Regulation of Expression, Activity and Localization of Fungal Chitin Synthases

Medical Mycology January 2012, 50, 2–17

Review Articles Regulation of expression, activity and localization of fungal chitin synthases

LUISE E. ROGG* , JARROD R. FORTWENDEL * † , PRAVEEN R. JUVVADI * & WILLIAM J. STEINBACH* † * Department of Pediatrics, Division of Pediatric Infectious Diseases, Duke University Medical Center, Durham NC, and †Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham NC, USA Downloaded from https://academic.oup.com/mmy/article/50/1/2/989229 by guest on 25 September 2021

The fungal cell wall represents an attractive target for pharmacologic inhibition, as many of the components are fungal-specifi c. Though targeted inhibition of β -glucan synthesis is effective treatment for certain fungal infections, the ability of the cell wall to dynami- cally compensate via the cell wall integrity pathway may limit overall effi cacy. To date, chitin synthesis inhibitors have not been successfully deployed in the clinical setting. Fungal chitin synthesis is a complex and highly regulated process. Regulation of chitin synthesis occurs on multiple levels, thus targeting of these regulatory pathways may represent an exciting alternative approach. A variety of signaling pathways have been implicated in chitin synthase regulation, at both transcriptional and post-transcriptional levels. Recent research suggests that localization of chitin synthases likely represents a major regulatory mechanism. However, much of the regulatory machinery is not necessarily shared among different chitin synthases. Thus, an in-depth understanding of the precise roles of each protein in cell wall maintenance and repair will be essential to identifying the most likely therapeutic targets. Keywords Fungi , cell wall , plasma membrane , secretion , synthesis , chitosome

Introduction Chitin is a linear homopolymer of β-1,4-linked N-acetylglucosamine (Glc-NAc). Chitin fi brils occur in The fungal cell wall is a complex cross-linked network of different conformations, both as long thin microfi brils and chitin, glucans, other polysaccharides as well as integral as short thick rodlets, suggesting that specifi c forms of proteins. The central core consists of glucans cross-linked chitin may be important in different structural roles [12]. to chitin, with various decorating polysaccharides depend- Chitin can also be deacetylated to chitosan, a more fl exible ing on the species (reviewed in [1]). Many of the compo- and soluble polymer, important in Saccharomyces cerevi- nents of the cell wall are fungal-specifi c, thus inhibition of siae ascospores and in the cell wall of Cryptococcus neo- these components represents a logical target for antifungal formans [13,14]. In addition, the pattern of cross-linking to agents. This approach has been validated by the echinocan- other polysaccharides varies depending on the type of chi- din class of antifungals that target β -1,3-glucan synthesis tin microfi bril as well as its subcellular location, consistent [2 – 6]. However, the fungal cell wall is a dynamic and with a highly organized and regulated cell wall structure developmentally plastic construction, capable of compen- [12,15]. Chitin biosynthesis is limited to fungi and insects, sating for loss of β-1,3-glucan by increased chitin deposi- thus chitin biosynthesis represents an as yet unexploited tion [7 – 11]. target for therapeutic intervention in fungal disease affecting humans. A number of chitin synthesis inhibitors have Received 21 December 2010; Received in fi nal revised form 4 March been identifi ed, the best characterized of which are the nik- 2011; Accepted 29 March 2011 komycins. Nikkomycins are natural products of Streptomy- Correspondence: William J. Steinbach, Department of Pediatrics, Divi- sion of Pediatric Infectious Diseases, Duke University Medical Center, ces tendae that are competitive inhibitors of chitin synthase Durham, NC 27710, USA. Tel.: ϩ 1 (919) 681 1504; Fax: ϩ 1 (919) 668 enzymes (reviewed in [16]). Enzyme kinetic studies in the 4859; E-mail: [email protected] genetically tractable yeasts have showed wide discrepancy © 2012 ISHAM DOI: 10.3109/13693786.2011.577104 Chitin synthesis regulation 3 in activity of the inhibitor against the various enzymes. In chitin synthases are non-viable except in the setting of both S. cerevisiae and Candida albicans, nikkomycin Z is acquisition of suppressor mutations, affi rming the essential much less active against the chitin synthases that fi ll near- nature of chitin in fungi [37,38]. essential roles in yeast cell wall biosynthesis [17– 19]. The chitin synthases are integral plasma membrane pro- Studies of nikkomycin Z treatment of murine models of teins that catalyze polymerization of UDP-Glc-NAc into candidiasis, histoplasmosis, blastomycosis, and aspergil- hydrophobic chitin chains that are then extruded through losis have discordant results. Nikkomycin Z monotherapy the cell membrane and incorporated into the cell wall of dimorphic infections showed improved survival and [39,40]. Chitin synthase (CHS) enzymes are encoded by some microbiologic cures [20 – 22]. In contrast, mice with members of a large gene family (Fig. 1A), suggesting the candidiasis did have better survival while on treatment, but possibility of both functional specialization as well as all relapsed off of therapy, suggesting that nikkomycin Z redundancy. Different chitin synthases produce chitin that

