Abstract
When Staphylococcus aureus strain 8325 was grown at 30°C and heat shocked at 40°C the rate of cell autolysis in buffer with or without Triton X-100 was reduced. Treatment of growing cells with other agents (CdCl2, ethanol, NaCl) known to induce heat shock proteins also resulted in cells that showed a decreased rate of autolysis. Heat shocked cells showed lower rates of freeze-thaw autolysin activity on purified cell walls, and isolated crude cell walls from heat shocked cells had lower rates of autolytic activity compared to controls. No differences in the peptidoglycan hydrolase activity profiles of control and heat shocked cells were detected by renaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is proposed that autolysins are damaged by heat shock and their targeting to the cell wall is impaired, possibly by complexing with heat shock proteins, which may also inhibit autolysin activity. Heat shock also inhibited the autolytic activity of methicillin-resistant and related-susceptible strains, and the possible relationship of this to the expression of methicillin resistance is discussed.
1 Introduction
In 1989 Young et al. [1] provided evidence that Escherichia coli heat shock proteins (Hsps) had an inhibitory effect on the lysis of the bacterium by the cloned E lysis protein of bacteriophage φX174. Mutants in five E. coli Hsp genes showed greater susceptibility to lysis by the cloned E protein than strains with intact copies of the genes. Extended protection against lysis was provided by overproduction of Hsps in cells containing a plasmid with the rpoH gene, which encodes the heat shock sigma factor, under the control of the tac promoter. In a later paper [2], cells induced to produce Hsps were resistant to lysis by a variety of β-lactam antibiotics. Hsp-mediated lysis resistance was abolished by a mutation in any of five heat shock genes. In 1990 we [3] provided the first description of the staphylococcal heat shock response. We have had a long-term interest in the autolysis of Staphylococcus aureus[4], and we wanted to see whether heat shock of this organism also inhibited autolytic activity.
We were also interested in exploring any relationship heat shock and autolysis might have to staphylococcal methicillin resistance, a resistance to β-lactam antibiotics not involving drug destruction. There are two classes of methicillin-resistant S. aureus strains in terms of their expression of methicillin resistance – homogeneous and heterogeneous. Only rare cells (1 in 103–107) in the population of heterogeneous strains grow in the presence of high concentrations of methicillin, whereas most cells in the population of homogeneous strains grow under these conditions [5]. Various physical and chemical factors have long been known to affect the expression of staphylococcal methicillin resistance [6]. A characteristic of heterogeneous strains is that growth at low temperatures, in the presence of NaCl or β-lactam antibiotics increase resistance expression, whereas high temperatures and low pH decrease resistance expression. Homogeneous methicillin resistant strains appear to have reduced autolytic activity compared to heterogeneous or susceptible strains [7, 8]. For these reasons it was of interest to investigate the effects of heat shock on autolytic activity in methicillin-resistant and -susceptible strains.
2 Materials and methods
2.1 Strains and growth conditions
S. aureus strains 8325, BB255, BB270 (heterogeneous methicillin-resistant), BB308 (femA mutant of BB270), SC4, and SC4mecC5 (heterogeneous resistant) were studied [9]. The organisms were grown in PYK medium supplemented with 1% (w/v) d-glucose [3].
2.2 Heat shock and measurement of autolysis
A starter inoculum was grown overnight with shaking (200 rpm) at 30°C in PYK+glucose medium. 100 ml of medium in a 250-ml Erlenmeyer flask was inoculated (2% v/v) and grown to an OD580 of about 0.6 at 30°C with shaking. For heat shocking, 50 ml of culture was transferred to a 250-ml Erlenmeyer flask prewarmed to 40°C and the culture was incubated with shaking at 40°C for 10 min. 25 ml of each culture was harvested by centrifuging (13 000×g, 4°C, 5 min). The cells were washed once by resuspension in cold distilled water and centrifuging, and were divided into two equal portions. One portion of the cells was resuspended in 0.05 M Tris-HCl pH 7.5 and the other in 0.05 M Tris-HCl containing 0.05% (v/v) Triton X-100 to stimulate autolysis [9]. The cell suspensions were incubated stationary at 30°C in Spectronic 20 (Bausch and Lomb, Rochester, NY) cuvettes, and the OD580 was measured at intervals. Hsps were also induced by 10-min treatments at 30°C with CdCl2 (25 μM), ethanol (680 μM) or NaCl (1.7 M) [3].
