Aspergillus fumigatus corneal infection is regulated by chitin synthases and by neutrophil-derived acidic mammalian chitinase.

Aspergillus fumigatus is an important cause of pulmonary and systemic infections in immune compromised individuals, and of corneal ulcers and blindness in immune competent patients. To examine the role of chitin synthases in Aspergillus corneal infection, we analyzed Aspergillus mutants of chitin synthase family 1 and family 2, and found that compared with the parent strain, the quadruple mutants from both families were more readily killed by neutrophils in vitro, and that both also exhibited impaired hyphal growth in the cornea. Further, inhibition of chitin synthases using Nikkomycin Z enhanced neutrophil killing in vitro and in vivo in a murine model of A. fumigatus corneal infection. Acidic mammalian chitinase (AMCase) is mostly produced by macrophages in asthmatic lungs; however, we now demonstrate that neutrophils are a major source of AMCase, which inhibits hyphal growth. In A. fumigatus corneal infection, neutrophils are the major source of AMCase, and addition of AMCase inhibitors or adoptive transfer of neutrophils from AMCase-/- mice resulted in impaired hyphal killing. Together, these findings identify chitin synthases as important fungal virulence factors and neutrophil-derived AMCase as an essential mediator of host defense.

Development of conditional cell lysis mutants of Saccharomyces cerevisiae as production hosts by modulating OCH1 and CHS3 expression.

The traditional yeast Saccharomyces cerevisiae has been widely used as a host for the production of recombinant proteins and metabolites with industrial potential. However, its thick and rigid cell wall presents problems for the effective recovery of products. In this study, we modulated the expression of ScOCH1, encoding the α-1,6-mannosyltransferase responsible for outer chain biosynthesis of N-glycans, and ScCHS3, encoding the chitin synthase III required for synthesis of the majority of cell wall chitin, by exploiting the repressible ScMET3 promoter. The conditional single mutants PMET3-OCH1 and PMET3-CHS3 and the double mutant PMET3-OCH1/PMET3-CHS3 showed comparable growth to the wild-type strain under normal conditions but exhibited increased sensitivity to temperature and cell wall-disturbing agents in the presence of methionine. Such conditional growth defects were fully recovered by supplementation with 1 M sorbitol. The osmotic lysis of the conditional mutants cultivated with methionine was sufficient to release the intracellularly expressed recombinant protein, nodavirus capsid protein, with up to 60% efficiency, compared to lysis by glass bead breakage. These mutant strains also showed approximately three-fold-enhanced secretion of a recombinant extracellular glycoprotein, Saccharomycopsis fibuligera ß-glucosidase, with markedly reduced hypermannosylation, particularly in the PMET3-OCH1 mutants. Furthermore, a substantial increase of extracellular glutathione production, up to four-fold, was achieved with the conditional mutant yeast cells. Together, our data support that the conditional cell wall lysis mutants constructed based on the modulation of ScOCH1 and ScCHS3 expression would likely be useful hosts for the improved recovery of proteins and metabolites with industrial application.

miR-71 and miR-263 Jointly Regulate Target Genes Chitin synthase and Chitinase to Control Locust Molting.

Chitin synthase and chitinase play crucial roles in chitin biosynthesis and degradation during insect molting. Silencing of Dicer-1 results in reduced levels of mature miRNAs and severely blocks molting in the migratory locust. However, the regulatory mechanism of miRNAs in the molting process of locusts has remained elusive. In this study, we found that in chitin metabolism, two crucial enzymes, chitin synthase (CHS) and chitinase (CHT) were regulated by miR-71 and miR-263 during nymph molting. The coding sequence of CHS1 and the 3'-untranslated region of CHT10 contain functional binding sites for miR-71 and miR-263, respectively. miR-71/miR-263 displayed cellular co-localization with their target genes in epidermal cells and directly interacted with CHS1 and CHT10 in the locust integument, respectively. Injections of miR-71 and miR-263 agomirs suppressed the expression of CHS1 and CHT10, which consequently altered chitin production of new and old cuticles and resulted in a molting-defective phenotype in locusts. Unexpectedly, reduced expression of miR-71 and miR-263 increased CHS1 and CHT10 mRNA expression and led to molting defects similar to those induced by miRNA delivery. This study reveals a novel function and balancing modulation pattern of two miRNAs in chitin biosynthesis and degradation, and it provides insight into the underlying molecular mechanisms of the molting process in locusts.

