Overnight cultures of strain S. aureus N315 were grown in TSB at 37⁰C to an OD600 of 1.0 with aeration. After addition of waldiomycin, the culture was incubated for an additional 10 min at the same temperature. The cells were then harvested (10 min;
5,400 ×g) and resuspended in the same volume of phosphate buffered saline (PBS) containing either 0.1% Triton X-100 or 200 ng/ml lysostaphin (Wako Pure Chemical, Osaka, Japan). These suspensions were then incubated at 30⁰C (Triton lysis assay) or 37⁰C (lysostaphin lysis assay) with aeration. Lysis was determined as the decrease in the OD600 over time. The results were presented as percentage lysis, calculated by dividing the measured OD600 by the initial OD600.
Biofilm formation assays
The strain S. aureus N315 was grown overnight in TSB, diluted in TSB plus 0.25% glucose to an OD600 of 0.1, and then distributed into a sterile, 96-well polystyrene plates (200 µl per well). Different concentrations of waldiomycin were added to the wells at 2 µl per well and mixed thoroughly. The plates were then incubated at 37⁰C for 24 hr. The wells were washed gently twice with PBS and air dried.
The adherent bacteria were stained with 0.1% crystal violet for 15 min at room temperature. The plates were then rinsed with PBS and air dried, and the stained biomass was resuspended in ethanol-acetone (80:20) for quantification. The absorbance of the solution was measured at 595 nm.
In vivo autophosphorylation assay
Overnight cultures of strain B. subtilis 168 transformed with pBsG-full were grown at 37⁰C in LB medium supplemented with chloramphenicol and 100 µM IPTG until the OD660 reached 0.3. After addition of waldiomycin, the cells were further grown for 1 hr at the same temperature. Cells from 2 ml aliquot of culture were then collected by centrifugation (2,300×g, 10 min, 4⁰C) and suspended into 200 µl of lysis buffer (100 mM NaCl, 50 mM Tris-HCl, pH 8.0). Then the cells were disrupted by using
Multi-beads shocker (Yasui Kikai, Osaka, Japan) at 2500 rpm, 60 sec ON, 60 sec OFF, 6 repeated cycles. Afterwards, the supernatant sample was mixed with 5×SDS-sample buffer and were electrophoresed at 4⁰C with a constant current of 20 mA on a 50 µM Phos-tag SDS-polyacrylamide gel (6% acrylamide, 100 µM MnCl2) for 150 min. After electrophoresis, the gel was soaked into 10 mM EDTA/Blotting buffer C (0.302% (w/v) Tris, 20% (v/v) methanol), thrice (10 min each), in order to remove Mn2+, and then soaked into Blotting buffer C for 10 min prior toWestern Blot analysis.
Western blotting
The samples were separated by electrophoresis and then transferred to a polyvinylidenedifluoride (PVDF) membrane (Immobilon-P membrane, Merck Millipore, Billerica, MA, USA). The PVDF membrane was blocked by 1% skim milk, probed with anti-WalK antibody as the primary antibody and enhanced chemiluminescence (ECL) peroxidase-labeled anti-rabbit antibody (GE Healthcare Bio-Science) as the secondary antibody. WalK was detected with LuminataTM Forte Western HRP (horseradish peroxidase) substrate (Merck Millipore), and signals were acquired with Image Quant Imager400 (GE Healthcare Bio-Science).
Results and Discussion
WalR regulon gene expression
Since we have shown that waldiomycin inhibited WalK in vitro, waldiomycin should exert an effect on the WalK/WalR system in vivo. Thus, the effect of this compound on in vivo expression of WalR regulon genes was analyzed using qRT-PCR.
In B. subtilis, WalK/WalR activates expression of yocH (cell wall hydrolase), yvcE (cwlO, cell wall endopeptidase), lytE (autolysin) and ydjM (cell wall-associated protein), while negatively regulates the expression of yoeB (modulator of autolysin activity) and yjeA (peptidoglycan deacetylase) (Bisicchia et al., 2007; Howell et al., 2003; Salzberg and Helmann, 2007, Stapleton et al., 2007). In S. aureus, WalR positively regulates the expression of cell wall metabolism genes including isaA and sceD (cellwall transglycosylase/muramidase), ssaA, SA2353, SA2097, SA0620, SA0710, and atlA (cell wall amidase), and lytM (cell wall glycyl-glycine endopeptidase) (Dubrac et al., 2007, 2008; Dubrac and Msadek, 2004). The cleavage sites for WalK/WalR regulon encoded cell wall hydrolases in S. aureus are indicated in Fig. 7.
