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Bacterial resistance to antibiotics has become a major problem in the management of bacterial infections. Discovering new antibiotics is one approach to overcome this problem. Bacterial RNA polymerase (RNAP), an essential enzyme in the process of transcription, is a target for only one class of antibiotics used in clinical practical, the rifamycins. Investigation of novel inhibitors of this enzyme to produce alternative RNAP inhibitors may contribute to the chemotherapy of bacterial infections.
In this study, the activity of rose bengal and compound (X) against S. aureus were determined through the minimum inhibitory concentration methods. The kinetics of cell deaths and cell lysis by rose bengal were investigated through time-kill experiments. In addition, mutation for resistance to rose bengal was also calculated to determine how easily bacteria develop resistance to these agents by mutation. Furthermore, detection of activities of rose bengal on membrane damage of S. aureus was investigated by using BacLight assay. Rifampicin was used as a comparative agent throughout. Finally, attempts are still in progress to purify RNA polymerase (RNAP) for screening assays to study activities of new RNAP inhibitors which will be performed based on commercially available KoolTM RNAP.
This study shows that rose bengal and compound (X) are active against S. aureus SH100 in the Minimum inhibitory concentration (MIC) tests. Rose bengal also demonstrates activities against six strains of rifimicin-resistance of S. aureus 8325-4. Rose bengal demonstrates bactericidal non-lytic activity against S. aureus SH100 and it acts as a dose-dependent agent in the time-kill experiments. The mutation frequency for development of resistance to rose bengal is roughly the same for rifampicin (2.7 - 10 - 8 and 6.3 - 10 - 8), respectively with no statistical difference. The membrane integrity experiments results reveal that rose bengal has effects on membrane integrity and damaged the bacterial membrane.
After discovering the antibiotic penicillin by Sir Alexander Fleming in the early 1920s, many other antibiotics were also discovered in the middle of 20th century, which has been called "The Golden Age" of antibiotics discovery (Chopra et al., 2002). However overuse of antibiotics for treating bacterial infection has resulted in the emergence of antibiotic resistant bacteria, which has become a major problem in the management of bacterial infections, and it seems to be return to "The Dark Age". In this project I aim to develop strategies for identifying and developing new inhibitors of bacterial RNA polymerase (RNAP), and especially that of Staphylococcus aureus.
1.1 Staphylococcus aureus
Staphylococcus aureus is a spherical bacterium, appears under microscope as gram-positive cocci in "grapelike" clusters. It is distinguished from other staphylococcal species by formation of the gold colonies and positive result of coagulase test (Lowy, 1998). S. aureus is found, as part of normal flora, on the skin and nasopharynx of many healthy individuals (Harris et al., 2002). S. aureus can be transmitted in many ways such as: contact with an infected person, from a person with a respiratory infection, an open lesion, or the hands or skin of an asymptomatic carrier. It has an ability to persist on contaminated objects for several days to more than a week making these objects a source of infection. It also can be transmitted by airborne particles (Robert & John, 2001).
S. aureus temporarily inhabit the nasal passages, skin and mucous membranes on about one-quarter of healthy people without causing any infection (Nathwani et al., 2008). However, if the organism has an opportunity to enter the body it can cause a variety of infections such as suppurative, superficial skin lesions (boils, furuncles, styes, impetigo). S. aureus can cause more serious infections such as pneumonia, mastitis, phlebitis, meningitis, and urinary tract infections. It is also a major cause of hospital acquired infection associated with surgical wounds and indwelling medical devices. S. aureus can release enterotoxins into food causing food poisoning. Moreover, it is responsible for toxic shock syndrome by releasing exotoxins (superantigens) into the blood stream. (Todar, 2008).
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infections that are becoming increasingly difficult to combat because of emerging resistance to all current antibiotic classes (Enright et al., 2002). MRSA is the main cause of antibiotic-resistant health care-associated infections worldwide (EARSS Annual Report, 2006). The cases of bacteraemia due to S. aureus in England, reported via the mandatory surveillance scheme in 2006, were 17,987 cases, which corresponds to a reporting rate of 35.4/100,000 population (Health Protection Agency, 2006). The proportion of S. aureus bacteraemias due to MRSA reported through the voluntary surveillance scheme appeared to plateau at ~42% between 2000 and 2002, while from 2003 to 2006, the proportions of S. aureus bacteraemias due to MRSA were 41.5, 39.7, 39.7 and 37.9%, respectively (Health Protection Agency, 2006).
1. 2 Antibiotic resistance of Staphylococcus aureus
There are two approaches for the bacteria to become resistant. Bacteria can obtain the resistance naturally or resistance can be acquired. Natural resistance is when the antibiotic has a lack of activity against the bacteria without genetic alteration. Acquired antibiotic resistance termed if the bacteria were susceptible to an antibiotic and become resistant. Acquired resistance occurs when the organism develops a mutation to be able to overcome the minimal inhibitory concentration (MIC) on each step to reach the levels unachievable using therapeutic doses (Haddadin et al., 2002).
