Characterization and expression patterns of a membrane-bound trehalase from <Emphasis Type="Italic">Spodoptera exigua</Emphasis>

Background  

The chitin biosynthesis pathway starts with trehalose in insects and the main functions of trehalases are hydrolysis of trehalose to glucose. Although insects possess two types, soluble trehalase (Tre-1) and membrane-bound trehalase (Tre-2), very little is known about Tre-2 and the difference in function between Tre-1 and Tre-2.     

Identification of 20-hydroxyecdysone late-response genes in the chitin biosynthesis pathway

Background

20-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 Findings

Here, 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.

Conclusions

We 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.     

Function and regulation of the acid and neutral trehalases of Mucor rouxii

Two different trehalose-hydrolysing activities, known as acid or non-regulatory trehalases, and neutral or regulatory trehalases, have been recognised in a number of fungal species. The true role of these apparently redundant hydrolases remained obscure for many years. However, recent evidence suggests that neutral trehalases would be specialised in the mobilisation of cytosolic trehalose, while acid trehalases would only hydrolyse extracellular trehalose. Results obtained with Mucor rouxii, a Zygomycete initially thought to posses only neutral trehalase activity, reinforced this hypothesis. M. rouxii grows efficiently in trehalose as the sole carbon source. Trehalose-grown or carbon-starved cells exhibit a high trehalase activity of optimum pH 4.5, bound to the external surface of the cell wall, in contrast with the neutral (pH 6.5) trehalase, which occurs in the cytosol. Other differences between the neutral and the acid trehalases are the temperature optimum (35°C and 45°C, respectively) and thermal stability (half-life of 2.5 min and 12 min at 45°C, respectively). The neutral trehalase, but not the acid trehalase, is activated in vitro by cAMP-dependent phosphorylation, stimulated by Ca²⁺, and inhibited by EDTA. It shows maximal activity at germination and decreases as growth proceeds. In contrast the activity of the acid trehalase is totally repressed in glucose-grown cultures and increases upon exhaustion of the carbon source, and is strongly induced by extracellular trehalose.     

Identification of GH15 Family Thermophilic Archaeal Trehalases That Function within a Narrow Acidic-pH Range

Two glucoamylase-like genes, TVN1315 and Ta0286, from the archaea Thermoplasma volcanium and T. acidophilum, respectively, were expressed in Escherichia coli. The gene products, TVN1315 and Ta0286, were identified as archaeal trehalases. These trehalases belong to the CAZy database family GH15, although they have putative (α/α)₆ barrel catalytic domain structures similar to those of GH37 and GH65 family trehalases from other organisms. These newly identified trehalases function within a narrow range of acidic pH values (pH 3.2 to 4.0) and at high temperatures (50 to 60°C), and these enzymes display K_m values for trehalose higher than those observed for typical trehalases. These enzymes were inhibited by validamycin A; however, the inhibition constants (K_i) were higher than those of other trehalases. Three TVN1315 mutants, corresponding to E408Q, E571Q, and E408Q/E571Q mutations, showed reduced activity, suggesting that these two glutamic acid residues are involved in trehalase catalysis in a manner similar to that of glucoamylase. To date, TVN1315 and Ta0286 are the first archaeal trehalases to be identified, and this is the first report of the heterologous expression of GH15 family trehalases. The identification of these trehalases could extend our understanding of the relationships between the structure and function of GH15 family enzymes as well as glycoside hydrolase family enzymes; additionally, these enzymes provide insight into archaeal trehalose metabolism.     

昆虫几丁质合成及其调控研究前沿

几丁质合成与降解是昆虫最重要的生理过程之一。本文根据国外和作者自己的研究,综述了昆虫几丁质合成及其调控研究进展。昆虫几丁质的生物合成通路始于海藻糖,终止于几丁质,其中共有8个酶参与。目前研究最多的为海藻糖酶和几丁质合成酶。昆虫存在2个海藻糖酶基因和2个几丁质合成酶基因。可溶性海藻糖酶基因对昆虫表皮的几丁质合成影响更大,而膜结合海藻糖酶基因则主要影响中肠的几丁质合成。几丁质合成酶A主要负责表皮和气管几丁质的合成,而几丁质合成酶B则负责中肠围食膜的几丁质合成。目前,昆虫几丁质合成的调控途径主要有两种:利用RNAi技术和几丁质合成抑制剂。     

