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

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 fa

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

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

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 post-

transcriptional 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)

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

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

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

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 Factors involved

1 Adhesins a) Pili (N-methyl-phenylalanine pilli

b) Polysaccharide capsule (glycocalyx) c) Alginate slime (biofilm)

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

b) Exotoxin A c) Lipopolysaccharide

7 Defense against immune

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)

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

Urine 32%

Trachea 26%

Wound 13%

Sputum 12%

Blood 10%

Abssess 2%

Cattether 2%

B.Fluids 3%

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)

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

(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

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 resistance-nodulation-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

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)

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

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

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

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)

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

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)

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

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)

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)

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

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

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 oprN in a mexT independent manner, most probably by some indirect effect (35)

mexEF-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

Table 2.2 Characteristics of RND efflux pumps in P aeruginosa (27)

Opero

n

Component Function Regulator Substrate(s)

Primary Secondary Antibiotics Additional

MexR NalD

NalC

Fluoroquinolon

es, β-lactams, β-lactamase inhibitors, tetracyclines, chloramphenic

ol, macrolides, novobiocin, trimethoprim, sulfonamides

Biocides (e.g., triclosan), detergents, dyes, HSL, aromatic hydrocarbon

es, β-lactams, tetracycline, chloramphenic

ol, macrolides, trimethoprim, novobiocin

Biocides (e.g., triclosan), detergents, dyes, aromatic hydrocarbon

s mexEF

-oprN MexE

MexF

OprN

MFP RND OMF

MvaT

Fluoroquinolon

es, chloramphenic

ol, trimethoprim

Biocides (e.g., triclosan), aromatic hydrocarbon

s mexXY

MFP

RND

MFP RND

es, β-lactams, tetracycline, aminoglycoside

s, macrolides, chloramphenic

ol

2.7 Regulators and factors affecting mexCD-oprJ expression in P aeruginosa

High levels of expression of mexCD-oprJ pump are observed in nfxB mutants where it contributes to the high resistance to antibiotics Two classes of nfxB mutants have been

studied, expressing moderate (type A, ~20 fold) or higher (type B, ~30 fold and higher) levels of the efflux system, with resistance levels corresponding to efflux gene expression Changes in susceptibility were higher for Type B than for Type A mutants and OprJ production was also higher in the former strain Type A mutants have four to eight times more resistance to ofloxacin, erythromycin, and new zwitterionic cephems,

i.e., cefpirome, cefclidin, cefozopran, and cefoselis, than the parent strain PAO1 Type B

mutants, on the other hand were more resistant to tetracycline and chloramphenicol, as well as ofloxacin, erythromycin, and the new zwitterionic cephems than PAO1, and were four to eight fold more susceptible to carbenicillin, sulbenicillin, imipenem, panipenem, biapenem, moxalactam, aztreonam, gentamicin, and kanamycin (38)

The above mentioned nfxB mutants have point mutations in the nfxB gene nfxB lies upstream of mexCD-oprJ in the genome and apart from being a negative regulator of the

pump it is also negatively autoregulates its own expression The influence of the cloned

nfxB gene on expression of a promoter mexC–lacZ fusion was assessed in E coli DH5α and it revealed that nfxB reduced the β-galactosidase activity by 65 fold confirming its role as a negative transcriptional regulator of mexCD-oprJ (39) Until now, NfxB is the

only well characterized regulator of the MexCD-OprJ pump in P aeruginosa

Overexpression of the MexCD-OprJ in nfxB mutants decreases Mex-AB-OprM and MexXY expression (40)

Since OprM is also expressed in nfxB mutants, to elucidate whether

MexAB-OprM or MexCD-OprJ is involved in the extrusion of certain agents, a study was

performed with strains in which mexA-mexB-oprM region was deleted from the chromosome of wild-type and nfxB mutants of P aeruginosa This study showed that

MexCD-OprJ system had a higher specificity in causing extrusion of the fluoroquinolones and fourthgeneration cephems and a lower specificity to cause the extrusion of tetracycline and chloramphenicol than MexAB-OprM (41)

A study performed by Li et al established that in the absence of a functional OprM, there was an increase in gene expression of inducible pumps MexCD-OprJ and

MexAB-MexEF-OprN However, increase in expression of mexCD-oprJ (which was not as high

as that for nfxB mutants) was not sufficient to functionally compensate for MexAB-OprM

in terms of antibiotic resistance Given the substrate overlap between the pumps, it was proposed that although there was no change in antibiotic resistance, MexCD-OprJ may effectively replace MexAB-OprM in the export of the unknown natural substrates of these pumps The compensatory changes seen in the inducible pumps when the main pump was knocked out indicated the possibility of a global regulation of these MDR

pumps in P aeruginosa (42)

