Disruption of cytoplasmic membrane-associated functions in Staphylococcus aureus by epicatechin gallate

Staphylococcus aureus is commonly encountered as a coloniser of skin and mucosa but gives rise to a range of infections when there is an opportunity for the bacteria to enter the body. It also makes a range of toxins that are responsible for food poisoning: if food workers carry the bacterium, it can enter the food chain and will survive even in salty foods due to its ability to tolerate salt. Staphylococci show a remarkable capacity to resist the action of commonly used antibiotics through the acquisition of resistance mechanisms. In particular, they can acquire the mecA gene, whose product penicillin binding protein (PBP) 2a allows it to survive in the presence of high concentrations of penicillins such as methicillin and oxacillin. Such methicillin-resistant S. aureus (MRSA) are a threat to patients entering hospitals, particularly for major surgery, and are increasingly acquired in the community. At one time these infections were confined to humans but in recent years have been encountered in companion animals, especially dogs and cats; in all likelihood the bacteria were initially acquired from humans. Animal and human MRSA infections are difficult to treat and new approaches to therapy and control are urgently needed, in particular those that reduce the rate of emergence of drug resistance. We have established that one of the major components of green tea, a relatively labile substance termed epicatechin gallate, or ECg, alters the properties of S. aureus in a way that should be beneficial to animals and humans. Although ECg has no effect on the growth rate of MRSA, it reduces their resistance to penicillins by a much as five hundredfold, raising the possibility that it can be used alongside conventional antibiotics to treat infections. As ECg modifies rather than kills the target bacteria, it will not apply the same degree of extreme selective pressure as currently used 'bactericidal' drugs and may slow the emergence of resistance to therapeutic combinations. Additionally, we have discovered that ECg prevents the secretion of toxins and enzymes that enable the organism to cause tissue damage and infection and that are responsible for the symptoms of food poisoning. ECg also prevents the formation of biofilms: these complex assemblages of bacteria form at interfaces and are a necessary prerequisite for colonisation of host surfaces and devices such as catheters and prostheses prior to tissue invasion. Unfortunately, the naturally occurring chemical is not ideal for therapeutic purposes due to its labile nature and moderate activity and we are making derivatives of ECg with a more attractive profile. These attempts are hampered by an incomplete understanding of how ECg exerts its anti-staphylococcal activities. We are beginning to learn that it possesses such wide-ranging properties because i inserts into the bacterial membrane, a structure that is home to many essential enzymatic cellular functions. We know that the compound changes the properties of the membrane in a way that will affect the proteins embedded in it. For example, penicillin resistance is due to cooperation between two PBPs, 2 and 2a, enabling the bacteria to continue to make cell wall in the presence of the drug. We will determine if ECg disrupts this complex at the site of cell division or if it simply detaches the complex in a way that allows continued synthesis at 'wrong' sites. It is becoming apparent that the fine detail of the lipid environment in the region of cell division is critical to the determination of efficient bacterial replication; we will examine the way in which ECg alters this parameter. Finally, the capacity of ECg to increase salt sensitivity of staphylococci means that it could become an effective agent for preserving food and preventing the elaboration of toxins associated with food poisoning. The bacteria pump out sodium ions using membrane-located proteins and we will examine the capacity of ECg to compromise this mechanism.