昆虫海藻糖酶的基因特性及功能研究进展

海藻糖酶(Treh)是昆虫能量代谢必不可少的一类酶, 亦是昆虫体内几丁质合成通路的第一个酶。其基因表达和酶活性直接与正常发育、 蜕皮、 变态以及繁殖等昆虫重要生理过程密切相关。目前已有多种昆虫的海藻糖酶基因被成功克隆, 从而发现昆虫海藻糖酶基因家族由多个成员组成。海藻糖酶基因所编码的蛋白大多数具有一个信号肽前导区, 部分蛋白拥有1~2个跨膜结构域, 根据是否具有跨膜结构, 可将其分为可溶性海藻糖酶(Treh1)和膜结合型海藻糖酶(Treh2)两类, 膜结合型海藻糖酶具有2个特有的标签序列, 即“PGGRFREFYYWDSY”和“QWDYPNAWPP”。海藻糖酶的主要功能是将胞外和胞内的海藻糖降解成葡萄糖, 为昆虫的生命活动提供能量。具体表现为两个方面, 一是参与昆虫几丁质合成途径, 从而调控表皮、 中肠等处的几丁质合成; 二是通过与激素的协同作用, 调控昆虫体内海藻糖和葡萄糖等糖类物质的浓度变化, 从而有效保护体内细胞的适应并渡过相应的逆境环境, 并提高其抗逆能力。鉴于海藻糖酶的重要功能, 其已成为害虫控制的潜在新靶标。不同类型海藻糖酶的功能研究及酶抑制剂的研发与应用将进一步推动害虫生物防治的发展。     

Trehalase transformation in silkworm midgut during metamorphosis

Summary The particulate trehalase from silkworm larval midgut was effectively solubilized by repeated freezing and thawing, and by incubation with snake venom and non-ionic detergents (Lubrol PX and WX and Triton X-100). With solubilization the activity was enhanced and the activation behaviour was dependent upon the developmental stage of silkworms, being highest (up to about 3-fold) at the spinning stage.When chromatographed on DEAE-cellulose columns separately, the enzyme solubilized by freezing and thawing and the soluble trehalases from feeding larval midgut were respectively eluted as single peaks, P I and P II. However, both P I and P II trehalases were demonstrated after solubilization of the particulate fraction from feeding larvae with Triton X-100, or after treatment of the midgut of spinning larvae by freezing and thawing.The apparent molecular weights of P I and P II trehalases as estimated by Sephadex G-200 chromatography were about 70,000 and 140,000, respectively. The optimum pH was 6.0 for P I and about 5.0 for P II trehalase. TheK _m values were about 1.0 mM for P I trehalase and 0.30 mM for P II trehalase.These results suggest that in feeding larval midgut there are two types of trehalase which are distinguishable from each other by intracellular localization, protein nature and kinetic properties. Furthermore, when the midgut undergoes metamorphosis, the P I enzyme found predominantly in feeding stages seems to be transformed to the P II enzyme via an intermediate form (Ppt-P II) with transitional properties.     

The Distribution of GYR- and YLP-Like Motifs in Drosophila Suggests a General Role in Cuticle Assembly and Other Protein-Protein Interactions

Background

Arthropod cuticle is composed predominantly of a self-assembling matrix of chitin and protein. Genes encoding structural cuticular proteins are remarkably abundant in arthropod genomes, yet there has been no systematic survey of conserved motifs across cuticular protein families.

Methodology/Principal Findings

Two short sequence motifs with conserved tyrosines were identified in Drosophila cuticular proteins that were similar to the GYR and YLP Interpro domains. These motifs were found in members of the CPR, Tweedle, CPF/CPFL, and (in Anopheles gambiae) CPLCG cuticular protein families, and the Dusky/Miniature family of cuticle-associated proteins. Tweedle proteins have a characteristic motif architecture that is shared with the Drosophila protein GCR1 and its orthologs in other species, suggesting that GCR1 is also cuticular. A resilin repeat, which has been shown to confer elasticity, matched one of the motifs; a number of other Drosophila proteins of unknown function exhibit a motif architecture similar to that of resilin. The motifs were also present in some proteins of the peritrophic matrix and the eggshell, suggesting molecular convergence among distinct extracellular matrices. More surprisingly, gene regulation, development, and proteolysis were statistically over-represented ontology terms for all non-cuticular matches in Drosophila. Searches against other arthropod genomes indicate that the motifs are taxonomically widespread.

Conclusions

This survey suggests a more general definition for GYR and YLP motifs and reveals their contribution to several types of extracellular matrix. They may define sites of protein interaction with DNA or other proteins, based on ontology analysis. These results can help guide experimental studies on the biochemistry of cuticle assembly.     

