Affiliation:
1. Department of Internal Medicine, Division of Infectious Diseases, School of Medicine, Virginia Commonwealth University, Richmond, Virginia, USA
2. Institute for Structural Biology and Drug Discovery, Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia, USA
Abstract
ABSTRACT
Resistance to methicillin and other β-lactam antibiotics in staphylococci is due to
mecA
, which is carried on a genomic island, staphylococcal cassette chromosome
mec
(SCC
mec
). The chromosomal excision and integration of SCC
mec
are mediated by the site-specific recombinase CcrAB or CcrC, encoded within this element. A plasmid-borne system was constructed to assess the activities of CcrA and CcrB in the excision and integration of SCC
mec
in
Escherichia coli
and
Staphylococcus aureus
. The excision frequency in
E. coli
mediated by CcrAB from methicillin-resistant
S. aureus
(MRSA) strain N315 was only 9.2%, while the integration frequency was 31.4%. In
S. aureus
the excision and integration frequencies were 11.0% and 18.7%, respectively. Truncated mutants identified the N-terminal domain of either CcrB or CcrA to be necessary for both integration and excision, while the C-terminal domain was important for recombination efficiency. Site-directed mutagenesis of the N-terminal domain identified S11 and R79 of CcrA and S16, R89, T149, and R151 of CcrB to be residues essential for catalytic activities, and the critical location of these residues was consistent with a model of the tertiary structure of the N terminus of CcrA and CcrB. Furthermore, CcrAB and CcrC, cloned from a panel of 6 methicillin-resistant
S. aureus
strains and 2 methicillin-resistant
Staphylococcus epidermidis
strains carrying SCC
mec
types II, IV, and V, also catalyzed integration at rates 1.3 to 10 times higher than the rates at which they catalyzed excision, similar to the results from N315. The tendency of SCC
mec
integration to be favored over excision may explain the low spontaneous excision frequency seen among MRSA strains.
IMPORTANCE
Spontaneous excision of the genomic island (SCCmec) that encodes resistance to beta-lactam antibiotics (methicillin resistance) in staphylococci would convert a methicillin-resistant strain to a methicillin-susceptible strain, improving therapy of difficult-to-treat infections. This study characterizes a model system by which the relative frequencies of excision and integration can be compared. Using a plasmid-based model for excision and integration mediated by the recombinases CcrA and CcrB, integration occurred at a higher frequency than excision, consistent with the low baseline excision frequency seen in most strains. This model system can now be used to study conditions and drugs that may raise the SCC
mec
excision frequency and generate strains that are beta-lactam susceptible.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
6 articles.
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