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. 4 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 7 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. 12 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. 25 Table 2.2 Characteristics of RND efflux pumps in P. aeruginosa (27) Opero n Component mexAB -oprM MexA MFP MexR MexB RND NalD OprM OMF mexCD -oprJ mexEF -oprN mexXY Function Regulator Primary Substrate(s) Secondary NalC MexC MFP MexD RND OprJ OMF MexE MFP MexF RND OprN OMF MFP MFP RND RND NfxB MexT MexS MvaT MexZ Antibiotics Fluoroquinolon es, β-lactams, β-lactamase inhibitors, tetracyclines, chloramphenic ol, macrolides, novobiocin, trimethoprim, sulfonamides Fluoroquinolon es, β-lactams, tetracycline, chloramphenic ol, macrolides, trimethoprim, novobiocin Fluoroquinolon es, chloramphenic ol, trimethoprim Additional Compounds Biocides (e.g., triclosan), detergents, dyes, HSL, aromatic hydrocarbon Biocides (e.g., triclosan), detergents, dyes, aromatic hydrocarbon s Biocides (e.g., triclosan), aromatic hydrocarbon s Fluoroquinolon es, β-lactams, tetracycline, aminoglycoside s, macrolides, chloramphenic ol 26 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). 27 Since MexAB-OprM is also expressed in nfxB mutants, to elucidate whether MexABOprM 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 MexABOprM, there was an increase in gene expression of inducible pumps MexCD-OprJ and 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 28 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 MexEFOprN 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 29 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). 30 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). 31 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 32 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) 33 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 timedependent 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 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. 34 3h 10h 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 35 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. 36 PAS and PAC domains Transmembrane Fig. 2.9 Redox Sesory GGDEF domain EAL domain Catalytic 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) 37 oprJ viiK mexD mexC nfxB PamorA 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). 38 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 Strain/Plasmid Description K767 PAO1 WT, prototroph K767 ∆morA K767 MorA deletion straina, Cmr K1119 K767 MexAB-OprM deletion strain, Cmr K1119 ∆morA K767 MexAB-OprM MorA double deletion straina, Cmr K767 MorA deletion strain with expression of MorA under control of native promoter in trans, Cmr , Cbr K767 ∆morA pUPMR Reference/ Source K. Poole et al., (2001) This study K. Poole et al., (2001) This study This study K1119 ∆morA pUPMR K1119 MorA deletion strain with expression of This study MorA under control of native promoter in trans, Cmr , Cbr K767 vector ctrl K767 with a promoterless lacZ at attTn7 siteb, Cmr This study K767 PmexCD K767 with a PmexCD -lacZ transcriptional fusion at attTn7 siteb, Cmr This study 39 K767 ∆morA PmexCD K767 MorA deletion strain with PmexCD -lacZ transcriptional fusion at attTn7 siteb, Cmr This study K1119 PmexCD K1119 with a PmexCD -lacZ transcriptional fusion at attTn7 siteb, Cmr This study K1119 ∆morA PmexCD K1119 MorA deletion strain with PmexCD -lacZ transcriptional fusion at attTn7 siteb, Cmr This study K767 ∆morA PmexCD pUPMR K767 ∆morA pUPMR with a PmexCD -lacZ transcriptional fusion at attTn7 siteb, Cmr, Cbr This study K1119 ∆morA PmexCD pUPMR K1119 ∆morA pUPMR with a PmexCD -lacZ transcriptional fusion at attTn7 siteb, Cmr, Cbr This study pUCP19 Broad-host-range vector; Cbr Schweizer H. P. 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. 