Chitin Biosynthesis Research Articles

Identification and characterization of two chitin synthase genes in African malaria mosquito, Anopheles gambiae

Chitin synthase (CHS) represents an attractive target site for combating insect pests as insect growth and development are strictly dependent on precisely tuned chitin biosynthesis and this pathway is absent in humans and other vertebrates. Current knowledge on CHS in insects, especially their structures, functions, and regulations is still very limited. We report the identification and characterization of two chitin synthase genes, AgCHS1 and AgCHS2, in African malaria mosquito, Anopheles gambiae. AgCHS1 and AgCHS2 were predicted to encode proteins of 1,578 and 1,586 amino acid residues, respectively. Their deduced amino acid sequences show high similarities to other insect chitin synthases. Transcriptional analysis indicated that AgCHS1 was expressed in egg, larval, pupal and adult stages whereas AgCHS2 appeared to be expressed at relatively low levels, particularly during the larval stages as examined by reverse transcription (RT)-PCR and real-time quantitative PCR. Relatively high expression was detected in the carcass followed by the foregut and hindgut for AgCHS1, and the foregut (cardia included) followed by the midgut for AgCHS2. Fluorescence in situ hybridization (FISH) and immunohistochemical analysis revealed new information including the localization of the two enzymes in the ommatidia of the compound eyes, and AgCHS2 in the thoracic and abdominal inter-segmental regions of pupal integument.

Physiological significance of alternatively spliced exon combinations of the single‐copy gene class A chitin synthase in the insect Ostrinia furnacalis (Lepidoptera)

Insect chitin synthase is an essential enzyme involved in chitin biosynthesis in insects. Chitin synthase A (CHSA) is expressed in different insect tissues during different developmental stages. CHSA contains alternative-splicing exons that allow tissue- and development-specific chitin synthesis. Here, we report that OfCHSA from the lepidopteran Ostrinia furnacalis contains two alternative-splicing exons, exons 2a and 2b and exons 19a and 19b. Although four combinations of these exons are theoretically possible, we found that transcripts containing exon 2a were dominant during most developmental stages, including embryonic development, larval-larval moulting, the larval-pupal transition and pupal-adult metamorphosis. Unexpectedly, 2b-containing transcripts were much more responsive to 20-hydroxyecdysone regulation than 2a-containing ones, suggesting that although OfCHSA isoforms encoded by 2b-containing transcripts are normally expressed at very low levels, they play unique roles. Spliced exons 2a and 2b have also been observed in Bombyx mori; therefore, this work provides new insights into the regulation of insect chitin synthase, particularly in lepidopteran insects.

The echinocandin B producer fungus Aspergillus nidulans var. roseus ATCC 58397 does not possess innate resistance against its lipopeptide antimycotic

Aspergillus nidulans var. roseus ATCC 58397 is an echinocandin B (ECB) producer ascomycete with great industrial importance. As demonstrated by ECB/caspofungin sensitivity assays, A. nidulans var. roseus does not possess any inherent resistance to echinocandins, and its tolerance to these lipopeptide antimycotics are even lower than those of the non-producer A. nidulans FGSC A4 strain. Under ECB producing conditions or ECB exposures, A. nidulans var. roseus induced its ECB tolerance via up-regulating elements of the chitin biosynthetic machinery and, hence, through changing dynamically the composition of its own cell wall. Importantly, although the specific β-1,3-glucan synthase activity was elevated, these changes reduced the β-glucan content of hyphae considerably, but the expression of fksA, encoding the catalytic subunit of β-1,3-glucan synthase, the putative target of echinocandins in the aspergilli, was not affected. These data suggest that compensatory chitin biosynthesis is the centerpiece of the induced ECB tolerance of A. nidulans var. roseus. It is important to note that the induced tolerance to ECB (although resulted in paradoxical growth at higher ECB concentrations) was accompanied with reduced growth rate and, under certain conditions, even sensitized the fungus to other stress-generating agents like SDS. We hypothesize that although ECB-resistant mutants may arise in vivo in A. nidulans var. roseus cultures, their widespread propagation is severely restricted by the disadvantageous physiological effects of such mutations.

