease LacY, the glycerol-3-phosphatetransporter GlpT and the
multidrug transporter EmrD all from E. Coli. The structural feature common to most
MFS members is the folding pattern consisting of two transmembrane domains that
surround a substrate translocation pore (68). The EmrD structure (Fig.2.4) has an interior
with mostly hydrophobic residues and displays two long loops extended into the inner
leaflet side of the cell membrane which serve to recognize and bind substrates directly

14


from the lipid bilayer (69). LmrP of L. lactis functions as a facilitated diffusion catalyst in
the absence of proton-motive force (70).
The SMR family is represented by EmrE of E. coli, which functions as a homodimer of a
small four-transmembrane protein (71). However, there are two opposing views regarding
the orientation of the two protomers within the dimer. While biochemical studies show
that the two protomers are inserted into the membrane in a parallel orientation (72), x-ray
crystallography suggests an antiparallel orientation (73). It has been shown that a
minimum activity motif of G90LxLIxxGV98 within the fourth transmembrane segment
mediates the SMR protein dimerization (74).
The MATE family is represented by NorM of Vibro parahaemolyticus and they confer
resistance by acting as H+- or Na + antiporters. Many of the bacterial MATE pumps have
been identified by expression in a heterologous, antimicrobial-hypersusceptible E. coli
and till now no crystal structures are available for any MATE transporters (17).
The structure of the S. aureus Sav1866 multidrug exporter (Fig 2.4) has provided insight
into ABC transporter mediated drug efflux. The outward-facing conformation of Sav1866
is triggered by ATP binding. In this state, the two nucleotide-binding domains are in
close contact and the two transmembrane domains form a central cavity through which
the drug is assumed to pass through. This cavity is shielded from the lipid bilayer and

cytoplasm but it is exposed to the external medium. On the other hand, the inward-facing
conformation is caused by dissociation of the hydrolysis products adenosine diphosphate
(ADP) and phosphate, and shows the substrate-binding site accessible from the cell

15


interior (75,76). The structure and molecular mechanism of RND pumps has been
explained in detail in section 2.6.

Fig. 2.4 Crystal structures of the multidrug efflux transporters exemplified by RND type
AcrAB-TolC (instead of AcrA, the complete structure of its homologue MexA is shown)
and MFS type EmrD of E. coli and ABC type Sav 1866 of S. aureus (17).
In comparison to the limited achievements in understanding the structure-function
relationships of the drug transporters, a significant amount of research has been put into
unraveling the regulatory pathways that govern the expression of these drug transporters.
In the case of bacterial efflux pump genes which are inducible, there are very few
instances in which translational control is the primary level at which expression is

16


controlled. Expression of a majority of the drug transporter genes known to be subject to
regulation is controlled by transcriptional regulatory proteins. These regulators include
both activators and repressors of target gene transcription, a process that can occur at
either the local or global level.
Local regulators of drug transporter genes include the E. coli TetR repressor of
tetracycline efflux genes and three regulators of MDR transporter genes, the B. subtilis
BmrR activator, the S. aureus QacR repressor and the E. coli EmrR repressor. Examples
of global regulators include the MarA, Rob and SoxS global activators in E. coli (78).

Two-component regulatory systems are increasingly being found to be linked with drug
efflux genes. In general, the regulators of bacterial drug transporter genes belong to one
of four regulatory protein families, the AraC, MarR, MerR, and TetR families. The
classification of the regulators into their respective families is based on similarities
detected within their DNA-binding domains. All drug transport regulators in general
possess α-helix-turn-α-helix (HTH) DNA-binding motifs, which are embedded in larger
DNA-binding domains that form a number of structural environments like three-helix
bundles and winged helix motifs (77). For the four local regulators of efflux pumps, the
portions of these proteins not involved in forming the DNA-binding domains are shown
to directly bind substrates of their cognate pumps. This, in turn, acts as a signal to
increase the synthesis of the respective transport protein(s) in response to these toxic
compounds (78).

