Chitin Synthesis Research Articles

The Transcription Factor CcRlm1 Regulates Cell Wall Maintenance and Poplar Defense Response by Directly Targeting CcChs6 and CcGna1 in Cytospora chrysosperma.

Maintenance of cell wall integrity is important for fungal cell morphology against external stresses and even virulence. Although the transcription factor Rlm1 is known to play major regulatory roles in the maintenance of cell integrity, the underlying mechanism of how Rlm1 contributes to cell wall integrity and virulence in phytopathogenic fungi remains unclear. Here, we demonstrated that CcRlm1 plays important roles in cell wall maintenance and virulence in the poplar canker fungus Cytospora chrysosperma. Among putative downstream targets, CcChs6 (chitin synthase) and CcGna1 (glucosamine 6-phosphate N-acetyltransferase) were found to be direct targets of CcRlm1 and shown to function in chitin synthesis and virulence. Furthermore, we found stronger induction of poplar defense responses when challenged with these gene deletion mutants. Collectively, these results suggest that CcRlm1 plays a critical role in the regulation of cell wall maintenance, stress response, and virulence by directly regulating CcChs6 and CcGna1 in C. chrysosperma. IMPORTANCE Cytospora chrysosperma causes canker diseases on woody plants, and the molecular basis of its infection is not well understood. This study shows that CcRlm1 is the major regulator of chitin synthesis and virulence of the poplar canker fungus. Our research contributes to further understanding the molecular basis of the interaction between C. chrysosperma and poplar.

Susceptibility of Culex pipiens L. in Sharkia Governorate, Egypt to Chitin Synthesis Inhibitors and their Biochemical Characterizations

Susceptibility of Culex pipiens L. in Sharkia Governorate, Egypt to Chitin Synthesis Inhibitors and their Biochemical Characterizations

Trichosporon asahii PLA2 Gene Enhances Drug Resistance to Azoles by Improving Drug Efflux and Biofilm Formation.

Trichosporon asahii is an opportunistic pathogen that can cause severe or even fatal infections in patients with low immune function. sPLA2 plays different roles in different fungi and is also related to fungal drug resistance. However, the mechanism underlying its drug resistance to azoles has not yet been reported in T. asahii. Therefore, we investigated the drug resistance of T. asahii PLA2 (TaPLA2) by constructing overexpressing mutant strains (TaPLA2OE). TaPLA2OE was generated by homologous recombination of the recombinant vector pEGFP-N1-TaPLA2, induced by the CMV promoter, with Agrobacterium tumefaciens. The structure of the protein was found to be typical of sPLA2, and it belongs to the phospholipase A2_3 superfamily. TaPLA2OE enhanced antifungal drug resistance by upregulating the expression of effector genes and increasing the number of arthrospores to promote biofilm formation. TaPLA2OE was highly sensitive to sodium dodecyl sulfate and Congo red, indicating impaired cell wall integrity due to downregulation of chitin synthesis or degradation genes, which can indirectly affect fungal resistance. In conclusion, TaPLA2 overexpression enhanced the resistance to azoles of T. asahii by enhancing drug efflux and biofilm formation and upregulating HOG-MAPK pathway genes; therefore, it has promising research prospects.

Synthesis and characterization of reversible thermosensitive methylated chitin and its application in zinc‐ion battery thermal runaway

