Date Published: September 13, 2019
Publisher: Public Library of Science
Author(s): Camilla Jensen, Kristoffer T. Bæk, Clement Gallay, Ida Thalsø-Madsen, Lijuan Xu, Ambre Jousselin, Fernando Ruiz Torrubia, Wilhelm Paulander, Ana R. Pereira, Jan-Willem Veening, Mariana G. Pinho, Dorte Frees, Michael Otto.
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.
Staphylococcus aureus is a commensal bacterium capable of causing a variety of both localized and invasive infections. Due to its ability to acquire resistance to all relevant antibiotics S. aureus remains a major clinical challenge worldwide . The most challenging antimicrobial resistance issue in S. aureus has been the dissemination of methicillin-resistant S. aureus (MRSA) strains that are resistant to almost all β-lactam antibiotics, one of the safest and most widely used classes of antibiotics ever developed . Early work on the mechanism of action of β-lactams culminated in the discovery that penicillin inhibits crosslinking of peptidoglycan (PG), the central component of bacterial cell walls . The enzymes mediating cross-linking of peptidoglycan strands, the targets of penicillin, were therefore designated penicillin binding proteins (PBPs). The realization that penicillin inhibits PG crosslinking led to the classical model in which penicillin-mediated cell lysis is believed to occur as a consequence of a mechanically weakened cell wall incapable of withstanding high intracellular turgor [3,4]. The killing effect of β-lactam antibiotics, however, has turned out to be more complex [5–9], and may even vary between bacteria, as the organization of PG synthesis and the number of PBPs differ widely between bacterial species . Spherical bacteria such as S. aureus have only one cell wall synthesis machine, and S. aureus encodes only four PBPs . Notably, MRSA and other Staphylococci have obtained resistance to β-lactams by horizontal acquisition of the mecA gene encoding an alternative PBP (PBP2a) that is resistant to inhibition by most β-lactams [12,13]. PBP2a mediated resistance additionally depends on several intrinsic factors that can be targeted by specific compounds to re-sensitize MRSA to β-lactams [14–16]. As an example, inhibitors of wall teichoic acid (WTA) biosynthesis, work synergistically with β-lactams to kill MRSA both in vitro and in in vivo models of infection, thereby opening a novel paradigm for combination treatment of MRSA . Indeed, a combination strategy pairing β-lactamase inhibitors with β-lactams has proven highly successful in restoring β-lactam efficacy against Gram-negative bacteria .
Because mis-coordination in activation of autolytic enzymes may have fatal consequences, regulatory checkpoints that coordinate the autolytic system with septum completion likely exist, however, little is known about these mechanisms. Here, we show that the widely conserved ClpX chaperone plays a temperature dependent role in staphylococcal cell division resulting in severe morphological changes at 30°C but not at 37°C. In wild-type S. aureus cells, splitting of daughter cells is not initiated prior to septum closure. In contrast, a substantial fraction of clpX cells displaying incomplete septa had initiated splitting of daughter cells indicating that the system responsible for coordinating autolytic splitting with septum completion has become dysregulated. In clpX cells displaying the premature splitting phenotype, septal PG synthesis did not progress inwards, demonstrating that clpX cells with premature split are unable to finalize the septum. The detrimental character of this defect likely prevents cells from undergoing further divisions, explaining why a large proportion of clpX cells are non-dividing and end up lysing. In support hereof, TEM pictures show that most clpX ghost cells were in the process of splitting despite having an incomplete septum. This is likely due to turgor pressure forces breaking the tip of the ingrowing septum where the cell wall is thin and mechanically weak . Hence, we assume that premature splitting is the underlying cause for the high rate of spontaneous lysis observed among clpX cells.