Next Article in Journal
From Microbial Ecology to Clinical Challenges: The Respiratory Microbiome’s Role in Antibiotic Resistance
Previous Article in Journal
Borrelia burgdorferi Strain-Specific Differences in Mouse Infectivity and Pathology
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Pathogens
Volume 14
Issue 4
10.3390/pathogens14040354
pathogens-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Echinococcus multilocularis Calreticulin Inhibits Lectin Pathway of Complement Activation by Directly Binding to Mannose-Binding Lectin

by
Yuxiao Shao
1,
Meng Xia
1,
Yinghui Song
1,
Yan Yan
1,
Xiaofang Dong
1,
Haoran Zong
1,
Bin Zhan
2,
Yanhai Wang
3,* and
Limei Zhao
1,*
1
Department of Pathogenic Biology, School of Basic Medical Sciences and Forensic Medicine, Baotou Medical College, Baotou 014040, China
2
Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX 77030, USA
3
Parasitology Research Laboratory, School of Life Sciences, Xiamen University, Xiamen 361102, China
*
Authors to whom correspondence should be addressed.
Pathogens 2025, 14(4), 354; https://doi.org/10.3390/pathogens14040354
Submission received: 4 March 2025 / Revised: 30 March 2025 / Accepted: 3 April 2025 / Published: 5 April 2025
(This article belongs to the Special Issue Immunity and Immunoregulation in Helminth Infections)
Browse Figures
Review Reports Versions Notes

Abstract

:
Alveolar Echinococcosis (AE) is a serious zoonotic disease caused by infection of Echinococcus multilocularis larvae. To survive within the host, E. multilocularis has developed a complex immune evasion mechanism including the inhibition of complement activation. This study focused on a calreticulin secreted by E. multilocularis (EmCRT) and its role in binding ability to human MBL and inhibiting MBL-mannose-mediated lectin pathway of complement activation. Results demonstrated the binding of recombinant EmCRT protein to both external and natural MBL in serum and the subsequent inhibition of MBL-mannose-initiated lectin pathway reflected by the reduced formation of complement intermediate products C3b and C4b. Fragment mapping determined that the MBL binding site was located within the S-domain of EmCRT. Combining with its role in inhibiting C1q-initiated classical complement activation in our previous study, the inhibition of MBL-mannose-initiated lectin pathway identified in this study suggests EmCRT plays an important role in the immune evasion of E. multilocularis alveolar larvae against host complement attack as a survival strategy within human tissue. This study supports the approach of using EmCRT as a good candidate for vaccine and drug development against E. multilocularis infection.

1. Introduction

Echinococcosis is a severe zoonotic disease caused by infection with the larval stage of the Echinococcus tapeworm. There are two types of echinococcosis: cystic echinococcosis (CE) caused by infection with Echinococcus granulosus and alveolar echinococcosis (AE) caused by infection with Echinococcus multilocularis [1,2]. Both AE and CE are serious diseases. Due to infiltrative growth characteristics of the larvae of E. multilocularis, cases of AE in humans and animals are more serious and difficult to treat than CE. Therefore, AE is also known as “malignant echinococcosis” or “worm cancer”. Human infections with E. multilocularis are usually caused by accidental ingestion of parasite eggs, which develop into larvae within the human body and migrate to the liver to form foci. The infection with E. multilocularis takes a heavy toll on the health and well-being of humans and their domestic livestock, responsible for infecting 29,600 individuals each year and causing nearly 17,000 mortalities worldwide. Cosmopolitan distribution of this ailments had led to losses of 3 billion dollars annually [3,4,5].
Tissue-dwelling E. multilocularis larvae in infected humans are exposed to a hostile immune microenvironment. As a survival strategy, the parasitic larvae have developed a relatively complex immune evasion mechanism. Understanding the immune evasion mechanism of E. multilocularis larvae in host could facilitate the development of vaccines or drugs to control the infection of Echinococcus and echinococcosis.
Parasitic helminths have evolutionally developed different levels of immune evasion mechanisms; one of them is to regulate the host immune system through the secreting of functional immunomodulatory proteins [6,7]. Since the complement system is the first line of innate immunity, playing a vital role in the immune defense against pathogen infections [8], helminths develop comprehensive strategies to inhibit or block host complement recognition and activation during invasion [9,10].
The complement system contains a collection of inactive components in the blood and tissues that become activated in response to infection and initiate the immune attack to the invaded pathogens [11]. There are three complement activation pathways: classical, lectin, and alternative [12,13]. The classical pathway is triggered by antigen–antibody immune complexes binding to C1q. The lectin pathway is initiated by the binding of mannose-binding lectin (MBL) to bacterial surfaces with mannose-containing polysaccharides (mannans) leading to the formation of the MBL/MASP-1/MASP-2 tri-molecular complex and subsequent cleavage of C4 into C4a and C4b [14]. Ultimately, three activation pathways lead to the formation of membrane attack complex (MAC) that lyses and kills the pathogen [7]. In addition, MBL is also involved in the regulation of phagocytosis, mediating cytotoxicity and apoptosis, and triggering the release of pro-inflammatory factors as well as the production of reactive oxygen species ROS as other means to kill invaded pathogens [15,16].
Calreticulin (CRT) is a multifunctional protein with its structure containing a globular N-terminal, proline-rich P, and acidic C-terminal domains with different functions associated with cell adhesion, calcium storage, and phagocytosis of apoptotic cells [17]. The N and P domains play a chaperone role in the endoplasmic reticulum, while the C-domain owns Ca2+ binding activity [18]. It has been demonstrated that CRT was conservatively expressed in helminth parasites with a strong regulating function on the human complement system as a strategy to evade attack by a complement-initiated innate immune response [19]. Our previous study has identified tissue-dwelling nematode Trichinella spiralis-produced CRT (Ts-CRT) strongly bound to human complement component C1q to inhibit C1q-initiated complement classical activation pathway [9]. The C1q-binding CRT was also identified in Haemonchus contortus, Trypanosoma cruzi, Necator americanus, and Brugia malayi [20,21,22,23], indicating CRT is a conserved protein secreted by different helminths to defend the complement-involved host first line immune attack. Further study identified that the C1q-binding site of Ts-CRT was located in the S-domain between the N and P domains (135–288 aa) [10]. In our previous study, we also determined that E. multilocularis-derived CRT (EmCRT) was also able to bind on human C1q to inhibit C1q-mediated complement activation and C1q-dependent non-complement activation of immune cells [24].
Except for the C1q-binding capacity to interfere with C1q-initiated classical pathway of complement activation, CRTs produced by some other parasites such as Trypanosoma cruzi [25] and T. spiralis [26] have also been shown to be able to inhibit the activation of the lectin pathway by binding to lectin pathway activation molecules such as MBL or ficolin as an alternative approach to inhibiting complement activation in order to survive in the host.
In this study, we investigated whether EmCRT was able to bind to MBL to inhibit lectin pathway of complement activation as another approach to defending complement-derived innate immune attack in addition to inhibiting C1q-initiated classic activation pathway. Our results showed that EmCRT was able to inhibit the activation of the complement lectin pathway by binding to human MBL, and the MBL binding region was mainly located in the S domain of EmCRT. The results identified in this study further confirm that E. multilocularis-expressed CRT plays a key role in evading the host immune response and facilitating parasite survival within the host.