40 3.3 Quantitative Beta-Galactosidase Assay β-Galactosidase is an enzyme encoded by the lacZ gene of the lac operon in E. Coli where it functions to cleave lactose into glucose and galactose, which are, in turn, used as carbon/energy sources in the cell. It is a large protein that exists as a tetramer. βgalactosidase (β-Gal) is resistant to proteolysis in cellular lysates and hence it is very stable and easy to assay. To monitor transcriptional activity, the promoter of the gene of interest is fused with the lacZ gene and the β-Gal levels are measured as readout for the promoter activity under various conditions. Promoter mexCD-lacZ fusions in the strains listed in Table 3.1 were used in the quantitative β-Gal assay. A schematic flow chart of the assay is represented in Figure 3.1. Ortho-nitrophenyl-β-D-galactopyranoside (ONPG) is used as the substrate and it is hydrolysed by β-Gal to ONP anion which produces a bright yellow color with a peak absorbance at 420 nm that can be quantified using a spectrophotometer. Preparation of the reagents is explained in Table 3.2. Table 3.2 Preparation of reagents for the β-Galactosidase assay* Component 1X PBS (Phosphate Buffered Saline) (Mg2+- and Ca2+-free) Composition 8 g NaCl (137mM) 0.2 g KCl (2.7mM) 1.44 g Na2HPO4 (10mM) 0.24 g KH2PO4 (2mM) H2O to 800 ml Adjust the pH to 7.4 Top up to 1000 ml with H2O Amount Storage 1L 22oC or 4°C 100mM Phosphate Buffer 1.61 g Na2HPO4.7H2O (0.06M) (or 0.85 g Na2HPO4.H2O) 0.55g NaH2PO4.H2O (0.04M) Adjust the pH to 7.0 100 ml 22o C 41 0.1% SDS 22o C Chloroform (CHCl3 ) 22o C 1x Z-buffer Note Prepare fresh before each assay. 1.61 g Na2HPO4.7H2O (or 0.85 g Na2HPO4.H2O) 0.55g NaH2PO4.H2O 1 ml 1M KCl 0.1ml 1M MgSO4 H2O to 80 ml Adjust the pH to 7.0 with NaOH or HCl. Top up to 100 ml with H2O 100 ml 4°C Add 270 µL 2-mercaptoethanol per 100 ml of 1x Z-buffer (50 mM final) prior to use. 22o C β -mercaptoethanol 0.04g ONPG ONPG (o-nitrophenyl-β-D10 ml 100mM Phosphate Buffer Galactopyranoside) stock solution Note Prepare fresh before each assay. 4 mg/ml Stop Buffer 5.3g Na2CO3 1 M Na2CO3 H2O to final vol. 50 ml * Adapted from the Invitrogen Kit 5 x 2 ml -20°C 50 ml 22o C or 4°C Sample Preparation: Overnight cultures were used to inoculate fresh LB broth to prepare the 3 biological replicates. Cultures were grown until the desired cell density (growth phase) based on absorbance measured at 600nm (OD600nm). Cells were harvested at 8000 rpm (RotorThermo scientific #3057) for 5 minutes. Growth medium was removed and cells are washed with 1X PBS. The pellet was then completely resuspended in 1X Z Buffer* and samples are maintained on ice. 100µl of this sample was then transferred to a tube containing 50µl of 0.1% sodium dodecyl sulfate (SDS) and 25µl of chloroform, vortexed 42 for 15s, and then maintained on ice for at least 20 min but no more than 50 min. The insoluble cell material was then pelleted by centrifugation at 14000g at 4°C for 5 min. The supernatant was transferred to a new micro centrifuge tube. Samples in duplicates were then incubated at 28°c for 5 minutes for equilibration and then assayed for βgalactosidase activity. Beta-galactosidase micro Assay: Cell lysates were prepared as described in sample preparation.10µl of the sample was added to a well containing 50 µl 1X Z Buffer with β- mercaptoethanol and 17 µl ONPG. The plate was covered and incubated at 37°C for 30 minutes. A faint yellow color should develop. Incubation time was recorded. 125 µl of stop buffer was added to stop the reaction by raising the pH of the solution to 11. Final volume was 192 µl. The absorbance was measured at 420 nm and 550nm using a 96-well plate reader. The reading at 420 nm is a combination of the absorbance by o-nitrophenol and light scattering by cell debris and the absorbance at 550nm corrects for the light scattering. Each well was read 3 times to get an average value. β-gal activity is expressed in Miller Units and it is calculated using the following formula: 1 Miller Unit = 1000 x [(Abs420 – (1.75 x Abs550))] / (t x V x Abs600) where, Abs420 - is the absorbance of the yellow o-nitrophenol, 43 Abs550 - is the scatter from cell debris, which, when multiplied by 1.75 approximates the scatter observed at 420nm, t - is the reaction time in minutes, v - is the volume of culture assayed in milliliters and Abs600 - reflects