Chitin in egg shells of Onchocerca gibsoni and Onchocerca volvulus

doi:10.1016/0166-6851(87)90090-9

Molecular and Biochemical Parasitology

Volume 25, Issue 3, October 1987, Pages 267-272

https://doi.org/10.1016/0166-6851(87)90090-9 Get rights and content

Abstract

Chemical analysis of adult females of Onchocerca gibsoni gave estimated chitin contents of 200–500 μg (g dry weight)⁻¹. Egg shells from both O. gibsoni and Onchocerca volvulus stained with Calcofluor white and with fluorescent wheat germ agglutinin as shown by fluorescent light microscopy, and bound gold-labelled wheat germ agglutinin as shown by electron microscopy, under conditions specific for chitin. The egg shells appeared as single electron dense layers from 50 to 85 nm in thickness. Purified chitinase digested these egg shells, leaving coiled microfilariae unattacked. We conclude that chitin is a major component of the egg shells.

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Cited by (34)

Versatile glycoside hydrolase family 18 chitinases for fungi ingestion and reproduction in the pinewood nematode Bursaphelenchus xylophilus
2016, International Journal for Parasitology
Citation Excerpt :
In nematodes, chitin has long been regarded as a component of the eggshell; the chitin forms a layer that provides mechanical support for the oval embryo (Schierenberg and Junkersdorf, 1992; Edgar, 1995). In particular, chitinases have been proposed to have a role in formation of the chitinous eggshell and egg hatching (Brydon et al., 1987). Soaking eggs of plant parasitic nematodes in dsRNA solution has been demonstrated as an efficient means to knock down genes that are highly expressed in eggs (Fanelli et al., 2005; Dalzell et al., 2010).
The glycoside hydrolase family 18 (GH18) of chitinases is a gene family widely expressed in archaes, prokaryotes and eukaryotes, and hydrolyzes the β-1,4-linkages in chitin. The pinewood nematode Bursaphelenchus xylophilus is one of the organisms that produces GH18 chitinases. Notably, B. xylophilus has a higher number of GH18 chitinases compared with the obligate plant-parasitic nematodes Meloidogyne incognita and Meloidogyne hapla. In this study, seven GH18 chitinases were identified and cloned from B. xylophilus based on genomic analyses. The deduced amino acid sequences of all these genes contained an N-terminal signal peptide and a GH18 catalytic domain. Phylogenetic analysis showed that the origin of B. xylophilus GH18 chitinases was independent of those from fungi and bacteria. Real-time quantitative reverse transcription PCR analysis indicated that GH18 chitinase genes had discrete expression patterns, representing almost all the life stages of B. xylophilus. In situ hybridisation showed that the mRNA of GH18 chitinase genes of B. xylophilus were detected mainly in the spermatheca, esophageal gland cells, seminal vesicle and eggs. RNA interference (RNAi) results revealed different roles of GH18 chitinase genes in B. xylophilus. Bx-chi-1, Bx-chi-2 and Bx-chi-7 were associated with reproduction, fungal cell-wall degradation and egg hatching, respectively. Bx-chi-5 and Bx-chi-6 may be involved in sperm metabolism. In conclusion, this study demonstrates that GH18 chitinases have multiple functions in the life cycle of B. xylophilus.
The presence of α-chitin in Tardigrada with comments on chitin in the Ecdysozoa
2016, Zoologischer Anzeiger
We used Fourier Transform Infrared Spectroscopy (FT-IR) to characterize for the first time chitin in the cuticle of a eutardigrade (Macrobiotus cf. hufelandi). Analysis of the isolated cuticles of single individuals and comparison with commercial α-chitin isolated from shrimp shell and β-chitin from squid pen revealed that the amide I band was split into two peaks characteristic for α-chitin. In the current literature cuticles containing α-chitin are considered as an apomorphic character of the Ecdysozoa (Cycloneuralia, Panarthropoda). This is a plausible assumption, although α-chitin has been unequivocally demonstrated only in the cuticle of the Panarthropoda, i.e. Onychophora, Tardigrada (this article) and Arthropoda, and in the Priapulida (Cycloneuralia), whereas chitin in the cuticle of the other cycloneuralian taxa either was not further specified or appears to be absent.
Isolation and characterization of endochitinase and exochitinase of Setaria cervi
2015, Parasitology International
Citation Excerpt :
Therefore, identification of new molecular targets and the characterization of parasite specific proteins/enzyme are of considerable importance to improve the treatment and control strategies against filariasis. Chitin (linear poly β-1,4-N-acetyl-d-glucosamine) is one of the most abundant polysaccharide in nature and can be found mainly in the cuticles, egg-shell [3–5], microfilarial sheath [6] and pharynx [7,8] of nematode parasites. The chitin metabolism has been proposed to be a parasite unique drug target, as the chitin has not been found in vertebrates [9,10].
Chitin metabolism has been shown to have a role in the development of parasitic nematodes including filarial parasites and the enzymes associated with chitin metabolism have been considered as potential vaccine and drug target. Chitinases are members of the enzyme superfamily of glycoside hydrolases, which are characterized by the ability to hydrolyze glycosidic bonds in chitin chain by either an endolytic or an exolytic mechanism. In the present study, we have demonstrated the chitinase (exochitinase and endochitinase) activity in different stages of Setaria cervi (bovine filarial parasite) and have also purified and characterized the endochitinase from microfilarial stage of the parasite. The chitinase activity has been detected in adult and microfilarial stages of S. cervi using the fluorescent substrates. The S. cervi adult stage was found to have high activity of exochitinase (28.72 ± 0.25 nmol/min/mg) while microfilarial stage showed high activity of endochitinase (24.40 ± 0.25 nmol/min/mg). Native polyacrylamide gel electrophoresis, followed by staining of enzyme activity with fluorescent substrates, revealed single isoenzymic form of exochitinase in adults and endochitinase in microfilariae of S. cervi. The endochitinase from S. cervi microfilariae was purified employing chitin affinity matrix and DEAE-Sephacel ion-exchange chromatography. The enzyme was purified about 55 fold with an enzyme recovery of 22.33%. The purified enzyme exhibited a doublet of protein bands on SDS-PAGE at 65–70 kDa. The closantel (chitinase inhibitor) strongly inhibited the enzyme activity of S. cervi microfilariae endochitinase with a Ki value of 4.3 ± 0.18 μM.
High prevalence of chitotriosidase deficiency in Peruvian Amerindians exposed to chitin-bearing food and enteroparasites
2014, Carbohydrate Polymers
