Genes associated with dimorphism and virulence of

Genes associated with dimorphism and virulence Candida albicans

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N.A.R. Gow, B. Hube, D.A. Bailey, D.A. Schofield, C. Munro, R.K. Swoboda, G. Bertram, C. Westwater, I. Broadbent, R.J. Smith, G.W. Gooday, and A.J.P. Brown

Abstract: Strategies for the analysis of a range of Candida albicat~sgenes, whose expression is regulated during the yeast to hyphal transition (dimorphism), including genes encoding putative virulence factors, are reviewed. T o help discriminate among genes whose products were the cause or consequence of dimorphism, temporal changes in the levels of the mRNAs of these and other genes were examined by northern analysis. The mRNA levels of most genes that were examined increased or decreased, transiently or persistently indicating complex alterations in gene expression during morphogenesis. Genes encoding four glycolytic enzymes were regulated transcriptionally during dimorphism but control experiments indicated no direct correlation with germ tube formation. Two chitin synthase genes (CHS2 and CHS3) and three aspartyl proteinase genes (SAP4-SAP6) were transcribed preferentially in the hyphal form, but in these cases hypha-specific expression was shown to be strain dependent o r medium dependent, respectively. A gene, HYRl (for Izyphal regulation), was isolated and found to exhibit strict hyphaspecific expression in a range of strains under a range of culture conditions. The ura-blaster protocol was used to generate disruptions in CHS2, CHSI, and HSP90 (for heat-shock protein). The homozygous CHS2 disruption did not affect the kinetics of germ-tube formation markedly but resulted in hyphae with a reduced chitin content. In contrast, homozygous null mutations in CHSl and HSP90 were apparently lethal because no homozygous null strains were isolated after integrative transformation of heterozygous mutants. The analysis of candidate genes for dimorphism and virulence of C. albicans through northern analysis and gene disruption should facilitate an understanding of these processes at the molecular level. Key words: Catzdida, dimorphism, gene regulation, virulence.

R6sum6 : Les auteurs prCsentent une revue des stratCgies utilistes pour analyser un ensemble de gbnes du Candida albicans dont I'expression est contr6lke au cours de la transition du stade levure au stade hyphe (dimorphisme), incluant des gbnes codant pour des prCsumCs facteurs de virulence. Pour tenter de faire la distinction entre les gbnes dont les produits sont la cause ou la consequence du dimorphisme, les changements temporels dans les teneurs en mARN de ces gbnes et d'autres ont CtC examinCes par analyse northern. Les teneurs en mARN d e la plupart des gtnes examinis augmentent ou diminuent, de faqon transitoire ou persistante, ce qui indique l'existence de modifications complexes dans l'expression des gbnes au cours de la morphogknkse. Les gbnes codant pour quatre enzymes glycolytiques sont contr6lCs par transcription au cours du dimorphisme mais des expkriences de verification indiquent qu'il n'y a pas de corrClation directe ou indirecte avec la formation du tube germinatif. Deux gknes de la synthase chitinique (CHS2 et CHS3) et trois genes de la protkinase de I'aspartyle (SAP4-SAP6) sont prCfkrentiellement transcrits dans la forme hyphe, mais dans ces cas on constate que l'expression spCcifique aux hyphes dCpend des souches ou du milieu, respectivement. Un gbne, le HYRl (contr6lant les hyphes), a CtC is016 et on dCmontre que son expression est strictement spkcifique aux hyphes, dans un ensemble de souches, sous un ensemble de conditions de culture. Une mCthode par Cclatement (ura-blaster) a CtC utilisCe pour amener des dislocations dans les gtnes CHS2, CHSI et HSP90 (de aheat-shock proteins : protCine de choc thermique). Au contraire, les mutations homozygotes nullifiantes chez les gtnes CHSI et HSP90 sont apparemment ICtales, puisqu'aucune souche homozygote inopkrante n'a CtC isolte aprbs transformation intigrative des mutants hCtCrozygotes. L'analyse des gbnes pouvant Ctre utilisCs pour Ctudier le dimorphisme et la virulence du C. albicans, par analyse northern et dislocation des genes devrait permettre d'amtliorer la comprChension de ces processus au niveau molCculaire. Mots cle's : Candida, dimorphisme, contralant les genes, virulence. [Traduit par la rkdaction] Received August 16, 1994.

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N.A.R. Gow,' B. Hube, D.A. Bailey, D.A. Schofield, C. Munro, R.K. Swoboda, G. Bertram, C. Westwater, I. Broadbent, R.J. Smith, G.W. Gooday, and A.J.P. Brown. Department of Molecular and Cell Biology, Marischal College, University of Aberdeen, Aberdeen AB9 1AS, Scotland, U .K.