the cell density. Notes: * To get the most accurate measure of activity, the absorbance at 420 nm (A420) should be in the range of 0.1 -.0.9. Difference volumes of Z-buffer and cells must be initially tried out so as to get an accurate absorbance reading. (Protocol from Marian Price-Carter) 44 Sample Preparation Strains containing PmexCD – lacZ fusion PmexCD Growth of cells till desired time point Enzyme Assay lacZ Early/mid/late log phase – Abs600 Mixed with substrate ONPG Pellet cells and PBS wash Incubation at Resuspension of pellet in Z buffer Stop buffer added 37oC ONPG (colourless) β-gal enzyme Permeabilization of cells using chloroform & SDS Spin down and supernatant contains enzyme ONP (yellow) + Galactose Strains with higher promoter mexCD activity yield more yellow colour Absorbance measured at 420nm and 550nm Fig. 3.1 Schematic flowchart for measuring promoter mexCD activity in strains using quantitative β-galactosidase assay 45 3.4 Qualitative β-galactosidase plate assay X-gal (bromo-chloro-indolyl-galactopyranoside) is another substrate for the βgalactosidase enzyme and it is cleaved to yield galactose and 5-bromo-4-chloro-3hydroxyindole. The latter is further oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue product. If X-gal and an inducer of β-gal (eg. isopropyl thiogalactoside (IPTG)) are contained within an agar medium on a culture plate, then strains with promoter-lacZ fusions will show up as blue colonies unlike control promoterless-lacZ strains which will remain colourless. We optimized this assay to visually observe the increase in promoter mexCD activity between K1119 and K1119∆morA. This assay is much easier to perform than the quantitative assay and hence can be adopted for any large scale screening of genes involved in the transcriptional control of mexCD-oprJ like random insertional mutagenesis. Once a limited number of mutants are selected, the increase/decrease in PmexCD can then be verified using the quantitative assay. 0.1 M stock solutions of IPTG were prepared in water and filter sterilized. 25mg/ml of Xgal was dissolved in DMSO (dimethyl sulfoxide) and protected from light. Both must be stored in the freezer. Different concentrations of IPTG and X-gal – LB agar plates were prepared and serially diluted overnight cultures of K1119 and K1119∆morA were plated out. The plates were left overnight and observed at different times so as to select the ideal X-gal IPTG plate and the specific time point at which the colour difference between the 2 strains was most distinct. 46 3.5 RNA extraction using TRIzol method Total RNA was extracted using TRIzol Reagent (Invitrogen Corp., Carlsbad, CA, USA) in accordance with manufacturer’s instructions. In brief, the strains were grown till a specific growth phase – early/late log phase and 10ml culture was spun down. The bacterial cell pellets were vortex-mixed with TRIzol and heated at 50oc for 10 min prior to RNA extraction to lyse the cells. Homogenization was followed by the removal of the high molecular weight DNA & polysaccharides. Phase separation was then performed by adding chloroform followed by centrifugation. The upper aqueous phase contains the RNA to which isopropanol was added. Finally, the RNA pellet was washed with 75% ethanol and dissolved in double autoclaved water and stored at -80 oc. Quality of RNA was assessed by measuring the 260/280 ratio of samples using a nano-drop and the optimal value should be approximately 2.0. The RNA samples were also run on gel to verify if the bands are intact and there is no degradation before proceeding to cDNA synthesis. 