The Sle1 Cell Wall Amidase Is Essential for β-Lactam Resistance in Community-Acquired Methicillin-Resistant Staphylococcus aureus USA300

Abstract

Most clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) strains have become resistant to β-lactams antibiotics through horizontal acquisition of the mecA gene encoding PBP2a, a peptidoglycan transpeptidase with low affinity for β-lactams. The level of resistance conferred by mecA is, however, strain dependent, and the mechanisms underlying this phenomenon remain poorly understood. We show here that β-lactam resistance correlates to expression of the Sle1 cell wall amidase in the fast-spreading and highly virulent community-acquired MRSA USA300 clone. Sle1 is a substrate of the ClpXP protease, and while the high Sle1 levels in cells lacking ClpXP activity confer β-lactam hyper-resistance, USA300 cells lacking Sle1 are as susceptible to β-lactams as cells lacking mecA. This finding prompted us to assess the cellular roles of Sle1 in more detail, and we demonstrate that high Sle1 levels accelerate the onset of daughter cells splitting and decrease cell size. Vice versa, oxacillin decreases the Sle1 level and imposes a cell separation defect that is antagonized by high Sle1 levels, suggesting that high Sle1 levels increase tolerance to oxacillin by promoting cell separation. In contrast, increased oxacillin sensitivity of sle1 cells appears linked to a synthetic lethal effect on septum synthesis. In conclusion, this study demonstrates that Sle1 is a key factor in resistance to β-lactam antibiotics in the JE2 USA300 model strain and that PBP2a is required for the expression of Sle1 in JE2 cells exposed to oxacillin.

Massive genome decay and insertion sequence expansion drive the evolution of a novel host-restricted bacterial pathogen

“…4E ). A similar effect was identified on sle1 encoding an N-acetylmuramyl-L-alanine amidase involved in staphylococcal cell separation and β-lactam resistance [32] [33], whereby IS insertion has disrupted the anti-sense regulation of Sle1 expression by a neighbouring gene in a growth phase-dependent manner ( Fig. 4F ).…”

“…For reference, the average number of SNPs between the S. aureus subsp. anaerobius isolates and the S. aureus RF122 isolate was 27,611 (27,650).…”

“…To test whether this organisation affected the regulation of an IS targeted gene in its native context containing a downstream gene and its promoter in antisense orientation with known overlapping transcription [25], we selected sle1 (aka aaa) encoding an N-acetylmuramyl-Lalanine amidase involved in staphylococcal cell separation [26] and essential for β-lactam resistance of S. aureus [27]. The gene downstream of sle1 is expressed as a single transcript from its own promoter allowing us to construct a set of reporters similar to the constructs prepared for mgrA in the plasmid pCN47 [20].…”

Identification and characterization of mutations responsible for the β-lactam resistance in oxacillin-susceptible mecA-positive Staphylococcus aureus

“…7 ). In addition, gene mutations identified in this study included many stringent response elements, and most of them were previously reported to be associated with conversion of hetero- to homo-resistance against β-lactam, such as genes associated with RNA polymerase (RNAP; rpoB 31 , 53 and rpoC 31 , 54 ), purine biosynthesis ( guaA 31 , prs 31 , 33 , hprT 31 , 33 , (p)ppGpp synthesis ( rel Sau 31 , 33 , 47 ), glycolysis ( pykA 31 , fbaA 31 , and fruB 33 ) protein quality control ( ftsH 31 , clpX 30 , 55 , and clpP 30 , 55 ), and tRNA synthase ( lysS 31 , and gltX 31 ). These results suggest that stringent-like response played an important role in β-lactam resistance in the oxacillin-resistant mutants of OS-MRSA.…”

Staphylococcal Biofilms: Challenges and Novel Therapeutic Perspectives

“…Hydrolases, such as AtlA/E (AtlA and AtlE in S. aureus and S. epidermidis , respectively) and Sle1 also participate in the initial stage of attachment ( Figure 1 C,I) [ 52 , 53 , 54 , 55 , 56 , 57 ]. AtlA/E is a bifunctional murein hydrolase with amidase and glucosaminidase activities that also plays an essential role during cell division [ 58 ]; while Sle1 is suggested to be involved in the same process [ 59 , 60 ]. Two possible explanations as to how hydrolases such as AtlA/E are involved during attachment have been suggested.…”