2. Materials and Methods

2.1. Serum

Normal human sera (NHS) were obtained from five healthy volunteers with signed consent under the protocol reviewed and approved by the Institutional Review Board (IRB) of Baotou Medical College (approval number: 2018026). Collected NHS was used as source of complement. C1q-depleted sera (C1qD) were purchased from Quidel (San Diego, CA, USA).

2.2. Expression of Recombinant Proteins of Full-Length EmCRT and Different Fragments

The DNA coding for the full-length EmCRT without signal peptide (18–395 aa) and the different domains [EmCRT-N (18–167 aa), EmCRT-P (168–325 aa), EmCRT-NP (18–325 aa), EmCRT-C (326–395 aa), and EmCRT-S (140–292 aa)] were originally amplified from the total cDNA of E. multilocularis metacestode [27] and cloned into prokaryotic expression vector pET-28a (Novagen, Darmstadt, Germany). The correctly cloned recombinant plasmids were transformed into BL21(DE3)-competent cells (TIANGEN, Beijing, China). The recombinant proteins with six His tags at N-terminus were expressed under induction of 1 mM IPTG overnight at 25 °C and purified with Ni2+ affinity chromatography (Beyotime Biotechnology, Shanghai, China), as described previously [24,26]. The concentration of purified recombinant proteins was determined by BCA protein concentration assay kit (Solarbio, Beijing, China).

2.3. EmCRT Binding to MBL

ELISA: To determine the binding ability of recombinant protein EmCRT (rEmCRT) to MBL, the 96-well plates were coated with different amounts of MBL (0, 0.5, 1, 2, 4, 8, 12, 15 μg/mL) (Sinobiological, Beijing, China) in 100 μL coating buffer (100 mM NaHCO3/Na2CO3, pH 9.6), and the control plates were coated with the same amount of bovine serum albumin (BSA). The wells were washed with 200 μL of 1 × PBST (1 × PBS + 0.05% Tween-20) and blocked with 200 μL of 3% BSA at 37 °C for 1 h. After another round of washing with PBST, 100 μL of binding buffer (20 mM Tris-HCl, 50 mM NaCl, 10 mM CaCl2, 0.05% Triton-X 100, pH 7.4) containing different amounts of rEmCRT (0, 0.2, 0.4, 1.2, 1.5 μM) was added to the wells for binding at 4 °C overnight. After being washed for three times, mouse anti-His-tag monoclonal antibody (BOSTER Biological Technology, Wuhan, China) at 1:5000 dilution in PBS was added into each well as the primary antibody, and HRP conjugated goat anti-mouse IgG (1:10,000, BOSTER Biological Technology, Wuhan, China) as the secondary antibody to detect the binding of rEmCRT to MBL. The chromogenic substrate TMB (Beyotime Biotechnology, Shanghai, China) was added, and the absorbance values were detected at OD450 with a microplate reader (Thermo Fisher, Waltham, MA, USA) after termination of the reaction.
Far western blot: Equal amounts of MBL and BSA (1 μg) were run SDS-PAGE on 12.5% gel and transferred to PVDF membrane (Merck, Darmstadt, Germany). After being blocked with 5% skimmed milk, the membrane was incubated with 15 μg/mL rEmCRT in binding buffer (20 mM Tris-HCl, 50 mM NaCl, 10 mM CaCl2, 0.05% Triton-X 100, pH 7.4, 1% skimmed milk) overnight at 4 °C. The binding of rEmCRT to MBL on the membrane was detected with mouse anti-His monoclonal antibody (1:1000, BOSTER Biological Technology, Wuhan, China) and HRP conjugated goat anti-mouse IgG (1:12,000, BOSTER Biological Technology, Wuhan, China).
To determine the specific fragment of EmCRT that binds to MBL, the same amounts of recombinant proteins of different fragments expressed in BL21(DE3) including rEmCRT-N, rEmCRT-P, rEmCRT-NP, rEmCRT-C, and rEmCRT-S were used in ELISA (0.5 µM) and Far western blot (1 µg), as described above.

2.4. Molecular Docking

Structural prediction of EmCRT 3D structure was performed using AlphaFold 3 (DeepMind, London, UK). Docking of human MBL (PDB: 1HUP) to the EmCRT model was performed using the professional protein–protein docking tool HDOCK v1.0 (Huazhong University of Science and Technology, Wuhan, Hubei).

2.5. Calcium-Binding Staining

The calcium-binding property of the purified recombinant proteins, rEmCRT, rEmCRT-N, rEmCRT-P, rEmCRT-NP, rEmCRT-C, and rEmCRT-S, were measured by staining with Stains-all (Sigma, St. Louis, MO, USA), a cationic carbocyanine dye that stains Ca2+-binding proteins as blue and non-Ca2+-binding proteins as red. BSA and non-relevant recombinant E. multilocularis ferritin (rEmFer) (expressed in the lab) were used as non-Ca2+-binding protein controls.