Expression of mexCD-oprJ was induced in response to clinical disinfectants like

benzalkonium chloride and chlorhexidine gluconate and other chemicals like tetraphenylphosphonium chloride, ethidium bromide, rhodamine 6G, and acriflavine but not in response to clinically relevant antibiotics (43) The fact that the pump is induced by

clinical disinfectants stresses on the important role that MexCD-OprJ plays in P aeruginosa strains resistance in the hospital environment where these disinfectants are

used on a regular basis Induction of mexCD-oprJ by membrane damaging agents (i.e.,

chlorhexidine) is dependent upon the stress response sigma factor AlgU (44)

To uncover the molecular mechanisms of substrate recognition by the multidrug resistance (MDR) pumps, a group isolated spontaneous mutations that had altered the

substrate specificity of the MexCD–OprJ pump from P aeruginosa Their results

indicated that the precise structure of the periplasmic loops of MexD determined the rate

of transport of the substrates by the pump (45)

The Type III secretion system (T3SS) is used by P aeruginosa to deliver toxins directly

into the cytoplasm of the host cell Overexpression of either MexCD-OprJ or

MexEF-OprN is associated with the impairment of Type III Secretion System in P aeruginosa

but overexpression of MexAB-OprM or MexXY had no effects on the system The overproduction of MexCD-OprJ/MexEF-OprN was shown to be linked with a reduction

in the transcription of the T3SS regulon due to the lack of expression of the exsA gene,

encoding the master regulator of the system (46)

2.8 Global regulators known to affect multidrug efflux pumps in bacteria

Due to the presence of numerous multidrug efflux systems in bacteria and their overlapping functions, there is a need for a well regulated expression of these pumps involving both local and global regulators Different global transcriptional regulators like

MarA, SoxS and Rob have been identified to be involved in regulating efflux pumps in E Coli In P aeruginosa, as described in the earlier sections an example would include the effect of global regulator MvaT on the MexEF-OprN system Deletion of mvaT resulted

in increased resistance to chloramphenicol and norfloxacin, but higher susceptibility to imipenem, and this was linked to the increased expression of mexEF-oprN (35)

Multiple-antibiotic-resistance (Mar) mutants of E coli show increased resistance to a wide range of structurally unrelated antibiotics and RND pump AcrAB plays a major role

in the antibiotic resistance phenotype of these mutants MarA of the marRAB operon is a global regulator known to affect several genes in E coli (24).

In Salmonella hadar, Rma plays an important role concerning antibiotic resistance via the

global regulation of several MDR efflux pumps The development of the resistance

phenotype was attributed to elevated expression of pump acrAB, and is also associated with that of pumps acrEF and mdtABC (47)

Hence, we can see the involvement of many global regulators in affecting the expression

of multidrug efflux pumps Our focus is on the effect of one such global regulator MorA

on the MDR pump MexCD-OprJ in P aeruginosa

2.9 3',5'-cyclic diguanylic acid (c-di-GMP) as a secondary messenger in bacteria

Cyclic diguanylate (c-di-GMP) is a bacterial secondary messenger of growing importance involved in the regulation of a number of complex physiological processes in the organism They relay signals received at the cell surface from the first messengers to the target molecules within the cell Secondary messengers have been studied primarily for their regulatory functions in eukaryotic cells and yet some play critical roles in regulating basic processes in bacteria Recently a number of complex systems have been identified for regulating the intracellular c-di-GMP concentration and an equally large number of regulatory targets of c-di-GMP have also been established (48)

c-di-GMP was first identified as an allosteric activator of cellulose synthase in the grape

associated bacterium Gluconacetobacter xylinus (49) The diguanylate cyclase (DGC) and

phosphodiesterase (PDE) enzymes capable of synthesizing and degrading c-di-GMP were

identified in G xylinus

Based on conserved amino acid residues, GGDEF was identified as the conserved protein domain with DGC activity and EAL as the conserved protein domain with PDE activity These protein domains are encoded in the genomes from diverse branches of the phylogenetic tree of bacteria (50) The large variety of known input signals and output of c-di-GMP metabolism, mediated via the GGDEF and EAL domains is represented in Figure 2.7 The GGDEF domain catalyzes the formation of c-di-GMP from two GTPs; the GG(D/E)EF motif itself is the active site c-di-GMP is hydrolyzed by EAL domain phosphodiesterase A (PDEA), resulting in the linear molecule 5′-pGpG, which is thought

to be biologically inactive and can be rapidly hydrolyzed by other PDEs in cell (48)

2.10 Phenotypes regulated by c-di-GMP and their roles in bacterial pathogenesis

While genes/proteins affected by c-di-GMP vary between bacterial species, one unanimous theme that has emerged is that c-di-GMP activates biofilm formation while inhibiting motility, thus regulating the transition between sessile and motile lifestyles Furthermore, c-di-GMP also affects the expression of virulence factors All these have major repercussions on the ability of various pathogens to cause disease (48)

