The cell wall of the filamentous fungus Aspergillus niger Damveld, R.A.
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(2) The cell wall of the filamentous fungus $VSHUJLOOXVQLJHU 5REEHUW'DPYHOG.
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(4) The cell wall of the filamentous fungus $VSHUJLOOXVQLJHU. . 3URHIVFKULIW Ter verkrijging van de graad van Doctor aan de Universiteit Leiden, op gezag van de Rector Magnificus Dr. D.D. Breimer, hoogleraar in de faculteit der Wiskunde en Natuurwetenschappen en die der Geneeskunde, volgens besluit van het College voor Promoties te verdedigen op dinsdag 14 juni 2005 te klokke 16:15 uur . door . 5REEHUWXV$QWRQLXV'DPYHOG . geboren te Vlaardingen in 1978.
(5) Promotie commissie Promotor:. Prof. Dr. C.A.M.J.J. van den Hondel. Co-promotor:. Dr. A.F.J. Ram. Referent:. Prof. Dr. H.A.B. Wösten (Universiteit Utrecht). Overige leden:. Prof. Dr. P.J.J. Hooykaas Prof. Dr. E.J.J. Lugtenberg Prof. Dr. H.P. Spaink Prof. Dr. S. Brul (Universiteit van Amsterdam) Dr. F.M. Klis (Universiteit van Amsterdam) Dr. J. Rether (BASF, Germany) Dr. R. Offringa. Cover:. Pictures of putative cell wall mutants (P. Hock). Printed by:. Ridderprint, Ridderkerk, The Netherlands. ISBN:. XX-XXXXXXX-X.
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(8) &RQWHQWV &KDSWHU &KDSWHU. &KDSWHU &KDSWHU &KDSWHU &KDSWHU &KDSWHU 6XPPDU\ 6DPHQYDWWLQJ &XUULFXOXPYLWDH 3XEOLFDWLRQV . General introduction. . The cell wall stress response in $VSHUJLOOXV QLJHU involves increased expression of the glutamine:fructose-6-phosphate amidotransferase-encoding gene (JID$) and increased deposition of chitin in the cell wall. Expression of DJV$, one of five 1,3-D-D-glucan synthaseencoding genes in $VSHUJLOOXV QLJHU is induced in response to cell wall stress. The $VSHUJLOOXV QLJHU MADS-box transcription factor RlmA is required for cell wall remodeling in response to cell wall stress. Characterisation of CwpA, a putative glycosylphosphatidylinositol anchored cell wall mannoprotein in the filamentous fungus $VSHUJLOOXVQLJHU. A novel screening method for the identification of genes involved in cell wall biosynthesis in $VSHUJLOOXVQLJHU. A novel GFP-based reporter method for the identification of cell wall integrity disturbing antifungal compounds. . Page 9 33. 59. 87. 119. 147 171. . 191. . 195. . 200. . 201.
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(13) ,QWURGXFWLRQ About 70.000 fungal species have been described and it is estimated that about 1.5 million species may exist (Hawksworth, 1991, 1995). Within the fungal group there is a large diversity in habitat preference. Some fungal species feed on dead and decaying organisms (saprophitic fungi), while others are found inside or on a living host (biotropic). Also the interaction between the fungus and the environment can be very diverse, ranging from beneficial (symbiotic) to harmful (pathogen). In our research we used the eukaryotic. ascomycete $VSHUJLOOXV QLJHU as model organism. $ QLJHU is a filamentous fungus (hyphal. growth) and distributed worldwide (Abarca HW DO 2004). The fungus can grow on a large diversity of substrates and is considered as a common food spoilage fungus (Pitt and Hocking, 1997). Over the last decade 16 fungal genomes have been sequenced, including. the genome of $ QLJHU, and more then 50 are in progress (Bernal HW DO., 2001). Although. most fungal genome sequences were made freely available, the genome of $QLJHU has not been published yet.. $QLJHUDVDSURGXFWLRQKRVW. $QLJHU has been used to produce citric acid for 80 years and is currently the primary. source of commercial citric acid production (Magnuson and Lasure, 2003). The enormous. secretion capacity of this fungus led to the use of $QLJHU as general production organism for. various food enzymes such as glucose oxidase, pectinase, D-amylase, and glucoamylase.. The fungus has the Generally Recognised As Safe (GRAS) status from the United States Food and Drug Administration (FDA) allowing its use in (food) enzyme production (Bigelis and Lasure, 1987).. $QLJHUDVDQRSSRUWXQLVWLFIXQJDOSDWKRJHQ. $VSHULOOXV species are very common fungi in the human environment. Airborne spores. can enter the human body by inhalation and even in non-immunocompromised patients the fungus can cause infection of the lungs, sinuses and other sites. At least 20 species of. $VSHUJLOOXV have been reported to cause human disease, including $QLJHU (Denning, 1998).. $QLJHU has also been reported to cause an infection of the outer ear (otomycosis) in tropical. and subtropical regions (Kaur HW DO., 2000). The fungus produces many secondary metabolites. Only one secondary metabolite, ochratoxin A, which is a nephrotoxic mycotoxin. produced by 3-10% of the $ QLJHU strains (Abarca HW DO., 1994) is considered harmful.. However $QLJHU is generally regarded as benign (reviewed by Schuster HWDO., 2002).. 11.