3.6 First strand cDNA synthesis from RNA Three μg of total RNA was converted to cDNA using the SuperscriptTM First-Strand Synthesis System for RT-PCR (Invitrogen) as per manufacturer’s instructions. The cDNA synthesis reaction is catalyzed by the SuperScript II reverse transcriptase (RT). Overview of the procedure is represented in Figure 3.2. Quality of cDNA was assessed by measuring 260/280 ratio and optimal value is around 1.7. It was then aliquoted and stored at -20 oc. 47 RNA + dNTPS + Random hexamers Denaturation @ 65°C for 5 min Other components added followed by SS II RT Terminate reaction 70°C for 15 min cDNA synthesis 42°C for 50 min Annealing @ 25°C for 10 min RNA removal by RNaseH : 37°C for 20 min Store cDNA @ -20°C Fig. 3.2 Overview of procedure for first strand cDNA synthesis from RNA 3.7 Reverse transcription polymerase chain reaction (RT-PCR) Primers for RT-PCR were designed with Gene Runner software. Reaction mix consisted of the cDNA template, 1μL each of forward and reverse primer, 12.5μL of Immomix (PCR master mix) and water. Thermal cycling conditions used were as follows: 950c for 7min, 30 cycles of 950c for 30s, 680c for 30s and 720c for 30s followed by 720c for 10 min. PCR products were run on 0.8% agarose gel. Housekeeping gene rpsL was used as an internal control to ensure that equal amounts of RNA from all strains are loaded in the wells. 48 3.8 Quantitative Real-Time PCR Primers for quantitative Real-time PCR were designed with Primer Express software (Applied Biosystems) and performed using the SYBR Green PCR Master Mix (Applied Biosystems) as per manufacturer’s instructions. The reaction mix of 50μL consisted of the cDNA template, nuclease free water, 2μL (10mM) each of forward and reverse primers and 25 µl of the SYBR Green PCR Master Mix. It was mixed well and aliquoted into tubes for the reaction. Absolute quantification was performed and it was ensured that the CT values fall within the optimal range. rpsL was used as internal control. The fold change in mexC levels was calculated for the mutant strains with respect to the wild-type strain using 2-∆∆Ct method. 3.9 Antimicrobial Susceptibility Testing MIC is defined as the lowest concentration (in mg/l) of the antimicrobial agent that prevents visible growth of a microorganism under defined conditions. In clinical practice, this in vitro parameter is used to classify the tested microorganism as clinically susceptible, intermediate or resistant to the tested drug. MIC determinations can be used for monitoring the development of antibiotic drug resistance. Agar dilution and broth dilution are the most commonly used techniques to determine the minimal inhibitory concentration (MIC) of antimicrobial agents. Initially we used the agar dilution method for determining the MIC. For agar dilution, solutions with defined numbers of bacterial cells were spotted directly onto a series of Mueller Hinton agar plates that contain antibiotics at an increasing concentration (2-fold). After incubation for 16-20h, the presence of bacterial colonies on the plates indicates growth of the organism 49 and the antibiotic plate where there was no visible growth was considered to be the MIC for that strain with respect to that antibiotic. However, we later switched our method to broth-microdilution which is based on the same principle but is more adaptable when a large number of samples and antibiotics need to be covered. Broth dilution uses liquid growth medium containing geometrically increasing concentrations (typically a 2-fold dilution series) of the antimicrobial agent, which was inoculated with a defined number of bacterial cells. It is performed in a 96-well plate and after incubation for 16-20hrs; MIC is defined as the well in which there is no visible turbidity. Preparation of bacterial suspension: The procedure was adopted from Nature Protocols The bacterial isolates to be tested were streaked onto nutrient-rich (e.g., Mueller–Hinton) agar plates without inhibitor to obtain single colonies. Plates were incubated overnight at 37oc. Different methods for the preparation of the innoculum can be used. We used direct colony suspension method. For each isolate, three to five morphologically similar colonies were selected from the fresh agar plate from and transferred into a sterile capped tube containing sterile Mueller Hinton II broth. It was vortexed and cultures were grown for about an hour till its turbidity was in the same range as that of the McFarland standard 0.5 which corresponds to a cell number of 1-2 X 108 cfu/ml. Broth Microdilution: All antibiotic stock solutions were prepared and filter sterilized before use. The series of 2 fold increasing concentrations of antibiotics were prepared in MHB and 50μL was added to the wells. To the sterility control well, 100 μL of MHB alone was added and to 50 the growth control well we add 50μL of MHB. The following formula was used to calculate the amount of antibiotic to be weighed for the stock solutions: W = (C X V) / P where, W - weight of antimicrobial agent in milligram to be dissolved; V- desired volume (ml); C - final concentration of stock solution (μ/ml); and P - potency of antibiotic given by the manufacturer (μg/mg). The bacterial suspension adjusted to 1 X 108 cfu/ml from previous step was vortexed and diluted by 1:100. The well containing the antibiotic solution and the growth control well was innoculated with 50 μL of the bacterial suspension. This resulted in the final desired cell concentration of 5 X 105cfu/ml. The original suspension was diluted and plated out on MHA plates to verify if the cell count is correct. MHA plates and the 96 well plates were incubated at 37oc for 16-20h. Observation of MIC: In order for the test to be valid, the MH agar plates for the cell count were checked to verify that the right number of cfu was used. It had to be determined if there is sufficient growth in the growth control well. We do not read the MIC value if the sterility control (no bacterial inoculum) is turbid. The MIC is defined as the well in which there is no defined turbidity observed. We observed the plates between 16-20 hrs both visually and also by measuring absorbance at 600nm using plate reader. The scheme for the antimicrobial susceptibility testing is represented in Figure 3.3. 51 Pure cultures of the bacterial isolates for which MIC needs to be determined Day 1 Preparation of bacterial suspension Streak onto non selective Mueller Hinton II agar plates overnight for single colonies Day 2 Colony suspension method 3-5 colonies inoculated in 5ml Mueller Hinton broth and incubate @ 37oc Adjust turbidity using a McFarland Standard 0.5 Antimicrobial susceptibility testing Prepare broth micro dilutions of the antibiotics in 96 well plate Suspension with 1–2 × 108 cfu/ml Dilute suspension to 5 X 105cfu/ml Plate out samples to check cell count and incubate @ 37oC Day 3 Ensure cell count is correct Inoculate broth micro dilutions of antibiotics and incubate plate @ 37oC Read MICs after checking growth and sterility control between 16-20 hrs Fig. 3.3 Scheme for the Antimicrobial Susceptibility Testing 52 CHAPTER 4 RESULTS AND DISCUSSION PART-I 4.1 Growth curve analysis and the effect of MorA on the steady state RNA levels of the MexCD-OprJ pump in wild type and mutant strains of K767 P. aeruginosa Introduction: In a previous study, a global gene expression profiling had demonstrated that over 80 genes were significantly affected by the loss of MorA, indicating its role as a high order or global regulator in P. aeruginosa PAO1. Interestingly, the most significantly affected genes were mexC, mexD and oprJ of the MexCD-OprJ pump (Dr. Choy’s Thesis). There was a twenty fold increase in mexC, nine fold increase in mexD and a 4 fold increase in oprJ respectively when morA was knocked out at early growth stages in PAO1. This was further validated using RT-PCR and Real time PCR and by performing promoter-fusion studies in PAO1. The promoter fusion studies will be discussed in the next section. Quantitative real time PCR results indicated that the expression of mexC was upregulated by 2-3 fold in PAO1 morA deletion strain as compared to the WT strain at early, mid and late log phases. Furthermore there was a gradual increase in mexC expression level from early to late log phase (Xu Yanting’s thesis). Since it was confirmed that MorA affects the steady state RNA levels of the pump in P. aeruginosa PAO1, we proceeded to look at the levels in WT and morA deletion strains of P. aeruginosa K767 (PAO1 prototroph) and also in K1119 (K767 mexAB-oprM deletion 53 strain). MexAB-OprM is the main constitutive pump in the organism and