1. 2. 1 Penicillin resistance
Penicillin-resistant S. aureus was recognized in 1942. By the late 1960s, approximately 80% of both community- and hospital-acquired staphylococcal isolates became resistant to penicillin (Lowy, 2003). In further study in 1997, 94% of 1,087 S. aureus isolates were found to be resistant to penicillin and ß-lactams susceptible to the action of ß-lactamase, such as ampicillin and ticarcillin (Lowy, 2003). Table 1 give a brief history of Staphylococcus aureus infection in the United States.
CA-MRSA, Community-acquired methicillin-resistant Staphylococcus aureus. Data from (So & Farrington, 2008).
Mechanisms of resistance: Staphylococcal resistance to penicillin occurs by two mechanisms, (i) inactivation the drug due to hydrolysis by ß-lactamase ,the enzyme encoded by the gene blaZ (Lowy, 2003), or (ii) in Methicillin-resistant Staphylococcus aureus (MRSA) ß -lactam resistance is caused by the expression of penicillin-binding protein 2a (PBP2a), encoded by the mecA gene. Penicillin-binding protein 2a (PBP2a) has low affinity for binding to most ß -lactam antibiotics so far introduced into clinical use (Kuwahara-Arai, 1996), thus preventing the drug induced inhibition of cell wall synthesis.
As shown in figure 1. in the presence of ß-lactam antibiotic penicillin, the DNA-binding repressor BlaI binds to the operator site leading to suppress RNA transcription from both blaZ and blaR1-blaI. Binding of penicillin to the membrane transducer BlaR1 stimulate BlaR1 autocatalytic activation. Active BlaR1 (or BlaR2) split BlaI into inactive fragments, allowing transcription of both blaZ and blaR1-blaI to start. Thus, the gene blaZ start synthesis ß-Lactamase which hydrolyzes the ß-lactam ring of penicillin rendering it inactive (Lowy, 2003). The mechanism of S. aureus resistance to methicillin is by expression of PBP2a which has a lower penicillin-binding affinity and higher rates of release of the bound drug. Presence of ß-lactam antibiotic stimulates MecR1synthesis that lead to inactivation of MecI and allowing synthesis of PBP2a.
1. 2. 2 Quinolone resistance
After introduced Fluoroquinolones in the 1980s the resistance to fluoroquinolones has emerged quickly among the methicillin-resistant strains because of increasing of the use of these antibiotics clinically (Hooper, 2002). This resistance is achieved by changes in aminoacids in the bacterial enzymes DNA gyrase and topoisomerase IV (particularly those in certain regions of each enzyme subunit called the quinolone-resistance-determining-region (QRDR)), that reduce quinolone affinity for both of its targets. The resistance may also achieved by the induction of a bacterial membrane pumps (NorA multidrug resistance efflux pump), that remove drug from the cell. It is thought to be that hydrophobicity of a fluoroquinolone molecule is one of the important properties that affect its transport by these pumps. Both resistance processes are affected by stepwise acquisition of chromosomal mutations that modify the target enzymes or increase the level of multidrug efflux pump expression (Hooper, 2002).
1.2.3 Vancomycin resistance
The first report of vancomycin intermediate-resistant S. aureus (VISA) is isolated in 1997 in a patient Japan. The vancomycin minimum inhibitory concentration (MIC) for this isolate was 8 µg/ml (Smith et al., 1999). However, high-level vancomycin-resistant S. aureus (VRSA) was isolated in June 2002 in Michigan, USA (MIC = 1,024 µg/ml) (Weigel et al., 2007).
Mechanisms of resistance in Vancomycin intermediate-resistant S. aureus (VISA): The changes in peptidoglycan biosynthesis appear to be the reason responsible for the reducing susceptibility to vancomycin in VISA. These changes in peptidoglycan biosynthesis are thought to represent the common pathway for the expression of vancomycin resistance (Lowy, 2003). It is assumed that reduced cross-linking of the cell-wall peptidoglycan leads to the exposure of more D-Ala-D-Ala residues and increase in free D-ala-D-ala side chains to which vancomycin can bind. As a result there are more D-Ala-D-Ala residues available to bind and trap vancomycin (Figure 2) which reduces the amount of vancomycin molecules to reach their target on the cytoplasmic membrane where the targets of vancomycin are located (Hanaki et al., 1998).
Mechanisms of resistance in vancomycin- resistance S. aureus to vancomycin (VRSA): VRSA strains resistance to vancomycin is suggested the possibility that the VRSA acquired vancomycin resistance via horizontal transfer of the vanA gene from an enterococcus (Lowy, 2003) that allows synthesis of a cell wall precursor that ends in D-Ala-D-Lac dipeptide rather than D-Ala-D-Ala. D-ala- D-lac termini has greatly reduced affinity for vancomycin by at least 1000-fold lower than for those ending in D-ala-D-ala (Hanaki et al.,1998). The novel cell wall precursor is synthesized, thus cell wall synthesis is not inhibited in the presence of vancomycin (Lowy, 2003) (figure 3).
1. 3 Antibiotics targets
There are many different classes of antibacterial each exerting a different type of inhibitory effect that specifically impact bacteria. Table 2. summarized the classes of antibiotics and their targets.
