Experiment 5: Bacterial proteome probing under the effect of drugs
Objective: To study the effect of selective drugs on protein expression profile in bacteria
A range of micro-organisms are prevalent in our environment and their habitation in human bodies at times leads to a range of infectious diseases that are a threat to mankind. Micro-organisms prevailing in air, soil and water can be infectious to humans and may enter the body via various channels. Bacteria, out of all these micro-organisms, multiply rapidly usually by asexual reproduction, via. binary fission and doubles its number every 20 minutes (approx.). Their random multiplication in the body leads to severe effects at the site of infection as well as in other tissues. Specific chemical compounds (natural, semi-synthetic and modern) have been used till date which arrest the bacterial growth or kill them and have been used as effective drugs for ages. These drugs are targeted to inhibit DNA replication, protein synthesis or they alter the cell wall permeability, efflux pumps and metabolic inhibitors. Certain drugs also inhibit the cell division and stop their growth. However, with the regular administration of these drugs, certain species of bacteria are becoming resistant to their action and hence becoming irrevocably prevalent in society.
New potential compounds are a current day need to act as drugs against certain bacterial species that pose a threat to the health sector of the society. New compounds are expected to target new molecules or pathways that can be used against the resistant species and selectively inhibit the bacterial growth. Bacterial cell division machinery is a promising target for antibacterial drug development discovery due to their conserved property amongst the prokaryotes. Potential drugs are known which can inhibit the cell division machinery of bacteria by inhibiting certain participant proteins. Studying the effects of differing concentrations of these drugs on bacterial populations may give preliminary results of their efficiency on bacteria. Comparative proteomic analysis of the treated protein extracts against the normals will result in a couple of proteins that are differentially expressed and can be selected for further research. The role of these proteins in different pathways will confirm the mechanism of action of these drugs and their direct/in-direct involvement in the mechanism of drug action.
Bacterial proteomics is gaining immense significance as it elucidates the protein details resulting from the presence or absence of specific drugs, media components or other compounds. Prospective drug targets are easily predicted by proteomic analysis on comparison with the untreated samples and the proteins altered by the action of drugs are automatically observed on the test sample profile when compared to the normal sample profile. The steps needed to identify these proteins are given below. To gain the complete understanding of this experiment, we suggest the study of the given sections in the following order:
- Establishing bacterial cultures
- Checking the IC50 and MIC of drugs under study
- Preparing the test and treated samples
- Protein extraction and quantification
- 1D: IEF
- 2D: SDS-PAGE
- Staining, destaining and scanning
- Image analysis using ImageMaster 2D platinum 7.0
I. Establishing bacterial cultures
Bacteria are the prokaryotic organisms that differ from each other based on their cell wall composition, growth patterns, structural similarity, etc. Culturing bacteria is a routine practice as well as an important process in numerous laboratories for having a structural and functional insight into the bacterial world. Proteomics studies require sufficient concentration of protein samples for analyzing the proteome of the samples and hence a great deal of sample is needed for extraction of proteins. Various researchers make use of the in-vitro culturing for target oriented ventures as well as basic science investigations. The selected strains of bacteria are cultured in liquid broth. The organisms multiply at a faster rate with the doubling time of approx. 20 minutes. On achieving the desired turbidity or optical density, the cultures are streaked on LB plates and allowed to grow. The colonies grown can be sub-cultured and maintained for future use. Bacterial Cell division is complex cytological process and is very important.Generally most of the bacterias reproduce through binary fission and as result cell cultures are doubled. DNA replicates by forming bubble at the replication fork , thereby other componets also get doubled for division. As the DNA replicates septum formation also strats at mid cell and gets divided into genetically identical daughter cells.
Fig.1: Schematic representationof bacterial cell division
II. Checking the IC50 and MIC of drugs under study
Effectiveness of drug action largely depends on the amount of drug administered to an organism or a study sample. The percentage of the number of samples surviving in a population on the administration of certain amount of drug decides its effectiveness and dosage. The IC50 stands for that concentration of the drug at which 50% of the population survives. It is the half maximal (50%) inhibitory concentration (IC) of a drug. MIC is the minimum inhibitory concentration of the drug that prevents any visual growth of the micro-organisms. IC50 is usually employed for investigating the action of a certain drug on a microbial population.
III. Preparing the test and control samples
Sample preparation accounts for one of the most crucial steps in any study, as for its complexity and quality will reflect on the acceptability of the results. The test samples are prepared by culturing selected bacterial strains in LB broth overnight and then the specific concentration (IC50) of drugs is added to the media and grown further till the cultures reach desired optical density and opacity. The control samples for this study are similar to the test samples in all parameters except for the absence of the drug in the media. Both the samples are cultured under sterile conditions and harvested by centrifugation which pellets down all the bacterial cells. These pellets are then processed further for protein extraction.
IV.Protein extraction and Quantification
The protein pellets are subjected to cell lyses to release the cellular material. The cellular components are subjected to phase separation to selectively separate the proteins from nucleic acids and lipids based on their properties and affinities towards different solvents. The separated proteins are precipitated and purified to remove the salts and other micro-contaminants and suspended in the respective buffers. Protein quantification becomes indispensable to identify the concentration of the purified proteins as specific minimum amount of proteins are required to be carried forward for 2-DE.