AbstractNovel water‐soluble methylated chitins (MCHs) were synthesized homogeneously in aqueous alkaline solution. The relatively mild reaction conditions resulted in the MCH with high degree of acetylation (DA >0.76). The chemical structure of the obtained MCHs was analyzed and the degree of methylation substitution (DS) and DA were determined by proton NMR in both D2O and 20% DCl/D2O. The MCH aqueous solutions (DS = 0.46 ~ 0.71) showed a reversible thermosensitive sol–gel–sol transition upon heating and cooling. The gel transition temperature of these MCHs (in the range of 15–85 °C) increased with increasing DS and decreasing polymer concentration. Thermal runaway has been an important safety issue impeding the development of high‐energy‐density zinc‐ion batteries. A smart thermosensitive reversible electrolyte was prepared based on this MCH for the aqueous zinc‐ion battery to prevent thermal runaway. When the temperature of zinc‐ion battery rises or even gets out of control, the thermosensitive electrolyte can quickly gel and inhibit the migration of zinc ions, resulting in increase of the internal resistance and realizing intelligent and efficient thermal self‐protection. Thus the novel thermosensitive methylated chitin shows promise for safe aqueous zinc‐ion batteries.

Untargeted UHPLC-MS Metabolomics Reveals the Metabolic Perturbations of Helicoverpa armigera under the Stress of Novel Insect Growth Regulator ZQ-8

According to the previous research of our group, we found compound ZQ-8 ((1S,2R,4S)-1,3,3-trimethylbicyclo [2.2.1]heptan-2-yl-4-(tert-butyl)benzoate). This compound showed a strong growth inhibitory effect on Helicoverpa armigera by inhibiting chitinase 2 and endochitinase. To further understand the mechanism of ZQ-8 interfering with the growth and development of H. armigera, ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) was utilized to analyze the metabolomics of the epidermis and viscera of H. armigera after ZQ-8 stress. The results revealed that the content of most metabolites was down-regulated after ZQ-8 treatment. Through the analysis of metabolic pathways, it was found that ZQ -8 mainly interfered with energy metabolism and amino acid biosynthesis pathways, which may be one of the important factors in which ZQ-8 caused the death of H. armigera larvae. Furthermore, ZQ-8 not only inhibits chitin degradation but also inhibits chitin synthesis in vivo. These findings provide new insights into a better understanding of the mechanism of action of ZQ-8.

Identification and functional characterisation of N-acetylglucosamine kinase from Helicoverpa armigera divulge its potential role in growth and development via UDP-GlcNAc salvage pathway

N-acetylglucosamine kinase (NAGK), a major enzyme of sugar-kinase/Hsp70/actin superfamily, catalyses the conversion of N-acetylglucosamine to N-acetylglucosamine-6-phosphate, the first step leading to the salvage synthesis of uridine diphosphate N-acetylglucosamine. Here, we present the first report on identification, cloning, recombinant expression and functional characterisation of NAGK from Helicoverpa armigera (HaNAGK). The purified soluble HaNAGK exhibited a molecular mass of ∼39 kDa with monomeric conformation. It catalysed the sequential transformation of GlcNAc into UDP-GlcNAc, indicating its role as the initiator of UDP-GlcNAc salvage pathway. HaNAGK exhibited ubiquitous expressions across all the developmental stages and major tissues of H. armigera. The gene was significantly upregulated (80 %; p < 0.01) by the moulting hormone 20-hydroxyecdysone and significantly downregulated (89 %; p < 0.001) by the chitin synthesis inhibitor novaluron, indicating its involvement in ecdysis and chitin metabolism. Furthermore, RNAi of HaNAGK caused poor weight gain, deformed insect bodies, aberrant metamorphosis and pronounced wing abnormalities in >55 % of surviving adults, while recording 7.79 ± 1.52 % and 24.25 ± 7.21 % mortality during larval and pupal stages, respectively. Altogether, the present findings suggest that HaNAGK plays a crucial role in the growth and development of H. armigera and thus, could be considered as a compelling gene of interest while formulating novel pest management strategies.

Aspergillus fumigatus transcription factor ZfpA regulates hyphal development and alters susceptibility to antifungals and neutrophil killing during infection.