Addition of waldiomycin to the culture of B. subtilis led to lowered expression of yocH (40%) and yvcE (13%) and increased expression of yoeB (16-fold) and yjeA (7-fold) within 5 min (Fig. 6A). As for S. aureus, addition of waldiomycin led to lowered expression of isaA (20%), sceD (30%), ssaA (20%), SA2353 (30%), SA2097 (40%) and SA0710 (60%) within 5 min (Fig. 6B). These results support the in vivo inhibition of the WalK/WalR system by waldiomycin. Although SA0620, atlA, and lytM are genes that are positively regulated by WalR, expression of SA0620 and lytM after waldiomycin treatment did not change, and that of atlA increased (Fig. 6B). We assumed that there may be some other regulatory factors besides WalR that may control the expression of these genes.
(A) (B)
Fig. 6. Transcriptional regulation of the WalR regulon by waldiomycin.
Waldiomycin was added to exponentially growing cultures of B. subtilis (A) and S.
aureus N315 (B). After 5 min incubation, total RNA was isolated and quantified by qRT-PCR. Relative results were expressed as the mean and standard deviation from triplicate experiments using primers specific for WalR regulon genes, gyrA and 16S rRNA (normalizing gene).
Relative Quantity
positive regulon negative regulon
Relative Quantity control0.5 µg/ml
1 µg/ml control
0.5 µg/ml 1 µg/ml
0 1 2 5 10 15 20
0 1 2 3 4 5
positive regulon
Genes Functions
isaA, sceD Cell wall transglycosylase/muramidase ssaA, SA2353, SA2097, Cell wall amidase
SA0620, SA0710,
AtlA Cell wall amidase/glucosaminidase
LytM Cell wall endopeptidase
Genes Functions
yocH Cell wall hydrolase yvcE Cell wall endopeptidase yoeB Modulator of autolysin activity yjeA Peptidoglycan deacetylase
Bacillus subtilis Staphylococcus aureus
Fig. 7. Diagram of S. aureus cell wall peptidoglycan and cleavage sites for WalR-regulon encoded cell wall hydrolases.
Peptidoglycan cleavage sites for WalR-regulon encoded cell wall hydrolases are indicated: a) Glycan strand glycosidic bond (glucosaminidase: atlA). b) Glycan strand glycosidic bond (lytic transglycosylases: sceD and isaA). c) N-Acetylmuramyl-L-alanyl amide bond (atlA amidase domain, ssaA, SA2353, SA2097, SA0710, SA0620), and d) Pentaglycine bridge glycyl-glycyl bond (lytM endopeptidase). Waldiomycin lowered the expression of those genes which are underlined. NAM=N-acetylmuramic acid, NAG=N-acetylglucosamine.
Effect of waldiomycin against cell morphology
Cell wall metabolism is involved in many cellular processes, and modifications of peptidoglycan degradation are linked to a wide range of phenotypes. A well-documented phenotype associated with a defect in autolysins is the propensity of planktonic cell suspensions to aggregate. As shown in our qRT-PCR results, waldiomycin inhibited the expression of several cell wall metabolism genes in S. aureus. We have tested the effects of this compound on S. aureus cell organization. As peptidoglycan hydrolases are involved in daughter cell separation, one would expect WalK/WalR inhibited cells to be aggregated in S. aureus instead of proper cellular dissemination. As shown in Fig. 8A,
o
OH OH
CH2OH
o
CH2OHo
o
CH3-CH-CO NH
CO-CH3
NH CO-CH3
L-Ala D-Gln L-Lys D-Ala D-Ala
(L-Gly)5 D-Ala L-Lys D-Gln
L-Ala CH3-CH-CO
o
NAM NAG---- ·
NAG NAM
o
---·Glucosaminidase
atlA Transglycosylase/Muramidase
isaA sceD
Amidase atlA ssaA SA2353 SA2097
SA0620 SA0710
Endopeptidase lytM
Waldiomycin
✂ ✂ ✂
✂
a b
c
d
waldiomycin-untreated S. aureus cells exhibited typically well separated morphology, whereas waldiomycin-treated cells aggregated and formed clusters of aggregated cells (Fig. 8B). This cell aggregation phenotype is consistent with the previously reported phenotypes of WalK/WalR starved cells (Dubrac et al., 2007).