Heterologous expression of an active chitin synthase from Rhizopus oryzae.

Chitin synthases are highly important enzymes in nature, where they synthesize structural components in species belonging to different eukaryotic kingdoms, including kingdom Fungi. Unfortunately, their structure and the molecular mechanism of synthesis of their microfibrilar product remain largely unknown, probably because no fungal active chitin synthases have been isolated, possibly due to their extreme hydrophobicity. In this study we have turned to the heterologous expression of the transcript from a small chitin synthase of Rhizopus oryzae (RO3G_00942, Chs1) in Escherichia coli. The enzyme was active, but accumulated mostly in inclusion bodies. High concentrations of arginine or urea solubilized the enzyme, but their dilution led to its denaturation and precipitation. Nevertheless, use of urea permitted the purification of small amounts of the enzyme. The properties of Chs1 (Km, optimum temperature and pH, effect of GlcNAc) were abnormal, probably because it lacks the hydrophobic transmembrane regions characteristic of chitin synthases. The product of the enzyme showed that, contrasting with chitin made by membrane-bound Chs's and chitosomes, was only partially in the form of short microfibrils of low crystallinity. This approach may lead to future developments to obtain active chitin synthases that permit understanding their molecular mechanism of activity, and microfibril assembly.

Cell wall composition plays a key role on sensitivity of filamentous fungi to chitosan.

Chitosan antifungal activity has been reported for both filamentous fungi and yeast. Previous studies have shown fungal plasma membrane as main chitosan target. However, the role of the fungal cell wall (CW) in their response to chitosan is unknown. We show that cell wall regeneration in Neurospora crassa (chitosan sensitive) protoplasts protects them from chitosan damage. Caspofungin, a ß-1,3-glucan synthase inhibitor, showed a synergistic antifungal effect with chitosan for N. crassa but not for Pochonia chlamydosporia, a biocontrol fungus resistant to chitosan. Chitosan significantly repressed N. crassa genes involved in ß-1,3-glucan synthesis (fks) and elongation (gel-1) but the chitin synthase gene (chs-1) did not present changes in its expression. N. crassa cell wall deletion strains related to ß-1,3-glucan elongation (Δgel-1 and Δgel-2) were more sensitive to chitosan than wild type (wt). On the contrary, chitin synthase deletion strain (Δchs-1) showed the same sensitivity to chitosan than wt. The mycelium of P. chlamydosporia showed a higher (ca. twofold) ß-1,3-glucan/chitin ratio than that of N. crassa. Taken together, our results indicate that cell wall composition plays an important role on -sensitivity of filamentous fungi to chitosan.

Modulation of Alternaria infectoria cell wall chitin and glucan synthesis by cell wall synthase inhibitors.

The present work reports the effects of caspofungin, a ß-1,3-glucan synthase inhibitor, and nikkomycin Z, an inhibitor of chitin synthases, on two strains of Alternaria infectoria, a melanized fungus involved in opportunistic human infections and respiratory allergies. One of the strains tested, IMF006, bore phenotypic traits that conferred advantages in resisting antifungal treatment. First, the resting cell wall chitin content was higher and in response to caspofungin, the chitin level remained constant. In the other strain, IMF001, the chitin content increased upon caspofungin treatment to values similar to basal IMF006 levels. Moreover, upon caspofungin treatment, the FKS1 gene was upregulated in IMF006 and downregulated in IMF001. In addition, the resting ß-glucan content was also different in both strains, with higher levels in IMF001 than in IMF006. However, this did not provide any advantage with respect to echinocandin resistance. We identified eight different chitin synthase genes and studied relative gene expression when the fungus was exposed to the antifungals under study. In both strains, exposure to caspofungin and nikkomycin Z led to modulation of the expression of class V and VII chitin synthase genes, suggesting its importance in the robustness of A. infectoria. The pattern of A. infectoria phagocytosis and activation of murine macrophages by spores was not affected by caspofungin. Monotherapy with nikkomycin Z and caspofungin provided only fungistatic inhibition, while a combination of both led to fungal cell lysis, revealing a strong synergistic action between the chitin synthase inhibitor and the ß-glucan synthase inhibitor against this fungus.