Hyphal growth is essential for host colonization during Aspergillus infection. The transcription factor ZfpA regulates A. fumigatus hyphal development including branching, septation, and cell wall composition. However, how ZfpA affects fungal growth and susceptibility to host immunity during infection has not been investigated. Here, we use the larval zebrafish-Aspergillus infection model and primary human neutrophils to probe how ZfpA affects A. fumigatus pathogenesis and response to antifungal drugs in vivo. ZfpA deletion promotes fungal clearance and attenuates virulence in wild-type hosts and this virulence defect is abrogated in neutrophil-deficient zebrafish. ZfpA deletion also increases susceptibility to human neutrophils ex vivo while overexpression impairs fungal killing. Overexpression of ZfpA confers protection against the antifungal caspofungin by increasing chitin synthesis during hyphal development, while ZfpA deletion reduces cell wall chitin and increases caspofungin susceptibility in neutrophil-deficient zebrafish. These findings suggest a protective role for ZfpA activity in resistance to the innate immune response and antifungal treatment during A. fumigatus infection.

Synergy among extracellular adsorption, bio-precipitation and transmembrane transport of Penicillium oxalicum SL2 enhanced Pb stabilization

As a potential bioremediation strain for Pb contamination, Penicillium oxalicum SL2 sometimes has secondary activation of Pb, so it is crucial to clarify its effect on Pb morphology and its intracellular response to Pb stress. We investigated the effect of P. oxalicum SL2 in medium on Pb2+ and Pb availability in eight minerals, and revealed the prioritization of Pb products. (i)Pb was stabilized within 30 days as Pb3(PO4)2 or Pb5(PO4)3Cl with sufficient phosphorus (P); (ii) under P deficiency but sulfur (S) sufficient, Pb was stabilized mainly in the form of PbSO4; (iii) under conditions of P and S deficiency, Pb was stabilized mainly in the form of PbC2O2. With the help of proteomic and metabolomics analysis, a total of 578 different proteins and 194 different metabolites were found to be matched in 52 pathways. Among them, the activation of chitin synthesis, oxalate production, sulfur metabolism and transporters improved the Pb tolerance of P. oxalicum SL2, and promoted the synergistic effect of extracellular adsorption, bio-precipitation and transmembrane transport on Pb stabilization. Our results fill the gap in the intracellular response of P. oxalicum SL2 to Pb and provide new insights into the development of bioremediation agent and technology for Pb contamination.

Eco-friendly deacetylated chitosan base siRNA biological-nanopesticide loading cyromazine for efficiently controlling Spodoptera frugiperda

Spodoptera frugiperda is a serious threat to various crops, such as corn and rice, and results in severe economic losses. Herein, a chitin synthase sfCHS highly expressed in the epidermis of S. frugiperda was screened, and when interfered by an sfCHS-siRNA nanocomplex, most individuals could not ecdysis (mortality rate 53.3 %) or pupate (abnormal pupation 80.6 %). Based on the results of structure-based virtual screening, cyromazine (CYR, binding free energy −57.285 kcal/mol) could inhibit ecdysis (LC50, 19.599 μg/g). CYR-CS/siRNA nanoparticles encapsulating CYR and SfCHS-siRNA with chitosan (CS) were successfully prepared, as confirmed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and 74.9 mg/g CYR was characterized in the core of CYR-CS/siRNA by high-performance liquid chromatography and Fourier transform infrared spectroscopy. Small amounts of prepared CYR-CS/siRNA containing only 1.5 μg/g CYR could better inhibit chitin synthesis in the cuticle and peritrophic membrane (mortality rate 84.4 %). Therefore, chitosan/siRNA nanoparticle-loaded pesticides were useful for pesticide reduction and comprehensive control of S. frugiperda.

Effects of three chitin synthesis inhibitors on egg masses, nymphs and adults of Halyomorpha halys (Hemiptera: Pentatomidae).