1. 4 Bacterial RNA polymerase
1. 4. 1 Structure and function of bacterial RNA polymerase
Bacterial RNA polymerase (RNAP) is essential enzyme for transcription which is essential for growth and survival of bacteria and is a final target in many pathways that control gene expression. In bacteria, RNAP is responsible for the synthesis of all species of RNAs in the cell (i. e. messenger RNA, ribosomal RNA, transfer RNA) (Lynch & Du, 2008). Bacterial RNAP exists in two forms (Figure 4): core and holoenzyme. The core enzyme has a relative molecular mass of around 400 kDa , and consists of five subunits: a-dimer (a 2), ß, ß', and ?. (Vassylyev et al., 2002). The core enzyme combines with Sigma factor (s) to form a complex that is referred to as the holoenzyme (Figure 4).
The functional cycle of RNAP consists of transcription initiation, processive transcription elongation, and transcription termination. During initiation, the core RNAP enzyme in bacteria binds to s factors to form holoenzyme that can binds the promoter DNA, forming the closed complex (RPc) in a process termed as promoter recognition. RPc involves sequence steps to the open complex (RPo), in which polymerase actively "melts" the DNA duplex to allow active-site access to the template strand (Geszvain & Landick, 2005). The holoenzyme then binds to two conserved hexamers in the promoter at nucleotide (nt) positions -35 and -10 to form a closed promoter complex. Then, it unwinds the double stranded DNA around the -10 result in the open promoter complex, and starts transcription using nucleoside triphosphate (NTPs) as both a primer and the substrates to produce RNA molecule with the DNA template strand in an RNA/DNA hybrid (Vassylyev et al., 2002). Once RNA polymerase has managed to synthesize about 9 - 12 nucleotides of RNA the polymerase shifts passes from the initiation to elongation mode of RNA synthesis (Alberts et al., 2002). After the synthesis of a 9 - 12 nt-long RNA, of which 8 - 9 are base-paired with the DNA template strand (RNA/DNA hybrid), the transcription complex passes from the initiation to elongation stage. This process is characterized by the escape of the RNAP from the promoter, the separate of s from the core, and the formation of an elongation complex (EC) (Vassylyev et al., 2002). The EC is transcribing at an average rate between 30 to100 nucleotide per second along the DNA template. Ending Transcription occurs when RNAP reaches the termination signal, in which an RNA hairpin in the nascent transcript, or is affected by the termination factor, making the RNA transcript and the DNA to be released resulting in freeing the core RNAP to begin additional round of transcription (Geszvain & Landick, 2005) (Figure 5).
1. 4. 2 Bacterial RNA polymerase as a drug target
Bacterial RNA polymerase can be an ideal source of drug targets and provide obvious opportunities for chemotherapy for the reason that bacterial RNA polymerase is central enzyme in the transcription cycle (Minakhin et al., 2001) and it is an essential enzyme for growth and survival of bacteria (Lynch & Du, 2008) and failure of this process is fatal for the cell. RNAP is also a proven target for antimicrobials because it is inhibited by Rifamycin class of antibacterial agents, the only class of RNAP inhibitors that have found their way into clinical use and has a broad spectrum of antibiotic activity against infection cause by Gram-negative and Gram-positive bacteria including staphylococcal disease (Artsimovitch & Vassylyev, 2006).
Validation of RNAP enzyme as a drug target comes from the fact that this enzyme, as described above, has multi-subunits that presents various targets (binding sites), for small-molecule inhibitors, that have not yet exploited for drug target identification (Darst, 2004). Consequently, there are several chances for inhibitors molecules to interfere with one of these structural units or functions of this molecular machine in a multiple ways. Moreover, comparisons between core of prokaryotic and prokaryotic RNAPs, the core of eukaryotic enzymes possesses up to ten additional subunits. As a result there are sufficient differences in the surface features and shape between prokaryotic and eukaryotic RNAPs (Chopra, 2007). Furthermore, regardless of the similarities of structural and functional of RNAP, however, bacterial RNAPs have different sequence homology regarding eukaryotic RNAPs, for example, eukaryotic RNAP are at least 102 to 104 times less susceptible to inhibition by rifampicin. This lack of homology makes the bacterial RNAP inhibitor attractive target for antimicrobial agents as it not target eukaryotic RNAPs (Villain-Guillot et al., 2007). O'Neill et al (2000) reported that RNAP inhibitors holomycin, thiolutin, ripostatin A, and corallopyronins, are candidates as alternative antibiotics to rifampinfor which resistance has already been recorded. Furthermore, there is no cross-resistance between these drugs and rifampin in S. aureus mutants.
The understanding of antibiotic mechanisms of action against RNAP, such as rifampicin, might help us to introduce new generations of antibiotics that can be more effective against bacteria.
1. 5 Rifamycin
The rifamycins (Rifs) were discovered in 1957 as fermentation from the organism Actinomycete, classified as Amycolatopsis mediterranei (Lal & Lal, 1994). The fermentation led to produce rifamycin B which has no or low antibacterial activity, but it can be converted into rifamycin SV which has much more antibacterial activity (Weherli & Staehelin, 1971) (Figure). The rifamycins have a broad spectrum of activity against Gram-positive bacteria much more than that to Gram-negative bacteria. Since resistance to rifampicin develops rapidly (Edelstein, 1991), thus the use of this drug in combination with other antibacterial agents is recommended (O'Neill et al., 2001).
