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Article

Research article

J. Microbiol. Biotechnol. 2020; 30(11): 1626-1639

Published online November 28, 2020 https://doi.org/10.4014/jmb.2006.06025

Copyright © The Korean Society for Microbiology and Biotechnology.

Regulation of Innate Immune Response to Fungal Infection in Caenorhabditis elegans by SHN-1/SHANK

Lingmei Sun1*, Huirong Li2, Li Zhao2, and Kai Liao3*

1Department of Pharmacology, Medical School of Southeast University, Nanjing 210009, P.R. China
2Key Laboratory of Developmental Genes and Human Disease in Ministry of Education, Medical School, Southeast University, Nanjing 210009, P.R. China
3Department of Pathology and Pathophysiology, Medical School of Southeast University, Nanjing 210009, P.R. China

Correspondence to:L.Sun
Phone: +86-25-83272525
E-mail: sunlingmei@seu.edu.cn
K.Liao
E-mail: liaokai0706@seu.edu.cn

Received: June 18, 2020; Revised: August 14, 2020; Accepted: September 8, 2020

Abstract

In Caenorhabditis elegans, SHN-1 is the homologue of SHANK, a scaffolding protein. In this study, we determined the molecular basis for SHN-1/SHANK in the regulation of innate immune response to fungal infection. Mutation of shn-1 increased the susceptibility to Candida albicans infection and suppressed the innate immune response. After C. albicans infection for 6, 12, or 24 h, both transcriptional expression of shn-1 and SHN-1::GFP expression were increased, implying that the activated SHN-1 may mediate a protection mechanism for C. elegans against the adverse effects from fungal infection. SHN-1 acted in both the neurons and the intestine to regulate the innate immune response to fungal infection. In the neurons, GLR-1, an AMPA ionotropic glutamate receptor, was identified as the downstream target in the regulation of innate immune response to fungal infection. GLR-1 further positively affected the function of SER-7-mediated serotonin signaling and antagonized the function of DAT-1-mediated dopamine signaling in the regulation of innate immune response to fungal infection. Our study suggests the novel function of SHN-1/SHANK in the regulation of innate immune response to fungal infection. Moreover, our results also denote the crucial role of neurotransmitter signals in mediating the function of SHN-1/SHANK in regulating innate immune response to fungal infection.

Keywords: SHN-1, innate immunity, neurotransmitter, Candida albicans, Caenorhabditis elegans

Introduction

Innate immune response is an important defense mechanism in animals and human beings against microbial infection. A large amount of evidence has implied the evolutionarily conserved property of the innate immune system between vertebrates, such as mammals, and invertebrates, such as Caenorhabditis elegans [1, 2]. Upon infection, C. elegans can potentially activate an inducible innate immune system or avoid pathogens [3-5]. After infection with certain bacterial or fungal pathogens, C. elegans can exhibit a rapid innate immune response as has been observed in other organisms [6-9]. An important advantage of the C. elegans-pathogen pathogenesis system is that it can reflect the different stages of mammalian infection despite the few commonalities between the known components of C. elegans and mammalian immunity; general defense strategies, such as recognition, signaling, and response, are conserved [10, 11]. Thus, the use of C. elegans as a powerful in vivo model animal for the study of infection and innate immune response to Candida albicans has the potential to teach us about the evolutionary origins of immunity and may reveal as yet uncharacterized aspects of mammalian defenses against infection [9].

C. albicans is the most common fungal pathogen of human beings in clinical settings [12-14]. Under normal circumstances, C. albicans is harmless to human beings. However, if the immune system is weakened or competing bacterial flora are eliminated in hosts, it can invade host tissues, colonize the host gastrointestinal tract, and even lead to life-threatening infections [7, 15, 16]. After exposure to C. albicans, some putative antimicrobial genes, including abf-2, cnc-4, cnc-7, and fipr-22/23, are induced and activated [17, 18]. Several important signaling pathways, such as the insulin and p38 mitogen-activated protein kinase signaling pathways, have been identified as being required for the regulation of innate immune response to C. albicans infection [17, 19, 20]. Nevertheless, the molecular mechanisms underlying innate immune response to C. albicans infection in C. elegans remain largely unclear.

In mammals, for example, rats, SHANK proteins have important functions in mediating proper protein localization at the postsynaptic density (PSD), and they form a complex by interacting with a variety of membrane and cytoplasmic proteins, such as GPAK, a PSD protein [21, 22]. The SHANK-associated RH domain-interacting protein SHARPIN is a key regulator of immune and inflammatory responses [21]. Moreover, SHANK regulates the proper localization of receptor proteins, including glutamate receptors, that are necessary to induce T-cell activation and modulate immune function [23, 24]. In C. elegans, SHN-1 is the homologue of SHANK, a scaffolding protein containing the PDZ (PSD-95, Dlg, and ZO-1) domain [25]. Herein, we determined the molecular basis of the involvement of SHN-1/SHANK in the regulation of innate immune response to fungal infection by using the C. elegans-C. albicans pathogenesis system to understand the role of SHN-1/SHANK in the regulation of innate immunity. Our data demonstrated the tissue-specific activities of SHN-1/SHANK in neurons and in intestine in the regulation of innate immune response to fungal infection. We further identified the specific signaling cascades mediated by SHN-1/SHANK in neurons in the regulation of innate immune response to fungal infection. Our study will be helpful for understanding the molecular mechanisms underlying the role of SHN-1/SHANK in the regulation of innate immunity after pathogen infection.

Materials and Methods

Strains and Media

C. elegans was maintained on nematode growth medium (NGM, 3 g/l NaCl, 2.5 g/l polypeptone, 5 mg/l cholesterol, 1 mM CaCl2, 1 mM MgSO4, 25 mM KH2PO4, and 17 g/L agar) plates seeded with Escherichia coli OP50 at 20°C as described [26]. C. elegans strains used in this study were wild-type N2, mutants of shn-1(ok1241), egl-19(tm8983), mgl-2(tm355), dyn-1(ky51), glr-1(n2461), zag-1(ok214), dat-1(ok157), ser-7(tm1325), dop-1(vs101), and glr-1(n2461)dat-1(ok157), and transgenic strains of Ex(Pshn-1-shn-1::GFP), shn-1(ok1241)Ex(Pges-1-shn-1), shn-1(ok1241)Ex(Pmyo-2-shn-1), shn-1(ok1241)Ex(Punc-14-shn-1), Is(Pges-1-shn-1), Is(Punc-14-shn-1), Is(Punc-14-shn-1);egl-19(tm8983), Is(Punc-14-shn-1);glr-1(n2461), Is(Punc-14-glr-1), and Is(Punc-14-glr-1);ser-7(tm1325). Age-synchronous populations of C. elegans (4 days old post egg lay) were used in this study. C. albicans SC5314 (wild-type strain) was grown in liquid yeast extract-peptone-dextrose (YPD, 10 g/l yeast extract, 20 g/l peptone, 20 g/l dextrose) broth or on brain heart infusion (BHI, 37 g/l BHI) agar containing kanamycin (45 μg/ml) at 30°C. Bacteria were grown in Luria Broth (LB, 10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl).