with its broad substrate profiles; the intrinsic resistance of the bacterium is mainly caused by efflux from this pump. Hence, in order to better characterize or study the inducible pumps like MexCD-OprJ, a mexAB-prM deletion strain is often employed. For example, in order to precisely understand the contribution of the inducible pump MexCD-OprJ to drug resistance in P. aeruginosa, a mexAB-oprM deletion was created in both wild type and nfxB mutants (which is known to overexpress the MexCD-OprJ pump) (41). Similarly, in order to better understand the effects of MorA on the genotype and phenotype linked to MexCD-OprJ pump, we included the K1119 (mexAB-oprM deletion) strain in our study. Mesaros et al. (2007) had evaluated the genotypic detection of the Mex efflux pumps in P. aeruginosa using methods like semi-quantitative RT-PCR, quantitative real time PCR and quantitative competitive RT-PCR (QC-RTPCR). For all experiments rpsL gene was used as a control. These tripartite pumps are encoded by an operon, and the expression analysis is usually performed with the first gene of the operon (mexA/mexC). The group had concluded that for the constitutive pumps (mexA and mexX), detection can be performed using real time and QC-RTPCR. A quantitative method is preferred, since these pumps are already transcribed at basal levels in the wild type and are overexpressed to variable levels (starting from 3-4 fold) in the mutants (62). For the inducible pumps (mexC and mexE) however, a semi-quantitative method was considered to be sufficient since the expression of these genes are significantly downregulated in WT strains but expressed to very high levels in mutants (100-1000X fold for nfxB and nfxC mutants) (63). In nfxB mutants, a minimal increase of atleast 20-30 fold in mexC expression is required to impact on the antibiotic resistance. Before we 54 proceeded to perform the RNA analysis, we did a growth curve study in K767 WT and K767∆morA morA to determine the early, mid and late log phases at which the various experiments will be done. 4.1.1 Growth curve analy analysis in K767 and K767 morA deletion strain Growth of the wild type K767 and morA deletion strain K767 ∆morA morA were monitored as described in section 3.2, by measuring absorbance at 600nm at regular intervals of time for a total period of 30 hours. Growth curve was generated by plotting time versus absorbance as represented in Figure 4.1 4.1. Similar to P. aeruginosa PAO1, where we know that MorA does not affect growth rate, in K767 strain also MorA had no effect on the growth rate. 7 Absorbance at600nm 6 5 4 C K767 3 K767 ΔmorA B 2 A 1 0 0 10 20 Time (hours) 30 40 Fig. 4.1 Growth curve for P. aeruginosa K767 and K767ΔmorA (morA deletion strain) Points on the growth curve represent A, B and C represent early, mid and late log phases respectively. 55 From the graph, the absorbance corresponding to the three log phases were as follows: a) Early log phase OD600nm = 0.9 b) Mid log phase OD600nm = 2.5 c) Late log phase OD600nm = 4.0 These time points in the log phase were used for promoter and steady-state RNA level studies. 4.1.2 MorA had effects on the steady state RNA levels of mexC in morA mutants as compared to K767 wild-type strains As discussed previously, we initially adopted semi-quantitative RT-PCR to visualize the difference in RNA steady state levels between the WT and morA KO strains. Strains were grown till early log phase and RNA was extracted using TRIzol reagent as described in section 3.5. RNA was converted to cDNA using SuperscriptTM First-Strand Synthesis System for RT-PCR (Invitrogen) as described in section 3.6. cDNA was stored at -20 o C. RT-PCR was performed using the method described in section 3.7. Since RT-PCR is an end-point assay, it was optimized with respect to cDNA concentration and cycling conditions so as to assess the difference in mexC RNA levels in a semi-quantitative manner. The bands observed for the wild type and morA mutants with respect to mexC and rpsL is shown in Figure 4.2. rpsL (30S rRNA), which is our internal control showed equal bands