1.5.1 Rifamycin mechanism of action
The antibacterial action of rifampicin comes from its affinity binding to, and inhibition of, the bacterial RNA polymerase (Campbell et al., 2001). Despite the various structural modifications made in rifamycins, the inhibition of DNA-dependent RNA polymerase seems to be the common mechanism for all anti-bacterially active rifamycins with no change in the principal mechanism of action (Floss & Yu, 2005). Generally, rifamycin prevent producing mRNA through the formation of a complex with the ß subunit of RNA polymerase (Lal & Lal, 1994), the binding site for rifampicin encoded by the rpoB gene, that is, the s subunit is not required for rifampicin binding (Naryshkina et al., 2001). Rifampicin binds to the ß subunit deep within the active-site, but about 12 Å away from the active site that contains a magnesium ion (Mg2+) (Darst, 2004) However, if the mRNA chain develops more than a few nucleotides the reaction will continuous in spite of presence of rifamycin or not and the elongation of the mRNA is completed (Lal & Lal, 1994). McClure and Cech (1978) also reported that rifampicin inhibit the formation of the first phosphodiester bond in RNA synthesis having an effect only on the binding of the first two triphosphates with no effect on the maximal velocity of their conversion into a dinucleoside tetraphosphate (McClure & Cech, 1978). Structural and functional studies of rifampicin bound to a bacterial RNAP holoenzyme, suggested that when rifampicin binding to RNAP, rifampicin clashes with the upstream base pair of the 3-base pair long RNA/DNA hybrid. As a result lower the affinity of the RNAP to the major catalytic Mg2+ ion, thus slowing down the catalytic reaction and subsequently facilitating its dissociation through the RNAP secondary channel (Artsimovitch & Vassylyev, 2006) (Figure 6). Rifs can be divided into two classes. The ansa backbone includes tails attached to C3 position gives rifampicin or rifapentin while that attached to C3/C4 position gives rifabutin or rifamexyl (Figure 7). These modes of attachment involved in determined the target of Rif action, accordingly, the C3 Rifs inhibit the formation of second phosphodiester bond, while theC3/4 compounds inhibit both, the first and the second bond formation. (Artsimovitch & Vassylyev, 2006).
It can be concluded that the mutations of rpoB to rifampicin resistance comes from decreased affinity of the RNAP to this antibiotic. This decreased affinity between antibiotic and target in the enzyme is the major mechanism of resistance in bacteria (Aubry-Damon et al., 1998), (Floss & Yu, 2005). Artsimovitch and Vassylyev (2006) summarized the substitutions in RNAP that confer resistance to rifampicin and they divided them into three categories (i) the "steric" substitutions prevent antibiotic binding through steric hindrance by reducing the space in the Rif binding site, (ii) the "affinity" substitutions lead to decrease rifampicin binding affinity to RNAP active site and (iii) the "allosteric" substitutions which may not reduce Rif binding, however, likely disrupt transmission of the allosteric signal.
1. 6 Rose bengal as RNAP inhibitor
Studies of new RNA polymerase inhibitors in more biological and molecular details such as mechanism of action , mutation of frequency , and the mechanisms by which bacteria confer resistance to the antimicrobial agent, could explain how some of these inhibitors bind to the RNAP, how they effect on transcription, and how resistance to an inhibitor is selective. As a result it could potentially lead to the design of a novel compounds targeting RNAP. Rose bengal is most frequently used as a biological stain and as a photosensitizing dye (Rasooly & Weisz, 2002). In this study I have attempted to explore this already proven in vitro RNAP inhibitor (Wu & Wu, 1973) (Figure 8), to build up complementary understanding on how it work on the bacterial RNAP and study its biological activity against the bacteria to increase our knowledge about this inhibitor and conclude whether it has characteristics to that could help to identify binding sites on the enzyme and therefore guide medical chemistry approaches in the design of new inhibitors.
1. 7 Objectives of the work
- To identify and characterize new bacterial RNAP inhibitors, which are designed and synthesised in the Department of Chemistry, Leeds University, against a range of bacterial species particularly Methicillin-resistant Staphylococcus aureus (MRSA).
- Evaluate these inhibitors microbiologically, genetically and biochemically to assess their potential to become drug candidates.
- Understanding these characteristics and the mechanism of action of these RNAP inhibitors may help to discover new generations of antimicrobials that can be considered as alternative drugs to semi-synthetic rifamycins, to which bacteria have already developed resistance (O'Neill et al., 2000).
To achieve these goals I will study the antimicrobial activity of these new inhibitors against S. aureus (e.g., MIC tests, time kill study). I also will determine frequencies of mutation for resistance to these inhibitors. Effects of inhibitors on macromolecular synthesis, (DNA, RNA, protein) and on bacterial membrane integrity will also be determined. For the compounds that give promising activities I will screen for RNAP inhibitor activity using in vitro methods. I will purify RNA polymerase from S. aureus using conventional procedures and test its susceptibility to chemical inhibitors. These inhibitors will be assessed for anti-staphylococcal activities using clinical isolates of MRSA. Inhibitors with anti-staphylococcal activities will also be screened against rifampicin-resistant S. aureus to distinguish any inhibitors that might be subject to existing mechanisms of resistance at the level of RNAP.