Halyomorpha halys, (brown marmorated stink bug, BMSB), is a high-concern invasive species causing severe damage to orchards in many countries outside its native Asian range. Management methods other than frequent sprays of broad-spectrum insecticides are needed to restore integrated pest management (IPM) practices in orchards. Chitin synthesis inhibitors are usually regarded as desirable options in IPM programs because of lower toxicity to beneficial insects and non-target organisms compared to neurotoxic insecticides. In this study, the activity of three chitin synthesis inhibitors (namely buprofezin, novaluron and triflumuron) was investigated on BMSB egg masses, 3rd instars and adults by means of laboratory bioassays. Novaluron and to a lesser extent triflumuron were detrimental to BMSB nymphs exposed to residues on potted peach plants. Novaluron caused high mortality among early instars emerged from sprayed egg masses. No significant differences were found between buprofezin and water control on eggs or 3rd instars. When sprayed on BMSB adults, none of the chitin synthesis inhibitors affected survival, fecundity or egg hatching. Given the activity on nymphs, but the lack of effects on adults, novaluron and triflumuron might be considered for field applications only as a tool in a wider management strategy along with other methods aimed at preventing the invasion of crops by BMSB adults. This article is protected by copyright. All rights reserved.

Mini-review highlighting toxic effects of triflumuron in insects and mammalian systems.

Pesticides have been used to kill pests such as insects, fungi, rodents, and unwanted plants. As these compounds are potentially toxic to the target organisms, they could also be harmful to human health and the environment. Several chronic adverse effects have been identified even after months or years of exposure. The adverse effects of pesticides on the agricultural ecosystem have been a matter of concern in recent decades. In this review, we present an overview of the studies, including our previous studies, monitoring currently used pesticides in the Tunisian agricultural soils that belong to the class of insect growth regulators (IGRs). Triflumuron (TFM) is a benzoyl phenyl urea insecticide belonging to the class of IGRs. TFM is widely used around the world to increase crop yield by protecting them from damage caused by insects. TFM works by inhibiting the synthesis of chitin, an essential part of the insect cuticle, making it susceptible to pathogens and deformities. Consequently, insects become more susceptible to pathogens and malformations. However, studies revealing its toxicity and its mode of action in mammalian systems remain very limited. The aim of this review is to better inform the community about the impact of TFM on crops, the environment, and human beings by summarizing its toxic effects.

Antifungal azoles trigger a xenobiotic detoxification pathway and chitin synthesis in Neurospora crassa

The presence of antifungal drugs is prompting the fungal microorganisms to react by mechanisms broader than the resistance. The fungi evolved mechanisms, by which they respond to various stress conditions, including the presence of antifungal compounds. In this work, we studied the response of model filamentous fungus Neurospora crassa to azole antifungals in the broader context of the adaptation mechanisms. We demonstrated the increase in expression of filamentous fungi-specific genes encoding cytochrome enzymes of CYP65 clan and plasma membrane-localized ABCC transporters. Azoles appear not to conjugate with glutathione. Surprisingly, the azoles caused changes in the hyphae organization and the amount of chitin in cell wall by the same manner that was thought to be echinocandin-specific. The response to individual azoles appeared to be influenced by the structure of azole compound (prochloraz – main outlier). Taken together, these findings demonstrate the importance of study of stress response mechanisms, specifically in filamentous fungi. Many aspects of the reaction within azoles seem to be similar, though specificities are occurring.

Relative Efficacy of Some Chitin Synthesis Inhibitors on the Mortality of Okra Jassid, &amp;lt;i&amp;gt;Amrasca biguttula biguttula&amp;lt;/i&amp;gt; (Ishida)

In this laboratory study, three chitin synthesis inhibitors (CSIs) <i>viz.</i> Tacoma 40SC (Buprofezin), Heron 5EC (Lufenuron) and Pyrifen 10.8EC (Pyriproxifen) were evaluated with three concentrations of each through different application methods <i>viz</i>. direct or topical, indirect or leaf-dip and combined (Direct + Indirect). Data on jassids mortality was collected at 3 and 7 days after treatment (DAT) application. Our present study showed that all of the CSIs had significant effect on the mortality of jassid. In all cases, mortality was clearly dose, time and application method dependent. It has been seen that higher concentrations were found to be more effective than lower concentrations. Moreover, significant level of mortality was found at 3DAT but reached to the peak level at 7DAT in case of all selected CSIs. Bioassay study has showed that all the selected CSIs became able to enter the insect body through contact as well as stomach action to disrupt moulting process by inhibiting chitin synthesis that confirmed the contact and systemic actions of the selected CSIs. Moreover, protocols developed in this study for jassids collection and their safe transferring inside the petridishes would be a useful and convenient approach for the researchers.