C. elegans Survival Assay

C. elegans survival analysis was performed as previously described [27]. C. albicans wild-type strain SC5314 was seeded on the plates containing brain heart infusion (BHI) and kanamycin (45 μg/ml). Age-synchronous populations of C. elegans were washed from NGM plates with M9 buffer (3 g/l KH2PO4, 6 g/l Na2HPO4, 5 g/l NaCl, 1mM MgSO4), and then added to the center of the C. albicans lawns for 4 h at 25°C. The fungal infection was started by adding 60 animals to each plate. After that, the examined C. elegans were transferred into a single well of a tissue culture plate (Corning, Inc.) containing 2 ml of liquid medium (80% M9 buffer and 20% BHI) and kanamycin (45 μg/ml). In the assay plates, fluoro-29-deoxyuridine (FUdR, 75 mg/ml) was added to prevent the growth of progeny. C. elegans were scored for dead or live every 24 h. C. elegans would be scored as dead if no response was detected after prodding with a platinum wire. Three replicates were analyzed for each experiment.

CFU Assay of C. albicans

The number of C. albicans CFU in C. elegans was quantified as described previously [20, 28]. C. elegans were infected with C. albicans lawns for 24 h. After washing with sterile M9 buffer for five times to remove the surface C. albicans, each group of 50 C. elegans was disrupted using a homogenizer, and then plated on a YPD agar containing kanamycin (45 μg/ml), ampicillin (100 μg/ml), and streptomycin (100 μg/ml). The plates were incubated for 48 h at 37°C. C. albicans colonies were counted to determine the CFU per nematode. Ten replicates were analyzed for each experiment.

Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)

Total RNA was extracted from C. elegans according to the manufacturer’s protocol in an RNeasy Mini Kit (Qiagen). Purity and concentration of RNAs were analyzed by the ratio of OD 260/280 in a spectrophotometer. cDNA was synthesized in a 12.5 μl reaction volume containing 625 ng total RNA, 0.5 mM reverse-transcript primers, 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 20 units of ribonuclease inhibitor, and 100 units of reverse transcriptase (Takara, China). The reaction mixture was incubated at 25°C for 5 min, followed by 42°C for 60 min. The reverse transcriptase was inactivated at 70°C for 15 min. Transcriptional quantification was determined by real-time PCR in an ABI 7500 real-time PCR system using Evagreen (Biotium, USA). The putative antimicrobial genes were selected as described [18]. The final results were expressed as relative expression ratio between targeted genes including the antimicrobial genes and reference tba-1 gene encoding a tubulin protein. All reactions were performed in triplicate. The related primers for targeted genes and reference gene were shown in Table S1.

DNA Constructs and Germline Transformation

To generate entry vector carrying promoter sequence, promoter region for shn-1 gene, ges-1 gene specially expressed in the intestine, myo-2 gene specially expressed in the pharynx, or unc-14 gene specially expressed in the neurons was amplified by PCR from wild-type C. elegans genomic DNA. The promoter fragment was inserted into the GFP expression vector pPD95_77 in the sense orientation. shn-1/C33B4.3c, or glr-1/C06E1.4 cDNA was amplified by PCR, and then inserted into the corresponding entry vector carrying the shn-1, ges-1, myo-2, or unc-14 promoter sequence. The designed primers for DNA construct generation were shown in Table S2. Germline transformation was performed by coinjecting a testing DNA (10-40 μg/ml) and a marker DNA of Pdop-1::rfp or unc-119(+) (60 μg/ml) into the gonad of C. elegans as described previously [29].

Fluorescence Microscope

The transgenic strain Ex(Pshn-1-shn-1::GFP) was infected with C. albicans lawns for 0, 12, or 48 h. After washing with sterile M9 buffer for three times, 50 worms were picked randomly to detect the GFP fluorescence. We used ImageJ software to analyze the images and calculated the relative fluorescence intensity.

RNAi Assay

We fed the C. elegans with E. coli strain HT115 (DE3) expressing double-stranded RNA that is homologous to a certain gene as described [4]. E. coli HT115 (DE3) grown in LB broth containing ampicillin (100 μg/ml) at 37°C overnight was plated onto NGM plates containing ampicillin (100 μg/ml) and isopropyl 1-thio-β-D-galactopyranoside (IPTG, 5 mM). L1 larvae were placed on the RNAi plates for 2 days until C. elegans became gravid. The gravid adults were transferred to fresh RNAi-expressing bacterial lawns to let them lay eggs so as to obtain the second generation of RNAi population. The eggs were allowed to develop into C. elegans for the subsequent assays. The RNAi efficiency was confirmed by qRT-PCR (data not shown).

Statistical Analysis

Data in the present study were presented as means ± standard deviation (SD). Graphs were prepared using Microsoft Excel software (Microsoft Corp., USA). Statistical analysis was performed using SPSS 12.0 software (SPSS Inc., USA). Differences between groups were determined using analysis of variance (ANOVA), and probability levels of 0.05 and 0.01 were considered statistically significant. In the C. elegans survival assay, the Kaplan-Meier method was used to calculate survival fractions and a statistically significant p value was calculated using a log-rank test.

Results

Effect of C. albicans Infection on shn-1 Expression

We first investigated the effect of C. albicans SC5314 infection on the transcriptional expression of shn-1. After C. albicans infection for 6 h, we observed a significant increase in the transcriptional expression of shn-1 (Fig. 1A). The increase in the transcriptional expression of shn-1 could be further detected in wild-type C. elegans after C. albicans infection for 12 or 24 h (Fig. 1A). By contrast, the transcriptional expression of shn-1 in wild-type C. elegans after C. albicans infection for 48 h was significantly decreased relative to that at pre-infection (Fig. 1A).

Figure 1. Response of shn-1 to C. albicans infection. (A) Effect of C. albicans infection on transcriptional expression of shn-1. The reactions were performed in triplicate. (B) Effect of C. albicans infection on expression of SHN-1::GFP. Arrowheads indicate the neurons. Pharynx (*) and intestine (**) were also indicated. Thirty animals were examined. Bars represent mean SD. **p < 0.01 vs 0 h.

In C. elegans, SHN-1 is expressed in various tissues, including neurons, the pharynx, and the intestine [25]. By using the transgenic strain Ex(Pshn-1-shn-1::GFP), we further revealed that SHN-1::GFP in neurons, the pharynx, and the intestine was significantly increased after C. albicans infection for 12 h (Fig. 1B). By contrast, after C. albicans infection for 48 h, the expression of SHN-1::GFP in neurons, the pharynx, or the intestine was significantly decreased (Fig. 1B).

