There is a big concern with bacterial resistance and this has increased significantly in recent years. One of these concerns is with staphylococci, particularly MRSA. The study of bacterial cellular proteins which play an important role in bacterial virulence might assist in improvement in development of new effective drug for treatment. There are many methods of protein extraction that can be employed in this study.
In 1880 Alexander Ogston discovered that certain abscesses in human are cause by cluster forming microorganism. And in 1882 he named the organism Staphylococcus. The term of Staphylococcus was synthesized from Greek word staphyle that means bunch of grapes and coccus means grain or berry this due to their ability to form grapes like clusters under microscopy. Staphylococci make up family of gram positive cocci that belong to Micrococcaceae (Crosslwy.2009).
They are spherical, non motile and form grape-like clusters ( Figure 1). They are facultative anaerobes (can grow in aerobe and anaerobe atmosphere) .They form bunches because they divide in two planes as opposed to their close relatives streptococci which, although they are similarly shaped, form chains because they divide only in one plane. They are gram positive cocci which resist decolourization by alcohol in Gram's method of staining and thus retain the crystal violet-iodine complex and appear purple; a characteristic of bacteria whose cell wall is composed of a thick layer of peptidoglyca and teichoic acid( Crossley.2009).
Staphylococci produce catalase enzyme that break down hydrogen peroxide H2O2 into water and oxygen and this can differentiate from the other group of gram positive cocci which is Streptococcus which cannot secrete catalase enzyme. Catalase is important virulence factor because H2O2 is microbicidal and its degradation limits the ability of neutrophil to kill ( Reuse & Bereswill.2009).There are three main species of staphylococcal human pathogens which are Staphylococcus epidermis, Staphylococcus saprophyticus and Staphylococcus aureus which is of greatest concern in the form of MRSA. Staphylococcus aureus or S.aureus is a bacterium that is commonly found in the environment and on the nose, skin and groin of healthy people. And it is the major human pathogen responsible for increasing the prevalence of community and hospital acquired infection. Staph aureus is one of the most virulent species that secretes coagulase enzyme that causes citrated plasma to clot by activating prothrombin to form thrombin and this will catalyze fibrinogen to fibrin clot so it is coagulase positive organism. This will help in distinguish from other coagulase negative organism such as Staph epidermis and Staph saprophyticus. Staph aureus are beta haemolytic due to the production of several haemolysins α toxin, β toxin, γ toxin and δ toxin. It can grow in a wide pH (4.8-9.4). It metabolises glucose, xylose, lactose, sucrose, maltose and glycol. Also it has ability to grow in a high salt medium due to its production of osmoprotectants and can tolerate 7.5-10% NaCl; this allows it to inhabit the skin. When it grows in mannitol salt agar it ferments mannitol to organic acids and form yellow zone around the colonies (Crossly, 2009).
Virulence factors and pathogenesis
The pathogenecity of S.aureus is determined by its ability to express multiple virulence factors ( Figure 2). These factors divide according to the cellular location and performed function. S.aureus causes disease ranging from mild infection to serious and potentially fatal systemic infection. These diseases divide into those which are mediated by toxins such as food poising and toxic shock syndrome. The second types are infections which may be cutaneous such as impetigo, folliculitis and wound infection. Other infection includes bacteraemia, endocarditis, septic arthritis and pneumonia (Salyers& Whitt.2002). S. aureus expresses many effectiveness virulence factors. There are surface proteins that encourage colonization of host tissues, invasins that promote bacterial spread in tissues (leukocidin, kinases, hyaluronidase), surface factors that inhibit phagocytic enhancing (capsule, Protein A), biochemical properties that enhance their endurance in phagocytes (carotenoids, catalase production), immunological disguises (Protein A, coagulase), membrane-damaging toxins that lyse eucaryotic cell membranes (hemolysins, leukotoxin, leukocidin ,exotoxins that destroy host tissues or expected raise symptoms of disease (SEA-G, TSST, ET) and inherent and acquired resistance to antimicrobial agents.