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Author to whom all correspondence should be addressed

Can. J. Bot. 73(Suppl. 1): S335-S342 (1995). Printed in Canada 1 Imprimt au Canada

Can. J. Bot. Vol. 73 (Suppl. I ) , 1995


Introduction Candida albicans is a normal commensal and a common opportunistic pathogen of the human host. Pathogenicity is a polygenic trait in this organism and likely virulence factors include the ability of the fungus to exhibit yeast-hyphal dimorphism, secretion of proteases and other hydrolytic enzymes, adherence to cell surfaces primarily via mannoproteins, and the ability to undergo rapid switching among a number of specific and differing phenotypes (Cutler 1991; Matthews 1994; Odds 1994). These virulence factors have been suggested on the basis of the presumed advantages conferred by such traits and with regard to natural isolates or mutagenised strains with altered phenotypic characteristics. However, the regulation of dimorphism and virulence in C. albicans is poorly defined at the molecular level partly because the organism is an asexual diploid and is intransigent to genetic analysis. Dimorphic and avirulent mutants have been created by chemical or UV mutagenesis, but these may harbour lesions at a multiplicity of unknown loci and their analysis has failed to produce unequivocal answers for example to the significance of dimorphism or protease production to virulence. Strains with well-defined mutations in specific putative virulence encoding genes have yet to be produced. Our aims are, therefore, to identify genes encoding essential functions in the yeast to hyphal transition and other possible virulence factors, and to create specific mutations in these genes to deduce their function and importance. To do this we are using two approaches. (i) To identify key genes in dimorphic regulation, we are developing screens for genes that are expressed differentially or whose expression regulates dimorphism. (ii) We are also cloning and analysing specific genes that encode activities that are likely to be important in the control of cell shape or interaction with the host. Here we describe the analysis of several genes whose expression is regulated differentially during dimorphism. We show that the formation of hyphae is accompanied by complex changes in the expression of genes encoding metabolic, biosynthetic, and hydrolytic enzymes and we describe strategies for functional analysis of such genes through the creation of specific mutant strains.

Molecular approaches towards isolation of genes involved in Candida virulence and morphogenesis Our approach to determine the role of a given gene in dimorphism and virulence is to isolate genes that may play a role in these processes, then examine their function. The primary step may (i) employ subtraction libraries or other screens that would select for genes that may have hypha-specific functions or (ii) involve searching for specific genes that encode activities that are suspected to be important for morphogenesis or pathogenicity. In addition we have examined the expression of genes of either type that have been cloned elsewhere to further understand the overall molecular changes occurring during dimorphism. Screening for genes that a r e regulated during dimorphism A cDNA library made from the mRNA of hyphal cells of C. albicans was screened with pooled sera from HIV-

positive patients with or without candidiasis (Gow et al. 1993; Swoboda et al. 1993, 1994a, 1994b). The sera were preadsorbed with protein extracts from Escherichia coli and the yeast phase of C. albicans to enrich for hypha-specific antibodies. From 40 000 clones screened, 83 positives were found, of which 10 were selected at random for further analysis. Sequencing of the 3 ' and 5' ends of the clones showed that two cDNAs encoded separate isolates of the pyruvate kinase gene (PYK1) (Swoboda et al. 1983), one encoded alcohol dehydrogenase (ADHI), and one encoded a ribosomal binding protein (RplO) (R.K. Swoboda, I. Broadbent, G. Bertram, G.W. Gooday, N.A.R. Gow, and A.J.P. Brown, unpublished data). None of these genes was expressed exclusively in the hyphal form but all were regulated during dimorphism. Failure to select for hypha-specific mRNA species during the immunological screening-procedure may-be attributed to incomplete removal of the yeast-specific antibodies at the preabsorption stage or because many hyphaspecific mRNAs are only expressed at low levels. Screening for genes involved in the regulation of putative virulence functions We have examined genes encoding proteinases, mannosyl transferase enzymes, and heat shock proteins because these proteins may have important roles in pathogenesis (Cutler 1991; Matthews 1994). Genes for the analysis of C. albicans secretory aspartyl proteinases were obtained from Hube et al. (1991 ; SAP]), Wright et al. (1992; SAP2), White et al. (1993; SAP3), Miyasaki et al. (1994; SAP4) and Monod et al. (1994; SAPS-SAP6). A gene encoding a-l,2-mannosyltransferase (MNTI), which in Saccharomyces cerevisiae is involved in 0-glycosylation (Hausler and Robbins 1992; Hausler et al. 1992) was isolated from a partial C. albicans genomic DNA library using the S. cerevisiae gene as a heterologous probe. Other putative mannosyltransferases have been identified by low stringency Southern analysis and by polymerase chain reaction (PCR) to conserved glycosyltransferase sequences (C. Westwater, E. Buurman, G. W. Gooday, A. J.P. Brown, and N.A.R. Gow, unpublished data). The analysis of these genes should enable us to gain a more detailed picture of the contribution of specific mannosyl residues in the various biological properties of C. albicans (adhesion, antigenicity, etc.) that are important for the host -fungus interaction. Antisera to a specific epitope of C. albicans HSP90 have been shown to confer a protective effect against Candida infections in animal models (Matthews et al. 1991). We have isolated a C. albicans HSP90 clone from the above cDNA library using the S. cerevisiae HSP90 gene as a probe, and then isolated the complete C. albicans HSP90 gene from a library provided generously by Mark Paton (Smith et al. 1992; R.K. Swoboda, G.W. Gooday, N.A.R. Gow, and A.J.P. Brown, unpublished data). The further analysis of the SAP1 -SAP6, MNTI, and HSP90 genes is described below. Gene expression and dimorphism The levels of a wide range of mRNAs have now been measured during germ-tube formation (Table l). To our knowledge, no C. albicans mRNA has been reported whose levels remain sufficiently constant during the dimorphic transition to use as an internal loading control for northern blots