2.6. Effect of rEmCRT on the Binding of MBL to Mannan

Microplates were coated with 50 μg/mL mannan (Sigma, St. Louis, MO, USA) in coating buffer at pH 9.6 overnight at 4 °C, then blocked with 3% BSA at 37 °C for 2 h. Total 1 μg of MBL was pre-incubated with different amount of rEmCRT or rEmCRT-S or rEmCRT-NP (0, 0.5, 1 μM) in binding buffer (20 mM Tris-HCl, 50 mM NaCl, 10 mM CaCl2, 0.05% Triton-X 100, pH 7.4) at 37 °C for 2 h before adding into the mannan-coated plates (100 µL). BSA (1 μM) was used as a control for the MBL binding reaction. The incubation was continued overnight at 4 °C. After washing with 1 × TBST containing 5 mM CaCl2, the inhibition of MBL binding to mannan was detected with mouse anti-MBL monoclonal antibody (1:3000, R&D System, Minneapolis, MN, USA) and HRP conjugated goat anti-mouse IgG (1:5000, BOSTER Biological Technology, Wuhan, China).
To mimic the natural environment of MBL in serum and exclude the C1q-involved classical complement activation pathway, C1qD serum at 1:50 dilution in binding buffer (20 mM Tris-HCl, 50 mM NaCl, 10 mM CaCl2, 0.05% Triton-X 100, pH 7.4) was pre-incubated with different amounts of rEmCRT or rEmCRT-S or rEmCRT-NP as described above. The reduced amount of MBL from C1qD serum binding to mannan on the plates was detected with anti-MBL monoclonal antibody.

2.7. C3b/C4b Deposition Experiments

Microplates were coated with mannan (50 μg/mL) overnight at 4 °C, then blocked with 5% BSA at 37 °C for 2 h. C1qD serum at 1:50 dilution in binding buffer (20 mM Tris-HCl, 50 mM NaCl, 10 mM CaCl2, 0.05% Triton-X 100, pH 7.4) was used as a natural source of MBL and pre-incubated with different amounts of rEmCRT or rEmCRT-S or rEmCRT-NP (0, 2, 4 μM) at 37 °C for 2 h. BSA (4 μM) was used as a non-relevant protein control. Total 100 μL of the incubation mixture was added to each well coated with mannan and incubated overnight at 4 °C. After washing with 1 × TBST containing 5 mM CaCl2, the C1qD diluted at 1:150 in 1 × Veronal Buffer (VB, Lonza, Basel, Switzerland) containing 0.1% gelatin, 0.05% Tween-20 was added (100 µL) as supplement of other complement components (without C1q) into each well of the plates and incubated at 37 °C for 1 h. The lectin pathway-activated C3b/C4b deposition was detected with rabbit anti-C3b monoclonal antibody (1:1000, BOSTER Biological Technology, Wuhan, China) and goat anti-C4b polyclonal antibody (1:3000, Abcam, Cambridge, UK). The HRP conjugated goat anti-rabbit IgG (1:1000, Affinity Biosciences, Liyang, China) or rabbit anti-goat IgG (1:8000, Affinity Biosciences, Liyang, China) were used as secondary antibodies, respectively.
To mimic the natural MBL in serum, after the same coating with mannan and blocking with BSA, a 1:100 dilution of NHS in binding buffer (20 mM Tris-HCl, 50 mM NaCl, 10 mM CaCl2, 0.05% Triton-X 100, pH 7.4) was pre-incubated with different amounts of rEmCRT or rEmCRT-S or rEmCRT-NP (0, 4, 8 µM) at 37 °C for 2 h before adding into plates coated with mannan at 100 μL per well for overnight at 4 °C. BSA (8 μM) was used as control for the binding. After washing with 1 × TBST containing 5 mM CaCl2, the other components of complement were supplemented with 1:150 with NHS in 1 × Veronal Buffer (VB, Lonza, Basel, Switzerland) containing 0.1% gelatin, 0.05% Tween-20 for incubation for 1 h at 37 °C. After washing, C3b and C4b deposition was detected in the same conditions as described above.

2.8. Data Analysis

All experiments were performed in duplicate and repeated at least once, and data were shown as meant ± standard deviation. The statistical analysis of the data was performed using the two-tailed Mann–Whitney test by GraphPad Prism 5 software (GraphPad Prism, Inc, San Diego, CA, USA). p < 0.05 was considered as statistically significant difference. We tested the data for normal distribution through the Kolmogorov–Smirnov test.

3. Results

3.1. Expression and Characterization of Recombinant EmCRT and Its Fragments

The structure of EmCRT and its functional domains is shown in Figure 1A based on the results from Ts-CRT [10]. The recombinant proteins of full-length EmCRT, EmCRT-N, EmCRT-P, EmCRT-S, and EmCRT-NP were successfully expressed in E. coli BL21 (DE3) and purified with immobilized metal affinity chromatography (IMAC). The purified recombinant proteins appeared as expected in SDS-PAGE based on the sequence size and possible molecular interaction (rEmCRT-N 20 kDa, rEmCRT-P 35 kDa, rEmCRT-S 35 kDa, rEmCRT-C 20 kDa, rEmCRT-NP 50 kDa, rEmCRT 58 kDa) and were recognized by anti-His antibody (Figure 1B) [27]. The Stains-all results revealed rEmCRT-P, rEmCRT-S, rEmCRT-C, rEmCRT-NP, and rEmCRT were stained blue, indicating their calcium-binding activities, while rEmCRT-N was stained red, indicating it does not contain calcium binding domain as control recombinant protein EmFer and BSA do (Figure 1C).

3.2. Binding of rEmCRT to Human MBL

The binding activity of rEmCRT to human MBL was examined by ELISA and Far western blot. ELISA results showed that rEmCRT was able to bind to human MBL coated on the plates in a dose-dependent pattern (Figure 2A(a)), but did not bind to control BSA (Figure 2A(b)). SDS-PAGE revealed a single strand of MBL was present under reduced condition with molecular weight of 33 kDa (Figure 2B(a)). Far western blot with anti-His antibody showed rEmCRT was able to bind to MBL transferred on PVDF membrane and did not bind to the BSA (Figure 2B(b)).

3.3. Predicted Structure and MBL Binding Region of EmCRT

The 3D structure of EmCRT was predicted and constructed using AlphaFold3 prediction, and the final 3D structure of EmCRT was optimized and adjusted using PROCHECK (Figure 3A). The interaction between EmCRT and MBL was analyzed and identified that amino acid residues located within the N domain and P domain of EmCRT were involved in binding to the MBL A-chain (Figure 3B). Specifically, amino acids Gly87 and Thr89, in the N domain, and Arg176, Asp178, Thr180, Ser194, Lys205, ASP209, Gln292, Ile293, Pro294, in the P domain, and Asp333 in the C domain interacted with LysA93, GlnA96, ThrA97, AlaA100, LysA104, GlyA115, LysA117, ArgA146, AsnA147, ArgA181, ThrA184, IleA228 of the MBL A chain. Amino acids Glu223, Pro280, Asn281, Pro282, Tyr284 in the P domain interacted with LysC170, ThrC179, GlyC180, AsnC181 of MBL C chain. Amino acid Glu300 in the P domain interacted with ArgB101 of MBL B chain. The interaction between EmCRT and MBL occurs mostly by hydrogen, electrostatic, and hydrophobic bonds. The docking predicted that MBL binding region in EmCRT is mainly located between Gly87 in the N-domain and Glu300 in the P-domain (Figure 3C). The docking results are consistent to the actual binding results shown in Figure 3.