(14) 7KHIXQJDOFHOOZDOO The shape of the fungus is determined by its cell wall. This component is essential to the fungus. By enclosing the cell, the cell wall protects the fungus from its environment and prevents it from lysing. The general view of the cell wall has changed through time from being a rigid structure, and able to withstand the turgor pressure, to a more dynamic one, being a structure that is able to adapt to various conditions (e.g. growth, development and stress). (Smits HWDO., 1999, Klis HWDO., 2002).. $UFKLWHFWXUHDQGFRPSRVLWLRQ The cell wall of most filamentous fungi and yeasts consists of three mayor components: chitin, glucans, and mannoproteins. The yeast cell wall composition and. architecture has been studied in most detail in 6DFFKDURP\FHVFHUHYLVLDH (Klis HWDO., 1998). The cell wall of this yeast is composed of: i) glucans (~ 60 % of the cell wall dry weight) which can be divided into the main polymer, E-1,3-glucan, which makes up ~ 55 % of the cell wall dry weight (~ 20 % alkali-soluble and ~ 35 % alkali-insoluble, chitin linked), and the second polymer, E-1,6-glucan that makes up ~ 5 % of the cell wall dry weight, ii) chitin, a E-1,4-linked homopolymer of N-acetylglucosamine residues, which is a minor component in the yeast cell wall accounting for only ~ 1–2 % of the cell wall dry mass, and iii) mannoproteins ( ~ 40 % of the cell wall dry weight). Also the cell wall architecture is well studied. The cell wall is a layered structure as shown by electron microscopy (Osumi, 1998). The inner, most electron dense layer consists mainly of E-1,3-glucan. The E-1,3-glucan is branched with E-1,6-linkages. at its branching points (Manners HWDO., 1973a and b). Both chitin chains, at the inner layer,. and E-1,6-glucosylated mannoproteins at the outer layer, are covalently linked to E-1,3-glucan. forming a supramolecular complex (Kollar HWDO., 1997). The chitin in 6FHUHYLVLDH is mainly found in bud scars and only a small amount of the total chitin content (2 %) is found in the. lateral walls (reviewed by Cabib HWDO., 1993).. The cell wall architecture of filamentous fungi, when observed under the electron microscope resembles the yeast cell wall architecture. It also has a layered structure. (Schoffelmeer HWDO., 1999). The inner electron dense layer is composed of chitin and glucan,. which are connected to the less electron dense layer composed of mannoproteins. The cell. wall composition of filamentous fungi (Fig. 1), including $ QLJHU, when compared to 6. FHUHYLVLDH is generally more chitin rich (10-15 %), and contains additional polymers like D-. 1,3-glucan or D-1,3-D-1,4 glucan polymers (10-35 % Fontaine HW DO., 2000, de Nobel HW DO.,. 2000a), components not found in 6 FHUHYLVLDH (Lipke and Ovalle, 1998) and &DQGLGD. DOELFDQV (Klis HWDO., 2001). The presence of the D-1,3-glucan polymer was reported in many fungal species (see for a complete list Grün, 2003). 12.
