β-lactam antibiotics interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Serendipitously we found that sub-lethal doses of β-lactams rescue growth and prevent spontaneous lysis of Staphylococcus aureus mutants lacking the widely conserved chaperone ClpX, and we reasoned that a better understanding of the clpX phenotypes could provide novel insights into the downstream effects of β-lactam binding to the PBP targets. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30°C, but not at 37°C, demonstrating that ClpX becomes critical for coordinating the S. aureus cell cycle as the temperature decreases. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, oxacillin antagonized the septum progression defects of clpX cells and prevented lysis of prematurely splitting clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with β-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, as did genetic inactivation of the gene encoding the septal autolysin, Sle1. Taken together, our data support a model in which Sle1 causes premature splitting and lysis of clpX daughter cells unless Sle1-dependent lysis is antagonized by β-lactams or by inhibiting an early step in WTA biosynthesis. The finding that β-lactams and inhibitors of WTA biosynthesis specifically prevent lysis of a mutant with dysregulated autolytic activity lends support to the idea that PBPs and WTA biosynthesis play an important role in coordinating cell division with autolytic splitting of daughter cells, and that β-lactams do not kill S. aureus simply by weakening the cell wall.

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.

20Most clinically relevant methicillin resistant Staphylococcus aureus (MRSA) strains have 21 become resistant to β-lactams antibiotics through horizontal acquisition of the mecA 22 gene encoding PBP2a, a peptidoglycan transpeptidase with low affinity for -lactams. 23The level of resistance conferred by mecA is, however, strain dependent and the 24 mechanisms underlying this phenomenon remain poorly understood. We here show 25 that β-lactam resistance correlates to expression of the Sle1 cell wall amidase in the fast 26 spreading and highly virulent community-acquired MRSA USA300 clone. Sle1 is a 27 substrate of the ClpXP protease, and while the high Sle1 levels in cells lacking ClpXP 28 activity confer β-lactam hyper-resistance, USA300 cells lacking Sle1 are as sensitive to β-29 lactams as cells lacking mecA. This finding prompted us to assess the cellular roles of 30 Sle1 in more detail, and we demonstrate that high Sle1 levels accelerate the onset of 31 daughter cells splitting and decrease cell size. Vice versa, oxacillin decreases the Sle1 32 level, and imposes a cell-separation defect that is antagonized by high Sle1 levels, 33 suggesting that high Sle1 levels increase tolerance to oxacillin by promoting cell 34 separation. In contrast, increased oxacillin sensitivity of sle1 cells appears linked to a 35 synthetical lethal effect on septum synthesis. In conclusion, this study demonstrates 36 that Sle1 is a key factor in resistance to β-lactam antibiotics in the JE2 USA300 model 37 strain, and that PBP2a is required for expression of Sle1 in JE2 cells exposed to oxacillin. 38 39 Importance 40 The bacterium Staphylococcus aureus is a major cause of human disease, and the global 41 spread of S. aureus resistant to β-lactam antibiotics (MRSA) has made treatment 42 increasingly difficult. β-lactams interfere with cross-linking of the bacterial cell wall, 43 however, the killing mechanism of this important class of antibiotics is still not fully 44 understood. Here we provide novel insight into this topic by showing that β-lactam 45 resistance is controlled by the Sle1 cell wall amidase in the fast spreading and highly 46 virulent MRSA USA300 clone. We show that Sle1 high levels accelerate the onset of 47 daughter cells splitting and decrease cell size. Vice versa, oxacillin decreases the Sle1 48 level, and imposes a cell-separation defect that is antagonized Sle1. The key finding that 49 resistance to β-lactams correlates positively to expression of Sle1 indicates that, in S. 50 aureus, the detrimental effects of β-lactam antibiotics are linked to inhibition of 51 daughter cells splitting. 52 53

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