(A) (B)
Fig. 8. Micrographs of S. aureus cells in the absence (A), or presence (B) of waldiomycin.
The S. aureus N315 strains were grown in TSB to an OD660 of 0.3 at 37⁰C.
Waldiomycin was added to the cultures and then incubated for an additional 1 hr at the same temperature with shaking. Cells were stained with Gram’s stain and observed under a light microscope at ×1,000 magnification. A: Waldiomycin untreated cells. B:
Waldiomycin treated cells.
Effect of waldiomycin against Triton X-100 and lysostaphin-induced cell lysis
Different regulatory mechanisms are involved in regulation of autolysis such as modifying cell wall peptidoglycan (the substrate of murein hydrolases) and regulating the expression of genes associated with cell wall hydrolase activities (Groicher et al., 2000; Rice et al., 2003; Zheng et al., 2007). Since the WalK/WalR system effectively regulates bacterial autolysis, waldiomycin should have an effect on cell lysis. We tested the effect of waldiomycin on Triton X-100, a nonionic detergent, and lysostaphin on the autolysis of S. aureus cells. As shown in Fig. 9A, waldiomycin-treated cells exhibited high resistance to Triton X-100-induced autolysis, since only 30% cell lysis was observed after 3.5 hr while 96% cell lysis occurred in waldiomycin-untreated cells. This effect may be the consequence of waldiomycin repressed expression of WalR regulon genes. The effect of WalK/WalR inhibition on the lysostaphin sensitivity was also examined. Lysostaphin is a glycyl-glycine endopeptidase that specifically cleaves the pentaglycine cross-bridges of staphylococcal cell wall, leading to rapid lysis of the
bacteria. As shown in Fig. 9B, only 39% cell lysis was seen for waldiomycin-treated cells after 30 min, whereas waldiomycin-untreated cells displayed a higher lysis rate, and 92% cells were lysed. These data showed that inhibition of WalK/WalR lead to increased lysostaphin resistance. This effect could be due to a modification of cell wall peptidoglycan composition resulting in lowered accessibility of the pentaglycine bonds targeted by lysostaphin. These cell lysis phenotypes are also consistent with the previously reported phenotypes of WalK/WalR depleted cells (Dubrac et al., 2007), and supports the fact that waldiomycin inhibits WalK in vivo.
(A) (B)
Fig. 9. Effect of waldiomycin on autolysis profiles of S. aureus cells mediated by Triton X-100 and lysostaphin.
The S. aureus N315 strains were grown in TSB to an OD600 of 1. After addition of waldiomycin at 6 µg/ml (3/4MIC), the culture was incubated for an additional 10 min.
Then the cells were pelleted and resuspended in PBS to test the effects of autolysis-inducing agents. Triton X-100 (A) and lysostaphin (B) mediated lysis of staphylococci was measured as the decline of OD600 over time. Autolysis of waldiomycin treated cells (open symbols) and untreated cells (closed symbols) were determined at 30⁰C in the presence of 0.1 % Triton X-100 and at 37⁰C in the presence of 200 ng/ml lysostaphin.
Effect of waldiomycin on S. aureus biofilm formation
According to the previous report of Dubrac et al. (2007), depletion of WalK/WalR system lead to impaired biofilm formation in S. aureus. We checked the effect of waldiomycin against S. aureus biofilm formation on a polystyrene surface. Contrary to our expectations, we found enhanced biofilm formation with increasing concentrations of waldiomycin. Sturdy biofilms were formed in the presence of higher concentration of waldiomycin as compared to the DMSO control. As shown in Fig. 10, the measured
absorbance was 0.823 in the presence of 3/4MIC of waldiomycin while it was 0.507 in the DMSO control well.
Autolytic activity of atlA gene has been linked to biofilm formation (Biswas et al., 2006; Heilmann et al., 1997). Since our qRT-PCR results revealed an increased expression of atlA gene due to addition of waldiomycin to the S. aureus culture (Fig.
6B), this may be the reason why waldiomycin lead to more robust biofilms in our biofilm formation assay.
Fig. 10. Effect of waldiomycin on biofilm formation of S. aureus N315.
Biofilm formation assays of S. aureus were performed on microtiter plates in TSB plus 0.25% glucose with increasing concentrations of waldiomycin. Quantifications of biofilms were performed by measuring the OD595 following crystal violet staining and resuspension inethanol-acetone (80:20).
