Acquisition of copper hyper-resistance is promoting increased bacterial survival in vivo.

Copper is highly toxic and used as an antimicrobial by human, animal, and plant defence systems to kill invading pathogens. Consequently all bacteria have copper resistance mechanisms to counteract copper toxicity to enable survival in vivo and in the environment. But we have identified a mechanism that can give bacteria hyper-resistance to copper.

The novel copper resistance mechanism (copXL) has been acquired by some strains of Staphylococcus aureus. CopX is an unusual copper efflux transporter, while CopL is a lipoprotein of unknown function. Our newly published data show that acquisition of CopXL confers resistance to extremely high levels of copper in which typical S. aureus cannot grow, and it promotes survival against the antibacterial killing by macrophages, which is part of the host's anti-bacterial defences. These results suggest that bacteria with copXL will have enhanced survival during infection.

Our studies show that proteins similar to CopL and CopX, are widespread in environmental bacteria, indicating that these genes have an essential but unknown function in environmental survival. Importantly they are not usually found in pathogenic bacteria. However, these genes are now spreading to several other bacteria that are increasingly problematic in human and veterinary medicine.

In S. aureus, the copXL genes are currently only found in the highly virulent and transmissible community acquired and livestock-associated methicillin resistant S. aureus (CA-, LA-MRSA) which have increased infectivity compared to typical S. aureus and they can cause disease in healthy humans with no prior exposure to healthcare settings. Additionally, copXL-like genes are spreading to several other antibiotic resistant opportunistic pathogens demonstrating increased infectivity, e.g. hospital associated vancomycin resistant Enterococcus faecium, methicillin resistant Staphylococcus haemolyticus associated with bovine mastitis, and methicillin resistant Staphylococcus pseudintermedius associated with animal infections.

Our hypothesis is that acquisition of copper hyper-resistance is driving the evolution of bacteria from being opportunistic pathogens, to becoming more virulent strains that no longer need to rely on a weakened immune system to cause infection.

The copXL genes are carried on mobile genetic elements that also encode genes for resistance to antibiotics. If copper hyper-resistance enhances bacterial survival by increasing survival against killing by copper, this will select for antimicrobial resistance genes on the same element as the copper resistance genes, even in the absence of other selective pressures such as antibiotics. This has major implications for our fight against antibiotic resistance and further justifies the need to understand copper hyper-resistance.

We do not know how these genes confer copper hyper-resistance, nor understand the full implications of acquisition of highly efficient copper resistance mechanisms for pathogen survival and retention of antibiotic resistance in the host. Therefore the aim of this fundamental bioscience proposal is to use S. aureus as a model organism to understand the mechanisms of action of copX and copL, and the biological consequences of copper hyper-resistance acquisition.

To do this we will use a multi-disciplinary approach involving biochemistry, genetics, microbiology, and in vivo models with S. aureus as a model system to:
1. Investigate the mechanisms of action of CopX and CopL.
2. Establish whether CopX and CopL enhance pathogen fitness in vivo.
3. Determine the importance of copper hyper-resistance for retention of antimicrobial resistance.

The results from this model system will be widely applicable for other systems where metal detoxification genes are co-encoded on mobile genetics elements with antibiotic resistance genes, and for other human infectious bacteria with copies of the copXL genes.

Copper can be toxic, and used as an antimicrobial by human, animal, and plant defences. We identified a novel copper hyper-resistance mechanism (copXL) in key strains of Staphylococcus aureus that promotes survival in macrophages. Homologues of copXL are widespread in environmental bacteria, suggesting an important but unknown function, but are not normally present in pathogens. However, copXL genes are being acquired by community acquired and livestock-associated methicillin resistant S. aureus, and other opportunistic pathogens becoming increasingly problematic in human and veterinary medicine, showing increased virulence.

Our hypothesis is that acquisition of copper hyper-resistance drives the evolution of bacteria from opportunistic pathogens to more virulent strains that no longer need to rely on a weakened immune system to cause infection.