Chitinases in Pneumocystis carinii pneumonia

Pneumocystis pneumonia remains an important complication of immune suppression. The cell wall of Pneumocystis has been demonstrated to potently stimulate host inflammatory responses, with most studies focusing on β-glucan components of the Pneumocystis cell wall. In the current study, we have elaborated the potential role of chitins and chitinases in Pneumocystis pneumonia. We demonstrated differential host mammalian chitinase expression during Pneumocystis pneumonia. We further characterized a chitin synthase gene in Pneumocystis carinii termed Pcchs5, a gene with considerable homolog to the fungal chitin biosynthesis protein Chs5. We also observed the impact of chitinase digestion on Pneumocystis-induced host inflammatory responses by measuring TNFα release and mammalian chitinase expression by cultured lung epithelial and macrophage cells stimulated with Pneumocystis cell wall isolates in the presence and absence of exogenous chitinase digestion. These findings provide evidence supporting a chitin biosynthetic pathway in Pneumocystis organisms and that chitinases modulate inflammatory responses in lung cells. We further demonstrate lung expression of chitinase molecules during Pneumocystis pneumonia.

Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods

Because of its importance to the arthropod exoskeleton, chitin biogenesis is an attractive target for pest control. This point is demonstrated by the economically important benzoylurea compounds that are in wide use as highly specific agents to control insect populations. Nevertheless, the target sites of compounds that inhibit chitin biogenesis have remained elusive, likely preventing the full exploitation of the underlying mode of action in pest management. Here, we show that the acaricide etoxazole inhibits chitin biogenesis in Tetranychus urticae (the two-spotted spider mite), an economically important pest. We then developed a population-level bulk segregant mapping method, based on high-throughput genome sequencing, to identify a locus for monogenic, recessive resistance to etoxazole in a field-collected population. As supported by additional genetic studies, including sequencing across multiple resistant strains and genetic complementation tests, we associated a nonsynonymous mutation in the major T. urticae chitin synthase (CHS1) with resistance. The change is in a C-terminal transmembrane domain of CHS1 in a highly conserved region that may serve a noncatalytic but essential function. Our finding of a target-site resistance mutation in CHS1 shows that at least one highly specific chitin biosynthesis inhibitor acts directly to inhibit chitin synthase. Our work also raises the possibility that other chitin biogenesis inhibitors, such as the benzoylurea compounds, may also act by inhibition of chitin synthases. More generally, our genetic mapping approach should be powerful for high-resolution mapping of simple traits (resistance or otherwise) in arthropods.

Physiological and Morphological Aspects of Aedes aegypti Developing Larvae: Effects of the Chitin Synthesis Inhibitor Novaluron

Population control of the dengue vector mosquito, Aedes aegypti, is difficult due to many reasons, one being the development of resistance to neurotoxic insecticides employed. The biosynthesis of chitin, a major constituent of insect cuticle, is a novel target for population control. Novaluron is a benzoylphenylurea (BPU) that acts as a chitin synthesis inhibitor, already used against mosquitoes. However, information regarding BPU effects on immature mosquito stages and physiological parameters related with mosquito larval development are scarce. A set of physiological parameters were recorded in control developing larvae and novaluron was administered continuously to Ae. aegypti larvae, since early third instar. Larval instar period duration was recorded from third instar until pupation. Chitin content was measured during third and fourth instars. Fourth instars were processed histochemically at the mesothorax region, stained with hematoxylin and eosin (HE) for assessment of internal tissues, and labeled with WGA-FITC to reveal chitinized structures. In control larvae: i) there is a chitin content increase during both third and fourth instars where late third instars contain more chitin than early fourth instars; ii) thoracic organs and a continuous cuticle, closely associated with the underlying epidermis were observed; iii) chitin was continuously present throughout integument cuticle. Novaluron treatment inhibited adult emergence, induced immature mortality, altered adult sex ratio and caused delay in larval development. Moreover, novaluron: i) significantly affected chitin content during larval development; ii) induced a discontinuous and altered cuticle in some regions while epidermis was often thinner or missing; iii) rendered chitin cuticle presence discontinuous and less evident. In both control and novaluron larvae, chitin was present in the peritrophic matrix. This study showed quantitatively and qualitatively evidences of novaluron effects on Ae. aegypti larval development. To our knowledge, this is the first report describing histological alterations produced by a BPU in immature vector mosquitoes.

