View of the protein essential for making drug-resistant bacteria
June 9, 2014 § Leave a comment
Drug resistance of bacteria to antibiotics is an alarming problem. Hospitals around the world are reporting that more than 50 percent of bacterial infections are cases of methicillin-resistant strains of Staphylococcus aureus (commonly known as MRSA). In a new paper in the Proceedings of the National Academy of Science, researchers report an analysis of a critical protein called RepA, which is required to maintain and propagate multidrug resistant plasmids from cell to cell. This study gives scientists a better idea of the molecular mechanisms at play in bacterial antibiotic resistance and may lead to new approaches of dealing with drug-resistant bacterial infections.
S. aureus carries circles of DNA called plasmids in addition to the bacterium’s central genome. These plasmids contain the genes that help the bacterium resist drugs. One bacterium can happily pass plasmids to a fellow bacterium, spreading antibiotic resistance throughout a bacterial colony.
One common feature of S. aureus plasmids is the highly conserved class of replication initiator proteins, known collectively as RepA. This class of proteins is needed for plasmid retention and production. The class has more than 100 members. Very little has been known about the structure and functions of these proteins, says Maria Schumacher at Duke University, who led the study: “They show no sequence homology to any structurally characterized protein.”
The lack of sequence homology and the frustrating inability to get the full-length protein to crystallize has hampered the structural elucidation of RepA over the years. So, to tackle this protein that has eluded structural analyses, Schumacher and colleagues took on multiple approaches. Their main tool was X-ray crystallography, but they also applied small-angle X-ray scattering, fluorescence polarization, atomic force microscopy, calorimetry and cell-based analyses. All together, the investigators were able to get some mechanistic and structural details about RepA.
Scientists know that RepA has two domains, one N-terminal and the other C-terminal, loosely connected to each other. Schumacher’s team founds that the RepA N-terminal and C-terminal domains show homology to the primosome protein DnaD, which is involved in the replication of the bacterium’s core genetic material, but that the RepA domains carry out different functions.
The N-terminal domain binds to the starting point for replication of the plasmid in a way that hasn’t been seen before; the structure of the N-terminal domain complexed with the DNA “reveal the first mechanism of origin handcuffing, which is a mechanism of copy number control,” states Schumacher. The C-terminal domain is responsible for recruiting a helicase.
While understanding plasmid replication by RepA is important for advancing fundamental research into DNA replication processes, Schumacher says the absolute need for RepA to produce multiresistance plasmids makes the work important from a clinical perspective. “Indeed, there are no related proteins in humans, which makes these proteins strong candidates for the development of highly specific and targeted inhibitor/drug compounds,” she says. “The successful design of such compounds necessitates structural and mechanistic insight.”