for the 4 strains indicating that equal amount of RNA was loaded in all wells. In K767 WT, it was clearly observed that mexC was weakly transcribed as shown in literature, since it is not a constitutive pump. K767 ∆morA showed slightly higher mexC RNA 56 levels than WT. K1119 and K1119 ∆morA showed the highest levels of mexC among the lot. A 1 2 3 4 5 B 1 2 3 4 5 Fig. 4.2 RT-PCR products for genes: A. rpsL (internal control) and B. mexC Lanes 1-4 represent strains: 1: K767; 2: K767 ∆morA; 3: K1119 (mexAB-oprM deletion); 4: K1119 ∆morA and lane 5 is the 1 Kb DNA ladder. In the absence of the constitutive pump MexAB-OprM in P.aeruginosa, it was shown previously using RT-PCR that the mexCD-oprJ gene expression increases (42). This explains the increase in mexC RNA levels observed in K1119. From the semiquantitative RT-PCR results, we can interpret that MorA has a negative effect on mexCDoprJ expression levels at early log phase similar to what was seen in PAO1 but to evaluate if the increase is significant we performed quantitative real time PCR. 57 4.1.3 Real-Time PCR results show a marginal effect of MorA on the steady state RNA levels of mexC The semi-quantitative results had indicated an increase in RNA levels of mexC in K767 ∆morA, K1119 and K1119 ∆morA compared to WT. The increase was more significant in the K1119 (mexAB-oprM deletion) background. Analysis of real-time PCR as a method to measure increase in gene expression of pumps in P.aeruginosa established that the use of thresholds—namely ≥3 for gene overexpression allows differentiation between low, probably meaningless, changes from those that may require reconsideration of the susceptibility pattern of the corresponding clinical isolate/strains (63). Real-time PCR was performed using the ABI 7000 Prism detection system as described in section 3.8. The method adopted for presenting the quantitative real-time PCR data was the comparative CT method (also known as the 2-∆∆Ct ) . The Ct is defined as the PCR cycle at which the fluorescent signal of the reporter dye crosses an arbitrarily placed threshold. The numerical value of the Ct is inversely related to the amount of amplicon in the reaction (i.e., the lower the Ct, the greater the amount of amplicon). The value of Ct is calculated as follows: CT = (Ct gene of interest – Ct internal control). The fold change calculated as 2-∆∆Ct is defined as the expression of the gene of interest (mexC) relative to the internal control (rpsL) in the test strain compared with the calibrator strain (K767 WT) (64). The fold change in mexC RNA levels for the various strains with respect to K767 WT at early log phase is provided in Table 4.1 and represented graphically in Figure 4.3. 58 Table 4.1 Fold change in mexC expression levels in mutants compared to that in K767 (WT) from quantitative real-time PCR at early log phase. (n=3) Strain Fold Change in mexC (2-∆∆Ct ) ± std error K767 (wild type) 1 K767 ∆morA 1.53 ± 0.12 K1119 (∆mexAB) 3.03 ± 0.46 K1119 ∆morA 8.99 ± 0.95 12 10 Fold – change in mexC RNA levels w.r.t K767 WT 8 3 fold 6 4 2 0 K767 K767 ΔmorA K1119 K1119 ΔmorA Fig. 4.3 mexC expression in the different strains compared to that in K767 (WT) at early log phase. Vertical lines on bars represent the standard error (n=3) As observed from graph, the increase in mexC expression levels in K767 ∆morA compared to K767 WT is 80 genes affected by MorA, the most significantly affected ones were those of the inducible multidrug-efflux pump MexCD-OprJ. We went on to understand the effect that MorA on this inducible pump at different growth stages and in the absence of the constitutive pump MexAB-OprM. The effect that MorA has on the promoter activity of MexCD-OprJ, the steady state RNA levels of the pump and on the multidrug resistance provided by the pump had been studied. At the gene expression levels, there was a clear distinction in the manner in which MorA was affecting mexCD-oprJ with respect to time and in the absence of MexAB-OprM. Promoter activity increased in the absence of MorA, indicating that MorA has a negative effect on the