In vivo inhibitory effect of waldiomycin against WalK HK
The effect of waldiomycin on WalR regulon gene expression, cell morphology, and autolysis strongly suggests that waldiomycin inhibits WalK and thereby represses the WalK/WalR system in cells. In order to examine whether waldiomycin actually inhibited in vivo WalK autophosphorylation, we employed Phos-tag SDS-PAGE to analyze the level of WalK autophosphorylation in B. subtilis. Phos-tag SDS-PAGE is a technique that is capable of separating phosphorylated and unphosphorylated forms of protein (Kinoshita et al., 2006). This technique has been successfully applied to the detection of phosphorylated RRs in vivo (PhoB, WalR and PhoP) (Fig. 11A) (Barbieri and Stock, 2008; Ishii et al., 2013; Wayne et al., 2012). As shown in Fig. 11B, we were able to separate the phosphorylated WalK from the non-phosphorylated WalK (lane 1).
OD595
Control 2 4 6 Waldiomycin (µg/ml)
When waldiomycin was added to exponential phase cells and incubated for 1 hr before Phos-tag SDS-PAGE analysis, the phosphorylated WalK bands decreased with increasing concentrations of waldiomycin (lanes 2-5). This result clearly showed that waldiomycin applied from outside the cell inhibited WalK autophosphorylation inside the cell.
(A)
(B)
Fig. 11. Effect of waldiomycin on in vivo autophosphorylation of WalK histidine kinase.
A) Basic structure of Phos-tag. B) Phos-tag SDS-PAGE. Overnight cultures of B.
subtilis 168 were grown with aeration to OD600 of 0.3 at 37⁰C. At this point, waldiomycin was added {Lane 1, 0 µg/ml; 2, 1 µg/ml; 3, 2 µg/ml; 4, 4 µg/ml; and lane 5, 8 µg/ml (MIC)} and the cultures were incubated for an additional 1 hr. Cells were harvested, disrupted, and analyzed by Phos-tag SDS-PAGE and Western blotting.
P-WalK, phosphorylated WalK.
P-WalK WalK
waldiomycin
1 2 3 4 5
CHAPTER IV
Effects of waldiomycin on WalK sensor histidine kinase localization to the cell division septum.
Introduction
The WalKR TCS plays critical roles in controlling cell wall and surface homeostasis and in responding to cell wall stresses in low-GC Gram-positive bacteria (Dubrac et al., 2008; Jordan et al., 2008; Winkler and Hoch, 2008). In Bacillus, Staphylococcus, and many other species, both the WalK HK and WalR RR are indispensable, and they cannot be depleted (Fabret and Hoch. 1998; Fukuchi et al., 2000; Martin et al., 1999). The WalKR regulons consist of various sets of genes that negotiate peptidoglycan biosynthesis, cell division, and the cell surface in different Gram-positive bacteria (Ahn et al., 2007; Bisicchia et al., 2007; Dubrac et al., 2007;
Fukuchi et al., 2000; Howell et al., 2003; Liu et al., 2006; Mohedano et al., 2005; Ng et al., 2003; Ng et al., 2005; Senadheera et al., 2005).
TCSs are the key components in cell cycle signalling in Caulobacter crescentus and in sporulation in B. subtilis, implying that they have been adopted to perform more crucial roles in cell division and growth (Piggot and Hilbert, 2004; Holtzendorff et al., 2006).
In this chapter, I illustrated the effects of waldiomycin against WalK histidine kinase localization to the cell division septum in growing cells of B. subtilis as well as S.
aureus by using immunofluorescence microscopy.
Materials and Methods
Culture media and growth conditions
B. subtilis cells were grown under aerobic conditions at 37⁰C in Luria-Bertani (LB) medium (1% polypeptone, 0.5% yeast extract, and 0.5% NaCl). S. aureus cells were grown in Trypticase Soy Broth (TSB) medium (1.7% Bacto tryptone, 0.3% Bacto soytone, 0.5% NaCl, 0.25% dipotassium phosphate, and 0.25% dextrose) at 37⁰C with aeration.
Slide preparation
Prior to immunofluorescent studies, microscope slide was prepared to absorb cells efficiently. A multiwell microscope slide (15-well multitest slides by MP Biomedicals)
was treated with 10 µl Poly-L-lysine solution {0.1% (w/v) in H2O} per well (Sigma-Aldrich) for 30 min at room temperature. The solution was removed with a Pipetman and the slide was washed once with H2O and air dried at room temperature.