Loss-of-Function Mutation of shn-1 Suppressed Innate Immune Response to C. albicans Infection

Under normal conditions, the loss-of-function mutation of shn-1 does not affect longevity (Fig. 2A). However, after C. albicans infection, the loss-of-function mutation of shn-1 caused the significant decrease in the survival of C. elegans (Fig. 2A). Meanwhile, the loss-of-function mutation of shn-1 increased the number of colony-forming units (CFU) of C. albicans in C. elegans (Fig. 2B). Therefore, the loss-of-function mutation of shn-1 might increase susceptibility to C. albicans infection.

Figure 2. Function of SHN-1 in the regulation of innate immune response to C. albicans infection. (A) Effect of shn-1 mutation on survival in C. albicans-infected C. elegans. Statistical comparison of the survival plots indicates that, after C. albicans infection, the survival of shn-1(ok1241) mutant was significantly different from that of wild-type C. elegans (p < 0.0001). Under normal conditions, the survival of shn-1(ok1241) mutant was not significantly different from that of wild-type C. elegans (p = 0.9875). Three replicates were performed. (B) Effect of shn-1 mutation on CFU of C. albicans in C. elegans. C. elegans were infected with C. albicans lawns for 24 h. (C) Effects of shn-1 mutation on expression levels of putative antimicrobial genes in C. albicans-infected C. elegans. C. elegans were infected with C. albicans lawns for 24 h. The reactions were performed in triplicate. Bars represent mean ± SD. **p < 0.01 vs wild-type.

We further utilized abf-2, cnc-4, cnc-7, and fipr-22/23 as putative antimicrobial genes to investigate the potential effect of the loss-of-function mutation of shn-1 on innate immune response to C. albicans infection. After C. albicans infection, the putative antimicrobial genes abf-2, cnc-4, cnc-7, and fipr-22/23 were induced in wild-type C. elegans [18]. Under normal conditions, the loss-of-function mutation of shn-1 did not significantly influence the expression levels of the examined putative antimicrobial genes (Fig. 2C). After C. albicans infection for 24 h, although the loss-of-function mutation of shn-1 did not affect the transcriptional expression of abf-2, we observed a significant reduction in the expression levels of cnc-4, cnc-7, and fipr-22/23 in the C. albicans-infected shn-1 mutant compared with those in the C. albicans-infected wild-type C. elegans (Fig. 2C). These results suggested that SHN-1 might be required for the regulation of innate immune response to fungal infection.

Tissue-Specific Activities of SHN-1 in the Regulation of Innate Immune Response to C. albicans Infection

We further examined the tissue-specific activities of SHN-1 in the regulation of innate immune response to fungal infection. After C. albicans infection, the intestinal or neuronal expression of shn-1 significantly increased the survival of the shn-1(ok1241) mutant and decreased the CFU of C. albicans in the shn-1(ok1241) mutant (Figs. 3A and 3B). By contrast, after C. albicans infection, the expression of shn-1 in the pharynx did not significantly affect the survival and CFU of C. albicans in the shn-1(ok1241) mutant (Figs. 3A and 3B).

Figure 3. Tissue-specific activities of SHN-1 in the regulation of innate immunity. (A) Tissue-specific activities of SHN-1 in the regulation of survival in C. albicans-infected C. elegans. Statistical comparisons of the survival plots indicate that, after C. albicans infection, the survival of shn-1(ok1241)Ex(Pges-1-shn-1) (p < 0.0001) or shn-1(ok1241)Ex(Punc-14-shn-1) (p < 0.0001) was significantly different from that of shn-1(ok1241), and the survival of shn-1(ok1241)Ex(Pmyo-2-shn-1) (p = 0.9719) was not significantly different from that of shn-1(ok1241). Three replicates were performed. (B) Tissue-specific activities of SHN-1 in the regulation of CFU of C. albicans in C. elegans. C. elegans were infected with C. albicans lawns for 24 h. (C) Tissuespecific activities of SHN-1 in the regulation of expression levels of putative antimicrobial genes in C. albicans-infected C. elegans. C. elegans were infected with C. albicans lawns for 24 h. The reactions were performed in triplicate. Bars represent mean ± SD. **p < 0.01 vs wild-type (if not specially indicated).

Considering the fact that the shn-1 mutant could decrease the expression levels of cnc-4, cnc-7, and fipr-22/23 in C. albicans-infected C. elegans as indicated above, we further selected these putative antimicrobial genes to examine the tissue-specific activities of SHN-1 in the regulation of innate immune response to C. albicans infection. After C. albicans infection, the intestinal or neuronal expression of shn-1 significantly increased the expression levels of cnc-4, cnc-7, and fipr-22/23 in the shn-1(ok1241) mutant (Fig. 3C). By contrast, after C. albicans infection, the expression of shn-1 in the pharynx did not significantly influence the expression levels of cnc-4, cnc-7, and fipr-22/23 in the shn-1(ok1241) mutant (Fig. 3C). These results suggested that SHN-1 could act in the intestine and neurons to regulate innate immune response to C. albicans infection.

Neuronal or Intestinal Overexpression of SHN-1-Enhanced Antifungal Immunity against C. albicans Infection

On the basis of the identified tissue-specific activities of SHN-1 in the regulation of innate immune response to fungal infection, we next examined the effect of the neuronal or intestinal overexpression of SHN-1 on innate immune response to C. albicans infection. Under normal conditions, the overexpression of intestinal or neuronal SHN-1 did not affect the longevity of C. elegans (Fig. 4A). After C. albicans infection, the neuronal or intestinal overexpression of SHN-1 increased survival and decreased the CFU of C. albicans in C. elegans (Figs. 4B and 4C). With the aid of putative antimicrobial genes, we further found that the neuronal or intestinal overexpression of SHN-1 significantly increased the expression levels of cnc-4, cnc-7, and fipr-22/23 after C. albicans infection, although the neuronal or intestinal overexpression of SHN-1 did not obviously influence the expression level of abf-2 after C. albicans infection (Fig. 4C). Under normal conditions, the neuronal or intestinal overexpression of SHN-1 did not significantly influence the expression levels of the examined putative antimicrobial genes (Fig. 4C). Therefore, the neuronal or intestinal overexpression of SHN-1 might enhance antifungal immunity to fungal infection.