may not be fungicidal in Candida [23]. In a model of inva- is localized to specifi c cell wall derived structures or devel- Downloaded from https://academic.oup.com/mmy/article/50/1/2/989229 by guest on 25 September 2021 sive aspergillosis, nikkomycin Z was not effective as opmental stages [12]. Phylogenetic analysis demonstrates monotherapy [24]. However, it did appear to show some the existence of seven classes of chitin synthases divided synergy with other agents, particularly echinocandins, rais- into two families (Fig. 1A). Class I, II and IV genes are ing the possibility of improved treatment with simultane- present in all fungi, whereas classes III, V, VI and VII are ous targeting of two cell wall components [24 – 27]. This specifi c to fi lamentous fungi and certain dimorphic species result is supported by in vitro data that demonstrates mark- (Fig. 1B). The number of putative chitin synthase genes edly enhanced cell wall damage to fungal cultures grown within each species varies, with three in S. cerevisiae , four in the presence of both chitin and glucan inhibitors as com- in C. albicans , seven in Wangiella dermatitidis and Neuro- pared to cultures grown with one or the other [28,29]. Chi- spora crassa and eight each in Aspergillus nidulans , A. tin synthase inhibitors have not been employed clinically, fumigatus and C. neoformans (Table 1). though there has been a recent resurgence of interest in Homology-based prediction of specifi c chitin synthase these agents, particularly for dimorphic fungal infections. function is highly imperfect, as individual classes do not In fact, the fi rst human phase I pharmacokinetic study of necessarily have the same function in different species. To nikkomycin Z was recently published and there are plans date, most research has employed classical genetic tech- for a phase II effi cacy trial of treatment of coccidioidal niques, primarily examining the phenotype of single and pneumonia [30,31]. multiple gene disruptions. Extension of mutant phenotypes In this review, we have focused on the regulation of the into functional descriptions is complicated by the existence chitin synthases, particularly in model species or medically of multiple enzymes with possible functional redundancy. relevant fungal pathogens, though signifi cant contributions In addition, extrapolation from yeast to fi lamentous fungi to our understanding of chitin synthesis regulation have is necessarily limited by the widely differing nature of their also been drawn from research of agricultural fungal patho- cell walls and life cycles. gens. The role of chitin in cell wall structure and pathoge- Class I CHS genes were the fi rst to be described, as they nicity has been recently reviewed [1,32]. are highly active in chitin synthase activity assays in vitro , though their actual contribution to chitin synthesis is rela- The role of individual chitin synthases cannot tively small [41,42]. Functionally, S. cerevisiae Chs1p acts as a repair enzyme at the site of cytokinesis, counterbalanc- be predicted based on homology ing the activity of chitinase in mediating separation, thereby Chitin is an important structural component in the septa maintaining cell wall integrity [43,44]. There are two class and cell walls of fungi, but depending on the fungal spe- I chitin synthase genes in C. albicans , CHS2 and CHS8 . cies, chitin can compose from a small minority up to nearly Both Chs2p and Chs8p appear to have modest effects on half of the cell wall dry weight [33]. In yeast, chitin is a overall growth and cell wall chitin content, and unlike S. minor cell wall component, totaling only 1 – 2% of the dry cerevisiae, no bud lysis defect has been elicited even when weight [34] and primarily found as a minor component in both genes are disrupted [42,45,46]. Mutation of the class the lateral walls as well as concentrated in the bud neck, I genes in A. nidulans , A. fumigatus and C. neoformans do bud scar and septa. The situation in fi lamentous fungi is not yield any obvious phenotypes, although mutation of different, as chitin tends to be a much larger overall cell CHS2 of W. dermatitidis results in decreased chitin syn- wall component (up to 40% or more of the dry weight) thase activity [47 – 50]. [35,36] and is more diffusely distributed in the cell wall Class II genes also tend to make a relatively small con- with increased deposition at the hyphal tips and septa. In tribution to total cellular chitin, but functionally are quite S. cerevisiae , in which chitin is a minor cell wall compo- important in S. cerevisiae and C. albicans , but not A. nid- nent, strains containing simultaneous disruption of all three ulans or N. crassa [47,51 – 53]. S. cerevisiae Chs2p acts in

Fig. 1 Chitin synthase gene functional domains. (A) Phylogenetic analysis of the chitin synthases of Aspergillus fumigatus , Aspergillus nidulans , Candida albicans , Cryptococcus neoformans , Neurospora crassa , Saccharomyces cerevisiae and Wangiella dermatitidis demonstrates seven classes of chitin synthases, divided into two families. Yeasts contain enzymes from classes I, II and IV, whereas fi lamentous fungi tend to contain at least one enzyme from each class. (B) The proteins encoded for by the class I, II and III genes tend to be smaller, and contain an amino terminal catalytic domain and multiple carboxytransmembrane domains. The proteins encoded by class IV, V, VI and VII genes tend to be larger and the catalytic domain tends to be more towards the carboxy terminal. The proteins encoded by class V and VII genes usually contain an amino terminal domain with some homology to myosin motor domains and a carboxy terminal with chitin synthase catalytic residues.

© 2012 ISHAM, Medical Mycology, 50, 2–17 Chitin synthesis regulation 5 1,83] [56 – 58, 73,145,147] [14,62,63,101, 104,106] Downloaded from https://academic.oup.com/mmy/article/50/1/2/989229 by guest on 25 September 2021 asexual structures asexual neck, plasma membrane with stress Mother-bud neck, septa, small cytoplasmic patches neck, septa, small cytoplasmic Mother-bud [12,45,64,65,83] deletions ND [49] deletion ND [49] chs chs3 deletions, cell wall stress deletions, cell wall patches, hyphal tip Bud-neck, septa, cytoplasmic [12,46,83] chs deletions, cell wall stress deletions, cell wall neck, vacuoles Mother-bud [12,45,52,81,83,154] deletions, cell wall stressors, hyphal deletions, cell wall induction ND Hyphal tips, forming septa, binds actin [78,138] chs ND ND [77,142] ND ND [75,141,142] chs Vegetative growth and septum formation growth Vegetative bud site, small and large bud vesicles, Endocytic Highest ND [49] brils Certain other conidiation conidiation, less virulent normal virulence virulence defective spore maturation defective stress sensitivity, decreased stress sensitivity, chitin VII ND ND ND [143] II Lethal I decreased chitin growth, Slower stress, hyphal induction Cell wall neck, peripheral patches, vacuoles Mother-bud [12,42,45,65, 8 I Bud lysis defect cycle Increased during sexual ND [17,37,41,43,44,54,90] IV Decreased rodlet chitin, decreased I IV None No long chain microfi Lower ND [49] II Aseptate cycle Decreased during sexual Bud neck during ring contraction [37,51,54,101, 102,117] III None Highest ND [49] IV chitin, Decreased cell wall VII None Lower ND [49] IV defects, lethal, growth Temperature V sensitivity Temperature Highest, certain other I,II sensitivity Temperature low Very ND [49] III sensitivity Temperature with Absent except I,IIsensitivity Temperature Highest ND [49] V VII Poor conidiation, abnormal hyphae poor Impaired hyphal growth, Changes in osmolarity Hyphal tips, septation sites, binds actin149,152,153] [69,74,78,136, VII ND ND ND [129] VI ND ND Hyphalapex,septa [97] V ND ND ND [129] IV None ND ND [70] I ND ND Hyphalapex,septa [97] II None ND ND [53] III Abnormal hyphae ND septa developing Hyphal apex, [61,98] IV Normal chitin or decreased Hyphae and conidiophores ND150] – [57,68,69,74,148 VI ND ND ND [151] III and Hyphal defects, poor growth VIchitin Decreased ND ND [77,141] I None Hyphae and conidiophores Hyphal tips, septation sites149] – [47,57,68,132,145 III No phenotype Increased with echinocandin ND [48,89,141] III ND ND ND [151] III defect growth Severe expressed Constitutively sites, Germ tubes, hyphal tips, septa, polarized growth II Decreased conidiation development Asexual Septation sites149] – [68,73,132, 133,144 IVphenotype No ND ND [141] IIphenotype No ND ND [48,141] V Decreased chitin, hyphal defects, I No phenotype Increased with echinocandin ND [48,89,141] chsF chsB chsA chsF chsB chsE chsA chsC chsC chsD chsG chsG chsD chs-7 chs-6 chs-5 chs-4 chs-3 chs-2 chs-1 csmA csmB CHS1 CHS2 CHS1 CHS3 CHS8 CHS1 CHS2 CHS2 CHS3 CHS4 CHS3 CHS5 CHS6 CHS7 CHS8 Characteristics of chitin synthase genes among model and pathogenic yeast and molds. Characteristics of chitin synthase genes among model and pathogenic yeast molds. chsEb

