Fungal Chitin Synthase Research Articles

Functional diversity of chitin synthases ofAspergillus nidulansin hyphal growth, conidiophore development and septum formation

Fungal chitin synthases are classified into seven classes. Recent advances in genome sequence analyses have revealed that there are more than seven chitin synthase-encoding genes in the genome of filamentous ascomycete fungi. To clarify the functional differences among these genes in the morphogenesis and hyphal tip growth, we have cloned six chitin synthase-encoding genes from Aspergillus nidulans and analyzed their functions. The results obtained suggest that chitin synthases are structurally and functionally divergent and play crucial roles in the growth and morphogenesis of A. nidulans.

Survey and expression analysis of five new chitin synthase genes in the biotrophic rust fungus Puccinia graminis

We have isolated and characterised the first set of chitin synthase genes from a rust fungus, a large group of economically highly important, obligately biotrophic plant pathogens. Puccinia graminis was used as a model organism for the rust fungi which are not well investigated on the molecular level today. One of the major structural components of most fungal cell walls is the chitin polymer which is synthesised by a family of enzymes called chitin synthases. In P. graminis, we have isolated five new chitin synthase genes from four different classes, chsII, chsIIIa, chsIIIb, chsIV, and chsV. The genes contain a high number of introns, unusual for other known fungal chitin synthases. The dinucleic stage of the fungus seems to contain two slightly different genes or alleles for four isoforms. One isoform, chsIIIa, seems to be expressed only in the youngest stages of fungal growth. Analysis of the derived proteins shows that together with other basidiomycete CHS, the pgtCHS form separate subgroups in the phylogenetic tree. This set of five rust chitin synthase genes, with some unusual features compared to known fungal chitin synthases, allows new insights into chitin synthase classification, and may help in the development of novel functional fungicides.

Synthesis of the C1-phosphonate analog of UDP-GlcNAc

We describe the first synthesis of the C1-phosphonate analog of UDP-GlcNAc, based on a new preparation of the corresponding glycosyl phosphonate. This C-glycosyl analog is shown to be a very weak inhibitor (Ki>10mM) of fungal chitin synthase, indicating that at least in this case the replacement of the anomeric oxygen with a methylene group is not an innocent substitution.

Insect chitin synthases: a review

Chitin is the most widespread amino polysaccharide in nature. The annual global amount of chitin is believed to be only one order of magnitude less than that of cellulose. It is a linear polymer composed of N-acetylglucosamines that are joined in a reaction catalyzed by the membrane-integral enzyme chitin synthase, a member of the family 2 of glycosyltransferases. The polymerization requires UDP-N-acetylglucosamines as a substrate and divalent cations as co-factors. Chitin formation can be divided into three distinct steps. In the first step, the enzymes' catalytic domain facing the cytoplasmic site forms the polymer. The second step involves the translocation of the nascent polymer across the membrane and its release into the extracellular space. The third step completes the process as single polymers spontaneously assemble to form crystalline microfibrils. In subsequent reactions the microfibrils combine with other sugars, proteins, glycoproteins and proteoglycans to form fungal septa and cell walls as well as arthropod cuticles and peritrophic matrices, notably in crustaceans and insects. In spite of the good effort by a hardy few, our present knowledge of the structure, topology and catalytic mechanism of chitin synthases is rather limited. Gaps remain in understanding chitin synthase biosynthesis, enzyme trafficking, regulation of enzyme activity, translocation of chitin chains across cell membranes, fibrillogenesis and the interaction of microfibrils with other components of the extracellular matrix. However, cumulating genomic data on chitin synthase genes and new experimental approaches allow increasingly clearer views of chitin synthase function and its regulation, and consequently chitin biosynthesis. In the present review, I will summarize recent advances in elucidating the structure, regulation and function of insect chitin synthases as they relate to what is known about fungal chitin synthases and other glycosyltransferases.