3.4. Mapping of MBL Binding Region in EmCRT

The MBL binding region on EmCRT was determined by the fragment mapping expression and MBL binding assays. ELISA with MBL coated on the plates demonstrated that the full-length EmCRT as well as fragments including rEmCRT-P, rEmCRT-S, rEmCRT-C, rEmCRT-NP were able to bind to MBL in a dose-dependent manner, with rEmCRT-S and rEmCRT-NP possessing the highest MBL binding ability similar to the level of full-length rEmCRT conferred, which is significantly higher than that of rEmCRT-P and rEmCRT-C (Figure 4A(a)). No significant binding of rEmCRT-N to MBL was observed. All rEmCRT and its fragments did not bind to control BSA (Figure 4A(b)). Far western blot results revealed that rEmCRT-S and rEmCRT-NP appeared to bind to MBL similarly to rEmCRT and significantly higher than that of rEmCRT-N, rEmCRT-P, and rEmCRT-C (Figure 4B), which was further illustrated by densitometry analysis of recognized bands with the reading of rEmCRT binding band set as 1.0 (Figure 4C). The results confirm that the MBL binding region is located in rEmCRT-S within rEmCRT-NP fragment.

3.5. Inhibition of MBL Binding to Mannan by rEmCRT and Its Binding Regions

To determine whether the binding of rEmCRT and its functional regions (rEmCRT-S and rEmCRT-NP) to MBL would interfere with the binding activity of MBL to its ligand mannan, ELISA with mannan coated on the plates was performed. The ELISA results demonstrated that rEmCRT, as well as its functional regions rEmCRT-S and rEmCRT-NP, were able to significantly inhibit the binding of MBL to mannan in a dose-dependent manner (Figure 5A). For further validation, we used C1q-depleted sera (C1qD) to mimic the natural environment of human serum containing the physiological level of MBL. It also showed that pre-incubation rEmCRT, rEmCRT-S, and rEmCRT-NP with C1qD (1:50) significantly inhibited the binding of MBL in the serum onto mannan coated on the plates at the similar level as external MBL shown in Figure 5B. The results showed that rEmCRT and the binding regions rEmCRT-S and EmCRT-NP were able to inhibit the binding of both external and physiological MBL to mannan in a dose-dependent pattern.

3.6. Inhibition of Lectin Pathway of Complement Activation by rEmCRT and Its MBL Binding Regions

To identify the effect of rEmCRT and its MBL binding regions on the activation of the complement lectin pathway, the depositions of C3b and C4b, the intermediate product of complement activation, were tested after the lectin activation pathway was initiated. At the first experiment, we used C1qD serum as physiological source of MBL to exclude the possible classical complement activation initiated by C1q. After pre-incubation with rEmCRT or rEmCRT-S or rEmCRT-NP, the C1qD serum containing MBL was added to plates coated with mannan. ELISA detected much less the formation and deposition of C3b and C4b on the plates, the intermediate complement products of MBL-mannose-initiated lectin activation pathway. The EmCRT-induced inhibition of lectin complement activation pathway was dose-dependent (Figure 6A). For further validation, we used normal human serum (NHS) as source of MBL to mimic the human physiological environment instead of using C1qD serum. The rEmCRT including rEmCRT-S and rEmCRT-NP inhibited the deposition of C3b and C4b at the similar level as shown above using C1qD serum (Figure 6B), indicating the EmCRT-triggered inhibition of complement activation is directly derived from MBL-mannose-initiated lectin pathway, not from C1q-initiated classical pathway.