2 MATERIALS AND METHODS
2. 1 Bacterial strains
- rsbU encodes a positive regulator of sigma factor sB which controls a general stress response of Staphylococcus aureus and may has a role in virulence (Horsburgh et al., 2002).
2. 2 Bacteriological media and culture conditions
Staphylococcus aureus strains SH1000 (derived from 8325-4 with rsbU replaced) (Horsburgh et al., 2002) and six different levels of Rif-resistance mutants of S. aureus 8325-4, R28 and R39 (low level); R34 and R44 (med level) and R24 and R51 (high level) was used in this study. The substitutions of amino acids of these mutants are described in table 3. Iso-Sensitest broth (ISB) and Iso-Sensitest agar (ISA) (Oxoid; Basingstoke, Hampshire, UK) were used routinely for all S. aureus strains culture during the MICs, time-kill kinetic, frequency of mutation for resistance and membrane damage studies for rose bengal and rifampicin. However, Brain Heart Infusion (BHI) broth was used for RNAP purification procedures from S. aureus (SH1000). Unless otherwise stated broth cultures were grown aerobically at 37°C with shaking at 220 rpm for 12-18 h and plate cultures were grown aerobically at 37°C for 12-18 h. For Cryogenic long-term storage S. aureus (SH1000) was stored in broth culture containing a final concentration of 80% (v/v) sterile glycerol at -80 °C.
2. 3 Antibiotics and chemicals
Rifampicin and rose bengal were purchased from Sigma-Aldrich Co. Ltd. (Poole, United Kingdom). Compound (X), which is presumptive RNAP inhibitor was received from Helmholtz-Zentrum für Infektionsforschung GmbH (Braunshweig, Germany). Rifampicin was dissolved in dimethyl sulfoxide (DMSO) which was diluted in distilled water (dH2O). Rose bengal and compound X were dissolved in dH2O. In all experiments rifampicin was used as a reference control.
2. 4 Minimum inhibitory concentration (MIC) determination
Minimum inhibitory concentration (MIC) test can be used to determine the concentration at which an antimicrobial agent must be present to inhibit a specific organism to find out the in vitro activity of new antimicrobials. MICs were determined by the agar dilution plate method and broth microdilution method recommended by British Society for Antimicrobial Chemotherapy (BSAC, 1991) guidelines. For the agar dilution plate method, ISA plates containing doubling dilutions of compound were prepared and inoculated with 106 cells using a 21-pin multi-point inoculator (Life Sciences International, Basingstoke, UK). Plates were allowed to air dry before aerobic incubation at 37 °C overnight. Broth microdilution method was performed in 96 well micro-titre plates with serial two- fold dilutions of the chemical in ISB. For both methods the MIC was defined as the lowest concentration of compound that completely inhibited visible growth of the organism after 18 to 24 h incubation at 37 °C.
2. 5 Time-kill assays
Time-kill experiments were performed by inoculating three flasks containing 50 ml of ISB each with 500 µl of overnight culture of S. aureus SH1000. Flasks were incubated in a shaking water bath at 37 °C. The cultures were allowed to grow to an optical density at 600 nm (OD600nm) of ~ 0.2, measured using a Jenway 6300 spectrophotometer (Essex, UK). When the cultures had reached an OD600nm rose bengal or rifampicin were added to the relevant culture at 1X and 4X and 4X and 16X their MIC values, respectively, in addition to control containing no chemical. Immediately after addition of the compounds, optical densities were measured and samples were taken for viable counting. Viable counts were performed in triplicate on ISB with an inoculum of 100 µl of neat sample or a dilution prepared in phosphate-buffered saline (PBS). Plates were incubated at 37 °C overnight and the number of visible colonies counted. Counts were expressed as colony forming units per ml. Optical density reading and viable counts were performed for every 60 min for 6 h. Rates of killing were determined by measuring the reduction in viable bacteria (log10 CFU/ml) at nought time, time 0 (when the compound was added), 1, 2, 3 ,4, 5, and 6 h with fixed concentrations of compound. The rate of killing by the inhibitors was established by plotting colony counts (logCFU/ml) against time (hours). Bactericidal effect was defined as a =3 log10 reduction in CFU/ml, while bacteriostatic effect was defined as a <3 log10 reduction in CFU/ml (Neuhausen et al., 2003).
2. 6 Determination of mutation frequencies
Mutation frequencies were determined for S. aureus stain SH1000 against rose bengal and rifampicin. A viable count was performed for SH1000 by plating 100 µl of a 10-6 dilution of three independent overnight cultures of SH1000 onto ISA in triplicate for each culture. ISA plates were prepared containing both compounds at 4 X MIC and 100 µl of neat overnight culture was plated, and 100 µl of overnight culture was diluted for total viable count determination on chemical-free medium. All plates were incubated at 37 °C overnight and the number of visible colonies counted. The results determined by the mean value obtained from triplicate of three independent overnight cultures. The mutation frequencies were calculated as the number of resistant mutants recovered as a fraction of total viable bacteria.