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(16) The cell wall surface of the fungal cell wall is covered with mannoproteins, determining the surface properties of the cell wall. These mannoproteins can be divided into different classes based on their linkage and extractability: i) SDS-extractable cell wall mannoproteins, which are bound to the cell via hydrogen bonds, ii) E-mercaptoethanol/DTT-extractable cell wall mannoproteins which are attached covalently to the cell wall via disulphide-bonds. (Cappellaro HWDO., 1994, 1998) or iii) cell wall mannoproteins that are covalently linked to the. glucan part (E-1,3- or E-1,6-glucan) of the cell wall. For 6FHUHYLVLDH two different classes of glucan-linked cell wall mannoproteins have been described, the protein with internal repeats (PIR)-class and the glycosylphosphatidylinositol (GPI) linked class. The first class of glucan-linked cell wall mannoproteins, consisting of members that. belong to the protein with internal repeats (PIR)-family (Toh-e HWDO., 1993) can be liberated. from the cell wall after mild-alkali treatment (Mrsa HW DO., 1997). The PIR proteins contain repeats that consist of a 19 amino acid sequence (core sequence: Q[IV][STGNH]DGQ[LIV]Q). and the number of repeats varies between the different PIR proteins (Toh-e HW DO., 1993).. Additionally, all PIR proteins contain an N-terminal signal peptide, a Kex2p protease cleavage. site, and a conserved cysteine motif (Klis HW DO., 2002). The Pir4p/Ccw5p contains only a. single PIR motif and removal of this motif results in the loss of covalent linkage to the cell wall, indicating that this sequence is required for the covalent linkage of Pir4p/Ccw5p to the. cell wall (Castillo HWDO., 2003). PIR proteins are most likely linked to the E-1,3-glucan part of the cell wall, but the exact way how PIR proteins are linked to the cell wall is still under. investigation (Mrsa and Tanner, 1999, Castillo HWDO., 2003).. The second class of glucan-linked cell wall mannoproteins are attached to the cell wall. through glycosylphosphatidylinositol (GPI) linkages (Lu HW DO., 1994, Montijn HW DO., 1994, Kapteyn HWDO., 1995, 1996). GPI-anchored proteins are found in all eukaryotes from fungi to. mammals. The general structure of the GPI anchor is well known, and the core structure is highly conserved. It consists of subsequently linked ethanolamine phosphate, trimannoside, glucosamine and inositol phospholipids (Ferguson and Williams, 1988 and Fig. 1). However some variation exists between different species (Ikezawa, 2002, Fontaine HWDO., 2003). The. GPI anchor synthesis takes place in the endoplasmic reticulum (ER) by a pathway consisting of ~ 10 reaction steps and in which ~ 20 proteins are involved (reviewed by Kinoshita and Inoue, 2000). The GPI-anchored cell wall proteins (GPI-CWPs) contain a hydrophobic sequence of 15-30 residues long, at their C-terminus that acts as a GPI-anchoring signal. GPI-anchor addition takes place in the ER where the hydrophobic domain is replaced by the. pre-assembled GPI-anchor (Orlean HW DO., 1997). After transport through the secretory pathway and arrival at the plasma membrane, the GPI-anchor is processed and attached to. 13.
(17) . !. Schematic representation of the fungal cell wall. The cell wall is composed of chitin, glucans. and mannoproteins. The proteins depicted represent: GPI-anchored plasma membrane proteins (1), GPIanchored cell wall proteins (2), and PIR proteins (3). An enlargement of the structure of a GPI-anchored plasma membrane protein (GPI-PMP) is shown. The boxed part within the enlargement is only found in GPI-PMPs. GPI-anchored cell wall proteins (GPI-CWPs) are processed in a still unknown way, removing the part only found in GPI-PMPs and linking them to the cell wall. Abbreviations used: plasma membrane (PM), glycoprotein (GlycP), ethanolamine (EtN), hydrofluoric acid (HF), pentomannose (Pman), glucosamine (GlcN), inositol (Ino), lipid moiety (LM), phosphate (P). Among fungi some variation has been reported, like the absence of chitin or 2003, and Grün, 2003). 14. ". -glucan. (Adapted from Ikezawa, 2002, Fontaine. . .,.