An An An An Ca An An An An Ca Af Af Af Af Af Af Af Table Table 1 Species Gene Af Class Mutant phenotype changes Transcriptional Protein localization References Sc Ca Ca Cn Nc Sc Cn Nc Sc Cn Cn Nc Cn Cn Nc Cn Nc Cn Nc Nc

construction of the primary septum after cytokinesis

; ND, [37,54]. C. albicans Chs1p is essential, owing to its role in producing both septal and lateral wall chitin [52]. The W. dermatitidis class II chs1Δ strain is unable to transition between growth types and yeast cells fail to divide normally, though rather than absent septa as seen in the yeast mutants, there is a broader neck with enriched chitin con- [76,84,137,156]

Wangiella dermatitidis Wangiella tent at the septa [55]. , Class III genes appear to be relatively important for Wd ; normal growth and morphology in Aspergilli. Both A. nidulans and A. fumigatus each contain two class III genes.

A. nidulans ChsB appears to play a role in synthesis of Downloaded from https://academic.oup.com/mmy/article/50/1/2/989229 by guest on 25 September 2021 chitin in the hyphal tips and conidia, owing to marked defects in hyphal morphology and poor conidiation when mutated [56 – 58]. Deletion of A. fumigatus chsG alone or both class III genes ( chsC and chsG) produces similar but Saccharomyces cerevisiae Saccharomyces , less marked defects, suggesting that the functions may be Sc ; related to those proposed in A. nidulans [59]. Disruption of the homologous genes in W. dermatitidis yields decreased chitin synthase activity but no apparent growth defects and the C. neoformans mutant has no phenotypes [49,60]. Mutation of N. crassa chs-1 alters hyphal morphology and dependent

Neurospora crassa Neurospora decreases overall activity [61]. ND [71,84] , Class IV chitin synthases are the bulk chitin synthases Nc ; in yeasts. The C. albicans and S. cerevisiae class IV genes

chs encode proteins that produce the majority of the total cell wall chitin, but loss of these genes does not produce equally dramatic phenotypes. S. cerevisiae CHS3 (also called CAL1 , CSD2 and DIT101 ) encodes the chitin synthase responsible for the majority of overall chitin content [14,62,63], similar to the role of C. albicans CHS3 [45,64 – Cryptococcus neoformans , 66]. Yeast class IV genes appear to function in the cell wall Cn deletions ND [55,84] ; compensatory response to cell wall stressors, as double

chs mutants with S. cerevisiae GAS1 , a β -1,3-glucanosyltrans- deletions ND [50,55,84] deletions ND [50,60,84,155] deletions ferase that modifi es cell wall cross-linking, and the Chs3p Other chs chs increases with stress, Constitutive, stressCell wall actin- hyphal tips, sites of expansion, buds, Yeast regulators CHS4 , CHS5 , CHS6 and CHS7 result in osmotic support-dependent viability [67]. Candida albicans

, However, the generalization of the class IV chitin syn-

Ca thases as bulk synthases cannot be extended to other fungi ; as the mutant phenotypes of the corresponding genes in fi lamentous fungi are not similar to the yeasts or even to each other. In A. nidulans , loss of chsD results in no defects in cell growth or morphology and no decrease or a modest decrease in chitin content [68,69] and in N. RIP Aspergillus nidulans Aspergillus mother-bud neck mother-bud clumping virulent , crassa , the chs-4 strain does not have any phenotypes

An [70]. Slightly more similar to the yeasts, the W. dermati- ; tidis chs4Δ strain contains decreased chitin [71]. In contrast, the C. neoformans chs3Δ strain is slow-growing and II I to separate, broader Failure phenotype No IIIphenotype No VI ND ND ND [137] IV Reduced chitin, abnormal yeast V defects, less growth Suppressive more sensitive to cell wall damage as well as producing enlarged abnormally shaped cells with increased chitin CHS1 CHS2 CHS3 CHS6 CHS4 CHS5 and reduced chitosan content [49]. The ability of this

mutant strain cell wall to retain melanin was reduced, sug- Aspergillus fumigatus Aspergillus , Wd Wd Wd Wd not done. Wd Wd Af gesting that melanin may associate with chitin microfi brils