Figure 4. Effect of neuronal or intestinal overexpression of SHN-1 on innate immune response to C. albicans infection. (A) Effect of neuronal or intestinal overexpression of SHN-1 on survival in C. albicans-infected C. elegans. Statistical comparisons of the survival plots indicate that, after C. albicans infection, the survival of C. elegans with shn-1 overexpression in the neurons or the intestine was significantly different from that of wild-type (p < 0.0001). Three replicates were performed. (B) Effect of neuronal or intestinal overexpression of SHN-1 on CFU of C. albicans in C. elegans. C. elegans were infected with C. albicans lawns for 24 h. (C) Effect of neuronal or intestinal overexpression of SHN-1 on expression levels of putative antimicrobial genes in C. albicans-infected C. elegans. C. elegans were infected with C. albicans lawns for 24 h. The reactions were performed in triplicate. Bars represent mean ± SD. **p < 0.01 vs wild-type.

Identification of the Potential Downstream Targets of SHN-1 in the Regulation of Innate Immune Response to C. albicans Infection

In mammals, more than 30 synaptic proteins can interact with SHANK proteins [30]. We searched the homologues of these interacting proteins in C. elegans (GAP-2/SynGAP, PIX-1/PIX, ABI-1/Abi1, STN-1/syntrophin, ATN-1/ACTN, GLR-2/AMPA ionotropic glutamate receptor, EGL-8/PLC-β, EPHX-1/ARHGEF, F42H10.3/NEBL, LET-413/Densin, DYN-1/Dynamin, SPC-1/α spectrin, GLR-1/AMPA ionotropic glutamate receptor, EGL-19/voltage-gated calcium channel, and MGL-2/metabotropic glutamate receptor) as the possible candidate targets of SHN-1. Under normal conditions, the loss-of-function mutation of shn-1 did not significantly affect the expression levels of F42H10.3, gap-2, pix-1, abi-1, stn-1, atn-1, glr-2, egl-8, ephx-1, and let-413, whereas the loss-of-function mutation of shn-1 significantly decreased the expression of dyn-1, spc-1, glr-1, and egl-19 and increased the expression of mgl-2 (Fig. 5A). Moreover, after C. albicans infection, the loss-of-function mutation of shn-1 significantly decreased the expression levels of dyn-1, spc-1, glr-1, and egl-19 and increased the expression of mgl-2 (Fig. 5B). These results implied that DYN-1, SPC-1, GLR-1, EGL-19, and MGL-2 might be the downstream targets of SHN-1 in the regulation of innate immune response to C. albicans infection.

Figure 5. Identification of potential downstream targets of SHN-1 in the regulation of innate immune response to C. albicans infection. (A) Effect of shn-1 mutation on expression levels of candidate targeted genes under normal conditions. (B) Effect of shn-1 mutation on expression levels of dyn-1, spc-1, egl-19, glr-1, and mgl-2 after C. albicans infection. (C) Effect of egl-19, mgl-2, dyn-1, or glr-1 mutation on survival of C. albicans infected C. elegans. Statistical comparisons of the survival plots indicate that, after C. albicans infection, the survival of egl-19(tm8983), mgl-2(tm355), or glr-1(n2461) was significantly different from that of wild-type (p < 0.0001), and the survival of dyn-1(ky51) was not significantly different from that of wild-type (p = 0.9182). Three replicates were performed. (D) Effect of RNAi knockdown of spc-1 on survival of C. albicans infected C. elegans. L4440(RNAi), empty vector RNAi. Statistical comparison of the survival plots indicates that, after C. albicans infection, the survival of spc-1(RNAi) was not significantly different from that of L4440(RNAi). (E) Mutation of glr-1, egl-19, or mgl-2 did not affect the longevity under normal conditions. Statistical comparisons of the survival plots indicate that, under normal conditions, the survival of egl-19(tm8983) (p = 0.9486), mgl-2(tm355) (p = 0.9122), or glr-1(n2461) (p = 0.9712) was not significantly different from that of wild-type. (F) Effect of glr-1, egl-19, or mgl-2 mutation on CFU of C. albicans in C. elegans. C. elegans were infected with C. albicans lawns for 24 h. (G) Effect of glr-1, egl-19, or mgl-2 mutation on expression levels of putative antimicrobial genes in C. albicans infected C. elegans. C. elegans were infected with C. albicans lawns for 24 h. The reactions were performed in triplicate. Bars represent mean ± SD. **p < 0.01 vs wild-type.

We further determined the potential roles of DYN-1, SPC-1, GLR-1, EGL-19, and MGL-2 in the regulation of C. albicans infection. After C. albicans infection, the mutation of egl-19 or glr-1 decreased survival, and the mutation of mgl-2 increased the survival of C. elegans (Fig. 5C). By contrast, after C. albicans infection, the mutation of dyn-1 did not significantly affect survival (Fig. 5C). Meanwhile, after C. albicans infection, the RNA interference (RNAi) knockdown of spc-1 did not significantly influence survival (Fig. 5D). Under normal conditions, the lifespan of the glr-1(n2461), egl-19(tm8983), or mgl-2(tm355) mutant was similar to that of wild-type (Fig. 5E). Moreover, the mutation of egl-19 or glr-1 increased the CFU of C. albicans, whereas the mutation of mgl-2 decreased the CFU of C. albicans (Fig. 5F). With the aid of putative antimicrobial genes, we further found that the mutation of egl-19 could significantly decrease the expression levels of abf-2, cnc-4, cnc-7, and fipr-22/23 after C. albicans infection, and the mutation of glr-1 could significantly decrease the expression levels of cnc-7 and fipr-22/23 after C. albicans infection (Fig. 5G). By contrast, the mutation of mgl-2 could significantly increase the expression levels of abf-2, cnc-4, cnc-7, and fipr-22/23 after C. albicans infection (Fig. 5G). Therefore, GLR-1, EGL-19, and MGL-2 might act as the potential downstream targets of SHN-1 in the regulation of innate immune response to fungal infection.

Identification of the Downstream Targets of Neuronal SHN-1 in the Regulation of Innate Immune Response to C. albicans Infection

We further attempted to identify the downstream targets of neuronal SHN-1 in the regulation of innate immune response to fungal infection. In C. elegans, egl-19, glr-1, and mgl-2 can be expressed in neurons [31-33]. Among the dysregulation of genes induced by shn-1 mutation after C. albicans infection, we found that the neuronal overexpression of SHN-1 significantly increased the expression levels of egl-19 and glr-1 after C. albicans infection (Fig. 6A). By contrast, after C. albicans infection, the neuronal overexpression of SHN-1 did not significantly affect the expression level of mgl-2 (Fig. 6A).