History of MRSA
During the past four decades, a type of bacteria has evolved from a controllable nuisance into a serious public health concern. This bacterium is known as MRSA stand for methicillin-resistant Staphylococcus aureus. It is one of the strains of staphylococci that are resistant to specific antibiotic which is methicillin. And it is one of the most common one which infect human. The semiynthertic and pencillinase resistance antibiotic methicillin was launched in 1959 to over come the problem of penicillin resistance S.aureus. The first clinical case of MRSA strain from UK was isolated in 1961. Many reports of MRSA out breaks in UK and Europe followed in 1960s and at the end of 1970s outbreak of MRSA had been reported from USA. MRSA is particular is considered a growing epidemiological and therapeutic problem , because they exhibit resistant to almost all other type of beta lactam antibiotic (Mayhall, 2004). The action of MRSA is similar to Staphylococcus aureus but the difference is that it is resistant to the antibiotics that are make it difficult to treat.MRSA can be categorized according to where the infection was acquired: hospital-acquired MRSA (HA-MRSA) or community -associated MRSA (CA-MRSA). HA-MRSA is acquired in the hospital setting and is one of many hospital-acquired infections exhibiting increased antimicrobial resistance. HA-MRSA has increased during the past decade due to a number of factors including an increased number of immunocompromised and elderly patients, advanced surgical operations and life support treatments, and failures in infection control measures such as hand washing prior to patient contact and removal of non-essential catheters( Brown & cookson. 2003).
Mechanism of resistance
Methicillin resistance needs the presence of the chromosomally localized mecA gene. The mecA gene is the gene responsible for methicillin resistance and is part of a mobile genetic component found in many MRSA strains called SCCmec (Figure 3). Up to now, there are at least five different SCCmec elements. These elements incorporate at the same time site in the chromosome by a mechanism involving site-specific recombination and excision from the chromosome at attBscc that is a part of an open reading frame of unknown function near the origin of replication. The genetic mechanism that is responsible for transfer of these mobile elements is unsure. Nevertheless, what we do know is that mecA gene in MRSA is responsible for the synthesis of penicillin binding protein2A. The MecA gene formula varies PBP2A in S aureus resulting in a loss of this target enzyme's susceptibility to penicillin. The acquisition of the mecA gene converts a b-lactam sensitive cell into one that is insensitive to penicillin (Rohrer et al., 2003) .
The development and skill of resistance in MRSA also requires the presence of other mechanisms. Horizontal gene transfer is a mechanism by which plasmids (resistant genes contained in small packets) found inside the cytoplasm of the bacteria has the ability to shift resistance genes between the same and different species (Cookson et. 2003). The three different mechanisms demanded in resistance are:
1) Conjugation is where there is cell-to-cell interaction.
2) Transformation is where genes from bacterial DNA in the external environment are acquired.
3) Transduction needs bacteriophages transferring DNA between two nearly related bacteria.
Bacterial Cell wall
The cell wall represents the outermost boundary of any bacteria cell. Unless if there is a glycocalyx or slime layer, which could be predominantly polysaccharide or protein. Because of its location it has many functions. It separates the interior of the bacteria from the outer environment in order to protect the cell from any harmful of surrounding area. Also it enables to transport substance and information inside and outside the cell and this it helps the cell to communicate with the environment. In addition it provides the bacteria its shape stability, insensitivity of osmotic shock, enables metabolism and growth, multiplication of cells. The elasticity of cell wall gives its considerable expansion (Seltmann& Holst, 2002).
In 1884 Christian Gram has successful in differentiating two large group of bacteria that categorized into two groups which are gram positive bacteria and gram negative bacteria and that by staining method called gram stain( Crossley,2009). Figure 4 shows the differences between gram positive and gram negative cell wall. Gram positive bacteria has many layers of peptidoglycan around 20-30 layers that gives thick and rigid structure and this thickness of this wall blocks the escape of the crystal violet-iodine complex when the cells are washed with alcohol or acetone during Gram staining. It doesn't possess an outer lipid membrane in contrast to gram negative bacteria that has one or few layers of peptidoglycan surrounding by a thin outer membrane composed of lipopolysaccharide (LPS). The region between the peptidoglycan and LPS layers is termed the periplasmic space it is a fluid or gel-like zone containing many enzymes and nutrient-carrier proteins. The crystal violet-iodine complex is easily lost through the LPS and thin peptidoglycan layer when the cells are treated with a solvent .The figure below shows the difference between gram positive and gram negative cell wall( Seltmann& Holst, 2002)..
This project is concerned with breaking the cell wall of staphylococci, so components of the cell wall and their function should be explained.
The cell wall of gram positive bacteria consists of peptidoglycan, secondary polymers teichoic acids, proteins and carbohydrates (Figure 5).
Structure; The cell wall of gram positive bacteria consist of a thick layer of peptidoglycan or other name of it is mucopeptide or murein and this contain of chains of disaccharides each composed of monosaccharides which are N acetylglucosamine (NAG) and N acetylmuramic acid (NAM) forming long chain which completely surround cytoplasm membrane.(Ghuysen&Hokenbeck.,1994).