Sequence of a cDNA and expression of the gene encoding a putative epidermal chitin synthase of Manduca sexta

Glycosyltransferases are enzymes that synthesize oligosaccharides, polysaccharides and glycoconjugates. One type of glycosyltransferase is chitin synthase, a very important enzyme in biology, which is utilized by insects, fungi, and other invertebrates to produce chitin, a polysaccharide of β-1,4-linked N-acetylglucosamine. Chitin is an important component of the insect’s exoskeletal cuticle and gut lining. To identify and characterize a chitin synthase gene of the tobacco hornworm, Manduca sexta, degenerate primers were designed from two highly conserved regions in fungal and nematode chitin synthase protein sequences and then used to amplify a similar region from Manduca cDNA. A full-length cDNA of 5152 nucleotides was assembled for the putative Manduca chitin synthase gene, MsCHS1, and sequencing of genomic DNA verified the contiguity of the sequence. The MsCHS1 cDNA has an ORF of 4692 nucleotides that encodes a transmembrane protein of 1564 amino acid residues with a mass of approximately 179 kDa (GenBank no. AY062175). It is most similar, over its entire length of protein sequence, to putative chitin synthases from other insects and nematodes, with 68% identity to enzymes from both the blow fly, Lucilia cuprina, and the fruit fly, Drosophila melanogaster. The similarity with fungal chitin synthases is restricted to the putative catalytic domain, and the MsCHS1 protein has, at equivalent positions, several amino acids that are essential for activity as revealed by mutagenesis of the fungal enzymes. A 5.3-kb transcript of MsCHS1 was identified by northern blot hybridization of RNA from larval epidermis, suggesting that the enzyme functions to make chitin deposited in the cuticle. Further examination by RT-PCR showed that MsCHS1 expression is regulated in the epidermis, with the amount of transcript increasing during phases of cuticle deposition.

Stage-specific expression of the chitin synthase DmeChSA and DmeChSB genes during the onset of Drosophila metamorphosis

Chitin, the major structural polysaccharide of arthropods, is an important constituent of the insect extracellular structures, cuticle and gut peritrophic matrix. Synthesis of cuticular chitin is strictly coordinated with the ecdysone-regulated molting cycle of insect development (the term “ecdysone” is used in this paper instead of “ecdysteroids” since the exact ratio of various hormonal forms changes during metamorphosis). Based on observed similarities between the fungal chitin synthases and other processive β-glycosyltransferases, we have identified the first insect chitin synthase genes, DmeChSA and DmeChSB (Database accession numbers: EMBL/GenBank/DDBJ A83122, A83126, AJ309488, AJ309489), from Drosophila melanogaster. Chromosomal localization has identified these genes close to and on either side of the centromere of the third chromosome. Partial cDNA clones of both genes have been isolated from a pupal cDNA library. To obtain the first insight into the transcriptional regulation of chitin synthesis, we have monitored the expression of DmeChSA and DmeChSB during the periods of the late-larval and prepupal ecdysone pulses that direct metamorphosis. Transcripts of either gene are barely detected prior to and during the late-larval ecdysone pulse. Once the late-larval ecdysone pulse is ceased completely, both DmeChSA and DmeChSB genes show a remarkable up-regulation.

Detection of chitin synthase class I and II type sequences in six different arbuscular mycorrhizal fungi and gene expression in Glomus intraradices

The arbuscular mycorrhizal (AM) fungus Glomus intraradices was grown in vitro with RIT-DNA transformed Daucus carota roots on plates divided into two compartments. One side of the plates contained only fungal hyphae and spores while the other contained mycorrhizal roots. Using this technique, the limitations of insufficient biological material available for molecular analysis of this obligate symbiont were overcome. Fungal material from the first compartment was used as a pure source of G. intraradices for genomic DNA extraction. PCR amplification as well as southern hybridization were conducted using this DNA. Sufficient pure genomic DNA and mRNA was obtained to carry out Southern analysis, achieve optimum PCR results with 25–35 cycles and conduct RT-PCR. Differential expression of chitin synthase class I and II was detected in G. intraradices. Using degenerate primers specific to fungal chitin synthase sequences, a single amplification product obtained after 25 cycles of PCR was cloned. Sequencing of this fragment revealed similarity to other fungal chitin synthase genes. PCR with these primers and additional primers allowed the amplification of chitin synthase fragments from spores of different isolates of G. intraradices as well as from G. mosseae, Gigaspora margarita, Acaulaspora scrobiculata, Scutellospora calospora and Entrophosphora colombiana. A total of 21 chitin synthase sequences from different species and isolates of various AM fungi were successfully amplified. Sequencing of these fragments permitted their classification into class I and II of the chitin synthase groups.

ChemInform Abstract: Synthesis and Structure—Activity Relationships of Novel Fungal Chitin Synthase Inhibitors.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Cloning and characterisation of a chitin synthase gene cDNA from the cultivated mushroom Agaricus bisporus and its expression during morphogenesis.

