Author: Harshini Balaga
Mentor: Dr. Vincent Boudreau, Ph.D. Postdoc University of California Berkeley
St. Francis College For Women
From past times to recent times antibiotic resistance has emerged as a global threat. Antibiotic resistance has mainly affected the progress in health care, food industries and ultimately life expectancy. Almost all regions of the world are suffering from these antibiotic resistant diseases. This is mainly due to the movement of people, animals and goods across borders and countries. The emergence of different species of bacterium like Staphylococcus aureus, Enterococcus faecium, Streptococcus pyogenes, Staphylococcus pneumonia and more by adapting various defense mechanisms, has lead to the development of resistance towards antibiotics like Penicillin and Methicillin. This resistance is mainly caused by the presence of two important genes called mec-A that codes the PBP2a protein. mec-A and PBP2a are known to confer resistance, but the mechanism of resistance remains unclear. Further study of the mechanism has the potential to develop a new generation of antibiotics. Here, we have investigated the structural biology basis of PBP2a antibiotic resistance and the contribution of the genetic background of resistant S. aureus strains. It has been found that PBP2a confers resistance through its Ser403 residue in its active site, but that PBP2a is not sufficient to drive resistance. Genetic studies have identified an additional gene responsible for conferring PBP2a-based resistance, that is the bla gene that codes a beta lactamase. The presence of bla genes along with mec-A is important for effective activity of the PBP2a protein, as the presence of mec-A alone showed insufficient activity of the PBP2a protein. Western blot analysis of PBP2a expression in different S. aureus strains determined, that the concentration of PBP2a protein was high in cells containing plasmids that carried both mec-A and bla genes and low concentration of PBP2a was found in cells containing plasmids carrying only the mec-A gene.
Antibiotic resistance is a property where a bacteria becomes resistant to antibiotics that are designed to kill them. Bacteria can escape inhibitory effects of drugs by acquiring certain mechanisms of resistance. There are several problems that are caused due to the persistent increase in the antibacterial resistance in bacteria. Antibiotic resistance occurs naturally by random mutations (Julin Davis and Dorothy Davis, 2010) through the process of natural selection and also can occur by selective pressure on the bacterial population (Alfredo Tello, et al. 2012). If a resistant gene is generated, it can be transferred from one strain to another by exchange of plasmids through horizontal gene transfer (HGT). During this process of acquiring resistance the antibiotics act as environmental pressure, due to which bacteria undergo certain mutations for its survival. When such strains proliferate, they produce progeny that are resistant towards various antibiotics by evolving certain resistance mechanisms.
Over usage of antibiotics, usage of broad spectrum antibiotics, incorrect diagnostics, unwanted prescription, wrong usage of antibiotics by patients and use of antibiotics in cattle feed for enhancing early growth, are mainly responsible for antibiotic resistance. It was discovered that the lexA gene is mostly responsible for these mutations (Charlie Ye Mo, 2016). A bacteria called Staphylococcus aureus is one of the major resistant pathogens, which is responsible for causing various diseases. Later on it became known as MRSA that was first detected in Britain in 1961, which is resistant to the methicillin antibiotic. Later in 1991, 4% of fatal cases of infections were recorded and rose to 37% by 1999 in the UK. In the USA 50% of the Staphylococcus aureus were resistant to tetracycline, penicillin, methicillin and erythromycin. Vancomycin used to be an effective drug at that period for Staphylococcus aureus but in the late 1990’s intermediate strains were detected with moderate resistance to Vancomycin at 4 μg/ml, which was named Vancomycin intermediate Staphylococcus aureus. In Japan in 1996 the first case was identified with a strain which is resistant to Vamcomycin at >16 μg/ml and the second case was found in the USA in 2002 (Susana Gardete and Alexander Tomasz, 2014 july).
In the late 1990’s new antibiotics like oxazolidinone and linezolid were found to be effective against MRSA. In 2003 Staphylococcus aureus resistance to linezolid was observed. CA-MRSA, community acquired MRSA, is the most common antimicrobial drug resistant pathogen found in US hospitals and became part of Endemic infections at that time. Along with MRSA there are other bacterial strains that are found to have resistance including Enterococcus faecium (penicillin resistant), in 1983, (vancomycin resistant) Enterococcus in 1987 and linezolid resistant Enterococcus in the late 1990’s. However group A Streptococcus pyogenes remained sensitive to penicillin. Later penicillin resistant Staphylococcus pneumonia emerged worldwide. The main gene responsible for beta lactamase and penicillin resistance is mec-A which encodes PBP2a proteins.