MoMih1 is indispensable for asexual development, cell wall integrity, and pathogenicity of Magnaporthe oryzae.

Asexual spore serves as essential inoculum of rice blast during the disease cycle, and differentiation of young conidium from conidiophore is intimately regulated by cell cycle. Mih1 encodes a dual-specificity phosphatase that involved in the G2/M transition of the mitotic cell cycle by regulating the Cdk1 activity in eukaryotes. Till now, the roles of Mih1 homologue, however, remain unclear in Magnaporthe oryzae. We here functionally characterized the Mih1 homologue MoMih1 in M. oryzae. MoMih1 is localized to both the cytoplasm and nucleus and can physically interact with the CDK protein MoCdc28 in vivo. Loss of MoMih1 led to delayed nucleus division and a high level of Tyr15 phosphorylation of MoCdc28. The MoMih1 mutants showed retarded mycelial growth with a defective polar growth, less fungal biomass, and shorter distance between diaphragms, compared with the KU80. Asexual reproduction altered in MoMih1 mutants, with both abnormal conidial morphogenesis and decreased conidiation. The MoMih1 mutants severely attenuated the virulence to host plants due to the impaired ability of penetration and biotrophic growth. The incapability of scavenging of host-derived reactive oxygen species, which was possibly ascribed to the severely decreased extracellular enzymes activities, were partially associated with deficiency of pathogenicity. Besides, the MoMih1 mutants displayed also improper localization of retromer protein MoVps26 and polarisome component MoSpa2, and defects of cell wall integrity (CWI), melanin pigmentation, chitin synthesis, and hydrophobicity. In conclusion, our results demonstrate that MoMih1 plays pleiotropic roles during fungal development and plant infection of M. oryzae.

Atrazine exposure can dysregulate the immune system and increase the susceptibility against pathogens in honeybees in a dose-dependent manner

Recently, concerns regarding the impact of agrochemical pesticides on non-target organisms have increased. The effect of atrazine, the second-most widely used herbicide in commercial farming globally, on honeybees remains poorly understood. Here, we evaluated how atrazine impacts the survival of honeybees and pollen and sucrose consumption, investigating the morphology and mRNA expression levels of midgut tissue, along with bacterial composition (relative abundance) and load (absolute abundance) in the whole gut. Atrazine did not affect mortality, but high exposure (37.3 mg/L) reduced pollen and sucrose consumption, resulting in peritrophic membrane dysplasia. Sodium channels and chitin synthesis were considered potential atrazine targets, with the expression of various genes related to lipid metabolism, detoxification, immunity, and chemosensory activity being inhibited after atrazine exposure. Importantly, 37.3 mg/L atrazine exposure substantially altered the composition and size of the gut microbial community, clearly reducing both the absolute and relative abundance of three core gram-positive taxa, Lactobacillus Firm-5, Lactobacillus Firm-4, and Bifidobacterium asteroides. With altered microbiome composition and a weakened immune system following atrazine exposure, honeybees became more susceptible to infection by the opportunistic pathogen Serratia marcescens. Thus, considering its scale of use, atrazine could negatively impact honeybee populations worldwide, which may adversely affect global food security.

