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 rep- resent an exciting alternative approach. A variety of signaling pathways have been impli- cated 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 affect- ing 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.