2. 7 Bacterial membrane damage
Bacterial membrane damage by rose bengal and rifampicin was initially examined by using the BacLight assay. The BacLight kit uses two nucleic acid stains, the green-fluorescent (SYTO 9) stain and the red-fluorescent (propidium iodide) stain. These stains have different ability to go through the membrane. When used alone, the SYTO 9 stain labels the nucleic acids in both live and dead bacteria, whereas propidium iodide penetrates only bacteria with destroyed membranes. When both stains are used, the propidium iodide stain reduces the SYTO 9. Thus, live bacteria with intact membranes fluoresce green, while dead bacteria with damaged membranes fluoresce red. S.aureus was grown overnight in ISB at 37°C with shaking at 220 rpm. The culture was diluted 1:100 in fresh ISB and grown to an optical density at 600nm (OD600) of 0.5 _ 0.6. The bacterial suspension was centrifuged at 10000-g for 15min, and the cell pellet was washed with sterilized deionised water. The cell pellet was resuspended to 1/10 of the original volume and then diluted 1:10 into either water or water containing test compounds at 4X the MIC.Samples were incubated at room temperature in a rocker for 10min, washed with sterilized deionised water and centrifuged at 10000-g for 10min and washed again with sterilized deionised water. The pellets were resuspended in sterilized deionised water in 4.5 ml cuvettes in triplicate. BacLight reagent was added to each cuvette, and then incubated for 15min in the dark room temperature. Finally, green fluorescence was measured at 530nm, and red fluorescence was measured at 645nm with a Perkin-Elmer LS 45 luminescence spectrometer. The ratio of green to red fluorescence which is an indicator of membrane integrity was normalized to the untreated control and expressed as a percentage of the control, where the negative control is 100% integrity and the positive control is 0% integrity.
2. 8 RNA polymerase isolation and purification
RNAP of S. aureus strain SH1000 was purified as described by Vassylyeva et al with minor modifications adopted by Amer Alomari, University of Leeds (data not published) as follow:
Preparation of cell pellets:
S. aureus cells were grown in BHI broth overnight. Two ml of overnight culture were used to inoculate 2 litres of BHI broth in a fermenter (BIOSTAT A plus, Sartorius, UK). The growth of the culture was monitored spectrophotometrically at 600 nm using Jenway 6300 spectrophotometer (Essex, UK) until the culture reached an OD600nm of 2-4. When the culture had reached an OD600nm of 2-4 the cells were harvested by centrifugation at 8000 - g. The pellets were washed with buffer (10 mM Tris-acetate pH 8.0, 14 mM Mg acetate, 1 mM dithiothreitol [DTT]) containing 1 M KCl, then followed by washing with the same buffer containing 50 mM KCl and stored at -80 °C.
The Pellets were thawed on ice for 30 min and fully resuspended to a final concentration of 0.5 g/ml in lysis buffer (40 mM Tris-HCl pH 7.7, 0.1 M NaCl, 10 mM 2-mercaptoethanol [2-ME]) containing protease inhibitor cocktail tablet (Roche, Switzerland), one tablet for 50 ml of lysis buffer. Lysostaphin was added to a final concentration of 20 µg/ml of the solution and the suspension incubated at 37°C for 25-45 min. The suspension was subjected to a high pressure using a French press for completely breaking bacterial cells "mechanically".
The crude cell lysate was centrifuged at 16000 - g and the supernatant precipitated by polymine P (0.5% final concentration), then centrifuged at 5500 rpm. The pellets were washed four times with lysis buffer containing 0.28 mM NaCl and extracted with the same buffer containing 0.9 NaCl. The extract was precipitated with ammonium sulphate (at 35% saturation), the pellets collected by centrifugation, redissolved in buffer A (20 mM Tris-HCl pH 7.7, 1 mM EDTA, 5 mM 2-ME, 5% glycerol) and centrifuged in a centrifugal concentrator tube at 4500 rpm. The resulting material was applied to SP-Sepharose column in high performance liquid chromatography (HPLC). To completely purify the enzyme it needs to be applied to a Mono Q column but I haven't achieved this step yet. Sp-Sepharose column are based on a matrix of 34 µm particles made from cross-linked agarose. The small particle size permits fast binding and dissociation even at high sample loads and flow rates which give high resolution separations. Stability of particle size and bed volumes, despite changes in ionic strength or pH, ensure fast separations at high flow rates. Mono Q column is highly efficient, pH-stable column designed for high performance ion exchange separations of proteins, peptides, and polynucleotides based on a hydrophilic material with small homogeneous particle sizes (10 µm). This small particle size allows fast binding and dissociation to facilitate high resolution while the monodispersity permits high flow rates at low back pressures.
3. 1 Minimum inhibitory concentrations (MICs)
All MIC values of tested compounds were determined using the broth microdilution method. However, the MIC of rose bengal and rifampicin against the strain SH1000 were obtained using both methods, the agar dilution plate method and broth microdilution method. MIC values for all the strains tested are presented in Table 4.