There are several problems caused by increasing antibiotic resistance that have become a major threat in various sectors including health, food, security, clinical sectors, agricultural sectors, It is most commonly prevalent among frontline workers like military officers, hospital staff and patients. It can affect anyone of any age, gender and any country. It occurs naturally, but misuse of antibiotics in humans and animals accelerate this process. Due to antibiotic resistance, the effectiveness of antibiotics is decreasing. As a result, it is becoming difficult to treat various diseases including pneumonia, tuberculosis, gonorrhea and salmonellosis.It leads to higher medical costs, longer hospital stays and increased mortality. Plasmids are one of the major important vectors which are responsible for spreading resistance among the strains in soil in agroecosystems. PBR3222 is one such resistant plasmid which carries genes for tetracycline and ampicillin resistance. HGT is one of the most common mechanisms by which bacteria either acquire or exchange resistance from other bacteria. Mobile genetic elements (MGE) like integrons (which are gene cassettes) usually carry antibiotic resistant genes to bacterial plasmids and transposons. Most of this resistance is due to the presence of certain proteins and genes, mainly the mecA gene which was originated from coagulase negative Staphylococci and is associated with a mobile genetic element called Staphylococcal chromosomal cassette mec (secmec) and integrates in the Staphylococcal chromosome. mecA encodes for the PBP2a protein, which is a transpeptidase and has a low affinity for beta lactam antibiotics like methicillin and tetracycline. PBP2a being a transpeptidase is involved in the continuous synthesis of the cell wall during antibiotic attack. PBP2d protein along with beta lactamases are mainly responsible for the broad spectrum resistance in MRSA.
The genes mec-A for the PBP2a protein and bla gene for beta lactamases are mainly responsible for resistance in bacteria like MRSA. To know the mechanism behind the resistance and to study the location and properties of the active site, the study of crystalline structures of PBP2a is very important (Lim and colleagues, 2002). For the crystallization process, cells were grown on m9 medium to which 50 g of protein solution containing thr, leu, met, selenomethionine were added per liter of culture. After 15 minutes, protein expression was induced and the colonies were subjected to further purification by hydrophobic affinity chromatography and gel filtration exchange chromatography by adding 0.1% thiodiglycol. Cells were lysed and native Staphylococcus aureus (Sau) PBP2a was extracted as a soluble protein by a purification process using a hydroxyapatite column.
Further purification was done by gel filtration chromatography on a sepharyle 1000 column apparatus, which is equilibrated with 5 mM NaHCO3. Extraction of PBP2a was carried out using a hanging drop vapour diffusion method. Then, exposure of PBP2a with chemical compounds like NaCl, PEG and HEPES at pH 7 led to the formation of crystals and the crystals were extracted by a cryopreservation technique in which the N terminal anchor of PBP2a was replaced with a methionine and was observed under electrospray mass spectrometry. When beta lactams are introduced, they inhibit beta lactam sensitive PBP’s. Strains containing PBP2a have low affinity for beta lactams, conferring antibiotic resistance by the continuous synthesis of the cell wall, which prevents the cell from lysis. Beta lactam antibiotics like penicillin and methicillin are substrate analogs of PBPs and catalyze the process of cell wall lysis and eventually lead to death. However,acquisition of mec-A genes by MRSA confers resistance. The mec-A gene is highly conserved among MRSA isolates with > 90% sequence identity and encodes the PBP2a protein. This PBP2a is resistant to beta lactam and does not have any sensitive analogs or homologs. It has a large molecular mass of 78 kD and belongs to class B PBPs which usually have broad spectrum for methicillin and other beta lactam antibiotics. This study on the crystal structure of PBP2a reveals the structural features responsible for its beta lactam resistance and provides important insights for the design of novel antibiotics against MRSA. Sau PBP2a has a transmembrane anchor which can be removed for the studying of beta lactam binding kinetics without affecting the beta lactam activity. The soluble derivative residues of PBP2a were determined by MAD method (Mean Absolute Deviation) using selenomethionine substituted protein. The extraction of this molecule has revealed structural conformations of the PBP2a protein and the location of active sites present within the non penicillin binding domain in the PBP2a. The active site along with the serine 403 at the N terminal helix of the alpha2 fold of Sau PBP2a along with a conserved oxidation hole containing serine 403 and Thr 600 as a nitrogenous backbone.During the acylation of beta lactam, the serine active site is mostly responsible for the process of inducing resistance towards the antibiotic. However, continuous acylation of serine molecules leads to dehydration. And may result in decreasing the activity of the active site, resulting in cell lysis and death. In order to make this process continuous, the serine 403 associated with the N terminal end of lys 406, present in beta lactam carboxylate enzyme. This forms hydrogen bonds, thus helping the serine present in the active site undergo deacylation, to further continue the process of cell wall protection by nucleophilic attack during the resistance mechanism.