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(19) E-1,6-glucan (Montijn HWDO., 1994, Kollar HWDO., 1997). GPI-CWPs are further characterized by the presence of a hydrophobic N-terminal signal sequence for import into the ER, and are often heavily O-glycosylated. The GPI-CWPS can be removed from the cell wall by enzymatic and chemical treatments. Both E-1,3- and E-1,6-glucanases liberate GPI-CWPs. from the cell wall (Kapteyn HWDO., 1996). In addition, GPI-CWPs can be extracted from the cell wall by treatment with hydrofluoric acid (HF), which cleaves the phosphodiester bonds in the GPI-anchor (Kapteyn HWDO., 1996, de Groot HWDO., 2004).. . &HOOZDOOELRV\QWKHVLV Chitin synthesis is catalysed by synthases that transfer N-actetylglucosamine (GlcNAc). from. UDP-N-acetylglucosamine. (UDP-GlcNAc). to. the. newly. synthesized. polysaccharide. In 6FHUHYLVLDH three chitin synthases, encoded by &+6, &+6, and &+6,. have been identified (reviewed by Cabib HWDO., 2001). In filamentous fungi also multiple chitin. synthases were identified (up to seven in $VSHUJLOOXVIXPLJDWXV) and have been reported to be involved in lateral wall biosynthesis, septum synthesis, and spore formation (Bulawa, 1993, Munro and Gow, 2001, Reviewed by Roncero, 2002). The hexosamine biosynthetic pathway leading from fructose-6-phosphate to UDP-N-acetylglucosamine, the chitin precursor, consists of five steps and is conserved in lower and higher eukaryotes, as well as. in bacteria. The rate of UDP-N-acetylglucosamine synthesis, and thereby the chitin synthesis, is determined by Gfa1p, a glutamine:fructose-6-phosphate amidotransferase, involved in the. formation of glucosamine-6-P from glutamine and fructose-6-phosphate (Lagorce HWDO., 2002,. Terashima HWDO., 2000).. E-1,3-glucan synthesis is catalysed by the E-1,3-glucan synthase. In 6FHUHYLVLDH this. is performed by the subunit Fks1p and the alternate subunit Fks2p (reviewed by Douglas, 2001). Fks1p and Fks2p are large proteins with 16 putative membrane spanning domains. (Mazur HWDO., 1995). These plasma membrane localized enzymes are thought to synthesise. the E-1,3-glucan intracellular and facilitate the translocation of the newly synthesized E-1,3glucan into the extracellular space (Inoue HWDO., 1996). ,QYLWUR synthesis of the polymer was. achieved by adding UDP-glucose, GTP, glycerol and bovine serum albumin to the purified protein at pH 8.0. The polymer is present in the cell wall as 1,3-linked E-1,3-glucan with some. E-1,6 linked branches. Some studies on E-1,3-glucan synthases from filamentous fungi (e.g.. 1HXURVSRUD FUDVVD, $VSHUJLOOXV QLGXODQV, $VSHUJLOOXV IXPLJDWXV, &U\SWRFRFFXV QHRIRUPDQV,. 3DUDFRFFLGLRLGHVEUDVLOLHQVLV) have been performed (Hrmova and Seliternnikoff, 1989, Kelly. HW DO., 1996, Beauvais HW DO., 1993, Thompson HW DO., 1999, Pereira HW DO., 2000). Rho1p, a. small GTPase, was identified to be the key regulator of Fks1p activity (Yamochi HWDO., 1994,. 15.
(20) Drgonova HW DO., 1996, Arellano HW DO., 1996), which in its turn can be stimulated by the addition of GTP.. The genes involved in E-1,6-glucan synthesis have been characterized in 6. FHUHYLVLDH, based on the resistance of E-1,6-glucan muntants towards killer toxin (Roemer HW. DO., 1994, Shahinian and Bussey, 2000, Levinson HWDO., 2002). However, the genes encoding. proteins that could act as E-1,6-glucan synthases have not been identified yet. E-1,6-glucan is. found in the walls of many fungi as a E-1,6-glucan polymer with E-1,3 branches and. responsible for linking cell wall proteins to chitin and E-1,3-glucan (Kapteyn HWDO., 1999).. The D-1,3-glucan polymer is synthesized by D-1,3-glucan synthases. The presence of. this polymer has been reported for many fungi among which $VSHUJLOOXVQLGXODQV(Bull 1970,. Zonneveld 1971, Zonneveld 1972), $ QLJHU (Johnston, 1965, Horisberger HW DO., 1972), $. IXPLJDWXV (Fontaine HW DO, 2000), &U\SWRFRFFXV QHRIRUPDQV (Reese and Doering, 2003),. +LVWRSODVPD FDSVXODWXP (James HW DO., 1990), %ODVWRP\FHV GHUPDWLWLGLV (Hogan and Klein,. 1994) and 3DUDFRFFLGLRLGHV EUDVLOLHQVLV (Borges-Walmsley HW DO., 2002). In $ QLJHU two. different D-glucan polymers have been identified. One of them, nigeran, was isolated as a. hot-watersoluble, linear, alternating D-1,4-1,3-glucan polymer (Barker HW DO., 1953, 1957). A. second D-glucan polymer, pseudonigeran, was extracted from $ QLJHU cell wall by alkaline. extraction. The structure of pseudonigeran was identified as a linear D-1,3-glucan polymer. with some (3-10 %) D-1,4-linkages (Johnston, 1965, Horisberger HWDO., 1972). In $QLGXODQV,. D-1,3-glucan synthesis has been mainly studied in relation to cleistothecium formation.. Zonneveld (1972, 1974) has proposed that D-1,3-glucan accumulates during vegetative. growth and is metabolised by an D-1,3-glucanase expressed during sexual development.. Surprisingly, deletion of an D-1,3-glucanase that is specifically expressed during sexual. development in Hülle cells, did not affect the formation of cleistothecia (Wei HWDO., 2001). In. &QHRIRUPDQV D-1,3-glucan has been shown to be required for the anchoring of the capsule. to the cell wall (Reese and Doering, 2003). The genes encoding the D-1,3-glucan synthases. were first identified in 6SRPEH (Hochstenbach HWDO., 1998, Katayama HWDO., 1999) and are. also identified in $QLJHU (Chapter 3).. &HOOZDOOUHPRGHOLQJ. As mentioned above the cell wall is a highly dynamic structure (Klis HWDO., 2002). The. fungus is obligated to respond to changes in its environment by altering the composition and architecture of its cell wall. Failure to adapt will result in lysis and subsequent cell death. The. cell wall remodeling mechanism has been most extensively studied in the yeast 6FHUHYLVLDH. To maintain the integrity of the cell wall, the fungus activates a signal transduction cascade 16.