2.3 Preparation of freeze-thaw autolysin extract
Freeze-thaw autolysin extract was prepared from heat shocked and control cells as described by Qoronfleh and Wilkinson [4].
2.4 Preparation of purified cell walls (PCW) and crude cell walls (CCW) and measurement of lysis
PCW and CCW were prepared from 1-l cultures in 2-l Erlenmeyer flasks from control and cells heat shocked for 30 min at 40°C as previously described [4, 9]. Lysis of PCW by freeze-thaw extract and autolysis of CCW were measured as described before [4].
2.5 Peptidoglycan hydrolase profiles of control and heat shocked cells
The peptidoglycan hydrolase activity profiles of control and heat shocked cells were studied by renaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with heat-killed Micrococcus luteus cells as described by Berger-Bächi et al. [10].
3 Results
3.1 Heat shock decreases the rate of autolysis of whole cells
In Fig. 1 it is shown that the cells of strain 8325 grown at 30°C and heat shocked for 10 min at 40°C had significantly slower rates of autolysis than control cells both in Tris-HCl alone or in buffer containing Triton X-100. In this experiment autolysis measurements were carried out at 30°C. Similar results were obtained when autolysis occurred at 40°C (data not shown). A very similar inhibition of autolysis was observed in heat shocked cells of methicillin-resistant strains BB270 and SC4mecC5, related methicillin-susceptible strains BB255 and SC4 respectively, and a FemA mutant (BB308) of BB270 [9](Table 1).
Autolysis of S. aureus strain 8325 with and without heat shock. Autolysis of control (◯,□) and heat shocked (•,?) cells in Tris-HCl buffer pH 7.5 (◯,•) and Tris-HCl buffer pH 7.5 containing 0.05% Triton X-100 (□,?).
Autolysis of S. aureus strain 8325 with and without heat shock. Autolysis of control (◯,□) and heat shocked (•,?) cells in Tris-HCl buffer pH 7.5 (◯,•) and Tris-HCl buffer pH 7.5 containing 0.05% Triton X-100 (□,?).
Triton X-100 stimulated autolytic activity of different S. aureus strains
| Strain | Conditions | Time (min) to reach 50% of initial OD580b |
| 8325 (Mcsa) | Control | 60 |
| Heat shock, 40ºC 10 min | 89 | |
| 680 μM ethanol 30°C 10 min | 82 | |
| 25 μM CdCl2 30°C 10 min | 130 | |
| 1.7 M NaCl 30°C 10 min | 114 | |
| 255 (Mcs) | Control | 22 |
| Heat shock, 40°C 10 min | 70 | |
| 270 (Mcra) | Control | 30 |
| Heat shock 40°C 10 min | 94 | |
| 308 (Mcs, femA mutant) | Control | 84 |
| Heat shock, 40°C 10 min | 103 | |
| SC4 (Mcs) | Control | 145 |
| Heat shock, 40°C 10 min | >180 | |
| Sc4 mec C5 | Control | 95 |
| Heat shock, 40°C 10 min | >180 |
| Strain | Conditions | Time (min) to reach 50% of initial OD580b |
| 8325 (Mcsa) | Control | 60 |
| Heat shock, 40ºC 10 min | 89 | |
| 680 μM ethanol 30°C 10 min | 82 | |
| 25 μM CdCl2 30°C 10 min | 130 | |
| 1.7 M NaCl 30°C 10 min | 114 | |
| 255 (Mcs) | Control | 22 |
| Heat shock, 40°C 10 min | 70 | |
| 270 (Mcra) | Control | 30 |
| Heat shock 40°C 10 min | 94 | |
| 308 (Mcs, femA mutant) | Control | 84 |
| Heat shock, 40°C 10 min | 103 | |
| SC4 (Mcs) | Control | 145 |
| Heat shock, 40°C 10 min | >180 | |
| Sc4 mec C5 | Control | 95 |
| Heat shock, 40°C 10 min | >180 |
aMcs=methicillin-susceptible; Mcr=methicillin-resistant.
bThe values represent the averages of two experiments.