© 2012 ISHAM, Medical Mycology, 50, 2–17 Chitin synthesis regulation 7 synthesized by specifi c chitin synthases [49], which has stages argues that chitin biosynthesis may not just serve as also been seen in C. albicans [72]. general cell wall structural support, but may also be closely Disruption of class V genes in A. nidulans , A. fumigatus regulated in ways that allow specifi c remodeling of the cell and W. dermatitidis produces fairly dramatic changes in wall as needed throughout growth. Similar studies have not growth and morphology, but the same is not seen in C. been done in fi lamentous fungi, as the vastly different neoformans [49,69,73– 76]. For instance, A. nidulans CsmA development program of fi lamentous fungi renders cell functions in hyphal growth as well as septal formation as wall development less tied to the cell cycle. evidenced by the mutant phenotypes and strains defi cient in A. fumigatus chsE have similar but less severe defects in hyphal growth [69,73 – 75]. The W. dermatitidis chs5 Δ Transcriptional compensation occurs strain has defective growth and morphology and even with disruption of chitin synthase genes, suggesting functional redundancy between ceases to be viable when grown without osmotic support Downloaded from https://academic.oup.com/mmy/article/50/1/2/989229 by guest on 25 September 2021 at elevated temperatures, with an accompanying decrease different chitin synthase proteins in virulence [76]. The large number of chitin synthase genes, particularly in Among class VI genes, disruption of A. fumigatus chsD fi lamentous fungi, raises the question of why so many results in moderately decreased total chitin content without genes are present: it may refl ect functional redundancy. In change in chitin synthase activity or accompanying defects both yeast and fi lamentous fungi, compensatory increases in growth or morphology [77]. in transcription have been found in response to disruption Loss of the class VII gene A. nidulans csmB results in of chitin synthase genes. Transcription of C. albicans the formation of ballooned and intrahyphal hyphae and CHS1 increases with disruption of nearly every other chitin increased tendency towards of lysis of subapical regions synthase, consistent with its functional importance, and [78]. A. nidulans Δ csmA Δ csmB is synthetically lethal sug- CHS2 is not upregulated in any of the mutants [83]. In W. gesting that the myosin motor domain-containing chitin dermatitidis , all other single gene deletions result in synthases play an important role in chitin synthesis in fi la- increased CHS5 transcription, though chs5 Δ does not cause mentous fungi species [78]. any changes in transcription of the other chitin synthase It is clear from the chitin synthase gene disruption stud- genes [84]. Thus, compensatory transcriptional increases ies that prediction of the function of individual genes is not of chitin synthase genes occurs in fungal strains carrying reliable based on homology to other genes of the same one chitin synthase gene disruptions, but the pattern of class from other fungal species. Importance of chitin syn- upregulation is mutation-dependent. thases may thus lie with their highly specialized roles in very specifi c aspects of fungal growth and development. Disruption of the cell wall results in increased Cell cycle and developmental stage alter chitin synthase gene transcription, mediated yeast chitin synthase transcription via several different signaling pathways In yeast, chitin synthase gene transcription changes The cell wall integrity (CWI) pathways encompass a num- throughout the cell cycle as well as during different devel- ber of compensatory cell wall changes that occur in opmental stages. Transcription of S. cerevisiae CHS1 peaks response to treatment with cell wall perturbing agents or in M/G1, CHS2 in M-phase and CHS3 after septum forma- mutations in cell wall synthetic or regulatory genes. Muta- tion and before division, which is consistent with the role tions in various cell wall genes result in global changes in of Chs1p in repair of the bud scar after cytokinesis and S. cerevisiae gene transcription, particularly among com- Chs2 in primary septum formation [14,79]. In C. albicans , ponents of a variety of cell signaling pathways, including transcription of CHS1 , CHS8 and CHS3 rise in G2, whereas the protein kinase C mitogen-activated protein (PKC- CHS2 does not demonstrate cell cycle-dependent changes MAP) kinase pathway, the Ca2 ϩ/calcineurin pathway and [80]. In terms of developmental stage differences in tran- a generalized global stress response pathway [11]. It is scription, C. albicans CHS1 is expressed at low levels in important to note that the specifi c response, including the early germ tube development and equally in yeast and pattern of alterations in gene transcription, varies depend- hyphal forms [45,81]. C. albicans CHS2 transcription ing on the specifi c stressor [85,86]. In C. albicans , the high increases during hyphal growth [45,81], consistent with its osmolarity glycerol (HOG) response MAP kinase pathway role in septum formation. C. albicans CHS3 expression and the Ca2 ϩ /calcineurin pathway have also been impli- increases in the transition from yeast to hyphal growth, and cated in the compensatory reaction to cell wall stressors then diminishes thereafter [45,82]. This linkage of individ- [83]. C. albicans CHS gene expression analysis in the set- ual chitin synthase transcription to specifi c developmental ting of various inducers of the CWI pathway results in

© 2012 ISHAM, Medical Mycology, 50, 2–17 8 Rogg et al . upregulation of different combinations of chitin synthases of gene transcription suggesting that protein is stable and depending on the treatment [83]. Mutations in components long-lived [90]. of the Ca 2 ϩ/calcineurin pathway results in increased tran- Studies utilizing chitin synthase activity assays must be scription of chitin synthases, though the pattern differs cautiously interpreted, given the likelihood that in vitro depending on which component is defi cient, whereas muta- conditions of the assay may not mirror in vivo conditions, tions in components of the PKC and HOG pathways cause possibly explaining why signifi cant discordance has been generally decreased CHS transcription [83]. found between total chitin synthase activity and total cel- C. albicans CHS1 transcription is regulated by PKC, lular chitin content. In addition, the existence of large chi- HOG and Ca 2 ϩ/calcineurin signaling. Specifi c promoter tin synthase gene families makes it diffi cult to identify the elements mediate transcriptional control by the PKC quantity and quality of chitin ascribable to individual chitin pathway, but deletion of consensus motifs for Ca 2 ϩ /cal- synthases. Historically, chitin synthases have been grouped