Sensitivity of Neurospora crassa to a marine-derived Aspergillus tubingensis anhydride exhibiting antifungal activity that is mediated by the MAS1 protein.

The fungus Aspergillus tubingensis (strain OY907) was isolated from the Mediterranean marine sponge Ircinia variabilis. Extracellular extracts produced by this strain were found to inhibit the growth of several fungi. Among the secreted extract components, a novel anhydride metabolite, tubingenoic anhydride A (1) as well as the known 2-carboxymethyl-3-hexylmaleic acid anhydride, asperic acid, and campyrone A and C were purified and their structure elucidated. Compound 1 and 2-carboxymethyl-3-hexylmaleic acid anhydride inhibited Neurospora crassa growth (MIC = 330 and 207 µM, respectively) and affected hyphal morphology. We produced a N. crassa mutant exhibiting tolerance to 1 and found that a yet-uncharacterized gene, designated mas-1, whose product is a cytosolic protein, confers sensitivity to this compound. The ∆mas-1 strain showed increased tolerance to sublethal concentrations of the chitin synthase inhibitor polyoxin D, when compared to the wild type. In addition, the expression of chitin synthase genes was highly elevated in the ∆mas-1 strain, suggesting the gene product is involved in cell wall biosynthesis and the novel anhydride interferes with its function.

Morphology engineering of Penicillium chrysogenum by RNA silencing of chitin synthase gene.

Chitin synthases, that catalyze the formation of chitin the major component of cell walls in most filamentous fungi, play crucial roles in the growth and morphogenesis. To investigate the roles of chitin synthase in Penicillium chrysogenum, we developed an RNAi system to silence the class III chitin synthase gene chs4. After transformation, mutants had a slow growth rate and shorter but highly branched hyphae. All transformants either were unable to form conidia or could form only a few. Changes in chs4 expression could lead to a completely different morphology and eventually cause distinct penicillin yields. In particular, the yield of one transformant was 41 % higher than that of the original strain.

Molecular cloning, characterization and mRNA expression of a chitin synthase 2 gene from the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae).

Chitin synthase (CHS), a potential target for eco-friendly insecticides, plays an essential role in chitin formation in insects. In this study, a full-length cDNA encoding chitin synthase 2 (BdCHS2) was cloned and characterized in the oriental fruit fly, Bactrocera dorsalis. The BdCHS2 cDNA had 4417 nucleotides, containing an open reading frame of 4122 nucleotides, which encoded 1373 amino acid residues with a predicted molecular weight of 158.5 kDa. Phylogenetic analysis with other insect CHSs suggested that BdCHS2 belongs to insect CHS2. The BdCHS2 transcript was predominately found in midgut but was detected at low levels in fat body, Malpighian tubules, integument, and trachea. Moreover, BdCHS2 was expressed in all developmental stages, and highly expressed in the feeding stages. There was a positive relationship between BdCHS2 expression and total chitin content during development. Furthermore, both the gene expression and chitin content in midgut decreased when the insect was fed for 24 h, then starved for 24 h, while they increased dramatically and rapidly under the condition of starvation for 24 h then feeding for 24 h. These results suggest that BdCHS2 may play an important role in regulating chitin content of the midgut, and subsequently affect the growth and development of B. dorsalis.

