Determination of cAMP and protein content in dormant chlamydospore and non-dormant chlamydospore of Duddingtonia flagrans
Feng-hui Wang1,2#, Bo-bo Wang2,3#, Xiao-jun Yang4, Yi-Bo Jia2, Shu-yue Tian2, Xin Li2, Xi-chen Zhang5, Yan-ming Wei1*, Jing Zhang2*, Kui-zheng Cai2*
Gansu Agricultural University, Lanzhou 730000, China; 2. Medical College of Yan’an University, Yan’an 716000, China; 3. Yan’an Key Laboratory of Zoonotic Parasitology Laboratory, Yan’an 716000, China; 4. School of Chemistry & Chemical Engineering, Yan’an University, Yan’an, 716000, China; 5. Key Laboratory of Zoonosis Research by Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China)
# These authors contributed equally to this study.
*Correspondence: Yan-ming Wei, Gansu Agricultural University, Lanzhou 730000, China.
E-mail: weiym@gsau.edu.cn
Phone: +86-0931-7632482
Fax: +86-0931-7632482
*Correspondence: Jing Zhang, Medical College of Yan’an University, Yan’an 716000, China.
E-mail: yadxzj@163.com
Phone: 86-0911-2332299
Fax: +86-0911- 2332299
* Correspondence: Kui-zheng Cai, Medical College of Yan’an University, Yan’an 716000, China.
E-mail: ckz000@126.com
Phone: 86-0911-2650158
Fax: +86-0911-2650158
Abstract:
Duddingtonia flagrans , a nematode-eating fungus, is an effective component of animal parasitic nematode biocontrol agents. In the dried formulation, the majority of spores are in an endogenous dormant state. This study focuses on dormant chlamydospore and non-dormant chlamydospore of D. flagrans to investigate the differences in cAMP and protein content between the two types of spores. In this study, cAMP and soluble proteins were extracted from the non-dormant chlamydospore and dormant chlamydospore of D. flagrans isolates SDH035 and DH055, respectively. The cAMP Direct Immunoassay Kit and Bradford protein concentration assay kit (Coomassie brilliant blue method) were used to detect the cAMP and protein content in two types of spores. Results showed that the content of cAMP in dormant spores of both isolates was significantly higher than that in non-dormant spores (p <0.05). The protein content of dormant spores in DH055 bacteria was significantly higher than that of non-dormant spores (p <0.05). In addition, the protein content of dormant spores of the SDH035 strain was slightly higher than that of non-dormant spores, but the difference was not significant (p >0.05). The results obtained in this study provide evidence for the biochemical mechanism of chlamydospore dormancy or the germination of the nematophagous fungus D. flagrans .
Keywords : Duddingtonia flagrans ; cAMP; protein; Dormant chlamydospore; Non-dormant chlamydospore
Introduction
Duddingtonia flagrans belongs to a species of nematophagous fungi, which produces three-dimensional viscous nets to capture nematodes. At present, D. flagrans is a member ofDuddingtonia , which has no sexual reproduction stage. The fungus was first described in 1948 [1], and during culture, the mycelium expands to form an intermediate chlamydospore. The chlamydospore is usually spherical, with an inner diameter of 24–32 μm and a wall thickness of 2 μm. Wang et al. (2015, 2019) reported that immature chlamydospore was elliptical with a smooth surface and few tubercles, whereas mature spore was spherical with many tubercles on the surface [2,3]. Chlamydospores were produced from the third day, and they increased with the extension of culture time. Compared with chlamydospores, the production of conidia, which can be produced within a week of culture, was small. The chlamydospore of this bacterium has an asexual reproductive structure. Given their high stress resistance, conidia are an important form of survival in nature and a source of laboratory inoculants. Many studies have shown that D. flagranschlamydospores that are taken by animals do not reproduce in the body, but they can still survive as feces are excreted from the body. When the external environment is suitable, the spores can germinate and produce predatory structures, thereby killing larvae in feces [4-8]. Chlamydospore feeding as a feed additive to animals has been shown to be effective in reducing infectious larvae (L3) on pasture under laboratory and field conditions [9-11]. Therefore, these fungi are an important biocontrol strain for the biological control of animal parasitic nematodes. Cyclic adenosine phosphate (cAMP) is an important second messenger in cells, which