Cell growth and fixation
Wild type B. subtilis 168 and S. aureus N315 strains were grown at 37⁰C with aeration up to OD525 of 0.3. At this point of growth, waldiomycin was added and the culture was incubated for an additional 10 min at the same temperature. After incubation, an aliquot of 500 µl culture was removed and 120 µl of formaldehyde solution was added to the cell suspensions, cells were fixed by keeping the mixture at room temperature for 20 min. Following this, the fixed cells were collected by centrifugation (14,000 rpm, 2 min, room temperature). The pellets of cells were washed twice with PBS and suspended in PBS to an OD525 nm of less than 0.4. Then 10 µl of cell suspensions was added to a Poly-L-lysine-treated multiwell glass slide and kept at room temperature for 30 min. The suspension was removed carefully with pipetman, and the plate was washed three times by the addition and removal of 20 µl of PBS. Finally, 10 µl of GTE buffer (25 mM Tris-HCl, pH 8.0, 10 mM EDTA, 50 mM Glucose) was added to the wells, and the slide was kept at room temperature for 10 min.
Cell Permeablization
To permeabilize the cells, 10 µl GTE buffer supplemented with 3 mg/ml lysozyme (B. subtlis) and 10 µl GTE buffer supplemented with 1 mg/ml lysostaphin (S. aureus) were added to the wells. To assure incomplete lysis, the enzymes were allowed to act for no longer than 10 min. The solution was removed, and the slide was washed 16 times with PBS to assure complete removal of enzymes. Following washing, the slide glass was submerged into ice-cold methanol for no longer than 5 min and subsequently air dried. Finally, 10 µl of PBS supplemented with 2% BSA (PBS-B) was added to the wells and kept at room temperature for 20 min, preparing the slides for antibody staining.
Fluorescence staining and visualization
The primary (WalK) antibody was diluted (1:25) in PBS-B and 10 µl of antibody solution was added to the wells and allowed to equilibrate at 4ºC overnight. The slides were then washed 16 times with PBS-B. Ten µl PBS-B was applied to the wells, the slides were kept at room temperature for 10 min. Secondary antibody {Alexa Fluor 488 (green)-conjugated antirabbit antibody} was then diluted (1:500) in PBS-B, and 10 µl of
secondary antibody solution was added to the wells and the slides were kept in the dark place for 3 hrs at room temperature. Then the slides were washed 16 times with PBS-B.
DNA was visualized by the addition of 20 µl of PBS supplemented with 0.5 µg/ml (B.
subtilis) and 0.25 µg/ml (S. aureus) 4′, 6-diamidino-2-phenylindole (DAPI). Following a 5-min incubation time in dark place, DAPI was removed and the cells were washed once with PBS. Then 1.5 µl of anti-bleaching buffer (20 % v/v PBS, 80 % v/v Glycerol and 0.1% w/v p-phenylenediamine) was added to the wells and the wells were concealed with a cover glass. The edges were sealed with nail polish, and the slides were kept at 4⁰C until use in fluorescent microscopy.
Fluorescence visualization of the cells was performed using a Carl Zeiss Axio Imager A1 microscope (a Plan-NEOFLUAR magnification, x100, numerical aperture 1.3). The sample pictures were taken with a charge-coupled-device (CCD) camera (AxioCam MRm; Carl Zeiss) driven by AxioVision software (version 4.6; Carl Zeiss).
Results and Discussion
Effect of waldiomycin on WalK sensor kinase localization to the division septum of B. subtilis cells.
I have shown here that waldiomycin inhibits activities of essential WalK HK in vitro as well as in vivo. In order to investigate the effect of this compound on localization of WalK to the cell division site in B. subtilis, phase-contrast (Fig. 12A) and DAPI-stained (Fig. 12B) images as well as immunostained images were shown (Fig.
12C). As the result, waldiomycin-untreated cells (DMSO control sample) exhibited WalK HK localized at the division septum of actively dividing cells. Here, WalK localization was evidenced as green colored and more concentrated appearance at the cell division site (Fig. 12C and D). In contrast, waldiomycin-treated cells displayed no WalK localization to the septa (Fig. 13C). This effect may be the consequence of inhibitory effects of waldiomycin against WalK HK activities and repression of the WalKR regulon genes expression by waldiomycin.