cineurin and HOG pathway transcriptional control does as zymogenic or non-zymogenic based on increased chitin Downloaded from https://academic.oup.com/mmy/article/50/1/2/989229 by guest on 25 September 2021 not correlate with the loss of expression [87]. Thus, the synthase activity in cell membrane extracts after addition basis for transcriptional control of CHS1 by these path- of proteases thus regulation may occur by proteolytic cleav- ways may not necessarily be through the classically age [91]. To date, no convincing candidate protease has defi ned downstream transcription factors. In addition, been identifi ed and it is unclear if proteolysis is a true transcriptional responses for other chitin synthases have regulatory mechanism in vivo. Expression of a truncated S. been studied, but whether the identifi ed responses are cerevisiae Chs2p result in higher protein levels in vivo , but mediated via stereotypical transcription factors remains in vitro activity from both the full-length and truncated pro- undefi ned at this time. teins are similar [92]. Heterologously expressed S. cerevi- Treatment of C. albicans with echinocandins, non-com- siae Chs2p can be hyperactivated by an extract containing petitive inhibitors of β -1,3-glucan synthesis, results in an undefi ned soluble yeast protease activity but this may increased transcription of all four chitin synthase genes not mirror actual in vivo conditions [93]. [88]. Conversely, ectopic activation of these pathways per- mits survival of C. albicans in the presence of otherwise Regulation of chitin synthase activity and lethal doses of echinocandins and suggests that these path- overall chitin deposition is affected by cell ways could provide a target for synergistic targeting of wall stress multiple cell wall components [88]. Mutations in the HOG, PKC and Ca 2 ϩ/calcineurin pathways prevent this upregula- A number of different disruptors of cellular homeostasis, tion, though disruption of each pathway produces a both directly active on the cell wall and others that are different pattern of transcriptional increases [88]. These more generalized, have been shown to alter chitin synthase pathways may each regulate subtly different aspects of cell activity and cell wall chitin content, synthesized by differ- wall compensatory responses. Similar echinocandin-medi- ent chitin synthases depending on the trigger. Mutation of ated transcriptional upregulation is seen for two chitin syn- the S. cerevisiae β-1,3-glucan synthase results in increased, thases in A. fumigatus, which is abrogated by calcineurin though delocalized, cell wall chitin associated with both inhibition or mutation [89], suggesting that inhibition of increased Chs1p and Chs3p activity [7]. Pharmacologic these compensatory pathways may represent a possible inhibition of glucan synthesis in C. albicans also produces future antifungal targeting strategy. increased chitin synthase activity and cell wall chitin content [88], demonstrating the dynamic ability of fungi to Post-transcriptional regulation of chitin respond to cell wall damage. A number of different cell biosynthesis is important for modulation membrane associated proteins have been implicated in these responses. S. cerevisiae Mid2p is a plasma membrane of chitin synthase activity associated cell wall sensor that mediates cell wall integrity Changes in chitin synthase gene transcription do not neces- via induction of Pkc1p MAP kinase signaling. Mutation of sarily correlate with changes in cellular chitin content or MID2 leads to decreased chitin deposition in response to chitin synthase activity, thus regulation of chitin synthesis cell wall stress, a response that is dependent on the pres- likely occurs both transcriptionally and post-transcription- ence of intact Chs3p [94]. Loss of GGP1/GAS1 , which ally. In S. cerevisiae , blocking CHS2 transcription results encodes a glycosylphosphatidylinositol-anchored plasma in quicker loss of Chs2p activity than loss of CHS2 mRNA, membrane glycoprotein, decreases cell wall glucan and suggesting that the Chs2p protein is short-lived [90]. S. increases chitin, dependent on Chs3p, suggesting a central cerevisiae CHS1 has cyclical mRNA transcription but role for CHS3 in mediating cell wall stress compensatory Chs1p activity is constant throughout the cell cycle [90]. responses [8,10]. In contrast, strains missing Bgl2p, a cell S. cerevisiae Chs3p has prolonged activity after cessation wall protein of unclear function; increase Chs1p activity as

© 2012 ISHAM, Medical Mycology, 50, 2–17 Chitin synthesis regulation 9 well as cell wall chitin content in a CHS3 -dependent different pools of vesicles, and these vesicles do not trans- manner and Chs1p activity [95]. Whereas many of the stud- port other cell wall biosynthetic enzymes [40,98]. Vesicles ies have suggested a central role for Chs3p in this compen- fuse with the membrane at sites of active cell wall growth satory response, other chitin synthases also may function such as septa and hyphal tips. Based on data from N. similarly raising the question of whether various cell wall crassa, this process is thought to occur in a two-step pro- disruptions acts through different signaling pathways to the cess, in which the enzyme moves through its specialized same end result. secretory apparatus to a collection of vesicles near the The same signaling pathways that have been shown to hyphal apex that may function as a reserve for supplying regulate chitin synthase transcription have been shown to growth [99,100]. From there, the vesicles move to the lead to alterations in chitin synthase activity and total cell plasma membrane. For yeast chitin synthases, some may wall chitin content. However, the net effects on transcrip- be recycled back into the vesicles whereas others are syn-

tion do not necessarily align with the changes in either thesized, localized and degraded in a cell cycle-dependent Downloaded from https://academic.oup.com/mmy/article/50/1/2/989229 by guest on 25 September 2021 activity or content, highlighting both the limitations of the manner [101,102]. assays and the likelihood that regulation of this process N. crassa chitin synthases cluster in vesicles near the occurs at multiple stages. In general, external cell wall hyphal apex, as well as occurring at lower levels in the stressors and blocking of signaling via pathways impli- hyphal tip plasma membrane [103]. Tagged N. crassa class cated in the response to these stressors result in increased I and VI chitin synthases have been traced from distal chitin synthase activity and/or increased chitin content. endomembranous compartments proximally to smaller Calcium treatment of C. albicans leads to increased Chs2p globular bodies and fi nally to vesicles that concentrate at and Chs8p activity and increased Chs3p-dependent chitin the Spitzenk ö rper [97]. Tagged class III chitin synthases content [83,88], again highlighting the lack of correlation partially co-localize with the other tagged chitin synthases between measurable activity and total content. This stress but are found in different vesicles, consistent with specifi c response is important for survival, as calcium and calco- secretory processes for individual chitin synthases [98]. In fl uor white pretreatment of the normally non-viable C. addition, this localization was dependent on actin but not albicans chs1 Δ chs3 Δ strain facilitates formation of a sal- microtubules [98], implicating cytoskeletal interactions in vage septum [88]. This reinforces the inherent complexity this process. of antifungal agent design and suggests the possibility that simultaneous inhibition of multiple targets may represent The yeast chitin synthase 3 protein secretory a more clinically effective mechanism. mechanism depends on multiple proteins Plasma membrane localization of chitin Detailed molecular biologic analysis of chitin synthase local- synthases occurs via specialized ization has been studied only in yeast. As a consequence, the secretion of some chitin synthases is fairly well understood, secretory pathways but it is not clear to what extent this can be generalized, given Plasma membrane extracts contain intrinsic chitin synthase the larger number of chitin synthase genes and the presence activity [39]. Membrane fractionation yields separate of classes specifi c to fi lamentous fungi. vesicular populations containing chitin synthase activity, Studies of S. cerevisiae Chs3p suggest that sequestra- one associated with intracellular specialized secretory ves- tion of chitin synthases to internal compartments and there- icles, deemed chitosomes, and the other associated with the after to the plasma membrane is a highly regulated process plasma membrane [40]. The presence of intracellular ves- (Fig. 2). Chs3p is normally polarized to a diffuse ring at icles is consistent with the existence of reserves of chitin the future bud site and the neck of small buds in a myosin- synthesis that may be accessible as a functional reservoir dependent manner [104]. A proportion of Chs3p is con- via regulated localization to the plasma membrane. How- tained in internal compartments that are endocytically ever, the processes that mediate the recruitment of chitin derived, suggesting that Chs3p may be shuttled into and synthases to the plasma membrane are as yet undefi ned. out of the membrane to titrate its activity [101]. The Rab Extension of fungal hyphae depends on the action of protein Ypt32p, a mediator of vesicle fusion with the cell wall biosynthetic enzymes, including chitin synthases, plasma membrane, regulates Chs3p delivery to the plasma at the hyphal apex [96]. Chitin synthase localization to the membrane [105]. Various cell wall stressors trigger the plasma membrane has been shown to occur via a highly redistribution of Chs3p from chitosomes to the plasma specialized secretory apparatus, distinct from the classic membrane, suggesting a mechanism through which fungi secretory pathway [97]. Transport appears to depend on may rapidly adjust chitin synthesis in response to stress certain cytoskeletal structures, especially the actomyosin [106]. The re-localization of Chs3p can be blocked by skeleton [98]. Different chitin synthases are transported via mutations in the regulatory GTPase Rho1p and the MAP