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(22) . $#!. The fungal cell wall integrity pathway (reviewed by Heinisch.
(23) . .,1999). In brief, the pathway. consists of plasma membrane localised sensor proteins (Wsc1-4p and Mid2), that mediate a signal through the Rho1p module and Pkc1p, resulting in the activation of the Slt2p MAPK signal transduction cascade. The MAPK Slt2p phosphorylates and thereby activates the Rlm1p transcription factor which upregulates the transcription of the Rlm1p target genes. Among these target genes are genes involved in cell wall biosynthesis. Abbreviations used: cell wall (CW), plasma membrane (PM), nuclear membrane (NM), and phosphorylation (P).. which results in the expression of genes able to alter the cell wall composition and architecture (Fig. 2.). This pathway is known as the Pkc1p, the Slt2/Mpk1p MAP kinase. signaling pathway or cell wall integrity pathway (reviewed by Banuett HWDO., 1998, Smits HWDO.,. 1999, Heinisch HW DO., 1999). Different environmental stimuli have been reported for 6. FHUHYLVLDH that activate the pathway: growth at elevated temperatures (Kamada HW DO., 1995),. hypo-osmotic shock conditions (Davenport HW DO., 1995, Kamada HW DO., 1995), the addition of. mating pheromones (Errede HW DO., 1995, Buehrer and Errede, 1997), the addition of agents. 17.
(24) that cause cell wall stress such as Calcofluor White, Congo Red, caspofungin or E-1,3glucanase (Ketela HW DO., 1999, de Nobel HW DO., 2000b, Reinoso-Martin HW DO., 2003, Garcia HW. DO., 2004), and actin depolymerisation agents (Harrison HW DO., 2001). The pathway is also. activated in mutants with impaired cell wall synthesis (Terashima HW DO, 2000, Lagorce HW DO., 2003) or in constitutively activated signaling mutants (Jung and Levin, 1999). Putative. sensors of the pathway are the transmembrane proteins Wsc1p-Wsc4p (Zu HW DO., 2001) and. Mid2p (Ono HW DO., 1994, Ketela HW DO., 1999, Green HW DO., 2003). The Wsc1p-Wsc4p proteins. interact through Tor2p with a guanine nucleotide exchange factor, Rom2p, to activate the. small GTPase Rho1p (Bickle HW DO., 1998, Sekiya-Kawasaki HW DO., 2002). Mid2p and Zeo1p also act as activators of Rho1p via a mechanism independent of Wsc1p and Rom2p (Sekiya-. Kawasaki HW DO., 2002, Green HW DO., 2003). One of the functions of Rho1p is the activation of. Pkc1p (Nonaka HW DO., 1995, Kamada HW DO., 1996). Pkc1p activates a linear MAPK-signaling. module consisting of the MAPKKK, Bck1p, (Costigan HW DO., 1992), the redundant pair of. MAPKK, Mkk1p and Mkk2p, (Irie HW DO., 1993) and the MAPK, Slt2p/Mpk1p, (Lee HW DO., 1993).. Activation of the PKC-pathway results in the phosphorylation of the threonine and tyrosine. residues in the TXY motif of Slt2/Mpk1p (Martin HW DO., 2000). One target of of Slt2p/Mpk1p is. the MADS-box transcription factor Rlm1p. Rlm1p was identified as a gene conferring. resistance to lethality of 0... % &('*). overexpression (Watanabe HW DO., 1995) and belongs to the. evolutionary conserved family of the MADS (Mcm1p-Agamous-Deficiens-Serum Response. Factor) box transcription factor proteins (Schwarz-Sommer HW DO., 1990).. Rlm1p is most closely related to the mammalian MADS-box MEF2 transcription. factors. The protein shows similar DNA-binding specificity LQ YLWUR CTA(T/A)4TAG (Dodou and Treisman, 1997). The transcriptional activation potency of Rlm1p is regulated through. phosphorylation by the protein kinase Mpk1p (Watanabe HW DO., 1997, Jung HW DO., 2002). In 6. FHUHYLVLDH, Rlm1p is localised in the nucleus irrespective of its activation or phosphorylation. status (Jung HW DO., 2002). Rlm1p and its binding sites have been shown to be required for the activation of genes involved in cell wall remodeling in response to cell wall stress (Jung and. Levin, 1999, Terashima HW DO., 2000). Indeed, genome wide expression analysis of the. response to different forms of cell wall stress in 6 FHUHYLVLDH has further indicated an. important role of Rlm1p in mediating the activation mechanism because of the presence of. putative Rlm1p binding sites in their promoters (Roberts HW DO., 2000, Lagorce HW DO., 2003,. Reinoso-Martin HW DO., 2003, Garcia HW DO., 2004, Boorsma HW DO., 2004). From these studies, it is also evident that the Pkc1p-Slt2p dependent pathway is not the only signaling pathway that. contributes to cell wall remodeling in yeast. Both the calcium/calcineurin pathway (Zhao HW DO.,. 1998, Yoshimoto HW DO., 2002) and the Hog1p-MAPK (reviewed by Hohmann, 2002) signaling. pathways are involved in the activation of genes implicated in maintaining cell wall integrity. 18.
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(26) In addition, the Heat shock transcription factor Hsf1p, which binds to the Heat Shock Element. HSE (Sorger, 1991, Fernandes HW DO., 1994) and Swi4p, a PKC1-regulated transcription factor, involved in G1/S specific gene expression was also reported to have an effect on the. up-regulation of genes after cell wall stress (Igual HW DO., 1996, Madden HW DO., 1997).. Little is known about the mechanism of cell wall remodeling in filamentous fungi. Compensatory reactions in response to cell wall stress have also been observed in. filamentous fungi (Gooday and Schofield, 1995, Sela-Buurlage, 1996, Kurtz HW DO., 1994,. Wang HWDO., 2002, Mellado HWDO., 2003). The compensatory mechanism has become evident. from morphological studies where cell wall becomes thicker and has an altered composition. as observed by electron microscopy after inhibition of E-1,3-glucan synthesis activity (Kurtz HW. DO., 1994). When microconidia of the filamentous fungus )XVDULXP VRODQL were allowed to germinate in the presence of sublethal concentrations of cell wall degrading enzymes. adaptation occurs since these germlings had become resistant to concentrations which for. non-challenged spores were lethal (Sela-Buurlage, 1996). Mellado HW DO. provide evidence. that interfering with chitin synthesis in $ IXPLJDWXV by deleting two chitin synthesis genes. FKV* and FKV(, results in aberrant cell morphology and increased levels of 1,3-D-D-glucan in. the cell wall of those mutants (Mellado HWDO., 2003).. The recent sequencing of many fungal genomes also revealed the presence of homologs of the cell wall integrity pathway in filamentous fungi (Table 1), however whether they perform a similar function requires further investigation. Only a small number of fungal. homologs have been isolated and studied in more detail: RhoA $ QLGXODQV (Guest HW DO.,. 2004), pkc1 7ULFKRGHUPDUHHVL (Morawtz HWDO., 1996), pkcA $QLJHU (Morawtz HWDO., 1996),. PCBCK1 3QHXPRF\VWLVFDUQLL (Vohra HWDO., 2004), MPKA $QLGXODQV (Bussink and Osmani,. 1999), Mkp1 3QHXPRF\VWLVFDUQLL (Fox and Smulian, 1999), Mpk1 &U\SWRFRFFXVQHRIRUPDQV. (Kraus HW DO., 2003), Mps1 1 FUDVVD (Xu HW DO., 1998, 2000). The presence of these highly homologous proteins in filamentous fungi suggests the existence of a similar cell wall integrity pathway in filamentous fungi.. +-,-./ $ !. Homologs of the cell wall integrity pathway 6from 4 01 filamentous fungi. The annotated genomes of 51 ,. 23 4 found in the genomes of 167 , 81 9 4946 , and :;1< 46 were 19.