Citation Excerpt :
In Quechua-Cusco community we found predominantly cases of Entamoeba histolytica and Ascaris lumbricoides. The main chitin-bearing parasites (based on published literature (Arroyo-Begovich, Carabez-Trejo, & de la Torre, 1980; Brydon, Gooday, Chappell, & King, 1987; Harrington, 2008; Lanuza, Carbajal, & Borrás, 1996; Wimmer, Schmid, Tag, & Hofer, 1998; Zhang, Foster, Nelson, Ma, & Carlow, 2005) present in the sample population were the protozoans Giardia, Entamoeba histolytica, and the macro-parasites Ancylostoma/necator, Ascaris, Enterobius vermicularis and H. nana. In contrast, lower parasite prevalence of mainly protozoan parasites characterizes the control population of urbanized Peruvians.
The human genome encodes a gene for an enzymatically active chitinase (CHIT1) located in a single copy on Chromosome 1, which is highly expressed by activated macrophages and in other cells of the innate immune response. Several dysfunctional mutations are known in CHIT1, including a 24-bp duplication in Exon 10 causing catalytic deficiency. This duplication is a common variant conserved in many human populations, except in West and South Africans. Thus it has been proposed that human migration out of Africa and the consequent reduction of exposure to chitin from environmental factors may have enabled the conservation of dysfunctional mutations in human chitinases. Our data obtained from 85 indigenous Amerindians from Peru, representative of populations characterized by high prevalence of chitin-bearing enteroparasites and intense entomophagy, reveal a very high frequency of the 24-bp duplication (47.06%), and of other single nucleotide polymorphisms which are known to partially affect enzymatic activity (G102S: 42.7% and A442G/V: 25.5%). Our finding is in line with a founder effect, but appears to confute our previous hypothesis of a protective role against parasite infection and sustains the discussion on the redundancy of chitinolytic function.
Cuticular chitin synthase and chitinase mRNA of whiteleg shrimp Litopenaeus vannamei during the molting cycle
2012, Aquaculture
Citation Excerpt :
World annual bioproduction of chitin was estimated at 100 × 109 t, being an important source of carbon for heterotrophic bacteria (Cauchie, 2002; Tharanathan and Kittur, 2003). Chitinous structures are found in several taxa as well as the cell wall of fungi (Roncero, 2002), egg shells of nematodes (Brydon et al., 1987) and exoskeleton and peritrophic matrix of insects (Merzendorfer and Zimoch, 2003) and crustaceans (Roer and Dillaman, 1984). Thus, the study of chitin metabolism is of biotechnological, ecological, and physiological relevance.
Chitin metabolism is of high relevance for shrimp growth because it has to be synthesized and cleaved in each molt. We studied chitin synthase and chitinase mRNAs from whiteleg shrimp Litopenaeus vannamei. For this, cDNA coding for cuticular chitin synthase (LvChS) and chitinase isoenzymes (LvChi1, LvChi2 and LvChi3) was amplified, sequenced and identified. In a qualitative analysis, LvChi1 and LvChi3 were detected only in the hepatopancreas and are probably involved in digestion of food chitin. LvChi2 transcript was found in pleopods, uropods, gills, eyestalk, and digestive tube; LvChi2 is likely to be involved in the hydrolysis of chitin from the exoskeleton and peritrophic membrane, but not in food chitin digestion. LvChS was found widely distributed in the organism, including the hepatopancreas. Thus, it seems to be involved in synthesis of chitin to build the exoskeleton and also the peritrophic membrane. Relative expression of LvChS and LvChi2 genes was evaluated by quantitative RT-PCR in the integument. These transcripts had a varying pattern of abundance during the molt cycle, based on the need of shrimp to synthesize or hydrolyze chitin from exoskeleton.
Chitin Biochemistry: Synthesis, Hydrolysis and Inhibition
2010, Advances in Insect Physiology
Citation Excerpt :
Along with its deacetylated form, chitosan, chitin is an important component of cell wall and septum structures as well as in spore formation in yeasts and most filamentous fungi. Chitin is present in calcified layers of mollusc shells and squid pens (Nagahama et al., 2008; Poulicek et al., 1986), in egg shell of nematodes (Brydon et al., 1987; Fuhrman and Piessens, 1985; Zhang et al., 2005) as well as in eukaryotic unicellular organisms like centric diatoms (Herth and Zugemaier, 1977, 1979), certain amoebae cysts (Arroyo-Begovich and Carabez-Trego, 1982; Van Dellen et al., 2006; Ward et al., 1985) and in protozoan parasites (Lanuza et al., 1996). Interestingly, chitin was found to be deposited in the unicellular alga, Chlorella, infected by a chlorovirus containing a gene encoding for a chitin polymerizing enzyme (Kawasaki et al., 2002).
Chitin is an abundant extracellular amino-sugar polymer that in association with other external biomacromolecules (carbohydrates, proteins) is an essential structural component in forming cell walls in fungi as well as exoskeletons and peritrophic membranes in arthropods. Chitin synthesis and degradation in insects are cyclical dynamic events, and since being crucial for facilitating growth and development, are strictly coordinated and regulated. Chitin formation and hydrolysis are complex, multi-faceted, interconnected and highly harmonized intracellular and extracellular cascades of biochemical and biophysical transformations. The list of processes, many of which are still poorly understood, include transcriptional, translational and post-translational modifications relative to the catalytic machinery; cellular movement of clustered catalytic units to and their eventual fixed spatial integration into plasma membranes; chitin polymerization and translocation of polymers across the cell membrane barrier; coalescence of nascent external chitin chains forming crystallites that subsequently associate with assorted proteins to form fibrillar supramacromolecular structures. Cloned and sequenced genes encoding the anabolic chitin synthase, and the catabolic chitinase, β-N-acetylglucosaminidase and chitin deacetylase combined with purification and characterization of the expressed enzymes as well as crystallographic and inhibition studies were instrumental in elucidating the intricate mechanisms of catalysis relative to chitin polymerization and degradation. The powerful tools of RNAi knockout methodologies were harnessed for functional analysis of a variety of chitin-related genes. Chitin research carries practical aspect as the physiologically crucial synthesis and hydrolysis of the polymer have been regarded as suitable, attractive and largely selective targets for interference and for producing effective pharmaceuticals and safe biopesticides.