Fig. 2 Transit of Chs3p from the ER to the plasma membrane. The movement of Saccharomyces cerevisiae Chs3p from the ER to the plasma membrane requires both direct and indirect interactions with a number of specialized proteins. The thick arrow represents the path followed by Chs3p. Thin arrows indicate proteins whose functions promote Chs3p transit. Chs7p is an ER protein that may facilitate Chs3p folding [108]. Pfa4p palmitoylates Chs3p [110]. Chs5p facilitates assembly of a multi-protein complex containing Chs6p that binds Chs3p to allow movement through the trans-Golgi [104,111,112,114,115]. Mutations in the signaling pathway genes PKC1 , RHO1 , and PIK1 , the vesicle fusion mediator YPT32, and cytoskeletal gene MYO2 prevent plasma membrane localization of Chs3p [104 – 106]. Generation of phosphoinositide phosphate via the Pik1p kinase is important for transport from the ER to the plasma membrane, with negative regulation of this process mediated via the Sac1p phosphatase [116]. kinase Pkc1p [106]. In addition, Chs3p has been shown to secretory stages. S. cerevisiae Chs5p and Chs6p are impor- be phosphorylated by Pkc1p and redistribution of Chs3p tant for Chs3p passage through the trans-Golgi from internal stores to the plasma membrane occurs with [104,111,112]. S. cerevisiae Chs1p is shuttled via the endo- expression of constitutively active Pkc1p [106]. In C. albi- cytosis pathway and its localization is not affected by loss cans, Chs3p is found at the mother-bud neck and in small of CHS6 , confi rming that regulation is not generalizable cytoplasmic patches during yeast growth, as well as at the between different chitin synthases [111,113]. Chs5p colo- septa and tips during hyphal growth [12]. This localization calizes with Chs3p in cytoplasmic patches, and loss of depends on the ability to alter the phosphorylation status CHS5 results in retention of Chs3p in these patches and of serine 139 [107]. However, loss of PKC1 does not affect absence of subsequent plasma membrane localization Chs3p localization in C. albicans , in contrast to the role [104]. Chs6p belongs to a four-member gene family in played in S. cerevisiae [106,107]. which the separate member proteins bind each other and Other proteins that play a role in mediating Chs3p local- Chs5p, after which they associate with Chs3p to facilitate ization have been identifi ed as a consequence of the marked export from the Golgi [114,115]. This specialized secretory drop in chitin content in these mutant strains. These proteins process is dependent on the actomyosin skeleton, as dis- are not chitin synthases per se , but instead interact with ruption of myosin results in delocalized Chs3p [104]. As Chs3p to regulate its traffi cking through the secretory with many other steps in the localization of chitin synthe- machinery and eventual localization to the plasma mem- sis, phospho-signaling plays an important regulatory role. brane. S. cerevisiae Chs7p is an endoplasmic reticulum (ER) Loss of Sac1p, a PI phosphatase and overexpression of membrane protein whose mutations results in ER retention Pik1p, a PI-4-kinase, results in abnormal accumulation of of Chs3p [108]. Deletion of C. albicans CHS7 also leads to Chs3p in vacuoles, thus this phosphorylation pathway may diminished chitin content and abnormal septation [109]. function as a regulator of transport from the Golgi [116]. Expression of S. cerevisiae CHS7 is limiting for Chs3p activity, and many of the developmental processes and exter- Chs3p interacts with an accessory protein as nal factors that increase cell wall chitin result in increased well as other scaffold proteins to maintain CHS7 transcription [108]. Pfa4p, a protein palmitoyltrans- activity and plasma membrane localization ferase, is also required for Chs3p to exit the ER, though Pfa4p and Chs7p appear to function independently [110]. Rather than regulating transit of Chs3p through its special- Other proteins have been identifi ed that are important ized secretory machinery, Chs4p is a Chs3p activator. Loss for transport of the class IV chitin synthases through later of CHS4 (also known as CAL2, SKT5 and CSD4 ) in

S. cerevisiae results in decreased cell wall chitin due to in increased Bni4p at the bud neck; Hsl1p may decrease diminished Chs3p activity [62,66,117 – 119], but does not the Bni4p-Glc7p interaction [125]. Assembly of the chitin mediate its activity via CHS3 transcription or translation ring and primary septum assembly is a complicated pro- [120]. Instead, it appears that a direct physical interaction cess that requires the coordinated interaction of a variety between farnesylated Chs4p and Chs3p is important for its of proteins, and future additional mechanistic understand- function [121– 123]. Chs3p can localize to the bud neck ing may permit identifi cation of possible novel therapeutic without Chs4p, but is inactive and is rapidly endocytosed; targets. thus Chs4p may function to maintain Chs3p at the correct Localization of Chs4p homolog proteins at other loca- plasma membrane site in a stable and activated form [122]. tions is important for normal asexual development. S. cer- Chitin synthase 3 is one of the components of a protein evisiae Shc1p is a Chs4p, homolog that is expressed complex at the mother bud neckthat positions the primary specifi cally during sporulation. Loss of SHC1 results in