4. Discussion

During the million years’ parasitism in host, helminth parasites have evolutionally developed strategies to evade host immune attack by downmodulating the host immune system through secreting a spectrum of molecules with immunomodulatory functions [28,29]. Understanding the immunomodulatory mechanism that parasitic helminths bear would not only facilitate the development of vaccines or drugs against parasite infection, but also help the design of therapeutic approaches to treat diseases of immunological disorders in humans [30,31]. Currently, the research focuses more on the activation of regulatory T cells [32] and suppressive macrophage populations that might be important in downmodulating immune responses [33]. The inhibition of complement activation has been identified as another approach for helminth to live concomitantly in the host, especially for those helminths living in the tissue, such as T. spiralis and E. multilocularis [9,24].
The complement system, an important component of innate immunity, is a highly complex biological response system involved in the direct lysis and killing of invasive pathogens, promoting phagocytosis and inflammatory response, therefore a crucial component of both the innate and adaptive immune responses [7,8]. Calreticulin (CRT) is a highly conserved Ca2+ binding protein involved in a range of biological processes such as wound healing, protein modification and folding, secretory pathway regulation, cell motility, cellular metabolism, protein synthesis, gene expression regulation, cell cycle regulation, and apoptosis [34]. It has been demonstrated that many helminths secrete calreticulin with the ability to inhibit host immune responses to facilitate the survival of the parasite within host. Among them, some helminth-expressed CRTs were able to bind to some complement components [19,35]. In our previous studies, we have identified that T. spiralis-produced CRT (Ts-CRT) could effectively bind to C1q to inhibit the C1q-initiated complement classical activation [9]. More specifically, the binding region of CRT to C1q was located within the S domain, which spans the N and P domains of Ts-CRT [10].
Recently, we identified that E. multilocularis alveolar larvae-derived CRT conferred the similar binding ability to human complement component C1q and inhibited the activation of the classical pathway of complement, as well as other non-complement-activated immune functions in which C1q is involved [24]. As a target recognition molecule of the lectin pathway, MBL is highly similar in structure and functions to C1q that initiates classical pathway [36]. A previous study confirmed that the diagnostic antigen C antigen on the surface of E. multilocularis contains a polysaccharide component, mainly galactose and N-acetylglucosamine residues [37]. These results indicate EmCRT is possibly involved in the inhibition of MBL-mannose-initiated lectin pathway of complement activation in addition to the C1q-involved classical pathway. We thus further explored the interaction of EmCRT with MBL and its role in the potential inhibition of lectin pathway of complement activation as an additional approach to evade host complement attack. In this study, we indeed demonstrated the binding of EmCRT to exogenous MBL (Figure 2) and to physical MBL in serum (Figure 5) as well, suggesting that EmCRT is able to inhibit the lectin pathway of complement through binding to MBL. Structural analysis and molecular docking of EmCRT with human MBL also exhibited the interaction and binding capacity between MBL and EmCRT (Figure 3). Further experiment confirmed that the binding of rEmCRT to external MBL or natural MBL in serum indeed inhibited the MBL-mannose-initiated lectin pathway leading to the decreased formation of complement intermediate products C3b and C4b (Figure 6).
It is a common strategy for parasites or other pathogens to produce molecules to inhibit lectin complement activation pathway. Dengue virus produced a nonstructural NS1 that is able to bind to MBL to inhibit its recognition by immune cells and the formation of the membrane-attacking complex on its membrane [38]. Candida tropicalis secreted asparagine protease 1 (SAPS1) to cleave MBL and inhibit the activation of the lectin pathway [39]. Klebsiella pneumoniae camouflaged its capsule components to avoid recognition by MBL and ficolins [40]. Similarly, flatworm Fasciola hepatica secreted a serine protease inhibitor (FhSrp) that inhibited MBL binding to its surface and the subsequent MBL-mediated activation of the lectin pathway [41]. Sarcoptes scabiei secreted SMIIPP-Ss D1 and I1, members of the protein-hydrolyzing inactive serine protease paralogues (SMIIPP-Ss) family, bound to the MBL and inhibited lectin pathway [42].
Our previous study has identified the C1q-binding site on Ts-CRT as located within the 154 amino acids of the S domain, between the N and P domains [10]. Considering the structural similarity between MBL and C1q [36], it is possible that EmCRT shares the similar binding site to C1q and MBL as well. To determine the actual MBL binding region on EmCRT, different fragments of EmCRT including EmCRT-N, EmCRT-P, EmCRT-S, EmCRT-C, and EmCRT-NP were expressed as corresponding recombinant proteins. The binding assays with different EmCRT fragments confirmed that rEmCRT-S possessed the similar MBL binding capacity to that of full-length rEmCRT and rEmCRT-NP, significantly higher than those conferred by other fragments (Figure 4). The MBL binding site within S-domain is consistent to the molecular docking analysis with strong interaction prediction between EmCRT and MBL A-chain within the amino acids between N domain and P domain of EmCRT (Figure 3B). The evidences of actual binding assays between EmCRT fragments and MBL and MBL-mannose-initiated lectin pathway assays demonstrated that EmCRT-S possesses the strongest binding capacity to MBL and highest inhibitory ability to lectin activation pathway (Figure 3, Figure 4, Figure 5 and Figure 6), further confirming the MBL binding region is located in S-domain of EmCRT.
All results revealed in this study demonstrated that E. multilocularis calreticulin was able to bind to MBL so as to block MBL-mannose-mediated lectin complement activation pathway. The MBL binding site on EmCRT is located within the S-domain of EmCRT. Combining with the inhibition of C1q-initiated classical complement activation identified in our previous study and the inhibition of MBL-mannose-initiated lectin pathway revealed in this study, we determine that EmCRT plays an important role in the immune evasion of E. multilocularis alveolar larvae against host complement attack as a survival strategy within human tissue. Thus, EmCRT could be a good candidate for vaccine and drug development against E. multilocularis infection [24,27]. This is supported by our previous study showing immunization with rEmCRT-induced protective immunity against infection of E. multilocularis in BALB/c mice. The immunological mechanism underlying the EmCRT-induced protective immunity and its potential therapeutic effect on established infection is under investigation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pathogens14040354/s1, Figure S1: (A) rEmCRT and its fragments recognized by anti-His mAb; (B) Identification of rEmCRT and its fragmentation by Stains-all. M: protein marker, 1: EmCRT-N, 2: EmCRT-P, 3: EmCRT-S, 4: EmCRT-C, 5: EmCRT-NP, 6: EmCRT, 7: EmFer, 8: BSA; Figure S2: (A) SDS-PAGE of MBL and BSA. M: protein marker, 1: MBL (1μg), 2: BSA (1 μg); (B) rEmCRT binding to human MBL by Far western blot. M: protein marker, 1: MBL (1 μg), 2: BSA (1 μg); Figure S3: (A) rEmCRT and its fragments binding to human MBL by Far western blot. M: protein marker, 1: MBL (1 μg), 2: BSA (1 μg); (B) SDS-PAGE of MBL and BSA. M: protein marker, 1: MBL (1 μg), 2: BSA (1 μg).

Author Contributions

L.Z. and Y.S. (Yuxiao Shao) designed and conceived the study. Y.S. (Yuxiao Shao) conducted the experiments. Y.S. (Yuxiao Shao), B.Z., M.X., Y.S. (Yinghui Song), Y.Y., X.D. and H.Z. analyzed the data. Y.S. (Yuxiao Shao) wrote the paper. L.Z., Y.S. (Yuxiao Shao), B.Z. and Y.W. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by grants from the National Natural Science Foundation of China (82360403), Inner Mongolia Youth Science and Technology Talent Support Program of Higher Education Institutions (NJYT23051), Inner Mongolia Natural Science Foundation (2023MS08035) and Baotou Medical College Scientific Research Fund Project (BYJJ-ZRQM 202302).

Institutional Review Board Statement

Ethics approval was attained from the Institutional Review Board Medical College (approval code: 2018026, date: 20 December 2018).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

Data included in this article are available from the corresponding author upon request.

