Essential Role of Chitinase in the Development of the Filarial Nematode Acanthocheilonema viteae

Abstract

Chitinases of pathogens have been proposed as potential targets of vaccines or specific inhibitors. We studied the genomic organization, transcript levels, developmental expression, and biological function of chitinases in the rodent filarial nematode Acanthocheilonema viteae, a model organism for human-pathogenic filarial worms. Characterization of nine genomic clones from an A. viteae phage library and Southern blot experiments revealed the existence of three different chitinase genes, two of which could theoretically yield functional transcripts. The deduced proteins of these genes had the common modular organization of family 18 chitinases. Northern blot experiments and rapid amplification of cDNA ends-PCR with adult worms and larval stages showed that only one gene is expressed, with high variation in transcript levels, as determined by real-time PCR. Chitinase transcript levels were lowest in the late male stage 4 larva (L4) and peaked in the stage 3 larva (L3), which was corroborated by Western blotting. RNA interference (RNAi) experiments showed that treatment of L3 and adult female worms with double-stranded RNA of chitinase inhibited molting of L3 worms and hatching of microfilariae. RNAi also led to the death of 50% of female worms, revealing the essential role of chitinase in the life cycle of filarial nematodes.Filarial nematodes are the causative agents of filariases, tropical diseases that afflict about 160 million people worldwide (27). Currently, there are neither safe and efficient drugs nor vaccines available to eliminate or prevent these infections, which makes the development of new control strategies a priority. Chitin, one of the most abundant polysaccharides in nature, is known to be present in the eggshell (7,14,26) and the microfilarial sheath (9) of nematodes and is an integral part of their pharynx (24, 30). Because chitin has not been found in vertebrates, enzymes associated with chitin metabolism might lend themselves as targets for the development of antihelminthic drugs and vaccines.Chitin is metabolized consecutively by two types of glycoside hydrolases, as follows: chitinase breaks down the ␤-1,4-glycosidic bonds of chitin to release N-acetylglucosamine dimers, which are then acted upon by N-acetylglucosaminidase. Enzymes of the glycosyltransferase family are responsible for chitin synthesis.Chitinases, in particular, have been proposed to have a role in remodeling processes during the molting of filariae and in the hatching of larvae from the eggshell (3, 5). However, the existence of large families of chitinases in the free-living nematode Caenorhabditis elegans suggests that these enzymes also fulfill other functions (17). The genome of this nematode contains 42 predicted glycoside hydrolase genes, comprising chitinase and N-acetylglucosaminidase genes (Carbohydrate-Active Enzymes server at http://afmb.cnrs-mrs.fr/CAZY). This raises questions regarding the large number of chitinases in nematodes and what particular roles these enzymes play in the worm's life cycle.Filarial ch...

Repositioning of an existing drug for the neglected tropical disease Onchocerciasis

“…Aside from a potential role of chitinase in ecdysis, chitin has also been found as a major component of the eggshells of O. volvulus (35). In a related filarial nematode, A. vitaea, which forms its eggshells in a similar manner, interference with chitinase activity was found to impact the L3 to L4 molt and led to the death of 50% of female worms (43). Affecting the female worms is a particularly advantageous approach as the female worm can live for up to 15 years, and continues to propagate its eggs and offspring, thus keeping the vicious lifecycle intact.…”

“…Although OvCHT1's exact metabolic role is not known, it was found to be expressed only in the infective L3 larvae with potential involvement in host transmission, molting, and important developmental processes in the parasite (14). Similarly, the A. vitae chitinase was shown to be essential for the molting process using RNAi (16). Yet, chitin has so far not been identified as a component of the cuticle of nematodes, and even for well-studied nematodes, such as C. elegans, it has only been described as a component of the pharynx (17).…”

“…Yet, chitin has so far not been identified as a component of the cuticle of nematodes, and even for well-studied nematodes, such as C. elegans, it has only been described as a component of the pharynx (17). Immunoelectron microscopy analysis has detected chitinase in the pharyngeal glands of L3 of A. viteae (16) and O. volvulus (14), structures that are proposed to contain a wide variety of proteins essential for the remodeling processes during molting and the final step of ecdysis, shedding of the old cuticle. In addition, chitinases are also found in developing eggs in utero and in microfilariae, thus, these enzymes may be appropriate targets for other developmental stages and other filarial parasites.…”

Identification of candidate infection genes from the model entomopathogenic nematode Heterorhabditis bacteriophora

“…Within H. bacteriophora , the presence of a chitinase seems obligate since the digestion of chitin would be necessary upon infection of a host insect. However, given its presence across several parasitic nematodes, it is possible a secreted chitinase may serve a more general role in parasitism [35]. …”

Nucleic acid transfection and transgenesis in parasitic nematodes

“…Very promising results have been obtained with filariae (Aboobaker and Blaxter, 2003; Lustigman et al 2004; Ford et al 2005, 2009; Pfarr et al 2006; Tachu et al 2008) and there have been some positive technical advances recently (Song et al 2010; Samarasinghe et al 2011). However, overall, a relatively small number of RNAi- sensitive targets have been interrogated successfully, and a relatively low efficiency in target knockdown and the consequent infrequency of informative phenotypes have hampered application of RNAi in many species of animal parasitic nematodes.…”