Figure 6. Identification of downstream targets of neuronal SHN-1 in the regulation of innate immune response to C. albicans infection. (A) Effect of overexpression of neuronal SHN-1 on expression levels of egl-19, mgl-2, and glr-1 genes after C. albicans infection. (B) Effect of egl-19 or glr-1 mutation on survival of C. elegans overexpressing neuronal SHN-1 after C. albicans-infection. Statistical comparisons of the survival plots indicate that, after C. albicans-infection, the survival of Is(Punc-14-shn-1);glr-1(n2461) was significantly different from that of Is(Punc-14-shn-1) (p < 0.0001), and the survival of Is(Punc-14-shn-1);egl-19(tm8983) was not significantly different from that of Is(Punc-14-shn-1) (p = 0.9182). Three replicates were performed. (C) Effect of glr-1 mutation on CFU of C. albicans in C. elegans overexpressing neuronal SHN-1. C. elegans were infected with C. albicans lawns for 24 h. (G) Effect of glr-1 mutation on expression levels of putative antimicrobial genes in C. elegans overexpressing neuronal SHN-1 after C. albicans-infection. C. elegans were infected with C. albicans lawns for 24 h. The reactions were performed in triplicate. Bars represent mean ± SD. **p < 0.01 vs wild-type (if not specially indicated).

We next focused on EGL-19 and GLR-1 to examine their interaction with neuronal SHN-1 in the regulation of innate immune response to fungal infection. In the transgenic strain overexpressing neuronal SHN-1, we found that the glr-1 mutation, but not the egl-19 mutation, could significantly decrease the survival of C. albicans-infected C. elegans (Fig. 6B). Moreover, the mutation of glr-1 significantly increased the CFU of C. albicans in C. elegans overexpressing neuronal SHN-1 (Fig. 6C). Considering that the overexpression of neuronal SHN-1 could affect the expressions levels of cnc-4, cnc-7, and fipr-22/23 and glr-1 mutation could affect the expressions levels of cnc-7 and fipr-22/23, we selected cnc-7 and fipr-22/23 as putative antimicrobial genes to investigate the interaction between GLR-1 and SHN-1 in the regulation of innate immune response to fungal infection. We found that the mutation of glr-1 significantly decreased the expression levels of cnc-7 and fipr-22/23 in C. elegans overexpressing neuronal SHN-1 after C. albicans infection (Fig. 6D). Therefore, GLR-1 might act as the downstream target of neuronal SHN-1 in the regulation of C. albicans infection.

Identification of the Downstream Targets of GLR-1 in the Regulation of Innate Immune Response to C. albicans Infection

In C. elegans, SER-7, DAT-1, ZAG-1, EGL-3, DOP-1, and USP-46 might act as the downstream targets of GLR-1 in regulating different biological events [34-39]. After C. albicans infection, glr-1 mutation did not significantly affect the transcriptional expressions levels of egl-3 and usp-46 (Fig. 7A). By contrast, after C. albicans infection, glr-1 mutation significantly decreased the transcriptional expression levels of ser-7, zag-1, and dop-1, and increased the transcriptional expression of dat-1 (Fig. 7A). After C. albicans infection, we further observed that although zag-1 or dop-1 mutation did not significantly affect survival, dat-1 mutation significantly increased survival and ser-7 mutation significantly decreased survival (Fig. 7B). In C. elegans, ser-7 encodes a metabotropic serotonin receptor, and dat-1 encodes a dopamine transporter. Moreover, dat-1 mutation significantly decreased the CFU of C. albicans, whereas ser-7 mutation significantly increased the CFU of C. albicans in C. elegans (Fig. 7C). Additionally, dat-1 mutation significantly increased the expression levels of abf-2, cnc-4, and cnc-7; however, ser-7 mutation significantly decreased the expression levels of abf-2, cnc-4, cnc-7, and fipr-22/23 (Fig. 7D). These results implied that DAT-1 and SER-7 might act as the potential targets of GLR-1 in the regulation of innate immune response to fungal infection.

Figure 7. Identification of downstream targets of GLR-1 in the regulation of innate immune response to C. albicans infection. (A) Effect of glr-1 mutation on the expression levels of ser-7, dat-1, zag-1, egl-3, dop-1, and usp-46 genes after C. albicans infection. (B) Effect of zag-1, dat-1, ser-7, or dop-1 mutation on survival of C. elegans after C. albicans infection. Statistical comparisons of the survival plots indicate that, after C. albicans infection, the survival of dat-1(ok157) or ser-7(tm325) was significantly (p < 0.0001) different from that of wild-type. Three replicates were performed. (C) Effect of dat-1 or ser-7 mutation on CFU of C. albicans in C. elegans. C. elegans were infected with C. albicans lawns for 24 h. (G) Effect of dat-1 or ser-7 mutation on expression levels of putative antimicrobial genes in C. elegans after C. albicans infection. C. elegans were infected with C. albicans lawns for 24 h. The reactions were performed in triplicate. Bars represent mean ± SD. **p < 0.01 vs wild-type.

Genetic Interaction between GLR-1 and SER-7 in the Regulation of Innate Immune Response to C. albicans Infection

We generated the transgenic strain of Is(Punc-14-glr-1) overexpressing the neuronal GLR-1 to examine the genetic interaction between GLR-1 and SER-7 in the regulation of innate immune response to fungal infection. After C. albicans infection, the neuronal overexpression of GLR-1 significantly increased survival (Fig. 8A), and decreased the CFU of C. albicans in C. elegans (Fig. 8B). Additionally, after C. albicans infection, the neuronal overexpression of GLR-1 significantly increased the expression levels of cnc-7 and fipr-22/23 (Fig. 8C), whereas the overexpression of neuronal GLR-1 did not significantly affect the expression levels of abf-2 and cnc-4 (data not shown). Moreover, we found that ser-7 mutation significantly decreased survival, increased the CFU of C. albicans, and suppressed the expression levels of cnc-7 and fipr-22/23 in the transgenic strain of Is(Punc-14-glr-1) (Fig. 8). Therefore, ser-7 mutation might suppress the innate immune response of the transgenic strain of Is(Punc-14-glr-1) to fungal infection. That is, SER-7 acted as the downstream target of neuronal GLR-1 in the regulation of innate immune response to fungal infection.

Figure 8. Genetic interaction between GLR-1 and SER-7 in the regulation of innate immune response to C. albicans infection. (A) Genetic interaction between GLR-1 and SER-7 in regulating survival of C. elegans after C. albicans infection. Statistical comparisons of the survival plots indicate that, after C. albicans infection, the survival of Is(Punc-14-glr-1) was significantly (p < 0.0001) different from that of wild-type, and the survival of Is(Punc-14-glr-1);ser-7(tm1325) was significantly (p < 0.0001) different from that of Is(Punc-14-glr-1). Three replicates were performed. (B) Genetic interaction between GLR-1 and SER-7 in regulating CFU of C. albicans in C. elegans. C. elegans were infected with C. albicans lawns for 24 h. (C) Genetic interaction between GLR-1 and SER-7 in regulating expression levels of putative antimicrobial genes in C. elegans after C. albicans infection. C. elegans were infected with C. albicans lawns for 24 h. The reactions were performed in triplicate. Bars represent mean ± SD. **p < 0.01 vs wild-type (if not specially indicated).