- the MIC using agar dilution plate method
- rifampicin showed same value for both methods
3.2 Time-kill assays
Time-kill curves were determined by plotting colony counts (logCFU/ml) against time for each inhibitor concentration. The results are the mean of three replicates (±S.D.).
3.2.1 Time-kill assessment of rifampicin (survival curve).
Figure 9 illustrates the bactericidal activity of rifampicin, obtained from time-kill experiments, at 4 and 16 X the MIC. The control continued to grow and reached 4.89 - 109 CFU/ml after 6 h. The cultures treated with 4 and 16 X the MIC shows a decrease in the number of colonies compared to the control. Both cultures treated at 4 and 16 times the MIC reached 8.33 - 105 and 8.67- 103 CFU/ml, respectively after 6 h of adding the antibiotic.
3.2.2 Time-kill assessment of rose bengal (survival curve).
Figure 10 revealed the bactericidal activity of rose bengal, obtained from time-kill assays, at 1 and 4 times the MIC. The untreated control continued to grow and reached 2.01 - 109 CFU/ml after 6 h. The cultures treated with rose bengal at 1 and 4 X the MIC shows an immediate decrease in viable cell numbers compared to the control. Both cultures treated at the two concentrations tested reached 3.00 - 105 and 1.33 - 103 CFU/ml, respectively after 6 h of adding the inhibitor.
3.3.1 Effects of rifampicin on culture turbidity.
(The OD600 scale has been altered in figure 11 b show the trends of the treated cultures more clearly)
Figure 11 demonstrates effects of rifampicin on OD600 of S. aureus SH1000 culture over 6 h. The data illustrated that the optical density at 600 nm (OD600) of the untreated control after 6 h is 6.192. The cultures treated with rifampicin at 4 and 16 X the MIC did not show any decreases in their optical density. As shown in figure 9, both cultures treated at the two concentrations tested became roughly stable in OD600, ranging from OD600 0.330 to 0.309 for 4 and 16 X the MIC, respectively.
3.3.2 Effects of rose bengal on culture turbidity
(The OD600 scale has been altered in figure 12 b show the trends of the treated cultures more clearly)
Figure 12 shows the effects of rose bengal on optical density of S. aureus SH1000 culture at 600 nm (OD600) over 6 h. The untreated control reached an OD600 of 6.776 after 6 h. The cultures treated with 1 and 4 X the MIC reached an OD600 of 0.335 and 0.321, respectively, and became stable, at different optical density points, after 1 h.
3.4 Mutation frequencies
The results obtained from the mutation frequency experiments indicated that the mutation frequency for development of resistance in S. aureus SH1000 to rose bengal was slightly less than for rifampicin at 4 x the MIC of the two compounds (2.7 - 10 - 8 and 6.3 - 10 - 8, respectively) as shown in table 5.
3.5 Membrane damage
Bacterial membrane damage of S. aureus SH1000 by rose bengal and rifampicin was examined by using the BacLight assay. As the BacLight data in Table 6 indicate, as expected along with the control, rifampin, a non-lytic agent, had no or little effect on membrane integrity and reduced the membrane integrity of S. aureus to >81%, whereas, rose bengal displayed more membrane effects on the membrane integrity and reduced the bacterial membrane to < 20 % integrity).
3.6 RNA polymerase isolation and purification
As clarified in the materials and methods, RNAP purification is still in its early stages of enzyme purification and there are additional steps have to be done to complete the procedures of the purification, on which I am still working up to now. What I have done so far is the process of extraction the enzyme accompanied with various cell components and proteins, as a complex mixture. Thus, this mixture requires more purification to get pure enzyme by using fast protein liquid chromatography which will be achieved by run SP-Sepharose column followed by Mono Q column. Once the enzyme is purified, screening assays to study activities of new RNAP inhibitors will be performed based on commercially available KoolTM RNAP kit.
4.1 Minimum inhibitory concentrations (MICs)
Minimum inhibitory concentrations (MICs) are used most often as a research tool to determine the in vitro activity of new antimicrobial agents. The MICs of the new compound tested (compound X) for the six different levels of RIF-resistance strains of S. aureus were 16 µg/ml for all strains apart from strain R24 which was 8 µg/ml, while all these strains showed a different levels of resistance against rifampicin (see results). Rose bengal was also active against the RIF-resistance strains and the MIC of rose bengal against these strains was lower than it was of compound (X). In view of expectation of compound (X) and rose bengal are RNAP inhibitor, therefore, they more likely have a similar mechanism of action to rifampicin. Consequently, because they shows activities against rifampin-resistant mutants, accordingly, these compounds are promising compounds in term of their activities against S aureus in which needs further studies as RNAP inhibitors.
