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.