And this NAM connected to tetrapeptide chain joined to one another by transpeptide bond or short polypeptide chain of amino acids and that by either direct peptide bonds bridges or indirect through pentaglycine cross-bridge ( Figure 7). The main chemical structure of peptidoglycan is remaining same although there is considerable variation in the details of composition in different species. The glycan chains are composed of alternating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), all linkages between sugars being ß,1->4. The tetrapeptide is connected at its N-terminus to the carboxyl group of mumaric acid and composed of L- and D- amino acids. When the interpeptide is formed the C- terminal D-ALA is removed ( Seltman & Holst., 2002) in (Figure 6).
The cross link peptide varies from species to species. As in staph aureus the thick cell wall of peptidoglycan have five glycines, L-Alanine, D- Glutamine, L-Lysine and two D-Alanine. They make up the linker between peptidoglycan monomers .
Synthesis and Function of peptidoglycan
New peptidoglycan synthesis takes place at the cell division machinery known as the divisome. In the divisome a bacterial enzymes called autolysins break the glycosidic bonds and the peptide cross-bridges that link the rows of sugars together. The bactoprenols transport the peptidoglycan monomers across the cytoplasmic membrane and helps insert them into the growing peptidoglycan chains to make the wall strong. As (Figure 9) shows that the synthesis involve such stages which starts in forming UDP-NAG by linkage between N-acetylglucosamine with uridine diphosphate and Some of the NAG is enzymatically converted to N-acetylmuramic acid (NAM) and form UDP-NAM. Five of amino acids are added to the UDP- NAM forming a pentapeptide. The two of D-alanine molecules enzymatically resulted from L-alanine. The NAM-pentapeptide is bind to the bactoprenol molecule in the cytoplasmic membrane, and then NAG is attached to the NAM-pentapeptide on the bactoprenol to form the peptidoglycan monomer (Barton ., 2005).
Peptidoglycan has such function as it is provides the rigidity of the cell. It helps in reshaping of the wall during cell growth and it assists in cell separation during division. If any damage occurs or inhibition of the synthesis the cells become distorted and lysis and that due to increase in internal osmotic pressure.
Staphylococcus aures is usually resistant to the lysis by murmidase lysozyme. And the thick wall of the gram positive is easy lysis by the endpeptidase lysostaphin that is type of glycosides which hydrolysis the glycine-glycine linkage in the glycan chain resulting in depolymerise the peptidoglycan leading to the breakdown in cell wall and lysis of the cell.
1- Teichoic acids
Structure; the cell wall contains of an anionic polysaccharides derivative called teichoic acid that bound to NAM in glycan chain that is important in cell viability( Figure 10). It is a phosphoodiester polymer (polyol phosphate polymer) of glycerol or ribitol linked by phosphate group. Ribitol teichoic acids associated with the cell wall and linked to the peptidoglycan through C6 hydroxyl group of N-acetylmumaric acid while the glycerol associated with inner of bacterial cell membrane and linked through glycolipid of cytoplasmic membrane. The substitute groups on the polyol chains could have D-alanine, N-acetylglucosamine, N-acetylgalactosamine and sugar glucose (Barton,2005).
Function ; the teichoic acids function is not clear but it helps to maintain the surface charge as negative charge because it contains phosphate group, control the activity of autolytic enzyme that act on peptidoglycan ( enzyme that break bonds in the peptidoglycan thereby causing lysis of the cells( Seltmann& Holst, 2002). Also it stabilizes the cell wall and makes it stronger and in addition it requires in demonstrating the electrical properties of the cell wall by establishing Mg ions concentration and chemical facilities of the cytoplasmic membrane. Function in peptidoglycan synthesis and play a role in competence of gram positive to undergo bacterial transformation (Barton, 2005)
The cell wall carbohydrates
They are the polysaccharides that bound to the peptidoglycan and associated to the cell wall. It is function unclear but it refers to mediates the communication with the elements in the environment and effect the chemical characteristics of the cell wall (Steen, 2005).
The cell wall proteins
Protein is the fundamental unit of all living cells. Bacterial proteins play an important role in different process like signalling, metabolism, bioenergetics and molecular transport. Bacteria are synthesis protein in order to interact and communicate with the environment. Most of the proteins are soluble enzymes but some of the proteins are ribosomal proteins. And these soluble cytoplasmic proteins are responsible in metabolism and control of all cell process. The cell wall of gram positive is the site in which the enzymes and proteins will react with the environment and needed for cell wall make up and breakdown. Also there are many layers of proteins that make S-layer in which it determine the shape of the cell, adhesion and protection. In addition these layers cover the cell during cell growth (Sara et al., 2000).