Full-length cDNA of a chitin synthase gene (chs1) was cloned from Agaricus bisporus by screening a cDNA library with a PCR amplified fragment of the chitin synthase gene. The chs1 contains an open reading frame of 2727 bp encoding a polypeptide of 909 amino acids and deduced molecular mass 102.3 kDa and pI 8.23. The central region of chs1 showed strong homology to other fungal chitin synthase genes with seven conserved domains. It belongs to the chitin synthase class III, analogous to chsB from Aspergillus nidulans, chsC and chsG from A. fumigatus and chs-1 from Neurospora crassa. It appears to be fruit body induced as the transcripts were higher in the developing mushroom compared to any mycelial stage.

Cloning and characterisation of a chitin synthase gene cDNA from the cultivated mushroom Agaricus bisporus and its expression during morphogenesis

Full-length cDNA of a chitin synthase gene ( chs1) was cloned from Agaricus bisporus by screening a cDNA library with a PCR amplified fragment of the chitin synthase gene. The chs1 contains an open reading frame of 2727 bp encoding a polypeptide of 909 amino acids and deduced molecular mass 102.3 kDa and p I 8.23. The central region of chs1 showed strong homology to other fungal chitin synthase genes with seven conserved domains. It belongs to the chitin synthase class III, analogous to chsB from Aspergillus nidulans, chsC and chsG from A. fumigatus and chs-1 from Neurospora crassa. It appears to be fruit body induced as the transcripts were higher in the developing mushroom compared to any mycelial stage.

Novel nikkomycin analogues: inhibitors of the fungal cell wall biosynthesis enzyme chitin synthase

A series of novel nikkomycin analogue inhibitors of the chitin synthase of fungal cell wall was synthesized and evaluated for their inhibitory activities. Among them, the compound having a phenanthrene group at the terminal amino acid was found to possess strong anti-chitin synthase activity.

Synthesis and structure–activity relationships of novel fungal chitin synthase inhibitors

A novel Candida albicans chitin synthase 1 (CaChs1) inhibitor, RO-41-0986 ( 1) was discovered by random screening. Systematic modification led to the identification of a highly potent CaChs1 inhibitor, RO-09-3024 ( 2), having strong antifungal activity against Candida spp. in vitro.

Differential expression of two Blumeria graminis chitin synthase genes

Abstract Two Blumeria graminis chitin synthase genes, designated BgChs1 and BgChs2 were cloned and characterized following the synthesis and use of degenerate PCR primers designed to the conserved regions of fungal chitin synthase (Chs) genes. Their sequences revealed high similarity with the Chs genes previously cloned from other fungi and placed BgChs1 and BgChs2 with the classes I and V, respectively. Each gene was present as a single copy within the barley powdery mildew genome. Semi-quantitative RT-PCR assays revealed BgChs1 to be up-regulated at both the primary germ tube (PGT) and appressorial germ tube (AGT) stages of differentiation whilst the BgChs2 transcript was up-regulated at the PGT stage. The B. graminisbeta-tubulin gene was used as a control for all RT-PCR reactions. The BgChs1 transcript was some 30 fold less abundant than the beta-tubulin transcript and BgChs2 was some 30 fold rarer than the BgChs1 transcript. The effects of the chitin substrate analogues nikkomycin Z and polyoxin D on conidial morphogenesis were assessed. These nucleoside peptide inhibitors did not affect germination but both polyoxin D and nikkomycin Z treatment led to a large population of abnormally swollen 'balloon-shaped' AGTs, whilst by 12 h after inoculation polyoxin treatment caused the swollen germ tubes to burst.

Synthesis and structure-activity relationships of novel nikkomycin analogs: inhibitors of the fungal cell wall biosynthesis enzyme chitin synthase.

A series of novel nikkomycin analogs, which inhibited chitin synthase, the fungal cell wall biosynthesis enzyme, has been synthesized and evaluated their inhibitory activities.

Isolation of csm1 encoding a class V chitin synthase with a myosin motor-like domain from the rice blast fungus, Pyricularia oryzae

A gene encoding a chitin synthase with a myosin motor-like domain ( csm1) was isolated from Pyricularia oryzae using a PCR fragment amplified from a fungal chitin synthase conserved region. The deduced amino acid sequence of csm1 is homologous to that of CsmA of Aspergillus nidulans (65% identity). The putative gene product of csm1 is consisted of the myosin motor-like domain and a chitin synthase domain as in A. nidulans csmA. The chitin synthase domain of its C-terminus was also homologous to Aspergillus fumigatus ChsE (61.4% identity) and Ustilago maydis Chs6 (48.6% identity) that encode class V chitin synthases. Northern analysis demonstrated that the csm1 was expressed throughout the mycelial growth of P. oryzae. This is the first report on the isolation of the gene encoding a class V chitin synthase with the myosin motor-like domain from P. oryzae.