septum (Fig. 3). Much of the work towards defi ning this decreased spore chitosan content, suggesting that Shc1p Downloaded from https://academic.oup.com/mmy/article/50/1/2/989229 by guest on 25 September 2021 complex in S. cerevisiae has focused on Bni4p, a scaffold may be the Chs4p homolog that interacts with Chs3p to protein that facilitates the interaction of septal fi laments to mediate synthesis of spore wall chitin that is subsequently an assortment of proteins at this complex, including the deacetylated to chitosan [127]. Overexpression of CHS4 Chs3p activator Chs4p. The chitin synthase is likely can compensate for deletion of SHC1, and overexpression recruited to the bud neck after the eventual septum posi- of SHC1 can partially compensate for loss of CHS4 , though tion has been determined, given the septin- and Bni4p- with imperfect Chs3p localization [127]. Interestingly, dependent localization of Chs3p and Chs4p [124]. Bni4p Chs3p is seen in the prospore membrane in strains defi cient directly interacts with the type 1 serine-threonine phos- in SHC1, thus Shc1p is important for Chs3p activity but phatase Glc7p; if this does not occur, then Bni4p does not not localization, in contrast to the importance of Chs4p for bind Chs4p and Chs3p is not localized to the bud neck both processes at the bud neck [128]. Phosphorylation also [125,126]. The targets for Glc7p activity at the bud neck appears to play a role in the regulation of chitin synthesis complex are not known; there is no evidence for activity during spore formation. Loss of Sps1p, a sporulation-spe- towards Chs3p, Chs4p or the septins, perhaps Bni4p itself cifi c kinase, leads to incorrect localization of Chs3p and may be the target. Bni4p is phosphorylated in a cell cycle- Shc1p at the prospore membrane [128]. Interestingly, dependent fashion with loss of the Hsl1p kinase resulting Sps1p and Chs3p co-localize in cytoplasmic puncta and at

Fig. 3 Localization of Chs3p to the bud neck. Localization of Chs3p to the mother-bud neck requires a protein complex containing septins, Bni4p, Glc7p and Chs4p [124– 126]. Chs3p can reach the bud neck without Chs4p [121– 123], but activity and sustained localization of Chs3p depends on all of the proteins. The target of the Glc7p phosphatase is not known, and the kinase Hsl1p may negatively regulate the interaction between Glc7p and Bni4p [125].

© 2012 ISHAM, Medical Mycology, 50, 2–17 12 Rogg et al . the prospore membrane, though it is not known if Sps1p Table 2 Amino acid sequence identity of Aspergillus fumigatus putative directly acts on Chs3p [128]. chitin synthase secretory and localization proteins compared to Candida Localization of chitin synthases, both in terms of main- albicans and Saccharomyces cerevisiae proteins. tenance in intracellular vesicles and subsequently at spe- C. albicans S. cerevisiae cifi c plasma membrane locations, appears to be affected by A. fumigatus Protein Identity Protein Identity multiple regulatory mechanisms. Transit through the secretory machinery requires the presence of multiple special- Afu6g02510 Chs5p 35% Chs5p 39% Afu3g12800 Chs6p 19% Chs6p 20% ized proteins [104,108,111,112,122]. After successful Afu1g12040 Chs7p 49% Chs7p 45% navigation of the secretory pathway, establishment of Afu3g05580 Chs4p 12% Chs4p 12% activity and maintenance at the appropriate membrane Afu6g02940 Chs4p 27% Chs4p 28% Afu8g05620 Chs4p 31% Chs4p 30% location depends on the presence of a complex of various Afu1g16680 Bni4p 18% Bni4p 16%

proteins, as demonstrated by the dependence of Chs3p Downloaded from https://academic.oup.com/mmy/article/50/1/2/989229 by guest on 25 September 2021 localization at the bud neck on the presence of Bni4p and Chs4p [122,124]. Finally, emerging research implicates at times that are consistent with a role in septation, both in phospho-signaling in the regulation of the chitin synthase yeast and fi lamentous fungi [12,101,132,133]. Studies in regulatory proteins as well as of chitin synthases, though S. cerevisiae suggest class II synthase proteins are rapidly the precise characterization of what step in localization is degraded and are closely tied to the cell cycle, presumably altered by changes in the phosphorylation status remain to to prevent ectopic synthesis of primary septa [101,102]. be identifi ed [106,107,116,128]. In addition, Chs3p is the Chs2p remains in the ER until mitotic exit occurs, and is only chitin synthase to have been studied in this degree of then transported to the membrane via Sec12p-dependent detail thus it is not clear if the regulatory mechanisms mechanisms [134]. The protein is found at the bud neck at described for this particular protein are relevant to other the end of mitosis and provides structural support, possibly chitin synthases. via chitin synthesis, that helps to maintain the stability of Some version of this regulated localization exists in actin-myosin ring during constriction [101,102]. As with other pathogenic fungi, as mutations in both the C. neo- Chs3p, phosphorylation is important for localization, formans class IV chitin synthase gene CHS3 and a chitin though in the case of Chs2p phosphorylation appears to synthase regulator homolog CSR2 possess similar signifi - prevent rather than promote plasma membrane localiza- cant growth defects [49]. The level of chitosan was mark- tion. Mutation of four putative cyclin-dependent kinase edly reduced in these mutants, raising the possibility that consensus sequence sites in Chs2p to mimic constitutive a chitin deacetylase may directly interact with this chitin phosphorylation or dephosphorylation either leads to reten- synthase [49]. tion of Chs2p in the ER even after exit from mitosis or an It is not known if chitin synthase secretion is similarly uncontrolled exit from the ER, respectively [93,135]. regulated in other pathogenic yeast and nothing is known about the possibility of localization regulation by activator Regulation of localization of fi lamentous proteins in fi lamentous fungi. There are putative proteins fungal chitin synthases depends on with relatively high homology to yeast Chs5p and Chs7p the cytoskeleton in both C. neoformans and A. fumigatus (Tables 2 and 3) [129,130]. Interestingly, in A. fumigatus, the gene Studies of the regulation of other classes of chitin syn- (Afu8g05620) that encodes the putative chitin synthase thases, particularly in fi lamentous fungi, have been more activator protein with the highest identity to C. albicans descriptive. Consequently, little is defi ned mechanistically. and S. cerevisiae Chs4p and C. neoformans Csr2 (31, 30 and 49%, respectively), is located immediately next to the Table 3 Amino acid sequence identity of Cryptococcus neoformans putative chitin synthase secretory and localization proteins compared to gene encoding the A. fumigatus class IV chitin synthase Candida albicans and Saccharomyces cerevisiae proteins. ChsF in a head-to-head confi guration, suggesting the possibility of coordinated regulation [131]. C. albicans S. cerevisiae C. neoformans Protein Identity Protein Protein Primary septal localization of yeast Chs2p is CNAG_04321 Chs5p 23% Chs5p 21% tied to the cell cycle CNAG_03325 Chs6p 18% Chs6p 19% CNAG_06898 Chs7p 39% Chs7p 52% Though they contribute only a minor portion of the overall Csr1 Chs4p 27% Chs4p 49% cellular chitin content, the class II yeast chitin synthases Csr2 Chs4p 38% Chs4p 41% are functionally important with a major role in primary Csr3 Chs4p 20% Chs4p 26% CNAG_02071 Bni4p 13% Bni4p 13% septum formation. These proteins localize in positions and

