Des Moines University, 3200 Grand Avenue, Des Moines, IA 50312, USA
Correspondence
Martin Schmidt
mschmidt{at}dmu.edu
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ABSTRACT |
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INTRODUCTION |
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There are three chitin synthase activities in S. cerevisiae, two of which are involved in cytokinesis (Shaw et al., 1991). At the beginning of the cell cycle, chitin synthase III activity (CSIII) lays down a ring of chitin at the presumptive bud site through which the bud emerges. At the end of mitosis, chitin synthase II activity (CSII) forms a primary septum from chitin between mother and daughter cell. Finally, after mother and daughter cells have separated through the action of a chitinase, chitin synthase I activity (CSI) acts on the daughter cell wall to repair damage caused by excessive chitinolysis.
Our understanding of the importance of chitin synthesis in S. cerevisiae has evolved over time. Based on tetrad analysis of heterozygous deletion mutants, it was first concluded that CSII was essential (Silverman et al., 1988). It was later shown that the apparent inviability of a CSII-deficient spore was due to a germination defect and that CSII was not necessary for growth and cytokinesis (Bulawa & Osmond, 1990
). A loss of CSII affects but does not prevent cytokinesis. Due to a lack of a primary septum, cells lacking the CSII catalytic moiety Chs2p fail to separate after cytokinesis and form cell clumps. These chs2 mutants achieve separation of mother and daughter cytoplasms by constructing a remedial septum, which is a bulky structure of cell wall material deposited at the bud neck. The completion of the remedial septum requires CSIII (Cabib & Schmidt, 2003
). It was shown that mutants defective in primary septum formation rely on CSIII for cytokinesis and survival. This correlation has been successfully exploited for the isolation of septation mutants by means of a synthetic lethality screen (Osmond et al., 1999
; Schmidt et al., 2002
).
This study was initiated after the surprising finding that in the presence of osmotic stabilizer a loss of both CSII and CSIII is tolerated. Based on this observation, a mutant lacking the catalytic moieties of all three known chitin synthase activities was constructed. After an initial period of fragility, the mutant acquires a suppressor and grows under normal culture conditions. The aim of this study was to show that growth and cytokinesis are possible in the absence of chitin.
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METHODS |
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Polyoxin D survival.
To assess the toxicity of polyoxin D, cells were grown overnight in synthetic complete medium at 30 °C to a titre of 12x106 cells ml1. Wet cell mass per ml was determined with an accuracy of 0·1 mg. The cultures were then diluted in fresh medium to 12 µg cells ml1, equalling 1x105 c.f.u. ml1 for YPH499 and 2x104 c.f.u. ml1 for YMS348s. To 0·1 ml of cells, polyoxin D was added at the specified concentrations and the cultures were incubated overnight at 30 °C with agitation. At the beginning and the end of the experiment, cells were counted in a haematocytometer, diluted appropriately and plated on solid YPD medium.
Strain construction.
All strains were constructed using the procedures described by Crotti et al. (2001). Transformation of yeast strains was achieved with a lithium acetate protocol (Burke et al., 2000
). To construct the chs1 chs2 chs3 triple deletant strain YMS348, ECY46 was sporulated and a chs1 : : HIS3 chs3 : : LEU2 spore was isolated. The resulting strain ECY46-1-20D was transformed with a chs2 : : TRP1 fragment and the transformed cells were incubated on Trp-omission medium containing 1 M sorbitol at 26 °C for 10 days. The chs2 : : TRP1 deletion was verified by PCR as described by Crotti et al. (2001)
. YMS348 cells with a suppressor (designated YMS348s) were isolated by streaking cells on Trp-omission medium without sorbitol and incubating at 30 °C. Strains used in this study are listed in Table 1
.
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Vital staining.
Staining of dead cells was achieved by washing cells once with water or 1 M sorbitol and incubating in 20 mg ml1 methylene blue in 50 mM KH2PO4 with or without 1 M sorbitol for 5 min at room temperature.
Cell wall digestion and nuclear staining.