Isolation of csm1 encoding a class V chitin synthase with a myosin motor-like domain from the rice blast fungus, Pyricularia oryzae.

A gene encoding a chitin synthase with a myosin motor-like domain (csm1) was isolated from Pyricularia oryzae using a PCR fragment amplified from a fungal chitin synthase conserved region. The deduced amino acid sequence of csm1 is homologous to that of CsmA of Aspergillus nidulans (65% identity). The putative gene product of csm1 is consisted of the myosin motor-like domain and a chitin synthase domain as in A. nidulans csmA. The chitin synthase domain of its C-terminus was also homologous to Aspergillus fumigatus ChsE (61.4% identity) and Ustilago maydis Chs6 (48.6% identity) that encode class V chitin synthases. Northern analysis demonstrated that the csm1 was expressed throughout the mycelial growth of P. oryzae. This is the first report on the isolation of the gene encoding a class V chitin synthase with the myosin motor-like domain from P. oryzae.

Molecular cloning and sequencing of a chitin synthase gene (CHS2) of Paracoccidioides brasiliensis

The nucleotide sequence of a chitin synthase gene (CHS2) of the dimorphic fungal human pathogen Paracoccidioides brasiliensis has been determined. The deduced amino acid sequence of Chs2p consists of 1043 residues and is highly homologous to other class II fungal chitin synthases. Computational structural analyses suggest very high similarity to other fungal chitin synthases with a highly variable region at the cytosolic amino-terminal region which may be related to its possible zymogenic nature, and the putative catalytic region close to seven membrane-spanning regions at the carboxyl terminus. The nucleotide sequence of CHS2 and its flanking regions has been submitted to GenBank under Accession Number Y09231. © 1998 John Wiley & Sons, Ltd.

Molecular cloning and sequencing of a chitin synthase gene (CHS2) of Paracoccidioides brasiliensis.

The nucleotide sequence of a chitin synthase gene (CHS2) of the dimorphic fungal human pathogen Paracoccidioides brasiliensis has been determined. The deduced amino acid sequence of Chs2p consists of 1043 residues and is highly homologous to other class II fungal chitin synthases. Computational structural analyses suggest very high similarity to other fungal chitin synthases with a highly variable region at the cytosolic amino-terminal region which may be related to its possible zymogenic nature, and the putative catalytic region close to seven membrane-spanning regions at the carboxyl terminus.

A Novel Fungal Gene Encoding Chitin Synthase with a Myosin Motor-like Domain

AcsmAgene that encodes chitin synthase with a myosin motor-like domain was isolated from the filamentous fungusAspergillus nidulans.Initially, we obtained thecsmAas a homolog of theAspergillus fumigatus chsE-partial fragment. A large open reading frame encoding a polypeptide of 1,852 a.a. was identified by determining the cDNA sequences. The chitin synthase conserved region was situated at the C-terminus and classified into class V as reported previously. On the other hand, the N-terminal region showed significant similarity to myosin motors and could not be classified into any types of myosins identifided so far. Thus, it is suggested that this is the first report of unconventional myosin fused to a metabolic enzyme. The finding of this new type of chitin synthase gene suggests that localization of chitin synthesis may be guided by association with cytoskeletal structures.

Yeast chitin synthases 1 and 2 consist of a non-homologous and dispensable N-terminal region and of a homologous moiety essential for function.

Predicted protein sequences of fungal chitin synthases can be divided into a non-homologous N-terminal region and a C-terminal region that shows significant homology among the various synthases. We have explored the function of these domains by constructing a series of nested deletions, extending from either end, in the CHS1 and CHS2 genes of Saccharomyces cerevisiae. In both cases, most or all of the sequences encoding the non-homologous N-terminal region (one-third of the protein for Chs1p and about one-fourth for Chs2p) could be excised, with little effect on the enzymatic activity in vitro of the corresponding synthase or on its function in vivo. However, further small deletions (20-25 amino acids) into the homologous region were deleterious to enzymatic activity and function, and often led to changes in the zymogenic character of the enzymes. Similarly, relatively small (about 75 amino acids) deletions from the C-terminus resulted in loss of enzymatic activity and function of both synthases. Thus, it appears that all the information necessary for membrane localization, enzymatic activity and function resides in the homologous regions of Chs1p and Chs2p, a situation that may also apply to other chitin synthases.