Diversity of the Bacterial and Fungal Microflora from the Midgut and Cuticle of Phlebotomine Sand Flies Collected in North-Western Iran

Background

Phlebotomine sand flies are the vectors of the leishmaniases, parasitic diseases caused by Leishmania spp. Little is known about the prevalence and diversity of sand fly microflora colonizing the midgut or the cuticle. Particularly, there is little information on the fungal diversity. This information is important for development of vector control strategies.

Methodology/Principal Findings

Five sand fly species: Phlebotomus papatasi, P. sergenti, P. kandelakii, P. perfiliewi and P. halepensis were caught in Bileh Savar and Kaleybar in North-Western Iran that are located in endemic foci of visceral leishmaniasis. A total of 35 specimens were processed. Bacterial and fungal strains were identified by routine microbiological methods. We characterized 39 fungal isolates from the cuticle and/or the midgut. They belong to six different genera including Penicillium (17 isolates), Aspergillus (14), Acremonium (5), Fusarium (1), Geotrichum (1) and Candida (1). We identified 33 Gram-negative bacteria: Serratia marcescens (9 isolates), Enterobacter cloacae (6), Pseudomonas fluorescens (6), Klebsiella ozaenae (4), Acinetobacter sp. (3), Escherichia coli (3), Asaia sp. (1) and Pantoea sp. (1) as well as Gram-positive bacteria Bacillus subtilis (5) and Micrococcus luteus (5) in 10 isolates.

Conclusion/Significance

Our study provides new data on the microbiotic diversity of field-collected sand flies and for the first time, evidence of the presence of Asaia sp. in sand flies. We have also found a link between physiological stages (unfed, fresh fed, semi gravid and gravid) of sand flies and number of bacteria that they carry. Interestingly Pantoea sp. and Klebsiella ozaenae have been isolated in Old World sand fly species. The presence of latter species on sand fly cuticle and in the female midgut suggests a role for this arthropod in dissemination of these pathogenic bacteria in endemic areas. Further experiments are required to clearly delineate the vectorial role (passive or active) of sand flies.     

Acid trehalase in yeasts and filamentous fungi: localization, regulation and physiological function

Yeasts and filamentous fungi are endowed with two different trehalose-hydrolysing activities, termed acid and neutral trehalases according to their optimal pH for enzymatic activity. A wealth of information already exists on fungal neutral trehalases, while data on localization, regulation and function of fungal acid trehalases have remained elusive. The gene encoding the latter enzyme has now been isolated from two yeast species and two filamentous fungi, and sequences encoding putative acid trehalase can be retrieved from available public sequences. Despite weak similarities between amino acids sequences, this type of trehalase potentially harbours either a transmembrane segment or a signal peptide at the N-terminal sequence, as deduced from domain prediction algorithms. This feature, together with the demonstration that acid trehalase from yeasts and filamentous fungi is localized at the cell surface, is consistent with its main role in the utilisation of exogenous trehalose as a carbon source. The growth on this disaccharide is in fact pretty effective in most fungi except in Saccharomyces cerevisiae. This yeast species actually exhibits a "Kluyver effect" on trehalose. Moreover, an oscillatory behaviour reminiscent of what is observed in aerobic glucose-limited continuous cultures at low dilution rate is also observed in batch growth on trehalose. Finally, the S. cerevisiae acid trehalase may also participate in the catabolism of endogenous trehalose by a mechanism that likely requires the export of the disaccharide, its extracellular hydrolysis, and the subsequent uptake of the glucose released. Based on these recent findings, we suggest to rename "acid" and "neutral" trehalases as "extracellular" and "cytosolic" trehalases, which is more adequate to describe their localization and function in the fungal cell.     