Correlation of proteome-wide changes with social immunity behaviors provides insight into resistance to the parasitic mite, Varroa destructor, in the honey bee (Apis mellifera)

BackgroundDisease is a major factor driving the evolution of many organisms. In honey bees, selection for social behavioral responses is the primary adaptive process facilitating disease resistance. One such process, hygienic behavior, enables bees to resist multiple diseases, including the damaging parasitic mite Varroa destructor. The genetic elements and biochemical factors that drive the expression of these adaptations are currently unknown. Proteomics provides a tool to identify proteins that control behavioral processes, and these proteins can be used as biomarkers to aid identification of disease tolerant colonies.ResultsWe sampled a large cohort of commercial queen lineages, recording overall mite infestation, hygiene, and the specific hygienic response to V. destructor. We performed proteome-wide correlation analyses in larval integument and adult antennae, identifying several proteins highly predictive of behavior and reduced hive infestation. In the larva, response to wounding was identified as a key adaptive process leading to reduced infestation, and chitin biosynthesis and immune responses appear to represent important disease resistant adaptations. The speed of hygienic behavior may be underpinned by changes in the antenna proteome, and chemosensory and neurological processes could also provide specificity for detection of V. destructor in antennae.ConclusionsOur results provide, for the first time, some insight into how complex behavioural adaptations manifest in the proteome of honey bees. The most important biochemical correlations provide clues as to the underlying molecular mechanisms of social and innate immunity of honey bees. Such changes are indicative of potential divergence in processes controlling the hive-worker maturation.

Effects of the Insect Growth Regulators Novaluron and Diflubenzuron on the Brown Marmorated Stink Bug

The brown marmorated stink bug (BMSB), Halyomorpha halys (Stål), is an invasive pest from China that causes economic damage to field crops, vegetables, and tree fruit. Due to its destructive potential, applications of broad-spectrum insecticides have escalated. Researchers are trying to identify options for controlling BMSB that have less of a negative impact on non-target species. Chitin biosynthesis inhibitors are more selective than the commonly used pyrethroids and organophosphates. They are active on the larval stage of the insect and are reported as having sublethal effects such as reducing adult fecundity. In our studies, bioassays were completed with chitin biosynthesis inhibitors novaluron and diflubenzuron to evaluate the effectiveness of these insecticides on adult mortality, nymphal growth, adult fecundity, and egg hatch. Our data indicate that treatments of novaluron at 362.2 g ai/ha or diflubenzuron at 280.2 g ai/ha effectively controlled BMSB nymphs. However, the insecticides were not effective at reducing egg hatch, adult fecundity, or adult life span. If novaluron or diflubenzuron are used in agriculture for BMSB control, then the nymphal stage should be targeted. Accepted for publication 9 November 2012. Published 12 December 2012.

