Section of Molecular Genetics and Microbiology, School of Biological Sciences, The University of Texas at Austin, Austin, TX 78712, USA1
Author for correspondence: Paul J. Szaniszlo. Tel: +1 512 471 3384. Fax: +1 512 471 7088. e-mail: pjszaniszlo{at}mail.utexas.edu
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ABSTRACT |
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Keywords: WdCHS, virulence, cell wall, morphological mutants, semi-quantitative RT-PCR
Abbreviations: ts, temperature-sensitive
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INTRODUCTION |
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Wangiella dermatitidis is a model fungus for the more than 100 other melanized fungal pathogens of humans (Szaniszlo et al., 1993 ). In this agent of phaeohyphomycosis, five chitin synthase structural (WdCHS) genes have been identified, characterized to various degrees, and found to encode five different isozyme (WdChsp) types: class II, WdChs1p (Mendoza, 1995
); class I, WdChs2p (Wang et al., 2001
); class III, WdChs3p (Wang et al., 2000
); class IV, WdChs4p (Wang et al., 1999
); and class V, WdChs5p (Liu et al., 2001
). Each of these genes has been disrupted singly and in a variety of combinations. Our systematic analyses of the disruption mutants (wdchs
), which are still in progress, have shown that the products of most WdCHS genes in some way contribute indirectly or directly to virulence. This occurs either by the imparting of a temperature sensitivity to the disruption strains, which then cannot grow at the elevated temperatures of infection (e.g. at 37 °C; Wang et al., 1999
; Liu et al., 2001
), or by the lowering of virulence without affecting the strains ability to grow at the higher temperature (Wang et al., 2001
; unpublished data). However, the comprehensive relationships among the expression of the five WdCHS genes at the transcriptional level have not been addressed. Here, we report that differential expression was detected among the WdCHS genes of W. dermatitidis. The patterns of differential expression observed suggest that compensatory mechanisms occur at the transcriptional level in response to a defective WdCHS gene, as well as in response to the exposure of the wild-type strains or two temperature-sensitive (ts) morphological mutants to the elevated temperature of an infected host.
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METHODS |
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RT-PCR.
The differential expression patterns of the five WdCHS genes were investigated by a semi-quantitative RT-PCR method, which was similar to those described by Noonan et al. (1990) , Ogretmen et al. (1998)
and Ogretmen et al. (2001)
. Primers for the RT-PCR were designed from non-conserved regions of each WdCHS gene, based on a multiple alignment of all five genes. The sequence of each primer pair and the expected size of each PCR product are listed in Table 2
. Primers 18S for the 18S rRNA gene and the 18S PCR Competimer (Ambion) were used as an internal control to ensure that an equal amount of total RNA was present in each of the different samples. The RT-PCR of a sample was done in one tube using the Access RT-PCR system (Promega). A typical RT-PCR reaction for 25 µl consisted of 1 µl MgSO4 (25 mM), 0·5 µl dNTP (10 mM), 2·5 µl buffer (10x), 1 µl forward and reverse primer (25 µM), 0·5 µl avian myeloblastosis virus RT, 0·5 µl Tfl DNA polymerase, 115 µl total RNA in appropriate dilution, and RNase-free water supplemented to 25 µl. The RT-PCR amplifications were carried out in a GeneAmp PCR System 9700 (Applied Biosystems) using the following cycling conditions: 48 °C for 1 h, 94 °C for 2 min, followed by different cycles of 94 °C for 30 s, 5058 °C for 1 min and 68 °C for 1 min 20 s, then an extra step of elongation at 68 °C for 5 min. The annealing temperature was 50 °C for WdCHS1, WdCHS3 and WdCHS4 amplifications, 55 °C for 18S and WdCHS5, and 58 °C for WdCHS2. Cycle numbers varied from 25 to 29. Both cycle number and template amount were carefully calibrated for each experiment to ensure that the RT-PCR was done within the exponential phase of amplification. For each sample, a parallel negative control having the same components, except avian myeloblastosis virus RT, as those used in the standard RT-PCR was subjected to amplification to ensure the absence of trace DNA contamination. The RT-PCR products were examined using 1·2% ethidium bromide/agarose gel electrophoresis, viewed under UV and finally analysed densitometrically with the AlphaImage 1220 documentation and analysis system (Carnock).
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RESULTS AND DISCUSSION |
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Compensatory expression of the five WdCHS genes in single wdchs disruption mutants
Five chitin synthase (WdCHS) genes have been cloned, characterized to different degrees, and disrupted singly and in various combinations. The absence of a dramatic decrease in chitin contents in any single mutant makes us assume that general compensation by at least one chitin synthase exists when one of the other WdCHS genes is defective. This suggested that study of the relative transcription levels of each of the remaining four WdCHS genes in the absence of one other might help confirm this hypothesis. For these experiments, cells of each wdchs mutant and of the wild-type were grown at 37 °C for 24 h. The RT-PCR results (Fig. 1b
) were evaluated after the densitometry data of each WdCHS mutant were normalized against those of the wild-type. The results (Fig. 2b
) showed that the expression levels detected again varied differentially depending upon which WdCHS gene was defective. These results also supported the notion that the product of some WdCHS genes might have a special role in cell proliferation and/or in maintaining the structural integrity of the cell wall. For example, WdCHS1 and WdCHS2 both appear to compensate somewhat for each of the other chitin synthase genes in their respective mutants. Furthermore, WdCHS1 disruption was compensated for only by higher expression of WdCHS2 (Fig. 2b
; compare data in vertical row 1), whereas WdCHS2 disruption appears to be compensated for by increased expression of WdCHS1, together with WdCHS3 and WdCHS4 (compare data in vertical row 2). These particular two datasets provide additional supporting evidence for the idea that WdCHS1 and WdCHS2 are functionally overlapping in vivo (Wang et al., 2001
). They may also help explain why WdCHS1 and WdCHS2 cannot both be defective in the same background for cell viability at 37 °C, in spite of the fact that they can grow weakly, albeit abnormally, at 25 °C (Zheng et al., 1997
). Taken together with the above observation that WdCHS1 and WdCHS2 responded only weakly to a temperature increase (Fig. 2a
), these data indicate that WdCHS1 and WdCHS2 play basic roles in cell proliferation. This hypothesis is further supported by our finding that the transcription levels for both WdCHS1 and WdCHS2 are the least abundant among those of the five WdCHS genes assayed, based on a relative abundance analysis by dot-blotting of RNA from cells grown at the restrictive temperature (Fig. 3
).