Regulation of expression, activity and localization of fungal chitin synthases.

The fungal cell wall represents an attractive target for pharmacologic inhibition, as many of the components are fungal-specific. Though targeted inhibition of ß-glucan synthesis is effective treatment for certain fungal infections, the ability of the cell wall to dynamically compensate via the cell wall integrity pathway may limit overall efficacy. 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.

The efficacy of a chitin synthesis inhibitor against field populations of organophosphate-resistant Aedes aegypti in Brazil.

The mosquito Aedes aegypti is the main focus of dengue control campaigns. Because of widespread resistance against conventional chemical insecticides, chitin synthesis inhibitors (CSIs) are considered control alternatives. We evaluated the resistance status of four Brazilian Ae. aegypti populations to both the organophosphate temephos and the pyrethroid deltamethrin, which are used in Brazil to control larvae and adults, respectively. All vector populations exhibited high levels of temephos resistance and varying rates of alterations in their susceptibility to pyrethroids. The effect of the CSI novaluron on these populations was also investigated. Novaluron was effective against all populations under laboratory conditions. Field-simulated assays with partial water replacement were conducted to evaluate novaluron persistence. Bioassays were continued until an adult emergence inhibition of at least 70% was attained. We found a residual effect of eight weeks under indoor conditions and novaluron persisted for five-six weeks in assays conducted in an external area. Our data show that novaluron is effective against the Ae. aegypti populations tested, regardless of their resistance to conventional chemical insecticides.

Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active.

Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA(-) cell lines are shown.

Stimulation of chitin synthesis rescues Candida albicans from echinocandins.

Echinocandins are a new generation of novel antifungal agent that inhibit cell wall beta(1,3)-glucan synthesis and are normally cidal for the human pathogen Candida albicans. Treatment of C. albicans with low levels of echinocandins stimulated chitin synthase (CHS) gene expression, increased Chs activity, elevated chitin content and reduced efficacy of these drugs. Elevation of chitin synthesis was mediated via the PKC, HOG, and Ca(2+)-calcineurin signalling pathways. Stimulation of Chs2p and Chs8p by activators of these pathways enabled cells to survive otherwise lethal concentrations of echinocandins, even in the absence of Chs3p and the normally essential Chs1p, which synthesize the chitinous septal ring and primary septum of the fungus. Under such conditions, a novel proximally offset septum was synthesized that restored the capacity for cell division, sustained the viability of the cell, and abrogated morphological and growth defects associated with echinocandin treatment and the chs mutations. These findings anticipate potential resistance mechanisms to echinocandins. However, echinocandins and chitin synthase inhibitors synergized strongly, highlighting the potential for combination therapies with greatly enhanced cidal activity.

Roles of the pH signaling transcription factor PacC in Wangiella (Exophiala) dermatitidis.

To study the function of the PacC transcription factor in Wangiella dermatitidis, a black, polymorphic fungal pathogen of humans with yeast-phase predominance, the PACC gene was cloned, sequenced, disrupted and expressed. Three zinc finger DNA-binding motifs were found at the N-terminus, and a signaling protease cleavage site at the C-terminus. PACC was more expressed at neutral-alkaline pH than at acidic pH. Truncation at about 40 residues of the coding sequence upstream of the conserved protease processing cleavage site of PacC affected growth on a nutrient-rich medium, increased sensitivity to Na(+) stress, decreased yeast growth at neutral-alkaline pH, and repressed hyphal growth on a nutrient-poor medium at 25 degrees C. Truncation at the coding sequence for the conserved signaling protease box of PacC impaired growth and reduced RNA expression of the class II chitin synthase gene at acidic pH. The results suggested that PacC is important not only for the adaptation of W. dermatitidis to different ambient pH conditions and Na(+) stress conditions, but also for influencing yeast-hyphal transitions in this agent of phaeohyphomycosis.

MoCRZ1, a gene encoding a calcineurin-responsive transcription factor, regulates fungal growth and pathogenicity of Magnaporthe oryzae.