Triton X-100 stimulated autolytic activity of different S. aureus strains
| Strain | Conditions | Time (min) to reach 50% of initial OD580b |
| 8325 (Mcsa) | Control | 60 |
| Heat shock, 40ºC 10 min | 89 | |
| 680 μM ethanol 30°C 10 min | 82 | |
| 25 μM CdCl2 30°C 10 min | 130 | |
| 1.7 M NaCl 30°C 10 min | 114 | |
| 255 (Mcs) | Control | 22 |
| Heat shock, 40°C 10 min | 70 | |
| 270 (Mcra) | Control | 30 |
| Heat shock 40°C 10 min | 94 | |
| 308 (Mcs, femA mutant) | Control | 84 |
| Heat shock, 40°C 10 min | 103 | |
| SC4 (Mcs) | Control | 145 |
| Heat shock, 40°C 10 min | >180 | |
| Sc4 mec C5 | Control | 95 |
| Heat shock, 40°C 10 min | >180 |
| Strain | Conditions | Time (min) to reach 50% of initial OD580b |
| 8325 (Mcsa) | Control | 60 |
| Heat shock, 40ºC 10 min | 89 | |
| 680 μM ethanol 30°C 10 min | 82 | |
| 25 μM CdCl2 30°C 10 min | 130 | |
| 1.7 M NaCl 30°C 10 min | 114 | |
| 255 (Mcs) | Control | 22 |
| Heat shock, 40°C 10 min | 70 | |
| 270 (Mcra) | Control | 30 |
| Heat shock 40°C 10 min | 94 | |
| 308 (Mcs, femA mutant) | Control | 84 |
| Heat shock, 40°C 10 min | 103 | |
| SC4 (Mcs) | Control | 145 |
| Heat shock, 40°C 10 min | >180 | |
| Sc4 mec C5 | Control | 95 |
| Heat shock, 40°C 10 min | >180 |
aMcs=methicillin-susceptible; Mcr=methicillin-resistant.
bThe values represent the averages of two experiments.
In order to provide some further evidence that the inhibition of autolysis was correlated with the induction of Hsps, cells were treated at 30°C with CdCl2, ethanol and NaCl. These treatments are known to induce Hsps in S. aureus and other bacteria [3]. Each of these chemical treatments led to inhibition of the autolysis of strain 8325 in 0.05 M Tris HCl containing 0.05% Triton X-100 (Table 1).
3.2 Heat shock decreases the activity of freeze-thaw extracted autolysin and the rate of autolysis of CCW
In order to probe further into the mechanism of autolysis inhibition, the autolytic activity of freeze-thaw extracted autolysin and CCW from heat shocked cells were compared with these preparations from control cells. Freeze-thaw preparation from heat shocked cells was significantly less active on PCW than the same preparation from control cells (Fig. 2). Furthermore, CCW from heat shocked cells had significantly less autolytic activity than CCW from control cells (Fig. 2). However, the peptidoglycan hydrolase profiles of heat shocked and control cells were very similar in renaturing SDS-PAGE with Micrococcus luteus cells as substrate (data not shown).
Lysis of PCW of S. aureus strain 8325 by freeze-thaw extract, and autolysis of CCW, from control and heat shocked cells. PCW in 0.05 M KH2PO4-K2HPO4 buffer pH 7.2 were incubated with freeze-thaw extract from control (◯) and heat shocked (•) cells. CCW isolated from control (□) and heat shocked cells (?) were suspended in 0.05 M KH2HPO4 buffer pH 7.2 and allowed to autolyze.