Functional analysis of a Hansenula polymorpha MNN2-2 homologue encoding a putative UDP-N-acetylglucosamine transporter localized in the endoplasmic reticulum

The Kluyveromyces lactis UDP-GlcNAc transporter (KlMnn2-2p) is responsible for the biosynthesis of N-glycans containing N-acetylglucosamine. A putative gene of Hansenula polymorpha encoding a KlMnn2-2p homologue, HpMNN2-2, was identified and investigated for its function. The deletion mutant strain of HpMNN2-2 (Hpmnn2-2Δ) showed increased sensitivity to geneticin, hygromycin B, and tunicamycin. However, the Hpmnn2-2Δ strain exhibited increased resistance to Calcofluor white, an inhibitor of chitin biosynthesis, along with a reduced chitin content. The localization of HpMnn2-2p at the endoplasmic reticulum-enriched membrane, different from the Golgi localization of a K. lactis homologue, further supports the involvement of HpMnn2-2p in cell wall chitin biosynthesis.

Saccharomyces cerevisiae chitin biosynthesis activation by N-acetylchitooses depends on size and structure of chito-oligosaccharides

BackgroundTo explore chitin synthesis initiation, the effect of addition of exogenous oligosaccharides on in vitro chitin synthesis was studied. Oligosaccharides of various natures and lengths were added to a chitin synthase assay performed on a Saccharomyces cerevisiae membrane fraction.FindingsN-acetylchito-tetra, -penta and -octaoses resulted in 11 to 25% [14C]-GlcNAc incorporation into [14C]-chitin, corresponding to an increase in the initial velocity. The activation appeared specific to N-acetylchitooses as it was not observed with oligosaccharides in other series, such as beta-(1,4), beta-(1,3) or alpha-(1,6) glucooligosaccharides.ConclusionsThe effect induced by the N-acetylchitooses was a saturable phenomenon and did not interfere with free GlcNAc and trypsin which are two known activators of yeast chitin synthase activity in vitro. The magnitude of the activation was dependent on both oligosaccharide concentration and oligosaccharide size.

A novel alternative splicing site of class A chitin synthase from the insect Ostrinia furnacalis – Gene organization, expression pattern and physiological significance

Insect chitin synthase A ( CHSA) catalyzes chitin biosynthesis in tissues that develop from ectoderm. Since only one gene copy encodes CHSA, we hypothesized that CHSA is very likely to exist as isoforms through alternative splicing, and the functions of these isoforms may be tissue-specific. Besides the known alternative splicing exons in the mid-ORF region, we report here the alternative exons ( OfCHSA-2a and OfCHSA-2b) of OfCHSA, the chitin synthase A from the lepidopteran pest Ostrinia furnacalis. Sequence analysis of the 5′ upstream region of the transcription start site indicated that presences of two independent promoters for controlling the expression of OfCHSA-2a/ b. Both OfCHSA-2a and OfCHSA-2b transcripts were preferentially expressed in the epidermis. During growth and development of O. furnacalis, OfCHSA-2a was mainly expressed during larval–larval molting and larval–pupal transformation, as well as in newly-laid eggs, while OfCHSA-2b was expressed only during the larval–larval molting. Gene silencing of OfCHSA-2a caused incomplete molting, while silencing of OfCHSA-2b exclusively influenced the head cuticle formation of the 3rd instar larval. Since O. furnacalis is phylogenetically close to the model insect Bombyx mori, the same undiscovered alternative splicing exon was also identified in BmCHSA by gDNA sequence alignment. This work may lead to greater understanding of the mechanism by which a single copy of the CHSA gene could fulfill various functions with tissue specificity.

Antifungal action of the oxathiolone‐fused chalcone derivative

AMG-148, an oxathiolone-fused chalcone derivative, exhibited in vitro antifungal activity against several strains of human pathogenic yeast, with minimum inhibitory concentration values within the range of 1-16 μg ml(-1) and a fungicidal effect was observed at higher concentrations. Presence of major drug-effluxing membrane proteins Cdr1p, Cdr2p or Mdr1p, did not affect substantially the fungistatic activity of this compound against clinical Candida albicans strains. Studies on the mode of action revealed that AMG-148 inhibited chitin and β(1→3)glucan biosynthesis and was in vitro an inhibitor of β(1→3)glucan synthase. Inhibition of chitin biosynthesis was responsible for fungistatic activity, while the fungicidal effect was a consequence of disturbance of β(1→3)glucan synthase function. The chalcone derivative may be a useful lead compound for the development of novel antifungal agents.