飞蝗可溶型海藻糖酶基因的序列分析及mRNA表达特性

海藻糖酶是海藻糖代谢过程中的一个关键酶, 在昆虫发育和能量调节中具有重要作用, 为进一步探讨海藻糖酶基因的功能, 本文分析了飞蝗Locusta migratoria一可溶型海藻糖酶基因的氨基酸序列并对其mRNA表达特性进行了研究。结果表明： 该海藻糖酶（TRE, GenBank登录号： FJ795020）不含有跨膜结构。系统进化树分析结果显示, 该酶与大豆蚜Aphis glycines、 豌豆蚜Acyrthosiphon pisum、 褐飞虱Nilaparvata lugens和灰飞虱Laodelphax striatella可溶型海藻糖酶具有较近的亲缘关系, 因此我们将该酶的基因命名为LmTre-1。对该基因在不同组织和发育时期表达量的荧光定量 PCR 分析表明： LmTre-1在卵发育前期、 中期的表达量都很低, 卵发育后期表达量显著提高; LmTre-1在5龄若虫和成虫被检测的组织部位中均有表达, 在体壁中的表达量最高, 其次是在脂肪体、 肌肉、 气管、 精巢及卵巢中; 5龄飞蝗刚蜕皮后LmTre-1在体壁中的表达量较高, 随着生长发育其表达量逐渐降低; LmTre-1在成虫发育期体壁中稳定高表达。LmTre-1的mRNA表达特性与几丁质合成酶1基因非常相似, 据此推测该基因可能与体壁几丁质的合成相关。本研究为深入探讨该基因的生理功能提供了重要的基础数据, 并为以海藻糖酶为杀虫靶标的农药筛选奠定实验基础。     

Regulation of soluble and membrane-bound trehalase activity and expression of the enzyme in the larval midgut of the bamboo borer Omphisa fuscidentalis

Diapausing larvae of Omphisa fuscidentalis contain soluble and membrane-bound trehalase in the midgut. Soluble trehalase activity accounts for three-fourths of the total trehalase activity in midgut homogenates. The exposure of diapausing larvae to juvenile hormone analog (JHA) induced pupation, accompanied by an increase in soluble trehalase activity at the beginning of the prepupal period. Injection of 20-hydroxyecdysone (20E) increased the level of soluble trehalase activity 5 days postinjection in a dose-dependent manner. In contrast, no increase in membrane-bound trehalase activity was observed under the same conditions. We cloned the cDNAs that encode the soluble and membrane-bound forms of trehalase in O. fuscidentalis trehalase-1 (OfTreh-1) and trehalase-2 (OfTreh-2), respectively. Treh-1 encodes a 581-aa protein while Treh-2 encodes a 648-aa protein with one putative transmembrane domain near the C-terminus. The mRNA expression level of Treh-1 was 27-fold higher than that of Treh-2 in diapausing larval midgut. Following the exposure of diapausing larvae to JHA, Treh-1 mRNA expression increased gradually until the prepupal period whereupon it increased dramatically; in contrast, the mRNA expression of Treh-2 remained at its initial level. Similarly, 20E upregulated Treh-1 expression but had no effect on Treh-2 expression. Taken together, these results suggest that an increase in the soluble trehalase activity at pupation is caused by upregulation of Treh-1 gene. Moreover, membrane-bound trehalase does not appear to be involved in the dynamic changes in the hemolymph trehalose concentration that occur during the larval-pupal transformation.     

Alpha-COPI coatomer protein is required for rough endoplasmic reticulum whorl formation in mosquito midgut epithelial cells

Background

One of the early events in midgut epithelial cells of Aedes aegypti mosquitoes is the dynamic reorganization of rough endoplasmic reticulum (RER) whorl structures coincident with the onset of blood meal digestion. Based on our previous studies showing that feeding on an amino acid meal induces TOR signaling in Ae. aegypti, we used proteomics and RNAi to functionally identify midgut epithelial cell proteins that contribute to RER whorl formation.

Methodology/Principal Findings

Adult female Ae. aegypti mosquitoes were maintained on sugar alone (unfed), or fed an amino acid meal, and then midgut epithelial cells were analyzed by electron microscopy and protein biochemistry. The size and number of RER whorls in midgut epithelial cells were found to decrease significantly after feeding, and several KDEL-containing proteins were shown to have altered expression levels. LC-MS/MS mass spectrometry was used to analyze midgut microsomal proteins isolated from unfed and amino acid fed mosquitoes, and of the 127 proteins identified, 8 were chosen as candidate whorl forming proteins. Three candidate proteins were COPI coatomer subunits (alpha, beta, beta''), all of which appeared to be present at higher levels in microsomal fractions from unfed mosquitoes. Using RNAi to knockdown alpha-COPI expression, electron microscopy revealed that both the size and number of RER whorls were dramatically reduced in unfed mosquitoes, and moreover, that extended regions of swollen RER were prevalent in fed mosquitoes. Lastly, while a deficiency in alpha-COPI had no effect on early trypsin protein synthesis or secretion 3 hr post blood meal (PBM), expression of late phase proteases at 24 hr PBM was completely blocked.

Conclusions

alpha-COPI was found to be required for the formation of RER whorls in midgut epithelial cells of unfed Aa. aegypti mosquitoes, as well as for the expression of late phase midgut proteases.