Nuclear receptor FTZ-F1 is required for larval-pupal molting by regulating ecdysteroidogenesis and chitin metabolism in Lasioderma serricorne

Fushi tarazu factor 1 (FTZ-F1) is a typical nuclear receptor transcription factor that plays crucial roles during the molting process in insects. However, the role of FTZ-F1 in Lasioderma serricorne, an important stored insect pest, remains unclear. In this study, the FTZ-F1 gene from L. serricorne was identified and cloned. The role of FTZ-F1 in regulating ecdysteroidogenesis and chitin metabolism was analyzed by RNA interference (RNAi) during larval–pupal transition of L. serricorne. The expression of LsFTZ-F1 varied during development and peaked in the final stage of fourth-instar larvae. The LsFTZ-F1 was mainly expressed in larval epidermis. The expression of LsFTZ-F1 was induced by 20-hydroxyecdysone (20E), but it was suppressed by silencing of ecdysone receptor genes LsEcR and LsUSP. RNAi-mediated knockdown of LsFTZ-F1 in the fourth-instar larvae significantly decreased 20E titers and the expression of 20E synthesis and signaling pathway genes. Suppression of LsFTZ-F1 disturbed the larval–pupal transition and resulted in 100% death, which could be rescued by supplementary 20E. The chitin contents of L. serricorne larvae treated with dsLsFTZ-F1 were decreased, as was the expression of chitin synthesis and degrading genes. Hematoxylin and eosin staining analysis indicated that LsFTZ-F1 depletion prevented the larval–pupal molt by inhibiting the synthesis of new cuticle. These results indicate that LsFTZ-F1 is critical for chitin metabolism and ecdysone synthesis during the larva to pupa molt, and that it is a promising RNAi target for L. serricorne control.

Trehalose-6-phosphate synthase regulates chitin synthesis in Mythimna separata.

Trehalose is a substrate for the chitin synthesis pathway in insects. Thus, it directly affects chitin synthesis and metabolism. Trehalose-6-phosphate synthase (TPS) is a crucial enzyme in the trehalose synthesis pathway in insects, but its functions in Mythimna separata remain unclear. In this study, a TPS-encoding sequence in M. separata (MsTPS) was cloned and characterized. Its expression patterns at different developmental stages and in diverse tissues were investigated. The results indicated that MsTPS was expressed at all analyzed developmental stages, with peak expression levels in the pupal stage. Moreover, MsTPS was expressed in the foregut, midgut, hindgut, fat body, salivary gland, Malpighian tubules, and integument, with the highest expression levels in the fat body. The inhibition of MsTPS expression via RNA interference (RNAi) resulted in significant decreases in the trehalose content and TPS activity. It also resulted in significant changes in Chitin synthase (MsCHSA and MsCHSB) expression, and significantly decrease the chitin content in the midgut and integument of M. separata. Additionally, the silencing of MsTPS was associated with a significant decrease in M. separata weight, larval feed intake, and ability to utilize food. It also induced abnormal phenotypic changes and increased the M. separata mortality and malformation rates. Hence, MsTPS is important for M. separata chitin synthesis. The results of this study also suggest RNAi technology may be useful for enhancing the methods used to control M. separata infestations.

Identification and Functional Characterization of Two Chitin Synthases in the Black Cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae).

The black cutworm, Agrotis ipsilon (Hufnagel), a seasonal migrant and a prolific generalist, can feed on nearly all vegetables and grain crops, causing considerable economic impacts on a global scale. Given its cryptic nature, A. ipsilon management has been extremely challenging. Chitin synthase (CHS), a key enzyme involved in chitin biosynthetic pathway and crucially important for the growth and development of insects, is the molecular target of chitin synthesis inhibitors, a group of broad-spectrum insecticides that is compatible with Integrated Pest Management practices. In this study, we investigated the potential of targeting chitin synthases to control A. ipsilon. As a result, two chitin synthases, AiCHS1 and AiCHS2, were identified and cloned from A. ipsilon. The temporal-spatial distribution study showed that AiCHS1 was predominantly expressed at the pupal stage and most abundant among tissues of head capsule and integument, while AiCHS2 was mainly expressed at the sixth instar larval stage and tissues of foregut and midgut. RNAi-based functional study confirmed gene silencing caused significant reduction in the expression levels of the corresponding mRNA, as well as resulted in abnormal pupation and mortality, respectively. Furthermore, under the treatment of lufenuron, a chitin synthesis inhibitor, A. ipsilon responded with an elevated expression in AiCHS1 and AiCHS2, while larvae showed difficulty in shedding old cuticle, and a cumulative mortality of 69.24% at 48 h. In summary, chitin synthases are crucial for chitin biosynthesis in A. ipsilon and can be targeted for the control (e.g., RNAi-based biopesticides) of this devastating insect pest.

