Modulation of beta-lactam resistance in methicillin-resistant Staphylococcus aureus by catechin gallates

Lead Research Organisation: School of Pharmacy
Department Name: Pharmaceutics

Abstract

The introduction of antibacterial drugs into medical practice, which began in the 1930s and continued in spectacular fashion with the mass production of penicillin in the early 1940s gave rise to the belief that infectious diseases could be controlled and, eventually, mastered. However, the initial widespread optimism that antibiotics would banish serious infectious disease to the ?dustbin of history? has proven to be premature; infections remain the second leading cause of mortality worldwide and the major killer in the developing world. The reasons for the failure to defeat the threat from infection are many and complex but the emergence of antibiotic resistance has had an enormous impact on our ability to combat infection. Whenever a new antibiotic has been introduced, resistance has always followed. Bacteria have quickly found the means to counteract this threat to their survival and have developed ways to pass on their resistance genes to other bacteria. This acquired antibiotic resistance is responsible for the emergence of multi-resistant strains ? called ?superbugs? by the media ? that are now commonplace in hospitals and increasingly found in community acquired infections. Typical of these is MRSA, which has become a persistent and common permanent inhabitant of hospitals in the United Kingdom and elsewhere. While it is still sensitive to a few expensive antibiotics, there are well-founded fears that this may not last, in which case MRSA infections will become untreatable. We have been researching ways to reverse antibiotic resistance in MRSA, making it again sensitive to inexpensive antibiotics such as methicillin and oxacillin. These antibiotics prevent bacteria from making the rigid wall that they need to survive ? MRSA subverts this action by altering the way it makes its wall. We have found that a component of tea called ?ECg? interferes with the MRSA subverting machinery and converts the bacteria to methicillin sensitivity. Thus, ECg might be used in combination with oxacillin to restore antibiotic sensitivity. Unfortunately, ECg is rapidly broken down in the body but we have changed its chemical nature to make it resistant to breakdown and we have modified the compound in other ways to increase its attraction as a therapeutic. We now wish to understand better how ECg works against MRSA ? we know it inserts into the bacterial membrane - as deeper understanding of the processes involved will enable us to refine our most promising compounds and bring them closer to clinical use.

Technical Summary

The incidence of nosocomial infections due to methicillin resistant Staphylococcus aureus (MRSA) has steadily increased over the last twenty years and the capacity of this organism to accumulate resistance genes is eroding our ability to contain the pathogen. In addition, MRSA strains are increasingly implicated in community-acquired infections. I am investigating a novel approach to the therapy of MRSA using compounds that inhibit the expression of drug resistance but have little or no effect on the viability or growth rate of these pathogens. Thus, (-)-epicatechin gallate (ECg) and other galloyl catechins have the capacity to restore the sensitivity of MRSA to inexpensive beta-lactam agents currently ineffective in the treatment of MRSA infections. I wish to determine the molecular mechanism of action of ECg and its synthetic analogues in order to provide a platform for the rational design of potent ECg-based chemotherapeutics. We have found that exposure of MRSA to ECg affects cell wall synthesis: cells separate poorly and have thickened walls due to disregulation of autolysins. Peptidoglycan cross-linking is reduced, there is some modulation of penicillin binding protein (PBP) activity and teichoic acid is released from the cell. It is probable that these changes result from the capacity of ECg, a naturally occurring molecule with very low intrinsic toxicity, to selectively intercalate into the staphylococcal membrane and perturb the orderly synthesis and turnover of the cell wall. ECg also reduces the transport out of the cell of a range of exoproteins associated with virulence, such as alpha-toxin and coagulase. To provide insight into the process of this phenotypic modification, I propose to examine the effect of galloyl catechins on the release and composition of teichoic acids from MRSA, to determine the nature of the interaction of galloyl catechins with the staphylococcal cytoplasmic membrane (CM) and with artificial membrane vesicles using a range of biophysical techniques, and to study the effect of ECg and synthetic analogues on PBP2/PBP2a complex formation at the septum of growing staphylococci. A deeper understanding of the nature of the interaction of these modifying agents with their primary cellular target, the CM, will considerably enhance our ability to develop lead structures based on ECg for potential therapeutic use.

Publications

10 25 50
 
Description BBSRC Research Grant
Amount £360,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2011 
End 03/2014
 
Description MRC Project Grant
Amount £681,606 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 05/2009 
End 09/2012
 
Description Evaluation of efficacy 
Organisation Mitsui Group
Department Polyphenon Pharma, Inc.
Country United States 
Sector Private 
PI Contribution Elucidation of the mechanism of action of ECg provided an opportunity to profile proprietary compounds owned by the company
Collaborator Contribution Provision of highly purified catechins and catechin gallates. Custom synthesis of tritiated ECg by Amersham International at a cost to Polyphenon's parent company, Mitsui Norin, of approximately £60000
Impact Provision of tritiated ECg allowed us to determine that the staphylococcal cytoplasmic membrane is the primary site of interaction with catechin gallates; this work is currently being drafted for submission.
Start Year 2007
 
Description PBP-ECg interactions 
Organisation Catholic University of Louvain
Country Belgium 
Sector Academic/University 
PI Contribution Provided the evidence that elimination of PBP2a distortion would enable us to focus on PBP delocalisation from the septum as the primary mechanism of action of ECg.
Collaborator Contribution Determined that ECg does not senstisie MRSA strains to beta-lactam drugs by distorting the molecular shape of penicillin binding protein 2a.
Impact Demostration that ECg does not significantly interact with PBP2a active site; manuscript in preparation.
Start Year 2009
 
Description PBP2 delocalisation 
Organisation University of Lisbon
Country Portugal 
Sector Academic/University 
PI Contribution We elucidated the interaction of ECg with the staphylococcal membrane, suggesting that PBP delocalisation was a possible explanation for the capacity of the molecule to sensitise MRSA strains to beta-lactam drugs
Collaborator Contribution Hosted postodctoral fellow to undertake investigation of delocalisation of penicillin binding proteins from the staphylococcal septum in the presence of ECg using a technique developed by Mariana Pinho in colaboration with Professor Jeff Errington
Impact Delocalisation occurred and will feature in a manuscript currently in preparation
Start Year 2007