Fig. 12. Immunofluorescence microscopy of growing cells of B. subtilis in the absence of waldiomycin (DMSO control sample).
(A) Phase-contrast image; (B) DAPI stained image; (C) Immnostained image; and (D) Enlarged image. Arrows indicate the cell division septa where WalK KHs are seen to be localized. Here, (A), (B) and (C) are the same images.
A B
C D
x Dx
Fig. 13. Immunofluorescence microscopy of growing cells of B. subtilis in the presence of waldiomycin (8 µg/ml).
(A) Phase-contrast image; (B) DAPI stained image; and (C) Immunostained image.
Effect of waldiomycin on WalK sensor kinase localization to the division septum of S. aureus cells.
The effect of waldiomycin on WalR regulon gene expression, cell morphology, and autolysis strongly suggests that waldiomycin inhibits WalK HK and thereby represses the WalK/WalR system in S. aureus cells. Therefore, we have also tested the effect of waldiomycin on WalK localization to the division septum of actively growing cells of S. aureus. As shown in Fig. 14C, WalK shown to be localized to the septum in S.
aureus when cells were not treated with waldiomycin (DMSO control sample). In contrast, waldiomycin-treated cells did not show such type of localization of WalK sensor HK to the septum (Fig. 15C). Here, we also observed that cells tended to be aggregated which we have already described in the previous findings of morphological changes of S. aureus cells induced by waldiomycin (Fig. 8B)
Fig. 14. Immunofluorescence microscopy of growing cells of S. aureus in the absence of waldiomycin (DMSO control sample).
(A) Phase-contrast image; (B) DAPI stained image; and (C) Immunostained image.
Arrows indicate the cell division septa where WalK KHs are observed to be localized.
Fig. 15. Immunofluorescence microscopy of growing cells of S. aureus in the presence of waldiomycin (8 µg/ml).
(A) Phase-contrast image; (B) DAPI stained image; and (C) Immunostained image.
For a bacterium, an abundance of cellular processes need to be accurately coordinated in pursuane of continuous growth as well as division as speedily as it does.
An intimidating challenge to this process is the need to arrange intrinsic events, DNA synthesis, septum formation, etc., with extracellular processes, the degradation and resynthesis of the cell wall at the division septum to guarantee cell separation.
According to the previous report of Fukushima et al. (2011), the WalK sensor HK organizes cell wall remodeling with cell division in Gram-positive bacteria by regulating the transcription of genes for autolysins and their inhibitors. In B. subtilis the WalK HK senses cell division and is enzymatically stimulated by associating with the divisome at the division septum.
The WalKR TCS is a component in this multifactorial signalling process (Bisicchia et al., 2007; Fukuchi et al., 2000). TCSs generally control gene expression and it is enlightening to examine the genes that WalR regulates (Bisicchia et al., 2007).
WalR∼PO4 positively accelerates the expression of genes for autolytic enzymes that cut at many distinct sites in the cell wall peptidoglycan matrix. Apparently these enzymes assemble the cell wall for restructuring and cell separation coinciding with the synthesis of the division septum. WalR∼PO4 is also a repressor of genes for proteins that suppress autolytic enzymes, yoeB, and that deacetylate peptidoglycan, yjeA, to impede the action of autolysins. In rapidly dividing cells, autolytic enzymes are necessary for cell division while these enzymes desire suppression in slow growth or non-growing conditions (Bisicchia et al., 2007). The balance of synthesis of autolysins and their inhibitors is regulated by the proportion of WalR∼PO4/WalR. This proportion must be higher in rapidly growing cells to aid autolysin synthesis, and low in non-growing cells to aid synthesis of autolysin inhibitors. Accordingly, WalR RR may be thought of as a homeostatic regulator tuned to the division state of cells. Since the phosphorylation state of WalR relies on the activity of the essential WalK sensor HK, WalK kinase becomes a negotiator of the state of the cell by responding to cellular signals. As a kinase of WalR RR, WalK is most functional when paired with the division septum (Fukushima et al., 2008).