plays a role in regulating physiological activities and material metabolism within cells. The content of cAMP in cells is small, which plays a variety of signal regulation and transduction roles. For eukaryotic fungi, cAMP has been shown to increase the frequency of appressorium differentiation of the entomopathogenic fungus Metarhizium anisopliae [12]. cAMP plays an important role in the growth, development, and pathogenicity of plant pathogenic fungi. cAMP also participates in the growth and development of plant pathogenic fungi, as well as regulates their pathogenicity and the germination of asexual spores [13]. Decreased intracellular cAMP levels affected the growth of Colletotrichum lagenarium and Magnaporthe grisea appressorium, which led to a decrease in fungal pathogenicity [14,15]. In C. gloeosporioides , kinase A, which is also known as cAMP-dependent protein kinase A, regulates its morphogenesis and plays an important in its pathogenicity [16]. cAMP is also required for the saprophytic germination and appressorium formation of C. gloeosporioides[17]. In Fusarium graminearum , the Ras GTPase guanine nucleotide exchange factor FgCdc25 regulates fungal development, deoxynivalenol (DON) production, and plant infection by modulating the cAMP and MAPK signaling pathways [18]. The transcription factors Tri6, Tri10, and AreA mediate DON synthesis by ammonium and cAMP signaling in F. graminearum [19,20]. Mutants deleted by the PKR regulatory subunit of cAMP-PKA in F. graminearum had severe defects, but they often produced spontaneous suppressors [18]. The cAMP/protein kinase A (cAMP/PKA) signaling pathway has been well studied because of its conserved and crucial roles in the pathogenesis and development of human pathogenic fungi [21-27]. In Candida albicans , their ability to switch between yeast and hyphal forms is regulated by multiple signaling cascades, including cAMP/PKA and other signaling pathways [28]. A protein kinase has two “inactivate” catalytic subunits in C. albicans , each bound to a regulatory subunit. In addition, environmental cues such as amino acids can trigger the cAMP/PKA signaling pathway by activating adenylyl cyclase to convert ATP to cAMP [21, 24]. Teasaponin can inhibit filamentation and biofilm formation by reducing the intracellular cAMP level in C. alicense [29]. To date, there have been no reports on the content of cAMP in the nematophagous fungi D. flagrans .
Fungal dormancy is the most sophisticated specific state that ensures the preservation of life under the action of unfavorable factors. Spore dormancy can be divided into endogenous dormancy and exogenous dormancy. Endogenous dormancy refers to the fact that the germination of spores is mainly influenced by internal factors such as their own permeability, self-inhibitory factors, and metabolic damage. The presence of water is not sufficient for spore germination in the case of endogenous dormancy, as this process is under the control of the cAMP system and other special autoinhibitory compounds, thereby delaying the exit of the spore from the dormant state [30]. Exogenous dormancy indicates that spore germination is mainly affected by external conditions such as temperature, humidity, oxygen, nutrients, environmental inhibitors, and symbiotic microorganisms. This dormancy is disrupted when the effect of an unfavorable factor ceases. During biological control preparations with the pachyspore of D. flagrans as the effective component, most of the spores are in endogenous dormant state. The dormant spores have a long shelf life when stored in a dry environment, which is conducive to the transportation, preservation, and promotion of the final product. The fungal cells in the state of biosis and the dormant cells are different in chemical composition. This difference is primarily reflected in the different contents of sugars such as trehalose, lipid, and triglycerides. Nitrogen-containing compounds such as amino acids and their derivatives have been less studied in spore dormancy and germination than non-reducing disaccharides. This study focuses on D. flagranschlamydospores and dormant chlamydospores as the research objects, aiming to explore the differences in cAMP and protein content between the two types of spores. The result of this study provides a basis for revealing the biochemical mechanism of the dormancy of the chlamydospore structure of this bacterium.