Acknowledgments

We appreciate Phadcalc (www.phadcalc.com) for the molecular docking simulation.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Stojkovic, M.; Junghanss, T. Cystic and alveolar echinococcosis. Neuroparasitol. Trop. Neurol. 2013, 114, 327–334. [Google Scholar]
  2. Yan, W.-L.; Meng, J.-X.; Li, X.-M.; Zhao, J.-P.; Zhang, M.; Wang, X.-Y.; Sun, Y.-Z.; Ni, H.-B.; Ma, H. Global Prevalence of Echinococcosis in Goats: A Systematic Review and Meta-Analysis. Foodborne Pathog. Dis. 2022, 19, 675–685. [Google Scholar] [CrossRef] [PubMed]
  3. Wang, Z.; Wang, X.; Liu, X. Echinococcosis in China, a Review of the Epidemiology of Echinococcus spp. EcoHealth 2008, 5, 115–126. [Google Scholar] [PubMed]
  4. Eckert, J.; Deplazes, P. Biological, Epidemiological, and Clinical Aspects of Echinococcosis, a Zoonosis of Increasing Concern. Clin. Microbiol. Rev. 2004, 17, 107–135. [Google Scholar] [CrossRef]
  5. Giraudoux, P.; Raoul, F.; Afonso, E.V.E.; Ziadinov, I.; Yang, Y.; Li, L.I.; Li, T.; Quéré, J.-P.; Feng, X.; Wang, Q.; et al. Transmission ecosystems of Echinococcus multilocularisin China and Central Asia. Parasitology 2013, 140, 1655–1666. [Google Scholar] [CrossRef]
  6. McSorley, H.J.; Hewitson, J.P.; Maizels, R.M. Immunomodulation by helminth parasites: Defining mechanisms and mediators. Int. J. Parasitol. 2013, 43, 301–310. [Google Scholar] [CrossRef]
  7. Heggi, M.T.; Nour El-Din, H.T.; Morsy, D.I.; Abdelaziz, N.I.; Attia, A.S. Microbial evasion of the complement system: A continuous and evolving story. Front. Immunol. 2024, 14, 1281096. [Google Scholar] [CrossRef]
  8. West, E.E.; Kemper, C. Complosome—The intracellular complement system. Nat. Rev. Nephrol. 2023, 19, 426–439. [Google Scholar] [CrossRef]
  9. Zhao, L.; Shao, S.; Chen, Y.; Sun, X.; Sun, R.; Huang, J.; Zhan, B.; Zhu, X. Trichinella spiralis Calreticulin Binds Human Complement C1q As an Immune Evasion Strategy. Front. Immunol 2017, 8, 636. [Google Scholar] [CrossRef]
  10. Shao, S.; Hao, C.; Zhan, B.; Zhuang, Q.; Zhao, L.; Chen, Y.; Huang, J.; Zhu, X. Trichinella spiralis Calreticulin S-Domain Binds to Human Complement C1q to Interfere with C1q-Mediated Immune Functions. Front. Immunol. 2020, 11, 572326. [Google Scholar] [CrossRef]
  11. Rothschild-Rodrigueza, D.; Causerb, A.J.; Brown, F.F.; Collier-Bain, H.D.; Moore, S.; Murray, J.; Turner, J.E.; Campbell, J.P. The effects of exercise on complement system proteins in humans a systematic scoping review. Exerc. Immunol. Rev. 2022, 28, 1–35. [Google Scholar]
  12. Lambris, J.D.; Ricklin, D.; Geisbrecht, B.V. Complement evasion by human pathogens. Nat. Rev. Nephrol. 2008, 6, 132–142. [Google Scholar] [CrossRef]
  13. Wallis, R.; Mitchell, D.A.; Schmid, R.; Schwaeble, W.J.; Keeble, A.H. Paths reunited: Initiation of the classical and lectin pathways of complement activation. Immunobiology 2010, 215, 1–11. [Google Scholar] [PubMed]
  14. Noris, M.; Remuzzi, G. Overview of Complement Activation and Regulation. Semin. Nephrol. 2013, 33, 479–492. [Google Scholar] [PubMed]
  15. Kalia, N.; Singh, J.; Kaur, M. The ambiguous role of mannose-binding lectin (MBL) in human immunity. Open Med. 2021, 16, 299–310. [Google Scholar] [CrossRef]
  16. Dobó, J.; Kocsis, A.; Farkas, B.; Demeter, F.; Cervenak, L.; Gál, P. The Lectin Pathway of the Complement System—Activation, Regulation, Disease Connections and Interplay with Other (Proteolytic) Systems. Int. J. Mol. Sci. 2024, 25, 1566. [Google Scholar] [CrossRef]
  17. Michalak, M.; Corbett, E.F.; Mesaeli, N.; Nakamura, K.; Opas, M. Calreticulin: One protein, one gene, many functions. Biochem. J. 1999, 344 Pt 2, 281–292. [Google Scholar] [CrossRef]
  18. Michalak, M.; Groenendyk, J.; Szabo, E.; Gold, L.I.; Opas, M. Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum. Biochem. J. 2009, 417, 651–666. [Google Scholar]
  19. Esperante, D.; Flisser, A.; Mendlovic, F. The many faces of parasite calreticulin. Front. Immunol. 2023, 14, 1101390. [Google Scholar]
  20. Naresha, S.; Suryawanshi, A.; Agarwal, M.; Singh, B.P.; Joshi, P. Mapping the complement C1q binding site in Haemonchus contortus calreticulin. Mol. Biochem. Parasitol. 2009, 166, 42–46. [Google Scholar]
  21. Ramírez, G.; Valck, C.; Molina, M.C.; Ribeiro, C.H.; López, N.; Sánchez, G.; Ferreira, V.P.; Billetta, R.; Aguilar, L.; Maldonado, I.; et al. Trypanosoma cruzi calreticulin: A novel virulence factor that binds complement C1 on the parasite surface and promotes infectivity. Immunobiology 2011, 216, 265–273. [Google Scholar] [CrossRef] [PubMed]
  22. Kasper, G.; Brown, A.; Eberl, M.; Vallar, L.; Kieffer, N.; Berry, C.; Girdwood, K.; Eggleton, P.; Quinnell, R.; Pritchard, D.I. Pritchard, A calreticulin-like molecule from the human hookworm Necator americanus interacts with C1q and the cytoplasmic signalling domains of some integrins. Parasite Immunol. 2001, 23, 141–152. [Google Scholar] [PubMed]
  23. Kaveri, S.V.; Yadav, S.; Gupta, S.; Selvaraj, C.; Doharey, P.K.; Verma, A.; Singh, S.K.; Saxena, J.K. In Silico and In Vitro Studies on the Protein-Protein Interactions between Brugia malayi Immunomodulatory Protein Calreticulin and Human C1q. PLoS ONE 2014, 9, e106413. [Google Scholar]