Many of the chitin synthases, irrespective of class, are vari- multiple proteins with a single approach. An in-depth ably seen at septa, tips, and reproductive structures con- understanding of the precise roles of each protein in cell fi rming the importance of chitin synthesis in the structural wall maintenance and repair will be essential to identifying integrity of the cell wall at a variety of locations and a the most likely therapeutic targets. variety of developmental stages [12,58,132,133]. Proteins Simultaneous targeting of multiple cellular pathways have also been seen in cytoplasmic patches suggestive of within the same pathogen is an accepted method to maxi- chitosomes and vacuoles, suggesting that controlled secre- mize therapeutic effi cacy. Inhibition of a single fungal cell tion along with possible recycling or degradation may be wall component has been shown to be effective for certain common regulatory motifs, though the regulatory proteins fungal infections, but cell wall compensatory responses may not generally be shared. may limit effi cacy. To date, chitin synthase inhibitors have Discovery of the class V and VII chitin synthases, spe- not been successfully employed clinically [16,140]. Regu-

cifi cally found in fi lamentous fungi, raised interesting lation of chitin synthesis occurs on multiple levels, thus Downloaded from https://academic.oup.com/mmy/article/50/1/2/989229 by guest on 25 September 2021 questions regarding the role of specifi c chitin synthases in targeting of these pathways may represent an alternative the promotion of hyphal growth, particularly given the gen- approach to achieving chitin synthesis inhibition. erally more dramatic growth defects seen in these gene class mutants. In addition, the combination of an amino Acknowledgements terminal myosin motor domain and a carboxy terminal chitin synthase domain suggested the possibility of physical LER is supported by a Biomedical Research Fellowship interaction with the cytoskeleton as a mechanism for reten- from The Hartwell Foundation funding. WJS is supported tion of these enzymes in specifi c membrane locations. through NIH 1R56AI077648-01A2. Studies in A. nidulans and W. dermatitidis have demonstrated actin binding by the myosin motor domain, and dependence on an intact actin network and actin binding Declaration of interest: The authors report no confl icts for localization as well as functionality [136 – 138]. In the of interest, except for WJS who received funding from plant fungal pathogen Ustilago maydis , studies have dif- Astellas Pharma. The authors alone are responsible for the ferentiated between the interactions important for delivery content and writing of the paper. of chitin synthases to the hyphal tip and those needed for localization to the apical plasma membrane [139]. U. may- References dis class V chitin synthase gene mcs1 depends on microtubules but not actin or an intact myosin motor domain for 1 Latg é J-P. The cell wall: a carbohydrate armour for the fungal cell. Mol Microbiol 2007; 66 : 279 – 290. long-range vesicular movement, but intact actin and a func- 2 Graybill JR. Hitting a new target with echinocandins: why chase tional myosin motor domain is needed for plasma mem- something else? Curr Opin Invest Drugs 2001; 2 : 468 – 471. brane localization [139]. 3 Ikeda F, Wakai Y, Matsumoto S, et al . Effi cacy of FK463, a new lipopeptide antifungal agent, in mouse models of disseminated candidiasis and aspergillosis. Antimicrob Agents Chemother 2000; 44 : Conclusions and future directions 614 – 618. 4 Matsumoto S, Wakai Y, Nakai T, et al . Effi cacy of FK463, a new Fungal chitin synthesis is a complex and highly regulated lipopeptide antifungal agent, in mouse models of pulmonary aspergil- process. Whereas transcription appears to be affected by a losis. Antimicrob Agents Chemother 2000; 44 : 619 – 621. variety of external and internal factors, these changes in 5 Tawara S, Ikeda F, Maki K, et al. In vitro activities of a new lipopep- mRNA levels do not necessarily correlate with changes in tide antifungal agent, FK463, against a variety of clinically important fungi. Antimicrob Agents Chemother 2000; 44 : 57 – 62. chitin synthase activity or cell wall chitin content. Multiple 6 Walsh TJ, Viviani MA, Arathoon E, et al . New targets and delivery signaling pathways have been implicated in chitin synthase systems for antifungal therapy. Med Mycol 2000; 38 : 335 – 347. regulation, at both transcriptional and post-transcriptional 7 Garc í a-Rodriguez LJ, Trilla JA, Castro C, et al . Characterization of levels. Recent research has suggested that localization of the chitin biosynthesis process as a compensatory mechanism in the chitin synthase localization likely represents a major regu- fks1 mutant of Saccharomyces cerevisiae . FEBS Lett 2000; 478 : 84 – 88. latory mechanism. Studies with the class IV chitin synthase, 8 Popolo L, Gilardelli D, Bonfante P, Vai M. Increase in chitin as an S. cerevisiae Chs3p, suggest that both transport through a essential response to defects in assembly of cell wall polymers in the specialized secretory apparatus and protein localization to ggp1 Δ mutant of Saccharomyces cerevisiae . J Bacteriol 1997; 179 : submembrane domains may be regulated. To date, regula- 463 – 469. tion of localization of other chitin synthases has not been 9 Ram AFJ, Kapteyn JC, Montijn RC, et al . Loss of the plasma membrane-bound protein Gas1p in Saccharomyces cerevisiae results in the studied in equal detail. At this point, much of the regulatory release of β 1,3-glucan into the medium and induces a compensation machinery is not necessarily shared among different chitin mechanism to ensure cell wall integrity. J Bacteriol 1998; 180 : synthases, thus it will be diffi cult to simultaneously target 1418 – 1424.

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This paper was fi rst published online on Early online on 4 May 2011.