c Methicillin-resistant Staphylococcus aureus (MRSA) has acquired the mecA gene encoding a peptidoglycan transpeptidase, penicillin binding protein 2a (PBP2a), which has decreased affinity for -lactams. Quickly spreading and highly virulent communityacquired (CA) MRSA strains recently emerged as a frequent cause of infection in individuals without exposure to the health care system. In this study, we found that the inactivation of the components of the ClpXP protease substantially increased the -lactam resistance level of a CA-MRSA USA300 strain, suggesting that the proteolytic activity of ClpXP controls one or more pathways modulating -lactam resistance. These pathways do not involve the control of mecA expression, as the cellular levels of PBP2a were unaltered in the clp mutants. An analysis of the cell envelope properties of the clpX and clpP mutants revealed a number of distinct phenotypes that may contribute to the enhanced -lactam tolerance. Both mutants displayed significantly thicker cell walls, increased peptidoglycan cross-linking, and altered composition of monomeric muropeptide species compared to those of the wild types. Moreover, changes in Sle1-mediated peptidoglycan hydrolysis and altered processing of the major autolysin Atl were observed in the clp mutants. In conclusion, the results presented here point to an important role for the ClpXP protease in controlling cell wall metabolism and add novel insights into the molecular factors that determine strain-dependent -lactam resistance.
dDaptomycin is a lipopeptide antibiotic used clinically for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. The emergence of daptomycin-nonsusceptible S. aureus isolates during therapy is often associated with multiple genetic changes; however, the relative contributions of these changes to resistance and other phenotypic changes usually remain unclear. The present study was undertaken to investigate this issue using a genetically characterized series of four isogenic clinical MRSA strains derived from a patient with bacteremia before and during daptomycin treatment. The first strain obtained after daptomycin therapy carried a single-nucleotide polymorphism (SNP) in rpoB (RpoB A 477 D) that decreased susceptibility not only to daptomycin but also to vancomycin, -lactams, and rifampin. Furthermore, the rpoB mutant exhibited pleiotropic phenotypes, including increased cell wall thickness, reduced expression of virulence traits, induced expression of the stress-associated transcriptional regulator Spx, and slow growth. A subsequently acquired loss-of-function mutation in clpX partly alleviated the growth defect conferred by the rpoB mutation without changing antibiotic susceptibility. The final isolate acquired three additional mutations, including an SNP in mprF (MprF S 295 L) known to confer daptomycin nonsusceptibility, and accordingly, this isolate was the only daptomycin-nonsusceptible strain of this series. Interestingly, in this isolate, the cell wall had regained the same thickness as that of the parental strain, while the level of transcription of the vraSR (cell wall stress regulator) was increased. In conclusion, this study illustrates how serial genetic changes selected in vivo contribute to daptomycin nonsusceptibility, growth fitness, and virulence traits. Staphylococcus aureus is a commensal bacterium that can cause a variety of both localized and more invasive infections. Due to its profound ability to acquire resistance to clinically relevant antibiotics, S. aureus remains a major clinical challenge worldwide (1). The most challenging antimicrobial resistance issue in S. aureus has been the evolution and dissemination of methicillinresistant S. aureus (MRSA) strains first in hospitals and later in the general community (1). MRSA infections are typically treated with last-line antibiotics, such as vancomycin and, more recently, daptomycin (DAP), a cyclic lipopeptide derived from Streptomyces roseosporus with bactericidal activity against a broad range of Gram-positive bacteria (2, 3). The mechanism of action of daptomycin has not been fully elucidated, but it is generally accepted that daptomycin inserts into the cytoplasmic membrane of Grampositive bacteria via a calcium-dependent pathway, leading to potassium efflux, membrane depolarization, and, eventually, cell death (2).The development of daptomycin nonsusceptibility (DAP-NS) in S. aureus seems to be a relatively rare phenomenon not involving horizontal gene transfer but, instead, taking place by a stepwise acqu...
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