  24. Xian, S.; Chen, L.; Yan, Y.; Chen, J.; Yu, G.; Shao, Y.; Zhan, B.; Wang, Y.; Zhao, L. Echinococcus multilocularis Calreticulin Interferes with C1q-mediated Complement Activation. Trop. Med. Infect. Dis. 2023, 8, 47. [Google Scholar] [CrossRef]
  25. Sosoniuk, E.; Vallejos, G.; Kenawy, H.; Gaboriaud, C.; Thielens, N.; Fujita, T.; Schwaeble, W.; Ferreira, A.; Valck, C. Trypanosoma cruzi calreticulin inhibits the complement lectin pathway activation by direct interaction with L-Ficolin. Mol. Immunol. 2014, 60, 80–85. [Google Scholar]
  26. Yi, C.; Shuai, S.; Jingjing, H.; Limei, Z.; Xinping, Z. Effect of Trichinella spiralis calreticulin on complement activation via the mannose-binding lectin pathway. Acta Parasitol. Med. Entomol. Sin. 2019, 26, 1–6. [Google Scholar]
  27. Chen, L.; Cheng, Z.; Xian, S.; Zhan, B.; Xu, Z.; Yan, Y.; Chen, J.; Wang, Y.; Zhao, L. Immunization with EmCRT-Induced Protective Immunity against Echinococcus multilocularis Infection in BALB/c Mice. Trop. Med. Infect. Dis. 2022, 7, 279. [Google Scholar] [CrossRef]
  28. Gazzinelli-Guimaraes, P.H.; Nutman, T.B. Helminth parasites and immune regulation. F1000Research 2018, 7, F1000. [Google Scholar]
  29. Hewitson, J.P.; Grainger, J.R.; Maizels, R.M. Helminth immunoregulation: The role of parasite secreted proteins in modulating host immunity. Mol. Bioche. Parasitol. 2009, 167, 1–11. [Google Scholar]
  30. Imai, S.; Fujita, K. Molecules of parasites as immunomodulatory drugs. Curr. Top. Med. Chem. 2004, 4, 539–552. [Google Scholar]
  31. Johnston, M.J.G.; MacDonald, J.A.; McKay, D.M. Parasitic helminths: A pharmacopeia of anti-inflammatory molecules. Parasitology 2008, 136, 125–147. [Google Scholar] [CrossRef] [PubMed]
  32. Taylor, M.D.; van der Werf, N.; Maizels, R.M. T cells in helminth infection: The regulators and the regulated. Trends Immunol. 2012, 33, 181–189. [Google Scholar] [CrossRef] [PubMed]
  33. Wu, Y.; Gao, H.; Yu, H.; Wang, X.; Li, H.; Jin, Q.; Zhu, X.; Li, Q.; Kong, N.; Tang, Y.; et al. Schistosoma japonicum cystatin alleviates paraquat poisoning caused acute lung injury in mice through activating regulatory macrophages. Ecotoxicol. Environ. Saf. 2024, 281, 116615. [Google Scholar] [CrossRef] [PubMed]
  34. Sawaya, A.P.; Vecin, N.M.; Burgess, J.L.; Ojeh, N.; DiBartolomeo, G.; Stone, R.C.; Pastar, I.; Tomic-Canic, M. Calreticulin: A multifunctional protein with potential therapeutic applications for chronic wounds. Front. Med. 2023, 10, 1207538. [Google Scholar] [CrossRef]
  35. Ramírez-Toloza, G.; Aguilar-Guzmán, L.; Valck, C.; Ferreira, V.P.; Ferreira, A. The Interactions of Parasite Calreticulin With Initial Complement Components: Consequences in Immunity and Virulence. Front. Immunol. 2020, 11, 1561. [Google Scholar] [CrossRef]
  36. Kjaer, T.R.; Le, L.T.M.; Pedersen, J.S.; Sander, B.; Golas, M.M.; Jensenius, J.C.; Andersen, G.R.; Thiel, S. Structural Insights into the Initiating Complex of the Lectin Pathway of Complement Activation. Structure 2015, 23, 342–351. [Google Scholar] [CrossRef]
  37. Chiaki Sato, K.F. Isolation and Characterization of a Diagnostic Polysaccharide Antigen from Larval Echinococcus multilocularis. Jpn. J. Med. Sci. Biol. 1994, 47, 65–71. [Google Scholar] [CrossRef]
  38. Thiemmeca, S.; Tamdet, C.; Punyadee, N.; Prommool, T.; Songjaeng, A.; Noisakran, S.; Puttikhunt, C.; Atkinson, J.P.; Diamond, M.S.; Ponlawat, A.; et al. Secreted NS1 Protects Dengue Virus from Mannose-Binding Lectin–Mediated Neutralization. J. Immunol. 2016, 197, 4053–4065. [Google Scholar] [CrossRef]
  39. Valand, N.; Brunt, E.; Gazioglu, O.; Yesilkaya, H.; Mitchell, D.; Horley, N.; Arroo, R.; Kishore, U.; Wallis, R.; Venkatraman Girija, U. Inactivation of the Complement Lectin Pathway by Candida tropicalis Secreted Aspartyl Protease-1. Immunobiology 2022, 227, 152263. [Google Scholar] [CrossRef]
  40. Sahly, H.; Keisari, Y.; Ofek, I. Manno(Rhamno)Biose-Containing Capsular Polysaccharides of Klebsiella pneumoniae Enhance Opsono-Stimulation of Human Polymorphonuclear Leukocytes. J. Innate. Immun. 2009, 1, 136–144. [Google Scholar] [CrossRef]
  41. Skelly, P.J.; De Marco Verissimo, C.; Jewhurst, H.L.; Dobó, J.; Gál, P.; Dalton, J.P.; Cwiklinski, K. Fasciola hepatica is refractory to complement killing by preventing attachment of mannose binding lectin (MBL) and inhibiting MBL-associated serine proteases (MASPs) with serpins. PLoS Pathog. 2022, 18, e1010226. [Google Scholar]
  42. Ribeiro, J.M.C.; Reynolds, S.L.; Pike, R.N.; Mika, A.; Blom, A.M.; Hofmann, A.; Wijeyewickrema, L.C.; Kemp, D.; Fischer, K. Scabies Mite Inactive Serine Proteases Are Potent Inhibitors of the Human Complement Lectin Pathway. PLoS Negl. Trop. Dis. 2014, 8, e2872. [Google Scholar]
Pathogens 14 00354 g001
Figure 1. EmCRT fragmentation and calcium binding ability. (A) Schematic diagram of EmCRT fragmentation of each functional domain. (B) Western blot showing 1 μg of rEmCRT and its fragments including rEmCRT-N (Line 1), rEmCRT-P (Line 2), rEmCRT-S (Line 3), rEmCRT-C (Line 4), rEmCRT-NP (Line 5), and rEmCRT (Line 6) recognized by moused anti-His monoclonal antibody. (C) Purified rEmCRT and its fragments (5 μg each) were stained with Stains-all in blue, indicating their calcium binding ability: rEmCRT-P (Line 2), rEmCRT-S (Line 3), rEmCRT-C (Line 4), rEmCRT-NP (Line 5), and rEmCRT (Line 6) stained in blue, while rEmCRT-N (Line 1), rEmFer (Line 7) as a E. multilocularis irrelevant protein control, and BSA (Line 8) control were stained in red, indicating these proteins have no calcium-binding activity. (Original Western blot and SDS-PAGE images of subfigures (B,C) can be found in Figure S1.)