The naturally occurring, abundant, polyphenol (-)-epicatechin gallate (ECg) has the capacity to abrogate beta-lactam resistance in multi-drug-resistant Staphylococcus aureus and is able to reduce the secretion of virulence effectors such as toxins and tissue-degrading enzymes, prevent the formation of biofilms and abolish halotolerance in resistant and susceptible strains of this species, making it potentially useful for the control of infections in animals and humans and for food preservation. The proposed study will provide a detailed understanding of the mechanisms responsible for these wide-ranging phenotypic modifications and support the design, synthesis and characterisation of structural analogues with enhanced bioactivities. We have established that ECg intercalates into the cytoplasmic membrane (CM), engenders physical and structural changes to the bilayer and delocalises penicillin binding protein 2 (PBP2) from the septal site of cell division, where it normally cooperates with PBP2a to facilitate continued cell wall synthesis in the presence of beta-lactams. We will determine if ECg disrupts the functional PBP2/2a complex such that PBP2a can no longer compensate for beta-lactam-mediated acylation of PBP2 or if it permits continued wall synthesis in the presence of oxacillin by a delocalised functional complex at sites remote from the division septum. We will investigate if ECg produces a sub-optimal lipid environment for key CM-anchored proteins by searching for lipid 'raft' domains and by determining the degree of phospholipid asymmetry across the inner and outer CM leaflets. The potential for distortion of PBP2a, leading to loss of resistance to active site acylation by oxacillin, following interaction of ECg with the CM will be examined. As halotolerance is due at least in part to a functional Na+/H+ CM-located antiporter system, the effect of ECg intercalation into the CM on the activity of this pump will be determined.

The Taylor group has been researching the interactions of epicatechin gallate (ECg) and multi-drug-resistant staphylococci since late 2001; the project was initially orientated towards resolution of hospital-acquired Staphylococcus aureus infections but it has become apparent that the impact of exposure to ECg has more far-reaching consequences. ECg has the capacity to prevent formation of biofilms on a variety of biological and non-biological surfaces, opening up the possibility for its use in a wide range of applications in the areas of food spoilage, contamination of food and beverage production plants and biofouling. Its capacity to prevent the elaboration of S. aureus toxins and abolish halotolerance in this food-borne bacterium presents an opportunity to introduce a mechanism-based approach to food safety issues involving staphylococci. Recent, worrying increases in the frequency of staphylococcal infections in companion and farm animals further increases the impact of our research in areas of concern highlighted by BBSRC, such as sustainable food supply. We may be able to provide cost-effective solutions for the control of these infections in a way that suppresses the rate of emergence of undesirable bacterial traits. Our previous work has provided insights into the complex events that occur after exposure of the bacteria to ECg and the proposal is now specifically orientated to address fundamental questions regarding the impact of ECg on the molecular processes involved in modification of the bacterial phenotype. We propose utilisation of novel biophysical techniques to give a truly interdisciplinary approach to the study of selective perturbation of the cell by this interesting natural product; the research will contribute to a holistic view of how staphylococci have evolved engineering solutions that underpin the problems of shape determination, maintenance of the internal cellular environment and cell division in the face of external insults. Industry, Policy, the public: Our entry into the disciplines of catechin chemistry and biology, traditionally dominated by Japanese scientists, has provoked much interest from both academic and industrial researchers in that country and has resulted in investment in our work from that quarter. We are keen to broaden our activities in a way that will support the exploitation of the properties of ECg for UK and European markets in the areas highlighted above. We have identified an agent that has the potential to control infections by a completely novel mechanism and ECg could be considered a lead for an alternative approach to treatment. Front-line antibiotics that perturb the bacterial cell wall by interfering with peptidoglycan synthesis have been the mainstay of treatments for seventy years and we now indicate how this essential structure can be further exploited for drug development. Catechin gallates are well-tolerated molecules that elicit effects on opportunistic bacterial invaders that will support the wellbeing of the general population; we are keen to communicate the benefits of these natural products to the wider public in the UK and our team has had considerable experience in media activities. Training: This proposal provides an opportunity for postdoctoral training in an interdisciplinary project involving biochemistry, molecular biology, membrane biophysics and natural product research in an area with great practical potential, coupled with state-of-the-art technologies at the cutting edge of bioscience. The PDRA will be embedded in a laboratory with a clear focus on staphylococcal research but also heavily involved in other research areas impacting on novel approaches to bacterial chemotherapy. This will provide an opportunity to interact with successful scientists in-house and to also spend periods in laboratories undertaking high quality research. The opportunities for cross-fertilisation of ideas and technologies are very high.