Introduction of cell wall lysis
Cell lysis or cellular disruption is a cell biology method for the release of biological molecules including organelles, proteins, DNA, RNA and lipids from inside a cell. As this project consider on the extract the protein through cell wall lysis of gram positive bacteria causing to release their protein component such peptidoglycan fragments and teichoic acids . The cell biology, molecular biology and immunological studies of the cellular proteins give essential information about bacterial pathogenesis in human and animals. Therefore developing of techniques and methods for examining distribution of biological macromolecules such as protein has been an important prerequisite for many of the advancement made in bioscience and biotechnology over past years.
In order to isolate intracellular proteins, cells must be disrupted. Disruption methods divide into chemical or enzymatic (non mechanical) and mechanical or physical techniques. An efficient protocol for cell disruption must be formulated to release the protein in soluble form from its cellular elements. Also it should be as gentle as possible to the protein, as extraction step is the beginning point for subsequent procedure. All these techniques depend on the starting material such as tissue, cell and blood. Also it looks for the source of organism for examples bacteria, viruses or plants (Mitra. 2003). The efficiency of lysis of microorganisms varies among species and the type of organism and its physiology play an important role in breakage of cell wall. As in general the gram negative bacteria are more easily in lysis than gram positive bacteria and this will be discussed later. As well as the old cells can be lysed more easily than younger cells.
Non mechanical methods are several techniques in which are confirmed that are useful and can be used in large scale. Also they are better as they give a prediction of releasing components because they are gentle in treatment, no need for shear or heat and inexpensive. Some of the techniques are enzymatic digestion which is gentle and digests cell wall although the cost of enzyme is moderate. Osmotic shock lysis is inexpensive, gentle and osmotic disruption of cell membrane .Freeze-thaw method is commonly used to lyse bacterial and mammalian cells. The technique involves freezing a cell suspension in a dry ice/ethanol bath or freezer and then thawing the material at room temperature or 37°C. This method of lysis causes cells to break as ice crystals form during the freezing process and then contract during thawing. Chemical and deteragents are gentle lysis and its mechanism depends on solubilization of the lipid bilayer ( Mitra, 2003).
For mechanical methods there are multi techniques which are showed successful in cell distribution for micro organisms and the mode of lysis is harsh to moderate and their mechanism depend on the shear force. Such as grinding with alumina or sand; cell wall are ripped off by micro roughness, grinding with glass beads; cell wall are ripped off by rapid vibration of glass, French press; in which the cells are forced through small orifice and very high pressure shear force disrupts cells and sonication or ultra sonic the cell is disrupted by shear forces and cavitations resulted from high pressure sound waves (Mitra, 2003).
This project uses two methods in order to extract protein from Staphylococcus aureus which are sonication and bead beater.
This method is an ideal one for breaking cell wall in a suspension. It is act of applying sound commonly ultrasounds energy to stimulate particles in a sample for many purpose. In biological application, it may be sufficient to disrupt a biological material as it is disrupt cell membrane and release cellular content and this process is called sonoportion. It works when a metal probe is lowered into the cell suspension with the high sound waves generated for a period of time. Then it produces shearing and cavitations that are rapid form collapses and turn up with bubbles in the cell suspension. These collapses of bubbles modify sonic energy into mechanical energy in the form of shock waves and break the cells membrane. The ultrasound process generates heat so the suspension should be placed on ice in order to avoid protein damage (William & Wilson, 2004).There are some good feature of sonicater. It is ideal for small samples (0.3-5 ml), easy to hold and feather-light, simple to use (just hold it in a vessel and depress the push-button power switch) and easy to clean. On the other hand there is disadvantage of the localized heating can occur leading to protein denaturation or aggregation so cooling the sample is essential.
A study done by Fyske et.al in 2003 in a rapid sonication method for lysis of gram positive bacteria i.e. bacillus cerues and this was evaluated with the real time of Polymerase chain reaction PCR as analysis for detection. The aim of the study was complete destruction of cell structure in order to release DNA without using any lysis solution and to avoid time consuming. It was established by the real time PCR that the maximum produce of DNA was existed after 3-5 min of sonication (Fayske et el., 2003).
Bead homogenization and grinding (Bead Beater)
Bead beater is a usual method that used in laboratories for cell disruption. The process depends on the vigorous agitation and that by adding beads which may be made of glass, ceramic, steel, zirconia or tungsten to the cell suspension and then mixing by vortex or other mixer. The action of the vortexed beads that it breaks the cells in order to release the cellular content of the cell. Cooling of the sample needed due to the heating that result from the extreme agitation (William & Wilson, 2004). Also there some feature of bead beater. It is rapid and highly efficient, simple to use, with disposable vials and beads, totally Sealed so no dangerous aerosols and very high energy shaking
A study done by Eun Taex Oh and Jae Seong So in 2002 in a rapid method for RNA prepration from gram positive bacteria. They used method that not requires lyzosome and proteinase K which is glass beads in order to break cell completely. They were success in isolate total RNA from different gram positive bacteria. As this method is rapid, simple and economic. The prepared RNA can be used as transcriptional analysis of various gram positive bacteria (Taex & Seong., 2002).