Genetic Interaction between GLR-1 and DAT-1 in the Regulation of Innate Immune Response to C. albicans Infection

In C. elegans, dat-1 is expressed in the neurons [40]. We generated the double mutant of glr-1(n2461)dat-1(ok157) to examine the genetic interaction between GLR-1 and DAT-1 in the regulation of innate immune response to fungal infection. We found that after C. albicans infection, dat-1 mutation significantly increased survival (Fig. 9A), decreased the CFU of C. albicans (Fig. 9B), and increased the expression levels of cnc-7 and fipr-22/23 in the glr-1(n2461) mutant (Fig. 9C). Therefore, GLR-1 acted as the upstream target of DAT-1 and antagonized its function in the regulation of innate immune response to fungal infection.

Figure 9. Genetic interaction between GLR-1 and DAT-1 in the regulation of innate immune response to C. albicans infection. (A) Genetic interaction between GLR-1 and DAT-1 in regulating survival of C. elegans after C. albicans infection. Statistical comparisons of the survival plots indicate that, after C. albicans infection, the survival of glr-1(n2461)dat-1(ok157) was significantly (p < 0.0001) different from that of glr-1(n2461) mutant. Three replicates were performed. (B) Genetic interaction between GLR-1 and DAT-1 in regulating CFU of C. albicans in C. elegans. C. elegans were infected with C. albicans lawns for 24 h. (C) Genetic interaction between GLR-1 and DAT-1 in regulating expression levels of putative antimicrobial genes in C. elegans after C. albicans infection. C. elegans were infected with C. albicans lawns for 24 h. The reactions were performed in triplicate. Bars represent mean ± SD. **p < 0.01 vs wild-type (if not specially indicated).

Discussion

Previous studies have suggested the important function of SHN-1 in the regulation of defecation rhythm behavior, pharyngeal pumping, and male fertility in C. elegans [25, 41]. Meanwhile, the RNAi knockdown of shn-1 did not induce lethality or developmental abnormality [25]. In this study, we further observed that, after C. albicans infection for 6, 12, or 24 h, the transcriptional expression of shn-1 and the expression of SHN-1::GFP were significantly increased (Fig. 1). In C. elegans, the loss-of-function mutation of shn-1 increased susceptibility to C. albicans infection as indicated by the reduction in survival (Fig. 2A). Additionally, the loss-of-function mutation of shn-1 increased the CFU of C. albicans in C. elegans (Fig. 2B). These results implied that after C. albicans infection, SHN-1 might be activated to mediate a protective mechanism in C. elegans against the adverse effects of C. albicans infection. Moreover, we observed that the transcriptional expression of shn-1 and the expression of SHN-1::GFP were significantly decreased after C. albicans infection for 48 h (Fig. 1). These observations implied at least two possibilities. One possibility is that long-term C. albicans infection might cause abnormality in defecation behavior and pharyngeal pumping under induction by the reduction in SHN-1. Another possibility is that the protective function of increased SHN-1 expression might be insufficient to further counteract the adverse effects of long-term C. albicans infection.

With the aid of putative antimicrobial genes, we found that the loss-of-function mutation of shn-1 might suppress the innate immune response of C. elegans to C. albicans infection (Fig. 2C). After C. albicans infection, the shn-1 mutant exhibited the decreased expressions levels of some putative antimicrobial genes (cnc-4, cnc-7, and fipr-22/23) compared with wild-type C. elegans (Fig. 2C). Moreover, the neuronal or intestinal overexpression of SHN-1 significantly increased the expression levels of these putative antimicrobial genes after C. albicans infection (Fig. 4C). In this study, we further found that the tissue-specific activity of SHN-1 in the neurons or in the intestine was required for the regulation of innate immune response to C. albicans infection (Fig. 3). By contrast, we did not detect the function of SHN-1 in the pharynx in the regulation of innate immune response to fungal infection (Fig. 3). Previous studies have suggested that the mutations of genes encoding SHANKs are closely associated with the occurrence of ASDs, such as autism or Asperger syndrome [30, 42, 43]. Our results further implied that neuronal SHN-1/SHANK might be required for the regulation of neuronal development and innate immunity. Moreover, our results also suggested the important function of intestinal SHN-1/SHANK in the regulation of innate immune response to fungal infection.

In mammals, the SHANK protein acts as a scaffold molecule and potentially interacts with some other proteins [30]. In C. elegans, among the homologues of proteins interacting with SHANK, we found that only GLR-1, EGL-19, and MGL-2 might function as the potential downstream targets of SHN-1 in the regulation of innate immune response to C. albicans infection. After C. albicans infection, the mutation of shn-1 altered the expression levels of glr-1, egl-19, and mgl-2 (Fig. 5B). Moreover, the mutation of glr-1, egl-19, or mgl-2 induced alterations in survival, the CFU of C. albicans, and the expression levels of putative antimicrobial genes in C. albicans-infected C. elegans (Figs. 5C, 5F, and 5G). Although the mutation of shn-1 also altered the expression levels of dyn-1 and spc-1 in C. albicans-infected C. elegans (Fig. 5B), the mutation of dyn-1 or the RNAi knockdown of spc-1 did not obviously affect the survival of C. elegans after C. albicans infection (Figs. 5C and 5D).

In mammals, SHANK can mediate the proper localization of glutamate receptors [23]. Ganor et al. found that AMPA ionotropic glutamate receptor (GluR3) is expressed in high levels in normal human T cells, human T leukemia cells, and mouse anti-myelin basic protein T cells [24]. The expression of GluR3 on T cells is necessary to induce T cell activation and modulate immune function [24]. In this study, we identified GLR-1, an AMPA ionotropic glutamate receptor and homologs of GluR3 in mammals [38], as a downstream target of neuronal SHN-1 in the regulation of innate immune response to C. albicans infection (Fig. 6). Nevertheless, we found that the mutation of shn-1 did not significantly affect the expression of glr-2, which encodes another AMPA ionotropic glutamate receptor (Fig. 5A). Additionally, the neuronal overexpression of SHN-1 did not significantly influence the expression of mgl-2 after C. albicans infection (Fig. 6A). mgl-2 encodes a metabotropic glutamate receptor in C. elegans [44]. These results suggested that neuronal SHN-1 could only act upstream of a certain glutamate receptor to regulate innate immune response to fungal infection.