17


2.5 Role of efflux pumps in the multidrug resistance of P. aeruginosa
There are various mechanisms of antibiotic resistance, which can be adopted by the
bacteria. Furthermore, it displays practically all known mechanisms of antimicrobial
resistance and often these mechanisms work together in bestowing the multidrug resistant
phenotype seen in this pathogen. These mechanisms include constitutive expression of
AmpC β-lactamase, production of plasmid or integron mediated β-lactamases from
different molecular classes, overexpression of efflux pumps that have a wide substrate
specificity, lower outer membrane permeability (loss of OprD proteins), synthesis of
aminoglycoside modifying enzymes and structural alterations of topoisomerases II and
IV determining quinolone resistance (23). Some of the major mechanisms are illustrated
in Figure 2.5.
Efflux is a general mechanism contributing to bacterial resistance to various antibiotics.
P. aeruginosa is known to possess intrinsic resistance to multiple antimicrobial agents
and also develops acquired multidrug resistance during antibiotic therapy. Majority of

this resistance is attributed to the multidrug efflux pumps present in these bacteria. In P.
aeruginosa, 428 such drug transporters are known to be present at a high density in its
genome. Among the major families of bacterial multidrug efflux transporters, clinically
relevant antimicrobials are primarily accommodated by the RND (Resistance Nodulation
Division) family. The four well characterized pumps coming under RND family and
major contributors to antibiotic resistance in P. aeruginosa are MexAB-OprM and
MexXY-OprM which are responsible for the intrinsic resistance and MexCD-OprJ and
MexEF-OprN which account for acquired resistance. We focussed on the inducible pump
MexCD-OprJ in this study.
18


Mutations in the regulators of these pumps are primarily responsible in increasing
expression of pumps which in turn, confer significant resistance to the respective
antibiotics. Overexpression of these RND pumps has been identified in the clinical
isolates of P. aeruginosa, and hence there is a great deal of interest in understanding the
regulation of these pumps. The regulators of these pumps itself are looked upon as
suitable antibiotic targets.
Both local and global regulators have been identified in various strains. The pump
components are encoded by an operon and often adjacent to the pump, the regulatory
gene is present. For example, in P. aeruginosa the MexCD-OprJ pump is encoded by an
operon and its negative transcriptional regulator nfxB is present next to it on the
chromosome. The only well known regulator of MexCD-OprJ in P. aeruginosa is the
negative transcriptional regulator nfxB and point mutations in nfxB lead to overexpression
of this pump and significantly higher antibiotic resistance. No other regulator has been
characterized in literature so far. Hence our study on understanding the regulation of
MexCD-OprJ pump in P. aeruginosa by the global response regulator MorA is novel in
this aspect.
Some pumps are regulated by two-component systems which intermediate the adaptive
responses of bacterial cells to their environment. Different global transcriptional

regulators like MarA, SoxS and Rob have been identified to be involved in regulating
efflux systems (24-26).

19


Fig.2.5 (A) Activity of antibiotics - fluoroquinolones and carbapenems in “wild-type”
susceptible P. aeruginosa expressing basal levels of AmpC, OprD, and nonmutated
fluoroquinolone target genes (gyrA, gyrB, parC, and parE) (27).
Fluoroquinolone molecules move through the outer membrane, peptidoglycan, periplasm,
and cytoplasmic membrane and interact with enzymes DNA gyrase and topoisomerase
IV targets, which are complexed with DNA in the cytoplasm. Carbapenem passes
through OprD, an outer membrane porin and interacts with penicillin binding proteins
(PBPs) situated on the outer cytoplasmic membrane.

20


Fig. 2.5 (B) Mutational resistance to fluoroquinolones and carbapenems involving
chromosomally encoded mechanisms expressed by multidrug resistant P. aeruginosa (27)
Fluoroquinolone resistance is mediated by (i) overexpression of RND efflux pumps
which export the drug molecules from the periplasmic and cytoplasmic spaces and/or (ii)
mutations in the fluoroquinolone target genes. The quinolone resistance determining
region (QRDRs) in target genes are highlighted in yellow.
Carbapenem resistance is mainly mediated by (i) decreased production or loss of
functional OprD in the outer membrane and/or (ii) overproduction of RND efflux pumps.
Minor changes in susceptibility are seen due to overexpression of AmpC, adding to the
resistance (27).