Analysis of cytokinesis by fixation and cell wall digestion was accomplished as previously described (Cabib & Schmidt, 2003). Cells were grown overnight in YPD medium to a titre of 12x107 cells ml1. Then 5 ml of cells were fixed by addition of formaldehyde to a final concentration of 5 %. After overnight incubation at 4 °C, fixed cells were washed once with PBS and suspended in 1 ml citrate/phosphate buffer (40 mM Na2HPO4 and 20 mM citric acid mixed 39/61 to obtain pH 6·5, 0·8 M sorbitol, 1 mM EDTA, 50 mM
-mercaptoethanol). After this step, mechanical stress was carefully avoided. One hundred microlitres of Glusulase (Perkin-Elmer Life Sciences) was added to the fixed cells, and cell walls were digested at 30 °C for 90 min with gentle shaking (60 r.p.m.). Spheroplast-like appearance of the digested cells indicated complete cell wall removal. Nuclei were stained by adding 1 µl of 0·1 mg ml1 Hoechst 33342 to the fixed, digested cells and incubating for 5 min at room temperature. Fluorescence was monitored with a standard DAPI filter set on a Zeiss Axioskop 2 fluorescence microscope.
Electron microscopy.
Cells were grown in 100 ml YPD medium to a titre of 13x107 cells ml1, spun down, washed once with water and resuspended in 1 ml 3 % glutaraldehyde, 0·1 M cacodylate containing 5 mM CaCl2 and 5 mM MgCl2. Cells were dispersed and embedded in agarose, cooled and cut. Blocks were fixed in 4 % KMnO4 for 1 h at room temperature, washed thoroughly with water and incubated with 0·5 % sodium metaperiodate for 15 min at room temperature. After washing with 50 mM potassium phosphate (pH 7·4), blocks were incubated in 50 mM ammonium phosphate (pH 7·4) for 15 min. After washing twice with water, blocks were placed overnight into 2 % uranyl acetate (pH 4·5) at room temperature in the dark. Blocks were dehydrated through a graded series of ethanol solutions (50100 %, v/v, at 40 °C) and left overnight in fresh 100 % ethanol at room temperature. They were then washed twice with 100 % ethanol and twice with propylene oxide before being embedded in Spurr resin. From these blocks, sections were cut and post-stained with lead citrate for 25 min.
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RESULTS |
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Chitin-free mutants become hyper-resistant to polyoxin D
The fungicidal effect of the chitin synthase inhibitor polyoxin D was examined in exponentially growing cultures (Fig. 2). In the WT, polyoxin D was fungistatic at 0·45 mg ml1 and resulted in 90 % killing at 0·75 mg ml1. In these experiments, the chitin-free mutant YMS348s was not at all inhibited by polyoxin D. In a separate experiment, cells were incubated with polyoxin D at 2 mg ml1, which resulted in 99·5 % killing of YPH499 cells but had no inhibitory effect on strain YMS348s. In order to determine whether the occasional survival of YPH499 cells at high doses of polyoxin D was due to chance or intrinsic resistance, three colonies of cells that had survived exposure to 2 mg polyoxin ml1 were incubated overnight with 1 mg polyoxin D ml1. All three strains previously exposed to polyoxin D were more resistant (75·1±33·9 % killing) than the control strain that had not been exposed (99·7 % killing).
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DISCUSSION |
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The apparent viability of a strain deleted in all of the known chitin synthase genes poses three questions. First, is there really no chitin synthesis in this strain? Second, how is cytokinesis achieved without the assistance of chitin? And third, how does the cytokinesis defect affect nuclear division and cell cycle progression?
The complete absence of chitin synthase activity in isolated membrane vesicles of YMS348s confirms that there are no more than three chitin synthases encoded in the genome of S. cerevisiae. This could be expected, although this is the lowest number encountered so far in fungi (see overview by Roncero, 2002). Chitin synthases can be identified based on genomic sequences because of highly conserved domains (Bowen et al., 1992
). In the S. cerevisiae genome, only the three known chitin synthases contain these domains, which made the existence of a fourth chitin synthase activity highly unlikely.
How is cytokinesis achieved in the absence of chitin? The data presented here indicate that the loss of chitin synthase activity causes a failure to synthesize a septum and leads to the formation of cell chains. Clearly, the synthesis of chitin does help with the timely completion of remedial septa. It has been suggested that the unique mechanism by which fungi divide contraction of an actomyosin ring coupled to synthesis of cell wall is necessary because a high cellular turgor pressure works against the advancing septa (Cabib, 2004). In the same way, reinforcement of remedial septa by chitin becomes necessary to overcome the turgor pressure (Cabib & Schmidt, 2003
). A chitin-deficient mutant initially grows only in the presence of 1 M sorbitol. This hyperosmotic growth condition is known to lower the intracellular turgor pressure, which lowers the resistance that the advancing septa must overcome. However, hyperosmotic growth conditions trigger an adaptive response that aims at increasing the intracellular pressure back to the equilibrium (reviewed by Hohmann, 2002
). Because of this physiological response, the actual cellular turgor pressure under hyperosmotic growth conditions is hard to predict.