We identified the plasma membrane dopamine transporter DAT-1[45] and the metabotropic serotonin receptor SER-7 [46] as the potential targets of GLR-1 in GLR-1-mediated signaling, which is involved in the control of innate immune response to fungal infection. The dysfunction of dopaminergic neurotransmission has been implicated in infection with the human immunodeficiency virus [47]. The homolog of SER-7 (serotonin type 7 receptor) in mammals has been discovered in human and rat immune tissues and has an important role in immune activation [48, 49]. Serotonin affects immune regulation including innate and adaptive immune system regulation [49]. In our study, after C. albicans infection, glr-1 mutation altered the expression levels of dat-1 and ser-7, and dat-1 or ser-7 mutation obviously influenced the fungal infection and innate immune response of C. elegans (Fig. 7). During the control of innate immune response to fungal infection, GLR-1 could positively regulate the function of SER-7 given that ser-7 mutation inhibited the resistance and innate immune response of transgenic strain Is(Punc-14-glr-1) to fungal infection (Fig. 8). Moreover, GLR-1 could also negatively regulate the function of DAT-1, given that dat-1 mutation suppressed susceptibility and increased the innate immune response of the glr-1 mutant to fungal infection (Fig. 9). Therefore, GLR-1-mediated serotonin signaling might regulate innate immune response of animals to fungal infection by differentially affecting the functions of SER-7-mediated serotonin signaling and DAT-1-mediated dopamine signaling.

In this study, although MGL-2 did not act as the downstream target of neuronal SHN-1 in the regulation of innate immune response to fungal infection (Fig. 6A), MGL-2 might function as an important downstream target of intestinal SHN-1 in regulating innate immune response to fungal infection. That is, neuronal SHN-1 and intestinal SHN-1 might regulate innate immune response to fungal infection via a different glutamate receptor.

In conclusion, here, we examined the role of SHN-1/SHANK in the regulation of innate immune response to fungal infection and its underlying molecular mechanism. Our results suggested that the mutation of shn-1 increased susceptibility to fungal infection and decreased innate immune response to fungal infection. Transcriptional expression of shn-1 and the expression of SHN-1::GFP were increased after C. albicans infection for 6, 12, or 24 h. In C. elegans, SHN-1 could act in the neurons and intestine to regulate innate immune response to fungal infection. In the neurons, we identified a signaling cascade of SHN-1/SHANK-GLR-1-SER-7/DAT-1 in the regulation of innate immune response to fungal infection (Fig. 10). Our results suggested the important function of activated SHN-1 in the neurons or the intestine in protecting C. elegans from the adverse effects of fungal infection. Given the conservation of general defense strategies, this study also will help us understand human innate immunity against fungal infection.

Figure 10. A diagram showing the molecular basis for SHN-1 in the regulation of innate immune response to fungal infection.

Supplemental Materials

Acknowledgments

This work was supported by grants from National Natural Science Foundation of China (81302814 and 81703574), Jiangsu Province Natural Science Foundation (BK20130640 and BK20140624), and the Fundamental Research Funds for the Central Universities.

Conflict of Interest

The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Response of shn-1 to C. albicans infection. (A) Effect of C. albicans infection on transcriptional expression of shn-1. The reactions were performed in triplicate. (B) Effect of C. albicans infection on expression of SHN-1::GFP. Arrowheads indicate the neurons. Pharynx (*) and intestine (**) were also indicated. Thirty animals were examined. Bars represent mean SD. **p < 0.01 vs 0 h.
Journal of Microbiology and Biotechnology 2020; 30: 1626-1639https://doi.org/10.4014/jmb.2006.06025

Fig 2.

Figure 2.Function of SHN-1 in the regulation of innate immune response to C. albicans infection. (A) Effect of shn-1 mutation on survival in C. albicans-infected C. elegans. Statistical comparison of the survival plots indicates that, after C. albicans infection, the survival of shn-1(ok1241) mutant was significantly different from that of wild-type C. elegans (p < 0.0001). Under normal conditions, the survival of shn-1(ok1241) mutant was not significantly different from that of wild-type C. elegans (p = 0.9875). Three replicates were performed. (B) Effect of shn-1 mutation on CFU of C. albicans in C. elegans. C. elegans were infected with C. albicans lawns for 24 h. (C) Effects of shn-1 mutation on expression levels of putative antimicrobial genes in C. albicans-infected C. elegans. C. elegans were infected with C. albicans lawns for 24 h. The reactions were performed in triplicate. Bars represent mean ± SD. **p < 0.01 vs wild-type.
Journal of Microbiology and Biotechnology 2020; 30: 1626-1639https://doi.org/10.4014/jmb.2006.06025

Fig 3.

Figure 3.Tissue-specific activities of SHN-1 in the regulation of innate immunity. (A) Tissue-specific activities of SHN-1 in the regulation of survival in C. albicans-infected C. elegans. Statistical comparisons of the survival plots indicate that, after C. albicans infection, the survival of shn-1(ok1241)Ex(Pges-1-shn-1) (p < 0.0001) or shn-1(ok1241)Ex(Punc-14-shn-1) (p < 0.0001) was significantly different from that of shn-1(ok1241), and the survival of shn-1(ok1241)Ex(Pmyo-2-shn-1) (p = 0.9719) was not significantly different from that of shn-1(ok1241). Three replicates were performed. (B) Tissue-specific activities of SHN-1 in the regulation of CFU of C. albicans in C. elegans. C. elegans were infected with C. albicans lawns for 24 h. (C) Tissuespecific activities of SHN-1 in the regulation of expression levels of putative antimicrobial genes in C. albicans-infected C. elegans. C. elegans were infected with C. albicans lawns for 24 h. The reactions were performed in triplicate. Bars represent mean ± SD. **p < 0.01 vs wild-type (if not specially indicated).
Journal of Microbiology and Biotechnology 2020; 30: 1626-1639https://doi.org/10.4014/jmb.2006.06025

Fig 4.

Figure 4.Effect of neuronal or intestinal overexpression of SHN-1 on innate immune response to C. albicans infection. (A) Effect of neuronal or intestinal overexpression of SHN-1 on survival in C. albicans-infected C. elegans. Statistical comparisons of the survival plots indicate that, after C. albicans infection, the survival of C. elegans with shn-1 overexpression in the neurons or the intestine was significantly different from that of wild-type (p < 0.0001). Three replicates were performed. (B) Effect of neuronal or intestinal overexpression of SHN-1 on CFU of C. albicans in C. elegans. C. elegans were infected with C. albicans lawns for 24 h. (C) Effect of neuronal or intestinal overexpression of SHN-1 on expression levels of putative antimicrobial genes in C. albicans-infected C. elegans. C. elegans were infected with C. albicans lawns for 24 h. The reactions were performed in triplicate. Bars represent mean ± SD. **p < 0.01 vs wild-type.
Journal of Microbiology and Biotechnology 2020; 30: 1626-1639https://doi.org/10.4014/jmb.2006.06025

Fig 5.