C1.3. Gow et al. Table 1. Summary of changes in overall levels of mRNA species during the first few hours after induction of hyphal growth by serum, N-acetylglucosamine, or pH -temperature. p

p

p

p

p


mRNAs CHSI,* HST7, GFAl PYKI, ADHI, GPMI, PGKl ACTI, TEF3, CYCI, PMAI, HSE20, RplO, MNTI, CHS2, CHS3, HSP90, HSP60, UBII-I, UBII-2* CHSI ,* SAP2 HYRI, SAP4,* SAPS,* SAP6,* CHSI*

p

Response to hyphal transition No significant change Transient decrease Transient increase Hypha-specific decrease Hypha-specific increase

NOTE: Some changes are medium dependent. Transient changes in the levels of mRNAs occur over the first 1-3 h following induction. ACT1 (actin; Swoboda et al. 19948); ADHl (alcohol dehydrogenase; Swoboda et al. 1 9 9 4 ~ ) CHSl-3 ; (chitin synthases; Chen-Wu et al. 1992; Schofield 1994); CYCl (cytochrome C; N.A.R. Gow, B. Hube, D . A . Bailey, D . A . Schofield, C. Munro, R.K. Swoboda, G. Bertram, C. Westwater, I. Broadbent, R.J. Smith, G.W. Gooday, and A.J.P. Brown, unpublished data); HST7 (homologue of STE7; Feldmann 1994); GFAl (glucosaminefructose-6-phosphate amidotransferase; N.A.R. Gow, B. Hube, D . A . Bailey, D . A . Schofield, C. Munro, R.K. Swoboda, G. Bertram, C. Westwater, I. Broadbent, R.J. Smith, G.W. Gooday, and A.J.P. Brown, unpublished data); GPMl (phosphoglycerate mutase; Swoboda et al. 19948); HSE20 (homologue of SEC20; N . A . R . Gow, B. Hube, D . A . Bailey, D . A . Schofield, C. Munro, R.K. Swoboda, G. Bertram, C. Westwater, I. Broadbent, R.J. Smith, G.W. Gooday, and A.J.P. Brown, unpublished data); HSP90 (heat shock protein 90; N.A.R. Gow, B. Hube, D.A. Bailey, D.A. Schofield, C. Munro, R.K. Swoboda, G. Bertram, C. Westwater, I. Broadbent, R.J. Smith, G.W. Gooday, and A.J.P. Brown, unpublished data); HYRl (hyphal regulated; N.A.R. Gow, B. Hube, D.A. Bailey, D . A . Schofield, C. Munro, R.K. Swoboda, G. Bertram, C. Westwater, I. Broadbent, R.J. Smith, G.W. Gooday, and A.J.P. Brown, unpublished data); MNTl (a-l,2-mannosyltransferase;N . A . R . Gow, B. Hube, D.A. Bailey, D . A . Schofield, C. Munro, R.K. Swoboda, G. Bertram, C. Westwater, I. Broadbent, R.J. Smith, G.W. Gooday, and A.J.P. Brown, unpublished data); PGKI (phosphoglycerate kinase; Swoboda et al. 1 9 9 4 ~ ) PYKl ; (pyruvate kinase; Swoboda et al. 1994~);RplO (ribosomal protein; R.K. Swoboda, I. Broadbent, G. Bertram, G.W. Gooday, N.A.R. Gow, and A.J.P. Brown, submitted); SAP2, SAP4-SAP6 (secretory aspartyl proteinases; Hube et al. 1994); TEF3 (translation elongation factor; Swoboda et al. 19948); UBll (polyubiquitin; Bailey et al. 1994). *Dependent on the conditions used to induce dimorphism.

