Regular ArticlesThe glycoside hydrolase 18 family chitinases are associated with development and virulence in the mosquito pathogen Pythium guiyangense
Introduction
Chitins, the second most abundant carbohydrate polymers only to cellulose in nature, exist in a wide range of aquatic and terrestrial organisms including viruses, bacteria, fungi, insects, plants and animals (McGowan and Fitzpatrick, 2017). They are the major structural components of exoskeleton of arthropods, insect peritrophic matrix, fungal cell wall and eggshell of nematodes (Berini et al., 2018). Chitinases (EC 3.2.1.14) are chitinolytic enzymes, characterized for hydrolyzing the β-1,4 linkage of chitin, which are most commonly spread across two families, 18 and 19 of glycoside hydrolases (GH) as described in CAZy (Carbohydrate-Active Enzymes) database and also includes small number of GH23 and GH48 (Adrangi and Faramarzi, 2013). These enzymes have received an increasing attention because of their biotechnological applications leading to a considerable amount of recent research which has provided comprehensive insight into their numerous biological roles (Adrangi et al., 2010). Bacteria on average are known to produce between two and four chitinases for supplying nitrogen and carbon as nutrients sources (Hamid et al., 2013). Unlike bacteria, filamentous fungi contain chitin as a major intrinsic cell wall component, and accordingly encode multiple (10 to 25) GH18 chitinases to participate in spore germination, tip growth, branching of hyphae and spore differentiation (Adams, 2004). Most entomopathogenic fungi also secrete extracellular chitinases as virulence determinant factors by degrading the insect’s chitin-containing cuticle as part of host defense mechanisms during infection of insects (Berini et al., 2018, Hamid et al., 2013, Hartl et al., 2012, Horner et al., 2012). The oomycete represents a class of filamentous eukaryotes whose cell walls has none or very small amounts of chitin, however significant animal and plant pathogens encode GH18 family to breakdown chitin in host cell walls (McGowan and Fitzpatrick, 2017). Mounting evidence also demonstrate that chitinases participate in remodeling processes during the molting and the hatching of larvae from the eggshell (Adam et al., 1996) and is involved in early embryonic development (Badariotti et al., 2007).
Pythium guiyangense X.Q. Su was discovered from Guizhou, China. It classically fell into the genus Pythium within kingdom Straminopila and phylum Oomycota (Su, 2006). Previous investigations revealed that it was highly infectious to a wide range of mosquito larvae, including several important infectious disease vectors, such as Aedes aegypti, Ae. Albopictus, Culex pipens quinquefasciatus, Cx. pipiens pallens, and Anopheles sinensis (Yu et al., 2008). To invade these hosts, zoospores of P. guiyangense need to adhere to the exoskeleton of mosquito larvae and penetrate cuticle to establish the infection process (Shen et al., 2019). Many studies on entomopathogenic fungi have led to the conclusion that a combination of physical force and secreted enzymes such as proteases and chitinases work synergistically to break down host cuticle and enable successful penetration (Aw and Hue, 2017, Scholte et al., 2004, St Leger and Wang, 2010). Chitin is a major component of the mosquito cuticle, constituting up to 40% of the dry mass on the mosquito species (Merzendorfer and Zimoch, 2003). Therefore, chitinases produced by mosquito pathogens play crucial role in successful infection of hosts. For example, the Chi2 gene from entomopathogenic fungi Metarhizium anisopliae and Bbchit1 from Beauveria bassiana, which both encode for endochitinases, are crucial for the penetration of the host cuticle and that over-expression of Bbchit1 increases the virulence of B. bassiana for aphids (Fang et al., 2005). However, the functions of GH18 family from P. guiyangense on mosquito invasion are still unknown because of limited data.
This study systematically identified and characterized GH18 chitinases in the genome of P. guiyangense, and studied the phylogenetic relationships, evolutionary dynamics of chitinases with other oomycetes and fungal pathogens. Biological functions of these chitinases on growth, development, stress response and virulence were further investigated in P. guiyangense. Our study provides valuable insight for understanding molecular mechanisms of chitinases in P. guiyangense and other entomopathogenic pathogens.
Section snippets
P. guiyangense strain and mosquito source
The P. guiyangense Su strain obtained from Dr. Xiaoqing Su (Guiyang Medical University, Guiyang, China) and Nanjing laboratory strain of Cx. Pipienspallens (Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, China) were used for conducting experiments. Both strains were maintained in the laboratory as described previously (Wang et al., 2019).
Bioinformatics analysis of GH18 chitinases
The genomes of P. guiyangense (Shen et al., 2019) and P. insidiosum (Rujirawat et al., 2018) were retrieved from GenBank. The genomes
Identification of GH18 chitinase genes
The identification of GH18 chitinase gene family was performed from 11 oomycete genomes to investigate the diversity and evolution of GH18 chitinase genes across oomycetes. GH18 chitinase genes were present in all the detected oomycetes, but the gene number varied across species (Fig. 1A). A total of 6 GH18 chitinase genes were identified in the P. guiyangense genome and were named as Pgchi1 to Pgchi6 for subsequent analysis. Sequence analysis revealed that two genes of each pair among 6
Discussion
The GH18 family of chitinases represents an ancient gene family with physiological, nutritional, pathogenesis and defense functions in a wide range of living kingdoms (Berini et al., 2018, Huang et al., 2012). Although many reports have extensively highlighted the importance of chitinases in microorganisms (fungi and bacteria), little is known in oomycete pathogens. In this study, we systematically predicted GH18 chitinases in the representative oomycetes, and 10 of the 11 detected oomycete
Declaration of Competing Interest
None declared.
Acknowledgements
This research was supported by grants from National Natural Science Foundation of China, China (31770157 to AX; 31625023 to DD) and Special Fund for Agro-scientific Research in the Public Interest, China (201503112 to DD).
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These authors contributed equally to this work.