Fig 2.7 Known input signals and output of c-di-GMP metabolism

Various domains N-terminal of GGDEF or EAL receive and transmit the input signals (left) and the output behaviour by variation of c-di-GMP concentration is shown on the right (51)

2.10.1 Regulation of motility by c-di-GMP

c-di-GMP is known to inhibit bacterial locomotion of various types, including swimming, swarming, and twitching motility The first major evidence of c-di-GMP regulation of

twitching motility was observed when there were mutations in P aeruginosa fimX gene,

which encodes a protein containing REC, PAS, GGDEF, and EAL domains (52)

Downregulation of flagellar motility by c-di-GMP also has been shown in Salmonella typhimurium and V cholerae It is becoming apparent that bacteria can regulate different

types of motility through regulation of DGC/PDE activity Since motility is important in

the earlier steps when pathogens colonise the host cells, the c-di-GMP-mediated regulation of this process is important for pathogenesis

2.10.2 Regulation of Biofilm Formation by c-di-GMP

Another important process regulated by c-di-GMP is the production of extracellular polysaccharides (EPS) especially those that constitute the extracellular matrix (ECM) for biofilm formation and support Biofilms are complex communities of microbial species that adhere to a surface and are often held together in a hydrated polysaccharide matrix c-di-GMP activates biofilm formation in a variety of bacteria, including many pathogens

such as P aeruginosa, Salmonella typhimurium, Vibrio spp., and Y pestis Biofilm formation in P aeruginosa is affected by various factors like EPS production,

chemotaxis, and quorum sensing An activation of the DGC WspR via loss of WspF is an

example in P aeruginosa where the increase in biofilm formation is due to increase in

EPS formation (53) Example of MorA stated above is also an example of a c-di-GMP mediated regulation of biofilm formation (54)

2.10.3 Regulation of Virulence Gene Expression

C-di-GMP modulation is known to directly modulate virulence properties and virulence

factor expression In V cholera, VieA response regulator/PDEA is involved in virulence

gene expression (55) Another example where c-di-GMP had an inhibitory effect on

virulence of Salmonella typhimurium involved the EAL domain-encoding gene cdgR

(56)

In P aeruginosa, mutations in two PDEA genes weakened pathogenicity of P aeruginosa in a murine model of pneumonia, and two mutations in GGDEF-EAL domain

proteins with degenerate GGDEF domains and predicted PDEA activity also reduced pathogenicity (57)

Although there is data that indicates that c-di-GMP represses virulence, there are exceptions where DGC enzymes have shown to contribute to infection Hence it appears that pathogenecity is controlled by c-di-GMP in a complicated manner invoving DGC, PDE and targets regulated by d-di-GMP

2.11 MorA – A membrane bound motility regulator in Pseudomonas and its effects

on biofilm formation

MorA was first identified in to be a novel membrane-bound protein in P putida, where it

controls the timing of flagella development and its loss led to changes in motility, chemotaxis and biofilm formation without affecting the growth rate or cell size (5)

In P aeruginosa, loss of MorA led to impairment of biofilm formation in a dependent manner similar to the case in P putida Initially the morA mutant formed a biofilm 70% smaller compared to PAO1 (P aeruginosa) wild type, but this difference

time-gradually narrowed down with time as illustrated in Figure 2.8 (A) Furthermore, there

was complete restoration of phenotype when the morA mutant was complemented with pUPMR (full-length morA gene cloned into pUCP19 vector) as shown in Figure 2.8 (B)

Thus, it was clear that MorA plays an important role in the early establishment stages of

biofilm in P aeruginosa PAO1

A B

Fig 2.8 MorA affects biofilm formation in P aeruginosa PAO1 (5)

A) Adherent cells in the biofilms formed on polystyrene surfaces at 3 and 10 h after inoculation were stained with crystal violet and examined by transmitted light microscopy

B) Biofilms were formed in polystyrene tubes, stained with crystal violet and the stain released in ethanol was quantitated by spectrophotometry at 595nm at various time intervals Plasmids pUCP19 and pUPMR are the vector control and the expression plasmid for PaMorA from its native promoter, respectively

2.12 MorA mediated regulation involves signaling via the secondary messenger cyclic diguanylate (c-di-GMP)

MorA is highly conserved in genomic context and structure among the various

Pseudomonas species with a sequence similarity of 58 - 93% Members of the

3h

10h

Pseudomonas MorA family share many features in common like (i) they are present as a

single copy in the genome, (ii) they possess 1-2 transmembrane domains, (iii) have a central sensory domain consisting of PAS-PAC motifs, and (iv) have a C-terminal catalytic domain consisting of GGDEF and EAL domains The various domains are represented in Figure 2.9 The N-terminal transmembrane region is more variable while the PAS-PAC motifs and GGDEF-EAL domains are highly conserved among the various species The domain architecture of the MorA family members in the various