(Delbruck and Ernst 1993; Swoboda et al. 1994a, 19946; R.K. Swoboda, I . Broadbent, G. Bertram, G.W. Gooday, N.A.R. Gow, and A.J.P. Brown, unpublished data). Most mRNAs showed a transient or persistent increase or decrease in level compared with ribosomal RNA although the precise temporal pattern was often medium or strain dependent. In addition, most protocols for inducing germ-tube formation involve resuspending stationary phase cells in fresh media and so many of the observed changes in mRNA levels may be attributed solely to alterations in the physiological status of the cells as they resume growth. Expression of genes for glycolytic enzymes The ADHl, GPMI, PGKI, and PYKl mRNAs, which encode glycolytic enzymes, all showed a transient decrease in their expression levels during the first hour following germ tube induction (Table 1). The levels then increased relative to the ribosomal RNA controls, in contrast to the ACT1 mRNA, which decreased transiently during germ tube . kinetics of the recovinduction (Swoboda et al. 1 9 9 4 ~ )The ery of the glycolytic mRNA levels did not correlate with the formation of hyphae and, hence, it was concluded that the changes in the levels of these mRNA levels were not related directly to yeast-hyphal morphogenesis (Swoboda et al. 1994~).

Chitin synthase genes There are three known chitin synthase genes in C. albicans (Au-Young and Robbins 1990; Chen-Wu et al. 1992; Sudoh et al. 1993). Because chitin is a major structural element in the fungal wall, the regulation of the expression of chitin synthase genes may be significant in the regulation of cell shape and, hence, dimorphism. However, northern analysis of the three C. albicuns CHS genes suggests that their transcriptional regulation is not involved directly in the regulation of dimorphism (Schofield 1994). Using restriction fragments from CHSl and CHS2, and a PCR product amplified from CHS3 as probes we performed a detailed northern analysis of these genes in a range of strains and environmental conditions (Schofield 1994). Levels of CHS2 and CHS3 mRNA increased transiently under four sets of culture conditions that promoted filamentous growth, and all three genes were expressed at higher levels at neutral pH. The concentration of CHSl in cells induced by serum (Gow and Gooday 1982) and during pH-temperature dimorphism (Buffo et al. 1982) increased gradually. In contrast, in N-acetylglucosamine medium (Mattia et al. 1982), CHSl mRNA levels decreased in cells forming germ tubes. Although all three genes were expressed in all the laboratory strains and clinical isolates examined, the pattern of expression differed in some cases. For example, an increase in CHS2 and CHS3 mRNA levels


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Fig. 1. Hyphal-specific expression of HYRI mRNA under two sets of experimental conditions. In the top panel an overnight culture of C. albicans 3153 grown in YPG at 25OC was used to inoculate four YPG cultures grown at 25 or 37°C in the presence or absence of 10% (vlv) bovine calf serum. Over 90% of the yeast cells formed germ tubes in the culture at 37OC in the presence of serum and approximately 10% formed germ tubes at 37°C in the absence of serum. No hyphae were formed in the other two cultures. Equal amounts of total RNA isolated from each culture at (1) 20, (2) 45, (3) 70, and (4) 120 min were subjected to northern blotting and probed with the HYRI cDNA. In the bottom panel an overnight culture of the same strain of C. albicans grown in a defined medium (Buffo et al. 1982) at 25°C at pH 4.5 was used to inoculate four cultures grown at either 25 or 37°C at pH 4.5 or 6.5. Over 80% of the yeast cells formed hyphae in the culture at 37°C at pH 6.5, and approximately 30% formed germ tubes at 37°C at pH 4.5. Evagination was by budding only in the other cultures. Equal amounts of total RNA isolated from each culture at (1) 30, (2) 60, (3) 120, and (4) 180 min were subjected to northern analysis using the HYRI cDNA as a probe.

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25C -serum

L_ 25C +serum

was still observed in strain CA-2 which exhibits filamentous growth in vivo (De Bernardis et al. 1993) but is incapable of forming germ tubes (