21

2.6 Structure, function and regulators of the RND (Resistance/Nodulation/Cell
Division) family of pumps in P. aeruginosa
While the primary active transporters of the ABC superfamily use ATP hydrolysis for
energy, the RND family is a secondary active transporter which utilizes the proton motive
force for export. RND pumps typically exist as a tripartite system with an outer
membrane factor (OMF), a cytoplasmic membrane (RND) transporter and a membrane
fusion protein (MFP) which links these two. Each of the three components is encoded by
genes present on an operon in the P. aeruginosa chromosome. While the P. aeruginosa
genome encodes efflux pumps from the five major families, majority of the pumps
belong to the RND family.
The three components of the pump assemble together into a complex that spans across
the entire membrane acting as a channel through which lipophilic and amphiphilic drugs
from the periplasmic space and cytoplasm are expelled out into the extracellular
environment as represented in Figure 2.6. There are ten RND systems in P. aeruginosa
out of which only four have been identified to contribute significantly to antibiotic
resistance when overproduced due to mutations. These include MexA-MexB-OprM and
MexX-MexY-OprM which are expressed in wild type cells and MexC-MexD-OprJ and
MexE-MexF-OprN which are inducible pumps in the MDR mutant strains (28).

22


Fig. 2.6 Structure and function of RND efflux pumps in P. aeruginosa
The pump extrudes antibiotics from periplasmic space and cytoplasm using proton
motive force (27).
Substrates of the Mex efflux RND pumps include antibiotics, biocides, dyes, detergents,
organic solvents, aromatic hydrocarbons, and homoserine lactones (29). Although they
have significant overlap in terms of substrate specificity they still contribute to unique

phenotypes based on their expression levels. Apart from the protective effects against

23


antimicrobials these pumps may also have a physiological role in P. aeruginosa (e.g.,
cell-to-cell communication and pathogenicity) (30).
MexAB-OprM was the first pump to be characterized in P. aeruginosa and it is the main
constitutive pump in wild type cells providing intrinsic resistance to a wide range of
antibiotics. It has an important role to play in beta lactam resistance and is unique in this
aspect as beta lactams are usually not pumped out by efflux. MexAB-OprM is
overexpressed in nalB mutants by 4-11 fold (mutations in MexR, a negative regulator of
MexAB-OprM) which display high antibiotic resistance (31). It is also overexpressed in
nalC and nalD mutants. The MexAB-OprM or OprM deletion strains in P. aeruginosa are
hypersusceptible to most antibiotics, especially carbenicillin and cephems to name a few
indicating its vital role in the organism.
The expression of MexAB-OprM has been shown to be growth-phase-dependent, with
maximum expression occurring at late log/ early stationary phases (32). This upregulation
has been attributed to a quorum sensing signal. The homoserine lactone - C4-HSL
synthesized as part of the rhl quorum sensing system and was shown to increase
expression of mexAB-oprM (33). The mexAB-oprM deletion strain K1119 in P.
aeruginosa, was found to be less invasive than the WT strain. This study strongly
suggested that the invasion determinants are predominantly exported by P. aeruginosa
via MexAB-OprM and partially by MexXY-OprM and other systems.
MexCD-OprJ is not expressed at high levels in wild type and so does not contribute to
intrinsic resistance. It is overexpressed by 20-70 fold in nfxB mutants, where it is known

24

to provide significant antibiotic resistance. Since it is the focus of our study, it is
discussed in detail in the following section.
MexEF-OprN is quiescent in wild type cells and is expressed in the nfxC type mutants.
These mutants show resistance to fluoroquinolones, chloramphenicol, trimethorpim and
imipenem (34). Unlike MexAB-OprM and MexCD-OprJ, this pump is not negatively
regulated. It is affected by MexT, a positive regulator of the pump. Interestingly, the
mexEF-oprN expression is also regulated by MvaT, a member of the histone-like
nucleoid structuring protein family, which acts as a global regulator of genes involved in
virulence, housekeeping, and biofilm formation. It was found that mvaT affected mexEFoprN in a mexT independent manner, most probably by some indirect effect (35).
MexXY-OprM was recently described in P. aeruginosa and lacks a linked outer
membrane gene and instead used OprM as its outer membrane component (36). It is
mainly linked to aminoglycoside resistance and mexZ is identified as negative regulator
of the pump. mexXY expression is induced when P. aeruginosa is grown in the presence
of tetracycline, erythromycin, and gentamicin and multiple pathways are involved in its
induction (37).
All characteristics of the Mex efflux pumps in P. aeruginosa including substrate profile,
components of the pump and its regulators are summarized in Table 2.2.

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