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NEW BRUNSWICK, N.J.—A research team at Rutgers University discovered a pair of genes present in some dangerous strains of Staphyloccocus bacteria that bestows the bacteria with resistance to copper, a frequently used antibacterial agent. The study, published in February, suggests that strains of the bacteria can acquire additional genes that promote antibacterial resistance, and identifies the function and structure of these genes in the hopes of opening new paths for the development of antibacterial drugs.
The antibiotic-resistant bacterium Staphylococcus aureus is one of the leading causes of serious, life-threatening infections in the United States. S. aureus bacteria live on skin and have become a scourge in hospitals and healthcare settings due to their high resistance to copper-based antibiotics. These bacteria include well-known antibiotic-resistant strains such as MRSA and VRSA.
The study aimed to examine why copper is so toxic to Staphyloccocus and how some strains achieve resistance. Previous studies have described two genes that appeared to play a role in copper detoxification.
“We created a mutant S. aureus strain lacking the described copper detoxification genes (CopAZ),” says Jeffrey M. Boyd, senior author of the study and associate professor in the Department of Biochemistry and Microbiology in Rutgers’ School of Environmental and Biological Sciences. “Interestingly, this mutant strain was as resistant to copper as the parent strain. This led us to hypothesize that our strain of S. aureus has an alternate copper detoxification mechanism. Using bioinformatic analyses, we identified a gene encoding a putative copper exporter (CopB), which was in an apparent operon with CopL.”
“When all four genes were removed from S. aureus, the resultant strain was very sensitive to copper,” he adds. “From there we further characterized CopB and CopL. CopB is a copper exporter, and CopL is an extracellular membrane bound copper-binding protein.”
The research suggests that it may be possible to inhibit the functions of CopB and CopL pharmacologically, causing S. aureus to become more sensitive to copper-based antibiotics that are the current standard of care.
According to study co-author Gaetano T. Montelione, a distinguished professor in the Department of Molecular Biology and Biochemistry at Rutgers–New Brunswick, the team also determined the three-dimensional structure of one of these genes, CopL, using Nuclear Magnetic Resonance (NMR) methods. This effort located the copper-binding region in a deep cleft on the surface of the protein. The highly conserved surface of the protein validated unambiguously that CopL is a copper-binding protein.
“Our 3D structure revealed that copper binds in a large, broad cavity at the interface of two structural domains of CopL,” explains Montelione. “This structural data provides guidance for the screening or designing of molecules, such as peptides, that could fit into this pocket and compete with copper binding, rendering CopL infective in suppressing the toxic effects of copper.”
“Using our 3D structure and the location of the copper binding site, we can initiate a program on CopL inhibitor discovery,” says Montelione.
This study is only beginning of this line of inquiry, with the Rutgers team already planning follow-up research aimed at understanding how copper is entering cells and killing S. aureus, determining the effects of CopL removal on pathogenesis in models of infection, investigating whether CopBL promotes survival on solid copper surfaces, learning more about the exact function of CopL, and the role of CopBL in the in the success of the arginine-catabolic mobile element containing S. aureus., according to Boyd.
“We have shown in this work that the apo CopL (with no copper bound) has a 3D structure very similar to the copper-bound CopL,” Montelione pointed out. “This opens the door for us to consider designing peptide, small-molecule and/or macrocyclic molecular libraries that can be screened to discover compounds that bind in the copper-binding pocket located between the two domains of apo CopL. We can screen for lead compounds using our NMR resonance assignments for CopL to identify molecules that bind in the copper-binding cleft, and also using the other spectroscopic methods outlined in the paper to screen for compounds that actually inhibit the copper binding function.”
“We can guide a more extensive medicinal chemistry discovery program aimed at creating bioavailable molecules that would suppress the copper-binding function of CopL of bacterial cells, making S. aureus—and potentially other pathogenic bacteria that use CopL—more sensitive to copper treatment,” Montelione added.
“To my knowledge, all strains of S. aureus are resistant to copper via CopAZ,” says Boyd. “However, not all strains also contain CopBL. The S. aureus lineage USA300, which is the primary community associated presenting S. aureus strain for physicians in the United States, contain CopBL.”
“Copper is an effective antimicrobial agent and strains defective in copper homeostasis have decreased survival in models of infection,” he notes. “These findings suggest that copper detoxification systems are viable antimicrobial targets.”