It is a serious of process meant to isolate a single type of protein from a complex mixture. Protein purification is critical for the characterization of the function, structure and interaction of the protein. The starting material is commonly a biological tissue or microbial culture. The several steps in the purifications process may free the protein from the matrix that holds it, separate the protein and non protein parts of mixture, at last separate the wanted proteins from all other proteins. Separation of one protein from all others is typically the hardest sense of protein purification. Also separation steps are affected by differences in protein size, physical- chemical attributes and binding affinity (Ahmed. 2004). Protein purification uses money, time, efforts and laboratory equipments therefore it is important to consider the reasons in which it is important. There are typical reasons such as to identify the function of the protein (e.g. enzyme), identify the structure of the protein, to use it as a part of research project and as commercial uses such as diagnostic or therapeutic (Roe. 2001)
Protein electrophoresis is a technique used to separate the different component proteins (fractions) in a mixture of proteins, base on the differences of the components that moves through a fluid-filled matrix under the influence of an applied electric. It can be subjected to one and two dimensional electrophoresis and followed by measurement of the intensity of the signals.
Gel electrophoresis separates molecules such as DNA, RNA or proteins into its individual polypeptides. This technique separates proteins according to their physical properties as they are moved through a gel by an electrical current. It provides much information about molecular weight, charges of protein, subunit structure of protein and the purity of the protein. It is simple and high reproducible. Also it has quantitative and preparative purpose due to the highly sensitive microanalytical methods and linear image analysis system (Garfin, 2003).
Gel is the key element in gel electrophoresis techniques and it determines the migration rate of protein holding it in a place at the end of the run until they appears by stain.
Polyacrylamide gel is the principle medium in protein electrophoresis. It has many features that make it well suited. The gel can be figured out in range of pore size and that useful for purifying proteins. The polymerization reaction easy, reproducible and can be cast in several shapes. Although the pore size is determines by the action of polymerization reaction. Also the polyacrylamide gels are hydrophilic and electrically neutral. They are transparent to light and do not bind to protein stain (Garfin, 2003).
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is an anionic detergent that breaks the non-covalent interactions in proteins. It is common, simple and reliable practical technique in which it estimates the number of polypeptides, compare of protein content and monitor the different fraction that obtained through chematographic or other purification methods. The limitation is that the protein should be denatured to their polypeptide chains because it is difficult to interpret the non denatured proteins in the gel. And that can be done with the isoelectric focusing that will be discussed later (Garfin, 2003).
Two- Dimensional gel electrophoresis (isoelectric focusing)
The rate of migration of the molecules depends on the net charge, size and shape of the molecules. Also it depends on the ionic strength, temperature and viscosity of the medium that are moving on. Proteins are amphoteric molecules; they can be positive, negative and zero charge. For every protein there is specific pH if the net charge is zero. This pH is called isoelectric focusing (IEF). If the pH above isoelectric point, a protein has negative charge so it moves towards the anode in an electric field. In contrast if the pH below isoelectric point the protein becomes positively charged and moves towards the cathode (William & Wilson, 2004). Although the proteins in IEF consist of multiple species on the other hand they are single species in SDS-PAGE. From the point of the electrophoresis; there are two most essential properties of protein which are electrophortic mobilities and isoelectric point. The electrophortic mobilities of proteins depend on the charge, size, and shape and influenced by pH, amount and type of counter ions. But the IEF depend only on the net charge.
Two- Dimensional gel electrophoresis is a powerful proteomic method for separating component mixture of protein into many more components. It was first introduces by O'Farrell in 1975. It separates complex protein from mixture such protein extracted from cells, tissues and other biological fluid samples into individual polypeptide chains. This separation is based on their isoelectric point in the first dimension and then using sodium dodeylsulfate (SDS) polycrylamid gel electrophoresis (PAGE) to separate these bases on the apparent size in the second dimension (Simpson.2003). Results are display as an array of protein spots not like protein bands that seen in one dimension electrophoresis.
Because 2-D electrophoresis is much more complex than ordinary polyacrylamide gel electrophoresis, and the identification of individual proteins is both complex and expensive. Although it is highly sensitive and available also it allows the visualization of hundreds of protein that helps in analytical purposes such detection of disease markers, discovery of drug and research of cancer.