4.2 Effects of rifampicin and rose bengal on growth (culture turbidity) and survival (time-kill) of S. aureus
Time-kill determination is useful method for examining the bacteriostatic and bactericidal activity of antimicrobial agents. (Pankuch et al., 1994). Time-kill data showed that rose bengal and rifampicin are bactericidal agents, since the number of viable bacteria surviving after 6 hours' exposure to both compounds; at 4 and 16X the MIC for rifampicin and 1 and 4X the MIC for rose bengal, were reduced by more than 3 log10 CFU/ml compared to the untreated control. Time-kill data reveals that rose bengal and rifampicin act as dose-dependent, since killing was greater following exposure to higher concentration than it to lower concentration, indicating that rose bengal and rifampicin are both dose-dependent against S. aureus SH1000. As described previously by Oliva et al. (2003), the data obtained from time-kill studies can be used to determine whether a compound is bactericidal-lytic or bactericidal-non-lytic. A bactericidal-lytic agent defined as that agent causing a loss of viability accompanied by a decreasing in culture turbidity, whereas a bactericidal-non-lytic agent cause a loss of viability with no correlation to decreasing culture turbidity. The results show that rose bengal and rifampicin are both bactericidal-nonlytic agents, since both compounds caused loss of viability with no decrease in culture turbidity.
According to these data, it is clear that rose bengal and rifampicin are both have a similar activity on growth and survival of S. aureus SH1000 suggesting that both compounds may have a similar mechanism of action. Since rifampicin is well defined as an RNAP inhibitor and rose bengal has already proven as in vitro RNAP inhibitor it probably has an effect on RNAP in the whole cell. Further study have to be done to confirm that rose bengal is inhibiting RNAP activity in intact cells (e.g. whole cell RNA labelling assays using radiolabelled precursors such as [3H]uridine).
4.3 Mutation frequency
The mutation frequency for development of resistance to rose bengal was slightly less than for rifampicin (2.7 - 10 - 8 and 6.3 - 10 - 8, respectively). This difference is not statistically significant as calculated by Student's t-Test (t = 0.33, p > 0.05, d.f. = 5).
Previous studies have shown that various mutations can occur within rpoB (which encodes the ß-subunit of RNA polymerase) (Lal & Lal, 1994) that confer different levels of resistance to rifampicin. Hence, before considering whether other RNA polymerase inhibitors might be developed, it is important to evaluate mutational changes and sequencing of mutations in the rpoB gene that lead to rifampicin resistance, as rifampicin resistance presents an applicable measure of overall mutation frequencies (Bjorkholm et al., 2001). Further studies on rose bengal for instance, MICs for new mutants and detection mutations by sequencing rpoB gene to investigate whether it display differences in the type of rpoB mutations compared to those of rifampicin have to be done. However, attempting to generate mutants was faced by complications since there was no growth when I inoculated a single colony from the agar plates to the broth media containing 4, 2, or 1 X the MIC. The reason for this phenomenon could be due to binding of rose bengal to the agar.
4.4 BacLight assays
Bacterial membrane damage was initially examined using the BacLight assay. Rose bengal produced higher effects on membrane integrity values 18% of the control and is considered to be a membrane-damaging agent. Whereas rifampicin had lower effect on membrane integrity with a little more integrity with 81% of the control. Although further studies to detect mechanism of action of these agents on plasma membrane, lysis of bacteria by these agents may results from one of the following mechanisms: (i) interference with peptidoglycan synthesis followed by autolysis, (ii) cell membrane Solubilised by a detergent-like action, or (iii) autolysis that results from membrane deenergization (Oliva et al., 2003). Further investigations have to be performed to examine which mechanism of these rose bengal is work. On the other hand, although Backlight technique showed that rose bengal cause membrane damage which suggests that rose bengal is lytic agent, in contrast, the data obtained from growth curve of rose bengal revealed that rose bengal is nonlytic agent. This contradiction could be explained by the possibility of rose bengal to interfere with Baclight stains which may lead to give an error in reading by luminescence spectrometer. The other possibility is that rose bengal may partially damage cell membrane, so that allows propidium iodide stain to penetrate the partially damaged membrane in Baclight assay, and allowing the intracellular components to release in turbidity experiment, explaining why the growth curve of the bacterial culture treated with rose bengal was constant referring to rose bengal as a nonlytic agent.
Since these explanations are just possibilities, the data from Baclight experiment can't be ignored, so, rose bengal in this study are considered as a membrane-damaging agent. For the reason that bacterial and eukaryotic cell membranes, in general, share the same structure and function, rose bengal is thought to have some toxicity against mammalian, unless it has selectivity for bacterial membranes. Consequently, as a result of this disadvantage, rose bengal was excluded for further studies to be candidate as an antimicrobial agent.
The main object of this work is to screening new RNAP inhibitors which are designed and synthesised in the Department of Chemistry, Leeds University, or obtained from other institutes, of which I am already starting examine, against a range of bacterial species particularly S. aureus.
Minimum inhibitory concentrations, time-kill studies, assessment of bacterial membrane damage and determination of frequencies of mutation for resistance to these compounds will be performed. Once one is given promising results, I will test it in vitro against RNAP of S. aureus. Purification of RNA polymerase holoenzyme from S. aureus SH100 will be continued for RNAP assays, to study the inhibition of RNAP activity by new compounds.
I will conduct whole cell RNA labelling assays using radioactive precursors to confirm that inhibitors of interest are inhibiting RNAP activity in intact cells. Inhibitors with anti-staphylococcal activity will be screened against rifampicin-resistant S. aureus mutants with defined mutations in rpoB. This screen will allow me to distinguish any inhibitors that might be subject to existing mechanisms of resistance at the level of RNAP.
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