Does variable exotoxin expression alter outcome in Panton Valentine Leukocidin associated S. aureus infections?
Lead Research Organisation:
University of Nottingham
Department Name: Sch of Molecular Medical Sciences
Abstract
In the UK, Methicillin-Resistant Staphylococcus aureus (MRSA) infections are associated with enormous personal and financial costs to the individual, their family and to the NHS (estimated at around #1 billion p.a.). Recently new Community-Acquired MRSA (CA-MRSA) strains have emerged which cause severe infections in healthy young people. CA-MRSA have a larger armament of toxins which disable human host defences and damage human tissues, including a toxin called Panton-Valentine Leukocidin (PVL).
Against this backdrop, the development of new classes of antibiotics has lagged far behind the urgent requirement for new drugs, in part because of the reluctance of major pharmaceutical companies to develop expensive new drugs that become rapidly obsolete through resistance. Consequently we need to gain better insights into the infection-specific lifestyle of bacteria if we are to discover new ways of preventing and treating infections.
S. aureus bacteria use chemical signals to make decisions as a group about when to produce toxins. We are seeking to understand what factors influence toxin production during an infection. Ultimately our aim is to develop drugs that will disable the production of toxins by interfering with this chemical signalling system. Such drugs are also less likely to promote drug resistance.
Against this backdrop, the development of new classes of antibiotics has lagged far behind the urgent requirement for new drugs, in part because of the reluctance of major pharmaceutical companies to develop expensive new drugs that become rapidly obsolete through resistance. Consequently we need to gain better insights into the infection-specific lifestyle of bacteria if we are to discover new ways of preventing and treating infections.
S. aureus bacteria use chemical signals to make decisions as a group about when to produce toxins. We are seeking to understand what factors influence toxin production during an infection. Ultimately our aim is to develop drugs that will disable the production of toxins by interfering with this chemical signalling system. Such drugs are also less likely to promote drug resistance.
Technical Summary
Staphylococcus aureus is a multi-antibiotic resistant pathogen causing diseases from minor to life threatening infections. Community-associated methicillin-resistant S. aureus (CA-MRSA) can be acquired without previous healthcare contact, causes skin and soft tissue infections, has become an important cause of necrotising pneumonia and is now epidemic in the USA. The enhanced capacity of CA-MRSA to cause disease is believed to depend on the production of secreted exotoxins including -toxin (Hla) and Panton-Valentine leukocidin (PVL) which, in turn, are regulated by the accessory gene regulator (agr) quorum sensing (QS) system. Through the release and sensing of auto inducing peptides (AIPs), the bacteria employ this system to respond collectively to changing environmental conditions, including potential threats to their survival. We hypothesise that variable exotoxin expression during an infection is regulated by agr, is an important determinant of outcome and therefore the agr system offers significant potential as a therapeutic target.
Work leading up to this project has identified synthetic AIPs with broad agr inhibitory activity. The efficacy of agr inhibitors will depend on both bacterial and host factors given the the variability in PVL production between PVL gene positive strains. PVL can be strongly induced by sub-inhibitory concentrations of oxacillin, of particular importance in the UK where flucloxacillin is the empiric antibiotic of choice for suspected staphylococcal infection. From our preliminary data, this induction could be suppressed by the inhibitory AIP suggesting that the antibiotic-induced elevation of PVL depends on agr.
In vitro laboratory work will focus on the mechanisms underlying variable and antibiotic-inducible agr-dependent PVL production, uncovering regulatory pathways which are currently not well understood. This will involve the use of ELISA, immunoblot and lux based bioluminescence reporter assays to screen clinical isolates for variable toxin expression, particularly in response to oxacillin and other environmental stimuli. Random transposon mutagenesis will be employed to identify the genes responsible for this enhanced expression. Identification of novel agr-dependent and independent regulatory elements controlling PVL expression are anticipated.
Variability in expression occurring during an infection and the impact of this on outcome will be assessed by using an in vivo skin abscess model and a reconstituted human skin model. Confocal scanning microscopy and non-invasive optical imaging will enable individual bacteria and genes to be labelled and monitored in real time. These models will allow us to test the efficacy of locally developed agr inhibitors, providing essential information about their potential value as future antibiotics.
Work leading up to this project has identified synthetic AIPs with broad agr inhibitory activity. The efficacy of agr inhibitors will depend on both bacterial and host factors given the the variability in PVL production between PVL gene positive strains. PVL can be strongly induced by sub-inhibitory concentrations of oxacillin, of particular importance in the UK where flucloxacillin is the empiric antibiotic of choice for suspected staphylococcal infection. From our preliminary data, this induction could be suppressed by the inhibitory AIP suggesting that the antibiotic-induced elevation of PVL depends on agr.
In vitro laboratory work will focus on the mechanisms underlying variable and antibiotic-inducible agr-dependent PVL production, uncovering regulatory pathways which are currently not well understood. This will involve the use of ELISA, immunoblot and lux based bioluminescence reporter assays to screen clinical isolates for variable toxin expression, particularly in response to oxacillin and other environmental stimuli. Random transposon mutagenesis will be employed to identify the genes responsible for this enhanced expression. Identification of novel agr-dependent and independent regulatory elements controlling PVL expression are anticipated.
Variability in expression occurring during an infection and the impact of this on outcome will be assessed by using an in vivo skin abscess model and a reconstituted human skin model. Confocal scanning microscopy and non-invasive optical imaging will enable individual bacteria and genes to be labelled and monitored in real time. These models will allow us to test the efficacy of locally developed agr inhibitors, providing essential information about their potential value as future antibiotics.