Ca(2+)-dependent signaling plays important roles in cellular development and metabolism in fungi. Pharmacological and molecular evidence clearly indicates that Ca(2+)-dependent signaling is required for infection-related development and pathogenicity in the rice blast fungus Magnaporthe oryzae. However, little information is available on downstream regulators in the Ca(2+)-dependent signaling pathway. To understand the role of a calcineurin-dependent transcription factor in the rice blast fungus, an ortholog of Saccharomyces cerevisiae CRZ1 in M. oryzae, MoCRZ1, was identified and functionally characterized. The Deltamocrz1 mutant exhibited impaired growth in the presence of Ca(2+) ions or cell wall perturbing agents. The Deltamocrz1 mutant also showed reduced conidiation and reduced pathogenicity, which is mainly due to a defect in host penetration. MoCRZ1 fused to EGFP was trans-localized into the nucleus in a Ca(2+)/calcineurin-dependent manner. The MoCRZ1 gene is also required for the calcineurin-dependent transcriptional induction of FKS1, a gene encoding a beta-1,3 glucan synthase, CHS2 and CHS4, genes encoding two chitin synthases, and PMC and PMR gene families encoding P-type ATPases in response to Ca(2+). These results suggest that MoCRZ1 is a downstream regulator in Ca(2+)-dependent signaling for pathogenicity in M. oryzae, and its biochemical mechanisms are well conserved among fungal species.

Differential trafficking and timed localization of two chitin synthase proteins, Chs2p and Chs3p.

The deposition of the polysaccharide chitin in the Saccharomyces cerevisiae cell wall is temporally and spatially regulated. Chitin synthase III (Chs3p) synthesizes a ring of chitin at the onset of bud emergence, marking the base of the incipient bud. At the end of mitosis, chitin synthase II (Chs2p) deposits a disk of chitin in the mother-bud neck, forming the primary division septum. Using indirect immunofluorescence microscopy, we have found that these two integral membrane proteins localize to the mother-bud neck at distinct times during the cell cycle. Chs2p is found at the neck at the end of mitosis, whereas Chs3p localizes to a ring on the surface of cells about to undergo bud emergence and in the mother-bud neck of small-budded cells. Cell synchronization and pulse-chase experiments suggest that the timing of Chs2p localization results from cell cycle-specific synthesis coupled to rapid degradation. Chs2p degradation depends on the vacuolar protease encoded by PEP4, indicating that Chs2p is destroyed in the vacuole. Temperature-sensitive mutations that block either the late secretory pathway (sec1-1) or the internalization step of endocytosis (end4-1) also prevent Chs2p degradation. In contrast, Chs3p is synthesized constitutively and is metabolically stable, indicating that Chs2p and Chs3p are subject to different modes of regulation. Differential centrifugation experiments show that a significant proportion of Chs3p resides in an internal compartment that may correspond to a vesicular species called the chitosome (Leal-Morales, C.A., C.E. Bracker, and S. Bartnicki-Garcia. 1988, Proc. Natl. Acad. Sci. USA. 85:8516-8520; Flores Martinez, A., and J. Schwencke. 1988. Biochim. Biophys. Acta. 946:328-336). Fractionation of membranes prepared from mutants defective in internalization (end3-1 and end4-1) indicate that the Chs3p-containing vesicles are endocytically derived. Collectively, these data suggest that the trafficking of Chs2p and Chs3p diverges after endocytosis; Chs3p is not delivered to the vacuole, but instead may be recycled.

Molecular cloning, expression, and functional analysis of the chitin synthase 1 gene and its two alternative splicing variants in the white-backed planthopper, Sogatella furcifera (Hemiptera: Delphacidae).