The copXL genes are carried on mobile genetic elements that also encode genes for resistance to antibiotics. We propose that copper hyper-resistance will provide a fitness benefit in a host by enhancing bacterial resistance to innate immunity, this will select for antibiotic resistance genes on the same element as the copper resistance genes, even in the absence of antibiotics. This has major implications for our fight against antibiotic resistance. However, we do not know how copXL confer copper hyper-resistance nor understand the full implications of acquisition of copper hyper-resistance for pathogen survival, and retention of antibiotic resistance.

To address this lack of understanding we will use a multi-disciplinary approach using S. aureus as a model organism (biochemistry, genetics, microbiology, and ex vivo and in vivo models) to:

1. Investigate the mechanisms of action of CopX and CopL.
2. Establish whether CopX and CopL enhance pathogen fitness in vivo.
3. Determine the importance of copper hyper-resistance for retention of antibiotic resistance.

This multi-disciplinary project will increase substantially the understanding of the molecular mechanisms of a copper hyper-resistance mechanism in environmental and pathogenic bacteria, and it will establish the biological implications of acquisition of highly effective copper mechanisms for pathogen survival in vivo. Furthermore it will provide new data on how antibiotic resistance can be acquired and retained in the absence of the selective pressure from antibiotics themselves.

IN WHAT GROUPS WILL THE IMPACT OF THIS RESEARCH BE FELT AND HOW?

Within SCIENTIFIC COMMUNITIES there will be an impact from the fuller understanding of the molecular mechanisms and biological effects of two barely described copper hyper-resistance proteins. The impact will include stimulation of further research of other pathogens with homologues of these proteins e.g. Enterococcus spp., other Staphylococcus spp. and Listeria monocytogenes. Also it will have impact in the AMR community from the increased awareness of the link between copper and antibiotic resistance genes on mobile genetic elements. The work will be high impact as environmental pollution and antimicrobial resistance are currently extremely topical as evidenced by the major interest in our recent publication on air pollution and bacteria (Hussey et al., 2017, 436 altmetric score, top 5% of all research outputs).

Among PHARMACEUTICAL AND HEALTH PROFESSIONALS there can be impact because the route to new drugs is from new targets, preferably within previously unconsidered biological systems. This project will increase understanding of the importance of metal hyper-resistance proteins for pathogenicity and how acquisition of copper hyper-resistance gives opportunistic pathogens an advantage in the host. Defining these mechanisms in vivo can be an essential first stage before undertaking a programme of drug discovery. Members of our team are already actively collaborating with drug discovery researchers.

HEALTHCARE AND ENVIRONMENTAL POLICY MAKERS, both in the UK and globally, will be impacted by this project because the increased understanding of the role of bacterial exposure to heavy metals in driving antibiotic resistance. It will provide a new type of evidence to support pollution intervention policy changes that have the twin effects of decreasing metal pollution while simultaneously reducing emergence of antibiotic resistance.
Project data outcomes will be of importance to DAC-listed countries, e.g, India and Bangladesh, where metal pollution of the environment and antibiotic resistance are major issues. Thus this project is compliant with OFFICIAL DEVELOPMENT ASSISTANCE (ODA) principles.

The GENERAL PUBLIC will benefit from the increased understanding of the risks inherent in environmental metal pollution and the impact this has on human and animal health. In addition, they will benefit from the transfer of knowledge through our public engagement activities.

RESEARCH STAFF working on the project, in Leicester and Newcastle, will benefit from training in molecular, chemical and infectious diseases methodologies including ex vivo and in vivo imaging system technologies. Additionally the named research co-investigator, Jo Purves will receive the added value of being a co-investigator which will support the advancement of her academic career and will extend her range of transferable skills e.g. managing impact and co-ordinating a large collaborative team. The successful outcome of this application will put us in a very strong position to obtain further funding. This inter-disciplinary collaborative project will give a unique opportunity for early stage scientists (Waldron & Purves) to work with different disciplines and countries, and the skills learned will enhance the career prospects of the researchers and increase the UK skills base.