Alternative splicing of POUM2 regulates embryonic cuticular formation and tanning in Bombyx mori.

Insect cuticle is an apical extracellular matrix produced by the epidermis, tracheal, hind- and foregut epithelia during embryogenesis and renewed during molting and metamorphosis. However, the underlying regulatory mechanism for embryonic cuticle formation remains largely unclear. Here, we investigate the function of the transcription factor POUM2 in the embryonic cuticular formation in Bombyx mori, a model lepidopteran insect. Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein-9-mediated knockout of POUM2 resulted in the defect of cuticular deposition, pigmentation, and sclerotization in the embryos. Differentially expressed transcripts analysis of 7-d-old embryos identified 174 up- or downregulated cuticular protein transcripts, 8 upregulated chitin degradation transcripts, 2 downregulated chitin synthesis transcripts and 48 up- or downregulated transcription factor transcripts in the POUM2-/- embryos. The expression levels of the key factors of the tyrosine metabolic pathway, such as tyrosine hydroxylase (Th), Dopa decarboxylase (DDC), and arylalkylamine N-acetyltransferase (aaNAT), were significantly decreased in the POUM2-/- embryos. POUM2 isoform POUM2-L specifically bound the POU cis-regulatory element (CRE) in the Th promoter and increased the transcription of Th, whereas POUM2-S could not bind the POU CRE, although it also increased the transcription of Th. Heterogeneous nuclear ribonucleoprotein Squid-1 directly bound the POUM2 pre-mRNA (messenger RNA) and inhibited the alternative splicing of POUM2-L to POUM2-S mRNA. These results suggest that POUM2 participates in the cuticular formation by regulating the chitin and cuticular protein synthesis and metabolism, and the cuticular pigmentation and sclerotization by regulating tyrosine metabolism during embryogenesis. This study provides new insights into novel function of POUM2 in embryogenesis.

Structural basis for inhibition and regulation of a chitin synthase from Candida albicans.

Chitin is one of the major components of the fungal cell wall. Chitin synthases (Chs's) catalyze chitin formation and translocation across the membrane. The detailed mechanism of chitin synthesis, translocation and assembly remains unknown due to the lack of high-resolution structures of chitin synthases. Chs's are targets of antifungal agents, including nikkomycin Z and polyoxin D. Nikkomycin Z has been clinically investigated to treat endemic fungal infectious disease (coccidioidomycosis). Polyoxin D has been widely used as a safe fungicide in organic agriculture. Lack of structural insights into the action of these inhibitors on Chs has hampered their further development for clinical usage because many fungal pathogens contain Chs's that are less sensitive to nikkomycins and polyoxins. We present the cryo-EM structures of Chs2 from the pathological fungi, Candida albicans (CaChs2), in the apo, substrate-bound, nikkomycin Z-bound, and polyoxin D-bound states. CaChs2 adopts a unique domain-swapped dimer configuration where a conserved motif in the domain swapped region controls enzyme activity. CaChs2 is also regulated by lipids. The chitin translocation tunnel is closed by the extracellular gate and plugged by a lipid molecule in the apo state to prevent non-specific leak. Analyses of substrate and inhibitor binding provide insights into the chemical logic of Chs inhibition, which can guide the optimization of Chs-targeted antifungal drugs.