Because the expression level of WdCHS5 changed little in response to the disruption of other WdCHS genes, it seems that the product of WdCHS5 is unique and may play roles very distinct from those of the other WdCHS genes. This possibility is enhanced by the knowledge that WdCHS5 has a nucleotide sequence that is divergent from those of the other four WdCHS genes when they are multiply aligned (data not shown), that its derived protein has a myosin motor-like domain, and, most importantly, by the observation that among the five different single wdchs mutants, only wdchs5
mutants showed loss of viability in stationary phase at 37 °C, and loss of virulence in a mouse model of acute infection (Liu et al., 2001
). On the other hand, WdCHS expression in a wdchs5
mutant was compensated for by each of the other four remaining WdCHS genes (Fig. 2b
; compare data in vertical row 5). These data support the idea that even though the other four WdCHS genes were induced to higher levels of transcription in the wdchs5
mutant, they are either not translated or their encoded proteins do not function in ways that can compensate for the loss of WdChs5p function and prevent eventual cell death at 37 °C.
Compensatory expression of the five WdCHS genes in two ts morphological mutants
Mc3 and Hf1 are two ts morphological mutants of W. dermatitidis. These mutants have special importance because whereas the wild-type produces mainly yeast cells at both 25 and 37 °C in rich media, the two ts mutants form multicellular (sclerotic) forms and hyphae, respectively, from yeast cells when shifted from the lower to the higher temperature in the same media (Roberts & Szaniszlo, 1978 ; Cooper & Szaniszlo, 1993
; Ye & Szaniszlo, 2000
). Thus, results with these ts mutants and the wild-type grown identically can be compared in such ways that any differences detected among them can be ascribed to changes in specific morphology and not to differences in environments. Previous results have shown that total chitin content and chitin synthase activity increased in the mutants compared to the wild-type when W. dermatitidis was grown in its multicellular form or as hyphae (Szaniszlo et al., 1983
; Wang & Szaniszlo, 2000
). The results here suggest that the higher chitin synthase levels and chitin increases noted previously may have resulted, in part, from the higher levels of expression of WdCHS1, WdCHS3 and WdCHS4 in the case of the ts multicellular-form mutant, Mc3, and of WdCHS1 and WdCHS3 in the case of the ts hyphae-producing mutant, Hf1. Although the results with WdCHS3 were not unexpected, because they were indicated previously by Northern analyses (Wang & Szaniszlo, 2000
), those with WdCHS4 and particularly WdCHS1 were not anticipated. Possibly they indicate special roles for the products of these genes in multicellular forms and hyphae. However, as yet, attempts to derive mutants with these genes disrupted in the Mc3 and Hf1 backgounds have all failed, possibly because some WdChsp defects in these backgrounds impart synthetic lethality. The results with WdCHS5 were also somewhat unexpected, at least for those in the Hf1 background. In this case, we had suspected that WdCHS5 (class V) would be the most highly expressed WdCHS gene in Hf1 at 37 °C, because the first class V chitin synthase was first isolated from A. nidulans (Fujiwara et al., 1997
), and then was found subsequently only among other filamentous fungi (Park et al., 1999
; Zhang & Gurr, 2000
; Liu et al., 2001
). However, WdCHS5 expression in Hf1 did not increase. Instead, it occurred at a level equal to those of the wild-type and Mc3, a result consistent with the recent finding that a wdchs5
mutant did not show any apparent morphological difference from the wild-type when both were cultured identically in a medium that induced hyphae.
Conclusions
The primary aim of this study was to investigate the hypothesis that compensatory expression of individual chitin synthase genes of W. dermatitidis occurs in response to stress stimuli. The secondary aim was to elucidate further the possible functional relationships among the WdCHS-encoded proteins. The results presented here showed that each of the five WdCHS genes exhibited a somewhat different expression pattern in response to the different growth situations tested, which supports the hypothesis that the expression of each one may have special, and sometimes distinct, roles in cell growth, viability and virulence. On the other hand, a general compensatory expression was evident in each single chitin synthase disruption mutant, which may explain the previous observations that chitin contents were not dramatically decreased in any single wdchs mutant, and that no single wdchs
mutant lost viability at 25 °C (Zheng, 1997
; Wang et al., 1999
, 2001
; Wang & Szaniszlo, 2000
; Liu et al., 2001
). We postulate that the general compensatory expression detected in W. dermatitidis in response to chitin synthase gene disruption and to exposure to other stresses might function as part of the cell wall integrity pathway in a manner similar to that recently described in S. cerevisae (Smits et al., 1999
). Support for this possibility is provided by very recent results that show that the transcripts of all five WdCHS genes are up-regulated in mutants defective in melanin biosynthesis (unpublished data). It is suspected that similar up-regulatory patterns of WdCHS transcription will be found that correlate with other cell wall defects.
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ACKNOWLEDGEMENTS |
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Received 15 February 2002;
revised 7 May 2002;
accepted 16 May 2002.