Lastly, we attempted to sum up the effect of waldiomycin on WalK sensor kinase localization to the division septum in actively growing cells of B. subtilis as well as S.
aureus (Fig. 16A and B). From our recent results, it could be postulated that in absence of waldiomycin, activity of the essential WalK HK is not hindered and therefore, the expression of WalKR regulons is not repressed. WalK HKs moved to the division sites of cells undergoing division state as well as localized to the septum which plays a vital role to initiate division septum formation as well as subsequent steps to complete cell
division process. Finally, two daughter cells are generated from a parent cell and afterwards the newly formed cells are well separated as single cell. In contrast, when waldiomycin was applied to the cells from outside at its MIC level of concentration (8 µg/ml) it targets WalK, inhibits its activity and therefore, septal localization of WalK HK is suppressed. Eventually, there is inhibition of the synthesis of autolysins necessary for cell division with cell wall restructuring. Subsequently, cell division process as well as cell separation are suppressed resulting in the formation of long filamentous cells (Fig. 16A).
As shown in Fig. 16B, when waldiomycin was added to S. aureus cells at a same concentration used in B. subtilis cells, by targeting WalK HK waldiomycin suppresses the essential WalK activity needed for cell division with cell wall remodeling. Therefore, the expression of genes for autolytic enzymes is hindered and thus inhibiting the formation of septum crucial for newly growing cells. The degradation and resynthesis of the cell wall at the division septum to assure cell separation is also suppressed by waldiomycin. Here we also revealed that cells aggregated instead of proper separation in case of S. aureus cells.
Taken together the results of our immunofluorescence microscopy, it can be predicted that when waldiomycin applied to the cells undergoing the state of active division process, there is perturbation of cell wall homeostasis and the WalKR essential signal transduction pathway needed for cell division process with cell wall remodeling in B. subtilis as well as S. aureus.
(A)
Cell elongation
WalK localization at cell pole Septum space
I. Phase-contrast image
II. DAPI stained image
III. Immunostained image
Waldiomycin
Cell division
WalK localization at septum is inhibited
Cell division is inhibited
Filamentous cells DNA
WalK at septum
(WalK is not localized)
(B)
I. Phase-contrast image
II. DAPI stained image
III. Immunostained image
WalK localized to the division septum
Each doublet is a pair of daughter cells
Waldiomycin
WalK localization at septum is inhibited Cell division
Single cell
Cell division is inhibited
Aggregated cells (WalK is not localized) DNA
Doublet
Fig. 16. Diagrammatic representation of WalK HK localization at the cell division septum and a model of the inhibitory effect of waldiomycin on cell division of B. subtilis and S. aureus. (A) B. subtilis and (B) S. aureus cells
CHAPTER V
SUMMARY
The highly conserved WalK/WalR TCS is specific to low G+C Gram-positive bacteria, including a number of important pathogens. An uncommon feature is that this system is necessary for viability in most of these bacteria. In our recent study, we have focused on the WalK HK inhibitor, waldiomycin which was discovered by screening metabolites from actinomycetes. Here, we applied in vivo and in vitro strategies to further characterize this novel WalK inhibitor, waldiomycin. We have shown that waldiomycin inhibited autophosphorylation of WalK HKs in vitro from B. subtilis, S.
aureus, E. faecalis, and S. mutans at a half-maximal inhibitory concentrations of 10.2, 8.8, 9.2, and 25.8 µM, respectively. Quantitative RT-PCR studies of WalR regulon genes have suggested that waldiomycin repressed the WalK/WalR system in B. subtilis as well as in S. aureus cells. Morphology of waldiomycin-treated S. aureus cells displayed increased aggregation instead of proper cellular dissemination. Furthermore, autolysis profiles of S. aureus cells revealed that waldiomycin-treated cells were highly resistant to Triton X-100- and lysostaphin-induced lysis. These phenotypes are consistent with those of cells starved for the WalK/WalR system, indicating that waldiomycin inhibited the autophosphorylation activity of WalK in cells. We confirmed that waldiomycin inhibits in vivo autophosphorylation of WalK by actually observing the phosphorylated WalK ratio in cells using Phos-tag SDS-PAGE. We also checked the effect of waldiomycin on WalK localization to the division septum of B. subtilis and S. aureus cells by using immunofluorescence microscopy. Our results revealed that WalK localized to the division septum in the actively dividing cells while cells treated with waldiomyicn did not show WalK localization to the septa.
Finally, our results suggest that waldiomycin targets essential WalK histidine kinases and inhibits autophosphorylation activity of this essential histidine kinase. It suppresses the WalR regulon genes expression. By inhibiting WalK HKs it inhibits the WalK/WalR essential signal transduction pathway involved in critical biological processes during cell growth and division processes including cell wall biosynthesis, daughter cell separation and cell wall turnover in gram-positive bacteria including MRSA (Fig. 17).