This serine-lysine bond promotes the PBP2a conformational change, thus providing unfavourable conditions for the antibiotic to attack the complex. The low affinity binding towards the beta lactam induces slow acylation, leading to a rate limiting step responsible for the beta lactam resistance in MRSA. In normal methicillin sensitive PBPs, higher acylation rates are one of the major reasons for the antibiotic sensitivity and lead to cell death.
Location and function of the mec-A gene
The mec-A gene is located on a mobile chromosomal cassette called secmec. In this paper (Katayama, et al. 2003), use of naive cells (strains without mec-A genes) and experienced hosts (methicillin susceptible strains in which mec-A was excised) was mainly done to demonstrate the expression of mec-A genes and PBP2a protein activity towards the beta lactam resistance. A plasmid called pYK-20 was used as a carrier for the mec-A gene and was introduced into experienced and naive strains. The excision of this mec-A gene from the methicillin resistant strain was done by CC5-1, CC5-2 restriction enzymes and were introduced into the different vector plasmids like pYK-644, pYK-20, Hind 3, Coln plasmid, pNR-5542 and pCN-2278 and some of the plasmids were introduced with both mec-A and bla genes. These plasmids were amplified using PCR and these plasmids were introduced into naive hosts and experienced hosts through the process of micropropagation and growth curves were measured to screen for the transformants containing the plasmid vectors. These cells were grown on nafcillin containing media to test for antibiotic resistance. Naive cells containing the plasmids that carried only mec genes showed weak growth of 2.5% and the naive cells containing plasmids which are introduced with both mec-A and bla genes showed resistance to nafcillin. Transformed experienced strains showed normal resistance as that of parents containing mec-A genes. Taken together, these experiments suggest resistance is driven by transformants containing the plasmids with both mec-A genes and bla genes, indicating that combined activity of mec-A and bla are required for resistance. As, the experienced strains showed resistance and the naive cells did not express due to the host barrier. Tranformants were then tested to analyse the expression of the PBP2a protein by western blot. After the electrophoresis separation the separated proteins are loaded onto the nitrocellulose membrane containing monoclonal anti PBP2a antibodies. The desired PBP2a proteins bind with the antibodies. After the washing the unwanted proteins are removed. The secondary labelled antibodies are introduced and bind to the desired protein-antibody complex and develop colour and the PBP2a are visualised as thick bands depending upon the concentration under autoradiography. It was observed that Colnex with pYK 20 showed the maximum PBP2a production indicating high resistance, containing a plasmid carrying both mec and bla genes.
Both PBP2a and beta Lactamases are important for antibiotic resistance, and the structural confirmation of Ser403 present in the active site of the PBP2a protein is most important to confer proper and effective resistance towards the antibiotics. As in the studies we observed that the cells with plasmids carrying both mec and bla genes showed more resistance than cells with plasmids containing a single gene. What this suggests is although PBP2a is required for antibiotic resistance, it is not sufficient to induce resistance. In fact, additional genes such as bla are also required to induce resistance. More studies and research on new antibiotics targeting Ser403 and the active site of PBP2a, possibly designing new antibiotics towards PBP2a and designing antibiotics that target bla gene products would be effective. Combinatorial antibiotic treatment targeting both PBP2a and bla may help in effective treatment towards these antibiotic resistant diseases.
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