(27) used.. 01
(28) 23 4 . (annotated) protein name. protein (size bp) Wsc1p (388) Wsc2pa (378). Wsc3p (556). Wsc4p (605) Mid2p (376). 20. 280 465 573 1017. gb|EAA62753.1| gb|EAA73352.1| gb|EAA28544.1| gb|EAA55414.1|. AN5660.2 NCU04170.1. 280 2076. gb|EAA62753.1| gb|EAA31492.1|. FG10435.1 NCU09267.1. 1763 1105. gb|EAA68209.1| gb|EAA35466.1|. AN4674.2. 304. gb|EAA60716.1|. MG01466.4 FG05656.1 NCU06981.1. 356 375 404. gb|EAA55815.1| gb|EAA75227.1| gb|EAA33385.1|. = >3?A@ B9C3D E3?9F G >3HJI9E K >AL*M E3F(FJE NO>3P9M @ FJI9E = >3?A@ B9C3D E3?9F K >AL*M E3F(FJE G >3HJI9E K >AL*M E3F(FJE = >3?A@ B9C3D E3?9F NO>3P9M @ FJI9E G >3HJI9E K >AL*M E3F(FJE. -. -. -. 2371. gb|EAA57731.1|. 2423 2509 ~2400. gb|EAA71932.1| gb|EAA31334.1| NA. MG03064.4 FG08572.1. 1281 1235. gb|EAA47821.1| gb|EAA71433.1|. AN4719.2 NCU00668.1. 1199 1251. gb|EAA60761.1| gb|EAA36572.1|. Rho1p (209). Rho1p(1) MG07176.4 FG04400.1 NCU08683.1. 193 193 195 200. gb|EAA62833.1| gb|EAA56821.1| gb|EAA72781.1| gb|EAA32796.1|. Pkc1p (1151). AN0106.2 FG09660.1 NCU06544.1 MG08689.4. 1083 1176 1142 1182. gb|EAA65284.1| gb|EAA75979.1| gb|EAA33015.1| gb|EAA51167.1|. FG06326.1. 1870. gb|EAA74943.1|. NCU02234.1 MG00883.4. 1786 1533. gb|EAA30411.1| gb|EAA49225.1|. AN4887.2. 1533. gb|EAA60965.1|. Mkk1p (508). NCU06419.1 MG06482.4 FG07295.1 AN4189.2. 587 515 524 502. gb|EAA28074.1| gb|EAA56511.1| gb|EAA77528.1| gb|EAA59288.1|. (2). Mpk1 (484). MpkA FG10313.1 MG04943.4 Mps1(3). 418 416 415 454. gb|AAD24428.1| gb|EAA70011.1| gb|EAA52251.1| gb|AAC63682.1|. Rlm1p (676). FG09339.1 AN2984.2 NCU02558.1 -. 681 605 625 -. gb|EAA76082.1| gb|EAA63555.1| gb|EAA36453.1| -. Bck1p (1478). organism. AN5660.2 FG03884.1 NCU00309.1 MG09221.4. FG08133.1 NCU05608.1 contig 2.1040+41. Rom2p (1356). Both Wsc1p and Wsc2p gave similar results. Rho1p, Guest and Momany, 2004. (1). accesion number. AN5982.2 Tor2p (2474). a. size (bp). = >3?A@ B9C3D E3?9F G >3HJI9E K >AL*M E3F(FJE NO>3P9M @ FJI9E NO>3P9M @ FJI9E G >3HJI9E = >3?A@ B9C3D E3?9F K >AL*M E3F(FJE = >3?A@ B9C3D E3?9F NO>3P9M @ FJI9E G >3HJI9E K >AL*M E3F(FJE = >3?A@ B9C3D E3?9F G >3HJI9E K >AL*M E3F(FJE NO>3P9M @ FJI9E G >3HJI9E K >AL*M E3F(FJE NO>3P9M @ FJI9E = >3?A@ B9C3D E3?9F K >AL*M E3F(FJE NO>3P9M @ FJI9E G >3HJI9E = >3?A@ B9C3D E3?9F = >3?A@ B9C3D E3?9F G >3HJI9E NO>3P9M @ FJI9E K >AL*M E3F(FJE G >3HJI9E = >3?A@ B9C3D E3?9F K >AL*M E3F(FJE NO>3P9M @ FJI9E. Score (bits). E-value. 70 54 48 47. 4.00E-12 3.00E-07 2.00E-05 5.00E-05. 70 51. 8.00E-12 3.00E-06. 50 49. 5.00E-06 1.00E-05. 75. 2.00E-13. 67 62 52. 7.00E-11 2.00E-09 1.00E-06. -. -. 2128. 0.0. 2102 2004 1888. 0.0 0.0 0.0. 501 487. e-141 e-137. 486 483. e-137 e-136. 297 290 288 233. 4.00E-81 5.00E-79 2.00E-78 1.00E-61. 479 462 453 451. e-135 e-130 e-127 e-126. 323. 1.00E-87. 322 319. 3.00E-87 1.00E-86. 293. 1.00E-78. 335 333 328 325. 7.00E-92 2.00E-91 8.00E-90 5.00E-89. 527 506 506 478. e-148 e-143 e-143 e-135. 105 105 104 -. 1.00E-22 2.00E-22 4.00E-22 -.
