The ClpX chaperone controls the Staphylococcus aureus cell cycle but can be bypassed by β-lactam antibiotics

The worldwide spread of Staphylococcus aureus strains resistant to almost all β-lactam antibiotics is of major clinical concern. β-lactams interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Here we show that sub-lethal doses of β-lactams stimulate the growth of S. aureus mutants lacking the widely conserved chaperone ClpX. S. aureus clpX mutants have a severe growth defect at temperatures below 37°C, and we reasoned that a better understanding of this growth defect could provide novel insights into how β-lactam antibiotics interfere with growth of S. aureus. We demonstrate that ClpX is important for coordinating the S. aureus cell cycle, and that S. aureus cells devoid of ClpX fail to divide, or lyze spontaneously, at high frequency unless β-lactams are added to the growth medium. 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. 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, β-lactams restored septum synthesis and prevented premature autolytic splitting of 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, underscoring a functional link between the PBP activity and WTA biosynthesis. The finding that β -lactams can prevent lysis and restore septum synthesis of a mutant with dysregulated cell division lends support to the idea that PBPs function as coordinators of cell division and that β -lactams do not kill S. aureus simply by weakening the cell wall. Author Summary The bacterium Staphylococcus aureus is a major cause of human disease, and the rapid spread of S. aureus strains that are resistant to almost all β-lactam antibiotics has made treatment increasingly difficult. β-lactams interfere with cross-linking of the bacterial cell wall but the killing mechanism of this important class of antibiotics is still not fully understood. Here we provide novel insight into this topic by examining a defined S. aureus mutant that has the unusual property of growing markedly better in the presence of β-lactams. Without β-lactams this mutant dies spontaneously at a high frequency due to premature separation of daughter cells during cell division. Cell death of the mutant can, however, be prevented either by exposure to β-lactam antibiotics or by inhibiting synthesis of wall teichoic acid, a major component of the cell wall in Gram-positive bacteria with a conserved role in activation of autolytic splitting of daughter cells. The finding that the detrimental effect of β-lactam antibiotics can be reversed by a mutation that affect the coordination of cell division emphasizes the idea that β-lactams do not kill S. aureus simply by weakening the cell wall but rather by interference with the coordination of cell division.

β -lactams interfere with cross-linking of the 2 3 bacterial cell wall, but the killing mechanism of this important class of antibiotics is not mutants have a severe growth defect at temperatures below 37°C, and we reasoned 2 7 that a better understanding of this growth defect could provide novel insights into how β -2 8 lactam antibiotics interfere with growth of S. aureus. We demonstrate that ClpX is  To further examine how β -lactams improve growth of the clpX mutant, we performed 2 4 8 SR-SIM analysis on oxacillin treated wild-type and clpX mutant cells grown at 30°C, as 2 4 9 13 described above (Fig 4). Interestingly, sub-lethal concentrations of oxacillin significantly 2 5 0 increased the fraction of phase 3 cells (closed septum): from 15 to 31% in the wild-type 2 5 1 (P < 0.001), and from 4 to 14% in the clpX mutant (P < 0.001). Moreover, oxacillin 2 5 2 significantly decreased the fraction of clpX cells (phase 2) that had initiated cell 2 5 3 separation prior to septum completion from 20% to 2%, and in line with this observation, 2 5 4 almost no lysed clpX mutant cells were observed (Fig 4b). Hence, oxacillin increases 2 5 5 the fraction of cells with complete division septa in both the wild-type and the clpX 2 5 6 backgrounds, and prevents premature splitting of clpX cells with incomplete division 2 5 7 septa. In contrast, asymmetrical ingrowth of septa is still readily observed in oxacillin 2 5 8 treated clpX mutant cells (Fig 4a and b).