Figure 5.Identification of potential downstream targets of SHN-1 in the regulation of innate immune response to C. albicans infection. (A) Effect of shn-1 mutation on expression levels of candidate targeted genes under normal conditions. (B) Effect of shn-1 mutation on expression levels of dyn-1, spc-1, egl-19, glr-1, and mgl-2 after C. albicans infection. (C) Effect of egl-19, mgl-2, dyn-1, or glr-1 mutation on survival of C. albicans infected C. elegans. Statistical comparisons of the survival plots indicate that, after C. albicans infection, the survival of egl-19(tm8983), mgl-2(tm355), or glr-1(n2461) was significantly different from that of wild-type (p < 0.0001), and the survival of dyn-1(ky51) was not significantly different from that of wild-type (p = 0.9182). Three replicates were performed. (D) Effect of RNAi knockdown of spc-1 on survival of C. albicans infected C. elegans. L4440(RNAi), empty vector RNAi. Statistical comparison of the survival plots indicates that, after C. albicans infection, the survival of spc-1(RNAi) was not significantly different from that of L4440(RNAi). (E) Mutation of glr-1, egl-19, or mgl-2 did not affect the longevity under normal conditions. Statistical comparisons of the survival plots indicate that, under normal conditions, the survival of egl-19(tm8983) (p = 0.9486), mgl-2(tm355) (p = 0.9122), or glr-1(n2461) (p = 0.9712) was not significantly different from that of wild-type. (F) Effect of glr-1, egl-19, or mgl-2 mutation on CFU of C. albicans in C. elegans. C. elegans were infected with C. albicans lawns for 24 h. (G) Effect of glr-1, egl-19, or mgl-2 mutation on expression levels of putative antimicrobial genes in C. albicans infected C. elegans. C. elegans were infected with C. albicans lawns for 24 h. The reactions were performed in triplicate. Bars represent mean ± SD. **p < 0.01 vs wild-type.
Journal of Microbiology and Biotechnology 2020; 30: 1626-1639https://doi.org/10.4014/jmb.2006.06025

Fig 6.

Figure 6.Identification of downstream targets of neuronal SHN-1 in the regulation of innate immune response to C. albicans infection. (A) Effect of overexpression of neuronal SHN-1 on expression levels of egl-19, mgl-2, and glr-1 genes after C. albicans infection. (B) Effect of egl-19 or glr-1 mutation on survival of C. elegans overexpressing neuronal SHN-1 after C. albicans-infection. Statistical comparisons of the survival plots indicate that, after C. albicans-infection, the survival of Is(Punc-14-shn-1);glr-1(n2461) was significantly different from that of Is(Punc-14-shn-1) (p < 0.0001), and the survival of Is(Punc-14-shn-1);egl-19(tm8983) was not significantly different from that of Is(Punc-14-shn-1) (p = 0.9182). Three replicates were performed. (C) Effect of glr-1 mutation on CFU of C. albicans in C. elegans overexpressing neuronal SHN-1. C. elegans were infected with C. albicans lawns for 24 h. (G) Effect of glr-1 mutation on expression levels of putative antimicrobial genes in C. elegans overexpressing neuronal SHN-1 after C. albicans-infection. C. elegans were infected with C. albicans lawns for 24 h. The reactions were performed in triplicate. Bars represent mean ± SD. **p < 0.01 vs wild-type (if not specially indicated).
Journal of Microbiology and Biotechnology 2020; 30: 1626-1639https://doi.org/10.4014/jmb.2006.06025

Fig 7.

Figure 7.Identification of downstream targets of GLR-1 in the regulation of innate immune response to C. albicans infection. (A) Effect of glr-1 mutation on the expression levels of ser-7, dat-1, zag-1, egl-3, dop-1, and usp-46 genes after C. albicans infection. (B) Effect of zag-1, dat-1, ser-7, or dop-1 mutation on survival of C. elegans after C. albicans infection. Statistical comparisons of the survival plots indicate that, after C. albicans infection, the survival of dat-1(ok157) or ser-7(tm325) was significantly (p < 0.0001) different from that of wild-type. Three replicates were performed. (C) Effect of dat-1 or ser-7 mutation on CFU of C. albicans in C. elegans. C. elegans were infected with C. albicans lawns for 24 h. (G) Effect of dat-1 or ser-7 mutation on expression levels of putative antimicrobial genes in C. elegans after C. albicans infection. C. elegans were infected with C. albicans lawns for 24 h. The reactions were performed in triplicate. Bars represent mean ± SD. **p < 0.01 vs wild-type.
Journal of Microbiology and Biotechnology 2020; 30: 1626-1639https://doi.org/10.4014/jmb.2006.06025

Fig 8.

Figure 8.Genetic interaction between GLR-1 and SER-7 in the regulation of innate immune response to C. albicans infection. (A) Genetic interaction between GLR-1 and SER-7 in regulating survival of C. elegans after C. albicans infection. Statistical comparisons of the survival plots indicate that, after C. albicans infection, the survival of Is(Punc-14-glr-1) was significantly (p < 0.0001) different from that of wild-type, and the survival of Is(Punc-14-glr-1);ser-7(tm1325) was significantly (p < 0.0001) different from that of Is(Punc-14-glr-1). Three replicates were performed. (B) Genetic interaction between GLR-1 and SER-7 in regulating CFU of C. albicans in C. elegans. C. elegans were infected with C. albicans lawns for 24 h. (C) Genetic interaction between GLR-1 and SER-7 in regulating expression levels of putative antimicrobial genes in C. elegans after C. albicans infection. C. elegans were infected with C. albicans lawns for 24 h. The reactions were performed in triplicate. Bars represent mean ± SD. **p < 0.01 vs wild-type (if not specially indicated).
Journal of Microbiology and Biotechnology 2020; 30: 1626-1639https://doi.org/10.4014/jmb.2006.06025

Fig 9.

Figure 9.Genetic interaction between GLR-1 and DAT-1 in the regulation of innate immune response to C. albicans infection. (A) Genetic interaction between GLR-1 and DAT-1 in regulating survival of C. elegans after C. albicans infection. Statistical comparisons of the survival plots indicate that, after C. albicans infection, the survival of glr-1(n2461)dat-1(ok157) was significantly (p < 0.0001) different from that of glr-1(n2461) mutant. Three replicates were performed. (B) Genetic interaction between GLR-1 and DAT-1 in regulating CFU of C. albicans in C. elegans. C. elegans were infected with C. albicans lawns for 24 h. (C) Genetic interaction between GLR-1 and DAT-1 in regulating expression levels of putative antimicrobial genes in C. elegans after C. albicans infection. C. elegans were infected with C. albicans lawns for 24 h. The reactions were performed in triplicate. Bars represent mean ± SD. **p < 0.01 vs wild-type (if not specially indicated).
Journal of Microbiology and Biotechnology 2020; 30: 1626-1639https://doi.org/10.4014/jmb.2006.06025

Fig 10.

Figure 10.A diagram showing the molecular basis for SHN-1 in the regulation of innate immune response to fungal infection.
Journal of Microbiology and Biotechnology 2020; 30: 1626-1639https://doi.org/10.4014/jmb.2006.06025

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