(29)
(30) (2). MpkA, Bussink and Osmani, 1999 Mps1, Xu ., 1999 Abbreviations used: not available (NA). (3). $LPRIWKHWKHVLV The fungal cell wall is an intriguing component of the cell. It provides the cell with the necessary support to prevent lysis, and it protects the cell from the harsh environment. Being an essential component of the cell, the cell wall is considered as an interesting target to. prevent fungal growth. It has been shown that the yeast 6FHUHYLVLDH is able to remodel its. cell wall architecture and composition in response to cell wall disturbing conditions in order to withstand cell wall threatening conditions. This remodeling mechanism has only been studied. in great detail in the yeast 6FHUHYLVLDHand knowledge about possible cell wall remodeling mechanisms in filamentous fungi is lacking. The goals of this thesis are (i) to provide evidence of for the existence of a cell wall remodeling mechanism in filamentous fungi and in. particular $ QLJHU (ii) to identify components of the signal transduction by which cell wall weakening is sensed and transduced into a transcriptional response and (iii) to develop cell. wall stress reporter systems to identify new cell wall related antifungal targets and to identify cell wall related antifungal compounds. Chapter 1 is a general introduction on the fungal cell wall. Both the composition of the fungal cell wall and the synthesis of the cell wall components are briefly discussed. The cell. wall integrity pathway in 6FHUHYLVLDH is discussed in detail, and evidence for the existence of a cell wall integrity pathway in filamentous fungi is presented. The recently sequenced genomes of several filamentous fungi were used to provide further evidence for the existence of a cell wall integrity pathway. Chapter 2 provides evidence for the presence of a remodeling mechanism in the. filamentous fungus $ QLJHU. An increased chitin content in the cell wall is observed in response to cell wall stress. An essential protein which determines the rate of chitin synthesis, named GfaA, is characterized in more detail. In chapter 3, a family of five D-glucan synthases is studied. The expression of the. genes encoding the members of this family, were monitored in response to cell wall stress.. The expression of the genes encoding the two members DJV$ and DJV( are induced in. response to cell wall stress indicating that increased levels of D-1,3-glucan is part of the the cell wall remodeling mechanism.. The output of the cell wall integrity pathway is mediated via a transcription factor,. named RlmA. The LQ YLYR binding-site of RlmA is studied together with the function of the protein in chapter 4. We show that the transcription factor is required for proper functioning of the remodeling mechanism. 21.
(31) Cell wall proteins have been shown to have diverse functions. However, the function of most cell wall proteins remains unknown. Most of them are thought to have a structural role and provide the cell wall protection and determine its surface properties. In chapter 5, a putative GPI-anchored cell wall protein CwpA is studied. Biochemical evidence is provided which shows that CwpA is linked to the cell wall via its GPI-anchor. In chapter 6 a new method for the isolation of cell wall mutants is described. The. screen was set up to select for mutants with an induced DJV$ expression. Since the induction. of DJV$ is proposed to be correlated with impaired cell wall integrity, it is expected to find. mutants with a weakened cell wall. Secondary screens were used to select mutants most suitable for complementation. This method could provide new anti-fungal cell wall targets. Chapter 7 describes the development of a Green Fluorescent Protein based cell wall stress reporter system. The method has been evaluated towards different known and unknown antifungal compounds and is a promising tool for the identification of new cell wall related antifungal compounds.. . 5HIHUHQFH/LVW. QR.
(32) ,- TS,
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(265) Umkm! . (1996). Activation of 1 q 1 #
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(300) _{mZZ U m! (1999). 0 9 qsr 64 6
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(330) _[O,-. .-Uj! (1997). Architecture of the yeast cell wall. Beta(1-->6)-glucan interconnects 1 q 1 ## mannoprotein, beta(1-->)3-glucan, and chitin. x1v , 17762-17775. m ,-Z-U-n\! O! U-]
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