Chitin synthase is responsible for chitin synthesis in the cuticles and cuticular linings of other tissues in insects. We cloned two alternative splicing variants of the chitin synthase 1 gene (SfCHS1) from the white-backed planthopper, Sogatella furcifera. The full-length cDNA of the two variants (SfCHS1a and SfCHS1b) consists of 6408 bp, contains a 4719-bp open reading frame encoding 1572 amino acids, and has 5' and 3' non-coding regions of 283 and 1406 bp, respectively. The two splicing variants occur at the same position in the cDNA sequence between base pairs 4115 and 4291, and consist of 177 nucleotides that encode 59 amino acids but show 74.6% identity at the amino acid level. Analysis in different developmental stages showed that expression of SfCHS1 and SfCHS1a were highest just after molting, whereas SfCHS1b reached its highest expression level 2 days after molting. Further, SfCHS1 and SfCHS1a were mainly expressed in the integument, whereas SfCHS1b was predominately expressed in the gut and fat body. RNAi-based gene silencing inhibited transcript levels of the corresponding mRNAs in S. furcifera nymphs injected with double-stranded RNA of SfCHS1, SfCHS1a, and SfCHS1b, resulted in malformed phenotypes, and killed most of the treated nymphs. Our results indicate that SfCHS1 may be a potential target gene for RNAi-based S. furcifera control.

Embryonic desiccation resistance in Aedes aegypti: presumptive role of the chitinized serosal cuticle.

BACKGROUND: One of the major problems concerning dengue transmission is that embryos of its main vector, the mosquito Aedes aegypti, resist desiccation, surviving several months under dry conditions. The serosal cuticle (SC) contributes to mosquito egg desiccation resistance, but the kinetics of SC secretion during embryogenesis is unknown. It has been argued that mosquito SC contains chitin as one of its components, however conclusive evidence is still missing. RESULTS: We observed an abrupt acquisition of desiccation resistance during Ae. aegypti embryogenesis associated with serosal cuticle secretion, occurring at complete germ band extension, between 11 and 13 hours after egglaying. After SC formation embryos are viable on dry for at least several days. The presence of chitin as one of the SC constituents was confirmed through Calcofluor and WGA labeling and chitin quantitation. The Ae. aegypti Chitin Synthase A gene (AaCHS1) possesses two alternatively spliced variants, AaCHS1a and AaCHS1b, differentially expressed during Ae. aegypti embryonic development. It was verified that at the moment of serosal cuticle formation, AaCHS1a is the sole variant specifically expressed. CONCLUSION: In addition to the peritrophic matrix and exoskeleton, these findings confirm chitin is also present in the mosquito serosal cuticle. They also point to the role of the chitinized SC in the desiccation resistance of Ae. aegypti eggs. AaCHS1a expression would be responsible for SC chitin synthesis. With this embryological approach we expect to shed new light regarding this important physiological process related to the Ae. aegypti life cycle.

Transcription and expression analyses of WdCHS5, which encodes a class V chitin synthase with a myosin motor-like domain in Wangiella (Exophiala) dermatitidis.

WdChs5p in Wangiella dermatitidis is a class V chitin synthase that is required for sustained cell growth at the temperature of infection (37 degrees C) and its encoding gene, WdCHS5, has a differential expression feature. Nuclear run-on and mRNA stability assays showed that increased WdCHS5 mRNA synthesis was the major factor responsible for the increased WdCHS5 transcript at 37 degrees C. Epitope tagging of WdChs5p in W. dermatitidis showed that the WdChs5p-myc protein had a differential expression feature that was similar to the differential transcription of the WdCHS5 gene. In conclusion, it is shown that transcriptional regulation is the first and probably the most important control point of the expression of WdCHS5.

Characterization of the chitin biosynthesis process as a compensatory mechanism in the fks1 mutant of Saccharomyces cerevisiae.

Deletion of the 1,3-beta-D-glucan synthase gene FKS1 in Saccharomyces cerevisiae induces a compensatory mechanism that is reflected in a significant increase in chitin synthase III (CSIII) activity, leading to high rates of chitin synthesis. Deregulation of CSIII activity is mainly due to the intracellular delocalization of Chs3p and Chs4p, the two main components of the CSIII active complex.