Identification of a female sex pheromone in Carcinus maenas

Despite major progress in our understanding of animal signaling systems, the identification of chemical signals in aquatic organisms is still in its infancy. Exemplary for this is the lack of structural knowledge of crustacean sex pheromones despite their initial description in crabs almost 40 yr ago. We hypothesized that species in which the reproductive event is linked to a short window of mating opportunities centered around the time of ecdysis would be ideal to purify and identify such a cue. Here we present the first identification of a crustacean sex pheromone, the female signal produced by the green crab Carcinus maenas from both conditioned seawater and female urine. We used a bioassay-driven purification scheme combined with a candidate approach focusing on excreted compounds that changed through the course of the female molt cycle. Uridine diphosphate (UDP), a nucleotide accumulated from UDP-N-acetylglucosamine during chitin biosynthesis, is the major component of the female crab pheromone and induced all key characteristics of male sexual behavior in bioassays (i.e. pre- and post-copulatory guarding of the female and initiation of mating) at a threshold of 10–5 M UDP in seawater. The identification of a nucleotide pheromone in crustaceans will enable future studies on signal specificity, biological function, biosynthesis, reception, and evolution as well as focus its potential use in pest control and aquaculture, thus allowing for a major expansion of crustacean chemical and behavioral ecology research that was hindered by the lack of structural knowledge.

Synthesis and Biological Properties of Dihydro-Oxadiazine-Based Heterocyclic Derivatives

Dihydro-oxadiazine and its derivatives have been demonstrated to be important heterocyclic scaffold platform with bioactive diversity, which present wide activities such as cardiovascular, antitumor, antibacterial, antimicrobial, acricidal, insecticidal, plant-growth regulating, chitin biosynthesis inhibitors and monoamine oxidase inhibition. Versatile features of dihydro-oxadiazine heterocycles have emerged, so the aim of the present paper was to review the recent advances of dihydro-oxadiazine-based heterocyclic derivatives mainly including synthesis and biological activities.

Echinocandins in Children

Echinocandins in Children

Chemical reactivity and antimicrobial activity of N-substituted maleimides

Several N-substituted maleimides containing substituents of varying bulkiness and polarity were synthesised and tested for antimicrobial and cytostatic activity. Neutral maleimides displayed relatively strong antifungal effect minimum inhibitory concentrations (MICs in the 0.5–4 µg ml−1 range); their antibacterial activity was structure dependent and all were highly cytostatic, with IC50 values below 0.1 µg ml−1. Low antimicrobial but high cytostatic activity was noted for basic maleimides containing tertiary aminoalkyl substituents. Chemical reactivity and lipophilicity influenced antibacterial activity of neutral maleimides but had little if any effect on their antifungal and cytostatic action. N-substituted maleimides affected biosynthesis of chitin and β(1,3)glucan, components of the fungal cell wall. The membrane enzyme, β(1,3)glucan synthase has been proposed as a putative primary target of N-ethylmaleimide and some of its analogues in Candida albicans cells.

Chitin Synthase, a Fungal Glycosyltransferase that Is a Valuable Antifungal Target

During the last 30 past years, more life-threatening fungal infections have appeared due to the increasing frequency of patients with weakened immune systems. Inhibition of fungal enzymes involved in the biosynthesis of sterols is considered to be a safe and effective option for antifungal therapy. However, the intensive use of sterol-biosynthesis inhibitors for years has resulted in resistance development. Consequently, the search for alternative therapeutics must be intensified. In this context, the biosynthesis of chitin, an essential component of the fungal cell wall that is absent in mammalian cells, was envisioned as a safe and selective therapeutic target. We present here recent successes in the inhibition of chitin synthase, the enzyme involved in the last step of the biosynthesis of chitin.