Pseudomonas species is illustrated in Figure 2.10 (5) Furthermore, in Pseudomonas species, P aeruginosa (PAO1) alone has the mexCD-oprJ-nfxB gene cluster present

adjacent to morA in the genome as shown in Figure 2.11 This is an interesting aspect, since we are studying the effects on MexCD-OprJ pump by MorA

The diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes capable of

synthesizing and degrading c-di- GMP were first identified in G xylinus and were found

to contain two conserved domains, termed GGDEF and EAL, based on conserved amino acid residues These domains were also found in many other bacterial proteins (50) These domains are known to play a role in the regulation of various processes including cell development, virulence, motility and cellulose biosynthesis.(58-60) The PAS-PAC motifs are also present in many prokaryotes in combination with GGDEF-EAL domains and are stipulated to act as sensors for light, redox potential or oxygen concentration (61)

Thus, MorA by virtue of its GGDEF/EAL domains is predicted to be involved in

signaling via this novel secondary messenger cyclic-di-GMP

Fig 2.9 Conserved domains of MorA present in the Pseudomonas species –PAS and

PAC domains, GGDEF and EAL domains (5)

P putida MorA

P fluorescens (Pf-5) MorA

P aeruginosa PAO1 MorA

Fig 2.10 Domain architecture of MorA family members in the various Pseudomonas

species

The three conserved regions of the predicted MorA proteins are (i) a transmembrane domain(s) (vertical bars), (ii) sensory PAS and PAC motifs, and (iii) catalytic GGDEF (DUF1) and EAL (DUF2) domains These domains were predicted by using the Simple Modular Architecture Research Tool (http://smart.embl-heidelberg.de) (5)

PAS and PAC domains

GGDEF domain

EAL domain

Transmembrane Redox Sesory Catalytic

PA4596

P.aeruginosa PAO1

Fig 2.11 Arrangement of operon mexCD-oprJ, its known negative regulator nfxB and

response regulator morA on the PAO1 chromosome (5)

viiK

CHAPTER 3

MATERIALS AND METHODS

3.1 Bacterial strains and plamids used in this study

P aeruginosa strain K767 and its various mutants were used in our study as described in Table 3.1 P aeruginosa strains were cultivated in Luria-Bertani (LB) (Difco) medium at

37oC with suitable antibiotics including chloramphenicol and carbenicillin However for antibiotic susceptibility testing alone, the medium used was Mueller Hinton II Broth/Agar (BBL™)

Table 3.1 Bacterial strains and plamids used in this study

Source

(2001) K767 ∆morA K767 MorA deletion straina, Cmr This study

K1119 K767 MexAB-OprM deletion strain, Cmr K Poole et al.,

(2001) K1119 ∆morA K767 MexAB-OprM MorA double deletion

K767 ∆morA

PmexCD

K767 MorA deletion strain with P mexCD -lacZ

transcriptional fusion at attTn7 siteb, Cmr

This study

K1119 PmexCD K1119 with a P mexCD -lacZ transcriptional

fusion at attTn7 siteb, Cmr

This study

K1119 ∆morA

PmexCD

K1119 MorA deletion strain with P mexCD -lacZ

transcriptional fusion at attTn7 siteb, Cmr

This study

K767 ∆morA

PmexCD pUPMR

K767 ∆morA pUPMR with a P mexCD -lacZ

transcriptional fusion at attTn7 siteb, Cmr, Cbr

This study

K1119 ∆morA

PmexCD pUPMR

K1119 ∆morA pUPMR with a P mexCD -lacZ

transcriptional fusion at attTn7 siteb, Cmr, Cbr

This study

et al., (1991) pUPMR Full-length P aeruginosa morA gene with

native promoter cloned into pUCP19; Cbr

Choy et al., (2004)

a: MorA markerless knockout strain was created using a morApa markerless KO cassette

on a sucide vector pK18mobsacB

b: Promoter mexCD- lacZ fusions were created by cloning the promoter sequence into

pUC18-mTn7T-Gm-lacZ vector (Tn7 transposon based vector) followed by transformation and its integration at attTn7 in the chromosome

Antibiotic resitance : Cmr – Chloramphenicol - 15µg/ml ; Cbr – Carbenicillin - 200µg/ml

3.2 Growth curve analysis of wild type and morA deletion strains

Growth of the wild type K767 and morA deletion strain K767 ∆morA were monitored by measuring the absorbance at regular intervals of time Overinight liquid culture from single colonies of the respective strains were used to reinnoculate 50ml of LB broth in conical flasks to start monitoring the growth Samples were drawn out from the culture flasks periodically over a total span of 30 hours and absorbance was measured using a spectrophotomoeter at a wavelength of 600nm Growth curve was generated using time versus absorbance plots