An Endogenous Staphylococcus aureus CRISPR-Cas System Limits Phage Proliferation and Is Efficiently Excised from the Genome as Part of the SCC mec Cassette

Abstract

CRISPR-Cas is an adaptive immune system protecting bacteria and archaea against mobile genetic elements such as phages. In strains of Staphylococcus aureus , CRISPR-Cas is rare, but when present, it is located within the SCC mec element, which encodes resistance to methicillin and other β-lactam antibiotics. CRISPR-Cas is an adaptive immune system that allows bacteria to inactivate mobile genetic elements. Approximately 50% of bacteria harbor CRISPR-Cas; however, in the human pathogen Staphylococcus aureus , CRISPR-Cas loci are less common and often studied in heterologous systems. We analyzed the prevalence of CRISPR-Cas in genomes of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in Denmark. Only 2.9% of the strains carried CRISPR-Cas systems, but for strains of sequence type ST630, over half were positive. All CRISPR-Cas loci were type III-A and located within the staphylococcal cassette chromosome mec (SCC mec ) type V(5C2&5), conferring β-lactam resistance. Curiously, only 23 different CRISPR spacers were identified in 69 CRISPR-Cas positive strains, and almost identical SCC mec cassettes, CRISPR arrays, and cas genes are present in staphylococcal species other than S. aureus , suggesting that these were transferred horizontally. For the ST630 strain 110900, we demonstrate that the SCC mec cassette containing CRISPR-Cas is excised from the chromosome at high frequency. However, the cassette was not transferable under the conditions investigated. One of the CRISPR spacers targets a late gene in the lytic bacteriophage phiIPLA-RODI, and we show that the system protects against phage infection by reducing phage burst size. However, CRISPR-Cas can be overloaded or circumvented by CRISPR escape mutants. Our results imply that the endogenous type III-A CRISPR-Cas system in S. aureus is active against targeted phages, albeit with low efficacy. This suggests that native S. aureus CRISPR-Cas offers only partial immunity and in nature may work in tandem with other defense systems. IMPORTANCE CRISPR-Cas is an adaptive immune system protecting bacteria and archaea against mobile genetic elements such as phages. In strains of Staphylococcus aureus , CRISPR-Cas is rare, but when present, it is located within the SCC mec element, which encodes resistance to methicillin and other β-lactam antibiotics. We show that the element is excisable, suggesting that the CRISPR-Cas locus is transferable. In support of this, we found almost identical CRISPR-Cas-carrying SCC mec elements in different species of non- S. aureus staphylococci, indicating that the system is mobile but only rarely acquires new spacers in S. aureus . Additionally, we show that in its endogenous form, the S. aureus CRISPR-Cas is active but inefficient against lytic phages that can overload the system or form escape mutants. Thus, we propose that CRISPR-Cas in S. aureus offers only partial immunity in native systems and so may work with other defense systems to prevent phage-mediated killing.