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Molecular and Biochemical Parasitology

Abstract

References (20)

A rapid method for the chemical estimation of filamentous fungi in plant tissue

Physiol. Plant. Pathol.

Chitin and yeast budding

J. Biol. Chem.

Chitin synthesis and sheath morphogenesis in Brugia malayi microfilariae

Mol. Biochem. Parasitology

Structure and chemistry of the egg shell of a nematode (Trichuris suum)

Tissue and Cell

Enzymes regulating glucosamine 6-phosphate synthesis in the zygote of Ascaris suum

Int. J. Parasitol.

Surface components of Onchocerca volvulus

Mol. Biochem. Parasitol.

Identification of Onchocerca

Bull. WHO

Filaria volvulus Leuckart, its distribution, structure and pathological effects

Parasitology

Investigations into the morphology and life-history of Onchocerca gibsoni

Aust. Inst. Trop. Med. Report for 1911

The microbial synthesis of cellulose, chitin and chitosan

Prog. Ind. Microbiol.

Cited by (34)

Versatile glycoside hydrolase family 18 chitinases for fungi ingestion and reproduction in the pinewood nematode Bursaphelenchus xylophilus

The presence of α-chitin in Tardigrada with comments on chitin in the Ecdysozoa

Isolation and characterization of endochitinase and exochitinase of Setaria cervi

High prevalence of chitotriosidase deficiency in Peruvian Amerindians exposed to chitin-bearing food and enteroparasites

Cuticular chitin synthase and chitinase mRNA of whiteleg shrimp Litopenaeus vannamei during the molting cycle

Chitin Biochemistry: Synthesis, Hydrolysis and Inhibition