Identification of 20-Hydroxyecdysone Late-Response Genes in the Chitin Biosynthesis Pathway

Background20-hydroxyecdysone (20E) and its receptor complex ecdysone receptor (EcR) and ultraspiracle (USP) play a crucial role in controlling development, metamorphosis, reproduction and diapause. The ligand-receptor complex 20E-EcR/USP directly activates a small set of early-response genes and a much larger set of late-response genes. However, ecdysone-responsive genes have not been previously characterized in the context of insect chitin biosynthesis.Principal FindingsHere, we show that injection-based RNA interference (RNAi) directed towards a common region of the two isoforms of SeEcR in a lepidopteron insect Spodoptera exigua was effective, with phenotypes including a high mortality prior to pupation and developmental defects. After gene specific RNAi, chitin contents in the cuticle of an abnormal larva significantly decreased. The expression levels of five genes in the chitin biosynthesis pathway, SeTre-1, SeG6PI, SeUAP, SeCHSA and SeCHSB, were significantly reduced, while there was no difference in the expression of SeTre-2 prior to 72 hr after injection of EcR dsRNA. Meanwhile, injection of 20E in vivo induced the expression of the five genes mentioned above. Moreover, the SeTre-1, SeG6PI, SeUAP and SeCHSB genes showed late responses to the hormone and the induction of SeTre-1, SeG6PI, SeUAP and SeCHSB genes by 20E were able to be inhibited by the protein synthesis inhibitor cycloheximide in vitro indicating these genes are 20E late-response genes.ConclusionsWe conclude that SeTre-1, SeG6PI, SeUAP and SeCHSB in the chitin biosynthesis pathway are 20E late-response genes and 20E and its specific receptors plays a key role in the regulation of chitin biosynthesis via inducing their expression.

A Selective Assay to Detect Chitin and Biologically Active Nano-Machineries for Chitin-Biosynthesis with Their Intrinsic Chitin-Synthase Molecules

A new assay system for chitin has been developed. It comprises the chitin-binding protein ChbB in fusion with a His-tag as well as with a Strep-tag, the latter of which was chemically coupled to horseradish peroxidase. With the resulting complex, minimal quantities of chitin are photometrically detectable. In addition, the assay allows rapid scoring of the activity of chitin-synthases. As a result, a refined procedure for the rapid purification of yeast chitosomes (nano-machineries for chitin biosynthesis) has been established. Immuno-electronmicroscopical studies of purified chitosomes, gained from a yeast strain carrying a chitin-synthase gene fused to that for GFP (green-fluorescence protein), has led to the in situ localization of chitin-synthase-GFP molecules within chitosomes.

Cloning and expression pattern of chitin synthase (CHS) gene in epidermis of Ectropis obliqua Prout

Ectropis obliqua Prout is a major pest in tea fields. Chitin synthase (CHS) plays an important role in biosynthesis of chitin and growth of the pest. A cDNA sequence encoding the CHS and its expression pattern during development of E.obliqua was investigated. The CHS cDNA sequence was 5496 bp nucleotides, with an open reading frame of 4692 bp encoding a protein of 1563 amino acids. It belonged to CHS-A member of CHS gene family. Alternative splice was found in the CHS-A cDNA and the alternatively spliced fragments were 177 bp and shared 65% identity with each other at the nucleotide level. The CHS-A expression was the strongest in the third and fourth instar larvae, during which the growth rate of E. obliqua larvae was the rapidest. Catalysis model of CHS-A enzyme in E. obliqua was also hypothesized according to the specific motifs and topological structure prediction of the protein. This study provided an important information for further research on development of RNA interference (RNAi) technology to control E. obliqua . Keywords: Ectropis oblique, tea pest, chitin synthase, gene cloning, RNAi, biological control African Journal of Biotechnology Vol. 9(33), pp. 5297-5308, 16 August, 2010