An appealing interpretation of the present results is that it is not the mechanical qualities that make chitin essential for the construction of remedial septa but rather its abundance. The presence of the suppressor enables yeast cells to form remedial septa even in the absence of chitin, which suggests that the function of chitin in these structures can be taken over by other cell wall components. As can be seen in the electron micrographs, the remedial septa contain significant amounts of cell wall material. The timely completion of these structures from glucan and mannoproteins alone may exceed the cell's biosynthetic abilities. This study shows that the suppressor changes the rate of synthesis rather than the ultrastructure of the remedial septa. The apparent rate of septum synthesis is a function of synthesis and degradation of cell wall material. Consequently, the suppressor either increases the deposition of septum material or decreases septum degradation by inhibiting enzymes such as Eng1p (Baladron et al., 2002). The molecular nature of the suppressor mechanism is still obscure and will be the subject of further studies. Unfortunately, classic genetic analysis is complicated by low mating efficiency, the germination defect of chs2 mutant spores and the rapid accumulation of suppressor in non-suppressed cultures.
How does a failure to close the septum affect nuclear division and cell cycle progression? Interestingly, completion of cell separation is not necessary for the initiation of a new cell cycle in S. cerevisiae. This is illustrated by the fact that a failure to assemble the septation apparatus or to contract the actomyosin ring at the bud neck gives rise to chains of incompletely separated cells, which nevertheless have properly divided nuclei (Bi et al., 1998; Vallen et al., 2000
; Luca et al., 2001
; Lim et al., 2003
). If the cells were arresting the cell cycle at the M/G1 border because of the abscission defect, it would cause the accumulation of large budded cells with separated nuclei (Hartwell et al., 1973
; Jaspersen et al., 1998
). This could not be observed in chitin-deficient mutants. The analysis of nuclear division in YMS348 reveals that even with mother and daughter cells not separating their cytoplasm, the cells continue the mitotic cycles which then leads to the formation of multinucleate cell chains.
Although a failure to form a septum in a timely manner does not arrest the cell cycle, it apparently does influence nuclear segregation. Analysis of nuclear distribution showed that binucleate cells can be found frequently in cell chains of YMS348. Binucleate cells are a consequence of incorrect positioning of the mitotic spindle. The proper positioning of the spindle is a process that depends on the interaction of cytoplasmic microtubules with the cell cortex (Cottingham & Hoyt, 1997; De Zwaan et al., 1997
; Lee et al., 1999
). In some way, growth in the absence of chitin disturbs this interaction. It goes beyond the scope of this study to satisfactorily explain the defect leading to nuclear missegregation. It should be noted, however, that the formation of bulky remedial septa is known to influence some cortical landmark proteins (Schmidt et al., 2002
), which makes it likely that the cortical component of the microtubule interaction is impaired in the chitin-deficient strain.
Finally, what does the possibility of life without chitin mean for medical mycology? Despite strong similarities in some areas, S. cerevisiae is very different from pathogenic fungi in many other respects. Especially the physical properties of the cell wall seem to be different, since S. cerevisiae is able to grow without chitin reinforcement of the lateral walls whereas even its close relative, Candida albicans, is not (Munro et al., 2001). In pathogenic fungi, chitin synthesis is a prime target for antifungal drugs, and several drugs with strong in vitro inhibitory potential are available. However, none of these drugs has been very effective in vivo (reviewed by Ruiz-Herrera & San-Blas, 2003
). This has been attributed to a variety of factors, including limited uptake of the inhibitor into the fungal cell and different susceptibilities of chitin synthases to different inhibitors, with no inhibitor being equally effective on all isozymes. The present study shows that growth of yeast in the absence of chitin is not only possible, but also leads to a hyper-resistance to chitin synthase inhibitors. Although S. cerevisiae and pathogenic fungi differ in important aspects of cell wall architecture, there might also be a similar resistance mechanism in pathogenic fungi which allows for growth even when chitin synthesis is strongly inhibited.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 29 March 2004;
revised 23 April 2004;
accepted 28 April 2004.
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