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These conclusions were supported when the oxacillin treated SA564 wild-type and clpX treatment conferred a number of well described morphological changes that were images support that oxacillin, even in concentrations well below the MIC value, prolongs 2 6 8 phase 3 and delays splitting of the septum.  To directly assess the impact of ClpX and oxacillin on progression of septal PG 2 7 3 synthesis, we used an established metabolic labeling method with fluorescent D-amino 2 7 4 acids (FDAAs) to visualize regions of new PG insertion [24,29,30]. PG synthesis was 2 7 5 followed at 30°C and 37°C by sequentially labeling cells with FDAAs of different colors, 2 7 6 thereby creating a virtual time-lapse image of PG synthesis [24,29,30]. Cells were first 2 7 7 pulse-labeled for 10 min with green nitrobenzofurazan-amino-D-alanine (NADA), followed by a 10-min pulse with the blue hydroxycoumarin-amino-D-alanine (HADA). Labeled cells were imaged by SR-SIM, and progression of PG synthesis was scored in 2 8 0 300 randomly picked wild-type and clpX mutant cells grown in the absence or presence 2 8 1 of oxacillin (Fig 5.,note that NADA is displayed in red). In the absence of oxacillin, PG 2 8 2 synthesis proceeded from phase 1 (no septa, PG synthesis takes place in the lateral 2 8 3 wall) to phase 2 (septal PG synthesis progresses inwards), and finally phase 3 (closed 2 8 4 septum, PG synthesis occurs in both septum and the lateral wall) in > 95% of wild-type 2 8 5 cells, as described in [24,25] (see Fig 5a). When the clpX mutant was grown at 37°C, localized with the NADA signal in the early septum ingrowth, and additionally, a ingrowth were indeed capable of septum progression and septum closure (green 2 9 5 15 asterisks in Fig 5a), the septal PG synthesis rate does not seem to be generally 2 9 6 reduced in the clpX mutant. Instead, the co-localization of the NADA and HADA in an 2 9 7 early septum ingrowth may reflect stalling of septum synthesis in a subpopulation of 2 9 8 clpX cells. Interestingly, in the presence of a sub-lethal concentration of oxacillin the 2 9 9 fraction of clpX cells displaying co-localization of NADA and HADA at the early-septum 3 0 0 ingrowth was reduced to 6 ± 2 % (Fig 5b). FDAAs only incorporate into newly synthesized PG and therefore premature splitting  The results presented so far suggest that oxacillin improves growth of an S. aureus clpX 3 1 7 mutant by stimulating septal PG synthesis and inhibiting premature splitting and lysis of 3 1 8 daughter cells. To investigate septal PG synthesis in cells with premature splitting, we 3 1 9 randomly picked 50 clpX cells grown at 30°C that had initiated septum formation during 3 2 0 incubation with NADA, and that displayed the characteristic morphology of premature  ClpX from diverse bacteria interacts directly with FtsZ suggesting that the ClpX 3 3 4 chaperone has a conserved role in assisting assembly/disassembly of the FtsZ polymer 3 3 5 [31-34]. We therefore reasoned that ClpX may regulate septum progression by [36]). Following closure, FtsZ undergoes a period of highly dynamic re-distribution, appear not to be affected by lack of ClpX activity. Next, we imaged the relative 3 4 6 localization of FtsZ and PG synthesis by sequentially labeling PG synthesis with FDAAs 3 4 7 as described above, except that tetramethylrhodamine 3-amino-d-alanine (TADA, red wall. Hence, our data supports the idea that FtsZ dynamics is not impeded in cells  Finally, we asked if the ability to rescue growth of an S. aureus clpX mutant is specific kill S. aureus [14,16], stimulated growth of the clpX mutant (Fig 7 and S7 Fig)  link between the PBP activity and WTA biosynthesis. Assembly of the bacterial cell division machinery is a highly coordinated process with 3 7 5 proteins recruited to the division site in a specific order and depending on the timely 3 7 6 interaction between a large number of proteins [37]. Here, we show that the widely 3 7 7 conserved ClpX chaperone plays an important role in staphylococcal cell division at 3 7 8 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 demonstrating that clpX cells with premature split are unable to finalize the septum. The lysing. In support hereof, TEM pictures show that most clpX ghost cells were in the 3 8 8 process of splitting despite having an incomplete septum. This is likely due to turgor 3 8 9 pressure forces breaking the tip of the ingrowing septum where the cell wall is thin and 3 9 0 mechanically weak [38]. Hence, we assume that premature splitting is the underlying 3 9 1 cause for the high rate of spontaneous lysis observed among clpX cells. Importantly, cells devoid of ClpX contain elevated levels of the two major autolysins  Therefore, premature splitting of clpX cells could simply be a consequence of excess 3 9 5 autolysins. However, whilst the elevated levels of SleI and Atl may contribute to the 3 9 6 20 premature splitting and spontaneous lysis of clpX cells, we believe that additional 3 9 7 factors are in play. This is based on the findings that i) premature splitting and lysis of 3 9 8 clpX cells is more frequent at 30°C than at 37°C, whereas autolysin levels are elevated 3 9 9 at both temperatures [20,21,39,40]; and, ii) the clpX phenotypes described here are not proposed to occur in two-steps: an initial FtsZ dependent slow step that may drive the with septum completion likely exist, however, little is known about these mechanisms.

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Remarkably, the growth and lysis defect of the clpX mutant was alleviated by sub-lethal cell lysis [5,9,42]. Here, we observed that oxacillin treatment of both S. aureus wild-type activation of autolytic enzymes and by stimulating late septal PG synthesis (Fig 8). Vice  In support of a central role of autolysins in the clpX phenotypes, we previously showed 4 5 2 that the fast-growing suppressor mutants arising when clpX cells are grown at 30°C  In our working model, we therefore propose that inhibition of WTA stimulates growth of 4 5 9 the clpX mutant by impeding premature split of clpX cells (Fig 8). In conclusion, we have shown that S. aureus cell division is temperature sensitive, and frequently has a fatal outcome because septal PG synthesis stalls and cell separation is 4 6 4 initiated prior to completion of the septum. Interestingly, these defects were prevented  Strains used in this study are listed in S2 Table. S. aureus strains were grown in tryptic of the medium. For solid medium, 1.5% agar was added to make TSA plates.  were determined by growing the relevant strains in a Bioscreen C instrument as 5 0 3 described above. The growth rates were automatically calculated as described before  26 plates were allowed to dry prior to the addition of 1 µg oxacillin discs (Oxoid) and 5 1 5 incubated at 37°C for 48 hours. or 8325-4ΔclpX/pCQ11ftsZ::eYFP) were grown overnight in TSB medium at 37°C and 5 2 0 cultures were diluted 100 times in fresh TSB medium and grown until an OD 600 of 0.1.