Lysis of PCW of S. aureus strain 8325 by freeze-thaw extract, and autolysis of CCW, from control and heat shocked cells. PCW in 0.05 M KH2PO4-K2HPO4 buffer pH 7.2 were incubated with freeze-thaw extract from control (◯) and heat shocked (•) cells. CCW isolated from control (□) and heat shocked cells (?) were suspended in 0.05 M KH2HPO4 buffer pH 7.2 and allowed to autolyze.
4 Discussion
When bacteria are heat shocked significant unfolding and aggregation of both newly synthesised and pre-existing proteins occurs [11]. The ‘classical’ heat shock regulon of E. coli produces a number of chaperones and proteases, under the control of the heat shock sigma factor encoded by rpoH, to prevent protein aggregation, promote proper folding, and remove terminally damaged proteins by proteolysis. In E. coli heat shock causes intracellular denaturation of proteins which aggregate to form a distinct fraction (fraction S), which co-sediments with the cell envelope [12]. Hsps are highly evolutionarily conserved and similar systems are believed to be present in all bacteria.
We have shown that heat shock of S. aureus results in lower autolytic activity in whole cells, freeze-thaw autolysin extract, and isolated CCW. Lower autolytic activity has been noted previously in S. aureus cells grown at higher temperatures (43 and 37°C) compared to cells grown at 30°C [13, 14]. The genes and proteins known to affect autolysis in S. aureus have recently been reviewed [15]. The major S. aureus autolysin, ATL [13, 16], is a hybrid enzyme containing amidase and glucosaminidase domains, and a signal sequence, that undergoes extensive post-translational processing. It is not clear why heat shocked cells and cells grown at higher temperatures have lower autolytic activities. Various explanations of this phenomenon may be proposed. It is possible that one or more of the S. aureus autolysins are thermolabile. Alternatively, thermal injury has been shown to result in a loss of Mg2+ from the S. aureus cell envelope [17], and lower Mg2+ in the cell wall could result in lower autolytic activity. However, the finding of lower autolytic activity in freeze-thaw autolysin extract and isolated CCW suggests that heat shock may interfere with the targeting of the autolysins to the cell wall. Possibly the autolysins are damaged by heat shock and accumulate in intracellular aggregates. In addition, the Hsps may complex with damaged autolysins and inhibit their activity, interfere with their export from the cell, and the heat shock proteases may degrade terminally damaged autolysins. The fact that no differences were observed in the peptidoglycan hydrolase activity profiles of heat shocked and control cells indicates that autolysins were not permanently denatured. In this assay gels are incubated overnight following electrophoresis to allow the peptidoglycan hydrolases time to renature.
The data of Young et al. [1] and Powell and Young [2] suggest that the Hsps themselves somehow inhibit lytic activity in E. coli. Overproduction of Hsps in non heat shocked cells resulted in decreased lytic activity of the E protein, and decreased β-lactam-induced lysis. Inhibition of autolysis in non heat shocked S aureus cells by ethanol, CdCl2 or NaCl treatments which induce Hsps [3], suggests that autolysin inhibition may involve the Hsps themselves. Long-term growth in the presence of NaCl yields cells that show increased autolytic activity [18].
How do our findings tie in to the expression of methicillin resistance? It has long been known that the expression of methicillin resistance is decreased at higher temperatures [6]. Our present work suggests that Hsp-mediated inhibition of autolysis is correlated with decreased expression of methicillin resistance. However, in the case of homogeneous methicillin-resistant strains decreased autolytic activity is associated with a high level of resistance expression [7, 8]. Conversely, in the case of heterogeneous strains an association between methicillin resistance and increased autolysis has been noted [9]. Further work will be necessary to resolve this apparent paradox.
Acknowledgements
We are grateful to Rozina Maredia, Debbie Bucz, Prerna Rajput and Bradley Shelton for their contributions to this study, and to Kevin D. Young for his comments on the manuscript.