promoter activity. MexCD-OprJ promoter activity in both strains (K767 and K1119 (mexAB-oprM deletion strain) were affected in a timedependent manner in the absence of MorA. The increase in promoter MexCD-OprJ activity was more pronounced at late log phase as compared to early log phase in both 79 strains, indicating that the negative effect that MorA has on promoter activity increases with time. Also the fold change in promoter levels was much higher in K1119 ∆morA (absence of MexAB-OprM), indicating that the negative effect of MorA on promoter activity is higher in the absence of constitutive pump. Transactivation with morA does not restore promoter activity to WT levels, implying that MorA is not a direct negative regulator of MexCD-OprJ but it indirectly impacts the pump. The effect of MorA on the RNA steady state levels of MexCD-OprJ followed a similar trend to promoter activity with respect to time and in the absence of pump MexABOprM. The increase in fold change between K1119 morA deletion strain and K1119, increased with time and these changes were small and reproducible. The fold increase was 8-fold which was not significant enough to impact antibiotic resistance as per literature, since inducible pump MexCD-OprJ is weakly transcribed in wild type cells and a fold change of 30-70 fold is required to impact resistance. However, in K767 morA deletion strain there was no increase observed in the RNA levels of the pump. Hence, in general at gene expression levels, the negative impact of MorA on MexCD-OprJ is more prominent in the absence of MexAB-OprM. Also in the absence of the constitutive pump, the inducible pump levels are higher indicating that the cell has an ability to maintain net levels of these efflux pumps. Finally, the antibiotic resistance phenotype study indicated that K767 had a 2 fold higher MIC than K767 morA deletion strain for antibiotics known to be pumped out by MexCDOprJ. Furthermore complementation with MorA restored the phenotype to WT levels, clearly establishing the fact that MorA was involved in affecting the antibiotic resistance phenotype with respect to MexCD-OprJ. Interestingly, there was no change in MIC 80 values between K1119 MorA deletion strain (mexAB-oprM deletion strain) and K1119, indicating that the presence of MexAB-OprM is required for the positive effect of MorA on the drug resistance phenotype. Also the changes observed at gene-expression levels of the pump clearly do not explain the changes seen at the phenotype level. Hence, MorA is affecting the drug resistance of the MexCD-OpJ pump in a post-transcriptional manner. This is similar to the regulation of Type II Secretion system by MorA in P. aeruginosa which also occurs in a post-transcriptional manner. This post-transcriptional effect on the pump by MorA may involve signaling via the secondary messenger c-di-GMP. Future work in this area will help in understanding some of these aspects. 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Regulation of Bacterial Drug Export Systems. Microbiology and Molecular Biology Reviews, Dec. 2002, p. 671–701. 88 [...]... 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... 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... 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... 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. .. 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 25 Table 2.2 Characteristics of RND efflux pumps... 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, ... 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... 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... 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) 33 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... 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) ... the time and strain dependent effects of MorA on the activity of promoter of MexCD-OprJ To study the effects of MorA on the steady state RNA levels of mexCD-oprJ To study the effect of MorA on. .. 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. .. 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