IEF or Iso-electric focusing includes the separation of the proteins based on their pI values on IPG strips or Immobilized pH gradient strips. The proteins are reduced by DTT and rehydrated on the IPG strips of selected pH range and length by passive or active absorption. The strips absorb and retain the proteins for further separation. These strips are subjected to increasing voltage to remove the salts in the initial run and finally separate the proteins where the proteins migrate on the strips based on their charge under the effect of high voltage and stop and concentrate at the pH values where their net charge is zero (Isoelectric pH).
Fig.2: Schematic representation of isoelectric focusing of proteins starts with the rehydration of strip with sample followed with placing the strip on the manifold, pouring the mineral oil in the lanes to carry out focusing.
Equilibration refers to reduction and alkylation of the cysteine residues which leads to the primary peptides and prevent them from folding back to form disulphide. Equilibration with buffer I (containing DTT) breaks all the disulphide linkages in between the peptides to yield primary peptide chains while equilibration with buffer II (containing IAA) leads to alkylation of the cysteine residues to prevent the re-formation of the disulphide linkages. This leads to the formation of linear peptides which are used further for SDS-PAGE.
VI 2D: SDS-PAGE
The second dimension or SDS-PAGE separates the previously separated proteins on the basis of their molecular weights with the High Molecular weight proteins migrating slower and Low molecular weight proteins migrating faster. The proteins are separated under denaturing conditions usually on a 12.5% gel. The gels are then stained with CBB (Coomassie Brilliant Blue) which interacts with protein residues by hydrophobic and ionic interactions. Overnight staining is sufficient to stain the most faint protein spot which is followed by destaining to remove excess of stain. The subsequent gels are scanned and saved for further analysis.
Fig3: The set unit for SDS-PAGE starts with a) Gel casting assembly b) Casting of gel c) placing the focused strip with sealing with agarose d) Setting up for separation by pouring tank buffer e) Typical experimental set-up unit for SDS-PAGE.
VIII.Image analysis using ImageMaster 2D Platinum 7.0
The gels are analyzed after equal cropping of images for detection of the number of spots on each gel. The spot boundaries are marked either automatically or manually to avoid any errors. The treated sample gels are compared with the control sample gels for the differential expression of the protein spots within the range of t-test and ANOVA values. This data is analyzed for target protein identification.
Mechanism of drug action: Drug action on prokaryotes differs based on the specific drug composition and its targets. Drugs/antibiotics act on different cell components or pathways and destroy the cells. There are 5 different modes by which the drugs attack on the bacterial cells. These may be:
1.Inhibition of cell wall synthesis: These drugs result in the failure of bacteria to cross - link the membranes, by disrupting the peptodiglycan cross-linkages.
2.Inhibition of protein synthesis: These drugs bind to the 30S or 50S ribosomal subunits and prevent the assembly of the translational complex and hence kill the bacteria.
3.Alteration of cell membranes: These drugs disrupt the cell membrane by puncturing or by other mechanism and kill the organism.
4.Inhibition of nucleic acid synthesis: These drugs bind or inhibit the enzymes involved in DNA replication and hence prevent DNA systhesis.
5.Antimetabolite activity: These prevent the synthesis of a couple of metabolites in the cell and hence affect the overall cell functions.
These drugs have been used for a long period which has led to the development of resistance in bacteria against these drugs. Thus, new drugs are required for managing these issues with new target mechanisms.
Totarol is a natural diterpenoid, which is extracted from the bark of Podocarpus family. It possesses anti-microbial, anti-oxidant, cytotoxic, anti-fungal, anti-plasmodial and anti-tumor activity. Totarol parent compound and its semi-synthetic derivatives are widely used in commercial products for skin and oral care. Understanding the mechanism of action of totarol has suggested that it has multiple actions such as inhibition of energy-coupled respiration transport, prevention of peroxidation of unsaturated fatty acids in lipid bilayer, hampering the oxidative phosphorylation by acting either as uncoupler or inhibiting the crucial enzymes. It is also found to inhibit the multidrug efflux pump, disturb the phospholipids bilayer integrity and recently, it was established that it inhibits bacterial cell division by inhibiting FtsZ polymerization. Totarol is a potential drug to combat the antibiotic resistant strains; however, its mechanism of action is not very clearly understood, and therefore, there is a need to apply latest high throughput genomic and proteomic approaches.
Fig 4 : Chemical structure of Totarol
Curcumin is a dietary polyphenolic compound having potential biological activities such as antibacterial property against Staphylococcus aureus, S. epidermidis and enterococcus, antioxidant activity, anti-proliferative activity and wound healing ability. Besides these properties, it can also show phototoxic effect against E.coli and S.typhimurium, antigenotoxic effect against DNA affecting drugs and also inhibit SOS response due to damage in DNA during cell division. It is extracted from rhizomes of Curcuma longa. Recently, it has been found that, curcumin is able to effect tubulin polymerization. Curcumin can inhibit the growth of many Gram positive as well as Gram negative organisms. Interestingly, FtsZ is homologous to tubulin. Researchers have started using curcumin to study its effect on Z-ring assembly. Curcumin inhibit the cytokinesis by inhibiting the FtsZ polymerization which leads to elongation of the cell. Curcumin treatment significantly reduces the Z-ring formation as well as frequency of Z-ring per cell. Curcumin hamper the Z-ring assembly by directly binding FtsZ monomers. Curcumin binding makes conformational change.
Fig 5 : Chemical structure of Curcumin