2 1
Cells were washed once in fresh TSB medium and spotted onto a TSB-polyacrylamide 5 2 2 (10%) slide incubated with TSB medium supplemented when appropriate with 0.008 μ g 5 2 3 ml -1 oxacillin or with 100 µM IPTG. Acrylamide pads were placed inside a Gene frame 5 2 4 (Thermo Fisher Scientific) and sealed with a cover glass as described before [46].

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Phase contrast images acquisition was performed using a DV Elite microscope (GE 5 2 6 healthcare) with a sCMOS (PCO) camera with a 100x oil-immersion objective. Images using Softworx (Applied Precision) software. Images were analyzed using Fiji 5 2 9 (http://fiji.sc). Each experiment was performed at least in triplicate. 69 micro colonies generation times using a custom R script (available at http://github.com/ktbaek/beta-  27 Time-lapse images of FtsZ-eYFP were acquired using a Leica DMi8 microscope with a 5 3 7 sCMOS DFC9000 (Leica) camera with a 100x oil-immersion objective and a Spectra X 5 3 8 (Lumencor) illumination module. Fluorescent images were acquired every 4 min with 5 3 9 400 ms exposure using a YFP filter cube (Chroma, excitation 492-514 nm, dichroic 520 5 4 0 nm, emission 520-550 nm). Images were processed using LAS X (Leica) and signal 5 4 1 was deconvolved using Huygens (SVI) software. Overnight cultures grown at 37°C were diluted 1:200 into 40 ml of fresh TSB and grown 5 4 5 at 30°C or 37°C to an OD 600 of 0.5. Bacteria (SA564, 8325-4, and JE2 and the clpX 5 4 6 mutant derived here from) from a 10-ml culture aliquot were collected by centrifugation 5 4 7 at 8,000 x g, and the cell pellets were suspended in fixation solution (2.5% fixed cells were further treated with 2% osmium tetroxide, followed by 0.25% uranyl Olympus Veleta camera with a resolution of 2,048 by 2,048 pixels. For quantitative 5 5 5 analysis, the images were acquired in an unbiased fashion by using the multiple image Exponentially growing S. aureus SA564 were collected by centrifugation, fixed in 2% kV. Sample preparation and SEM imaging was performed at CFIM. channel specific optical transfer functions and noise filter settings ranging from -6 to -8. Laser specifications can be seen in S3 Table. SR-SIM was performed at CFIM. To address progression of the cell cycle, exponential cultures of S. aureus were 5 8 0 incubated for 5 min at room temperature with the membrane dye Nile Red, the cell wall  Table). Samples were 5 8 2 placed on an agarose pad and visualized by SR-SIM as described above. 300 cells  analysis was performed on two biological replicates. less than 15% septum ingrowth were scored as "early", while cells with more than 15% 5 9 0 septum ingrowth were scored as "late") and whether the ingrowth was asymmetrical or 5 9 1 showed signs of premature splitting. The latter was based on staining with Van-FL. This 5 9 2 analysis was performed on two biological replicates. To evaluate localization of PG synthesis, exponential cultures of S. aureus (SA564 or 5 9 6 8325-4) were pulse labeled with FDAAs; cells were initially incubated 10 minutes with displayed the characteristic morphology of premature splitting were selected randomly. 6 0 7 PG synthesis was followed by assessing. from an IPTG-inducible promoter were analyzed using sequentially labeling with FDAAs 6 1 3 as described above (incubation with TADA for 10 minutes followed by HADA for 10 6 1 4 minutes). Cells were grown at 30°C in the presence of 50 µM IPTG (at higher IPTG 6 1 5 concentrations cell division defects were observed in the wild-type strain). Statistical analysis was done using R statistical software. Student's t-test was used to 6 1 8 assess significant differences in growth in the absence or presence of a tested We would like to thank Professor Simon Foster (University of Sheffield) for the generous 6 2 7 gift of FDAA's and the FtsZ-eYFP fusion plasmid. We would like to thank Ewa Kuninska   (a) SA564 wild-type and SA564ΔclpX were plated at 37°C and tested for susceptibility  Note that different scales were used on the two axes due to the difference in growth SA564 wild-type and clpX when grown at 30°C as described above. Numbers above significantly improved growth rate (P < 0.05). The P values were obtained by comparing 6 5 3 the growth rates at each concentration to the growth rate without antibiotics and were 6 5 4 calculated using Student's t-test.  Localization of FtsZ relative to PG synthesis was analyzed by sequentially labeling S. with TADA (red) for 10 minutes followed by washing and labeling with HADA (blue) for  and grown at 37°C until mid-exponential phase. These cultures were then diluted into indicate standard deviations. Note that different scales were used on the two axes due to the difference in growth between the WT and clpX mutant: values for the clpX mutant vertical axis to allow easy comparison of growth between the two strains.