Figure 1. EmCRT fragmentation and calcium binding ability. (A) Schematic diagram of EmCRT fragmentation of each functional domain. (B) Western blot showing 1 μg of rEmCRT and its fragments including rEmCRT-N (Line 1), rEmCRT-P (Line 2), rEmCRT-S (Line 3), rEmCRT-C (Line 4), rEmCRT-NP (Line 5), and rEmCRT (Line 6) recognized by moused anti-His monoclonal antibody. (C) Purified rEmCRT and its fragments (5 μg each) were stained with Stains-all in blue, indicating their calcium binding ability: rEmCRT-P (Line 2), rEmCRT-S (Line 3), rEmCRT-C (Line 4), rEmCRT-NP (Line 5), and rEmCRT (Line 6) stained in blue, while rEmCRT-N (Line 1), rEmFer (Line 7) as a E. multilocularis irrelevant protein control, and BSA (Line 8) control were stained in red, indicating these proteins have no calcium-binding activity. (Original Western blot and SDS-PAGE images of subfigures (B,C) can be found in Figure S1.)
Pathogens 14 00354 g001
Pathogens 14 00354 g002
Figure 2. rEmCRT binding to human MBL. (A) ELISA results showing rEmCRT (0, 0.2, 0.4, 1.2, 1.5 μM/well) was able to bind onto MBL coated on the plates (0, 0.5, 1, 2, 4, 8, 12, 15 μg/mL) detected by anti-His monoclonal antibody. (a) Nothing bounds to BSA coated on the plates (0, 0.5, 1, 2, 4, 8, 12, 15 μg/mL). (b) Data are expressed as mean of OD450 ± SDs of three independent experiments (*** p < 0.0005 compared to BSA-coated plates). (B) Far western blot showing rEmCRT was able to bind onto MBL transferred on PVDF membrane. Separation of 1 μg MBL by SDS-PAGE under denaturing condition (a). Far western blot with anti-His antibody (1:1000) (b). Lane 1: 1 μg MBL. Lane 2: 1 μg BSA. (Original Western blot and SDS-PAGE images of subfigures (B) can be found in Figure S2).
Figure 2. rEmCRT binding to human MBL. (A) ELISA results showing rEmCRT (0, 0.2, 0.4, 1.2, 1.5 μM/well) was able to bind onto MBL coated on the plates (0, 0.5, 1, 2, 4, 8, 12, 15 μg/mL) detected by anti-His monoclonal antibody. (a) Nothing bounds to BSA coated on the plates (0, 0.5, 1, 2, 4, 8, 12, 15 μg/mL). (b) Data are expressed as mean of OD450 ± SDs of three independent experiments (*** p < 0.0005 compared to BSA-coated plates). (B) Far western blot showing rEmCRT was able to bind onto MBL transferred on PVDF membrane. Separation of 1 μg MBL by SDS-PAGE under denaturing condition (a). Far western blot with anti-His antibody (1:1000) (b). Lane 1: 1 μg MBL. Lane 2: 1 μg BSA. (Original Western blot and SDS-PAGE images of subfigures (B) can be found in Figure S2).
Pathogens 14 00354 g002
Pathogens 14 00354 g003
Figure 3. Predicted protein–protein molecular docking of EmCRT with MBL (PDB:1HUP) 3D model. (A) 3D model obtained by homology modelling of EmCRT shows the N domain (cyan), the P domain (rosy red), the C domain (yellow). (B) Predicted MBL binding regions on EmCRT are located in the N domain and P domain with detail of interacting amino acids between EmCRT and MBL showing on the right panel. (C) Interface analysis of EmCRT and MBL binding regions showing the interacting amino acids and their forces of action.
Figure 3. Predicted protein–protein molecular docking of EmCRT with MBL (PDB:1HUP) 3D model. (A) 3D model obtained by homology modelling of EmCRT shows the N domain (cyan), the P domain (rosy red), the C domain (yellow). (B) Predicted MBL binding regions on EmCRT are located in the N domain and P domain with detail of interacting amino acids between EmCRT and MBL showing on the right panel. (C) Interface analysis of EmCRT and MBL binding regions showing the interacting amino acids and their forces of action.
Pathogens 14 00354 g003
Pathogens 14 00354 g004
Figure 4. Binding of recombinant proteins of EmCRT and its fragments to MBL. (A) ELISA showing the binding of rEmCRT and the expressed recombinant fragments (0.5 μM/well) to MBL coated on the plates (0, 2, 4, 8, 12, 20 μg/mL) (a) but not binding to BSA coated on plates (0, 2, 4, 8, 12, 20 μg/mL). (b) The bound recombinant proteins were detected by anti-His monoclonal antibody. Data are expressed as mean of OD450 ± SDs of three independent experiments (*** p < 0.0005, ns, not significantly different compared to plates coated with BSA). (B) Far western blot was performed to detect rEmCRT and its fragments (15 μg/mL) bound onto MBL (1 μg) transferred on membrane, visualized by anti-His antibody (1:1000). SDS-PAGE of MBL and control BSA was shown on the right panel. (C) Densitometry reading of recognized bands. The binding ability of rEmCRT-S and rEmCRT-NP was similar to rEmCRT. (* p < 0.005, *** p < 0.0005, ns, no significant difference compared to bands incubated with rEmCRT). (Original Western blot and SDS-PAGE images of subfigures (B) can be found in Figure S3).
Figure 4. Binding of recombinant proteins of EmCRT and its fragments to MBL. (A) ELISA showing the binding of rEmCRT and the expressed recombinant fragments (0.5 μM/well) to MBL coated on the plates (0, 2, 4, 8, 12, 20 μg/mL) (a) but not binding to BSA coated on plates (0, 2, 4, 8, 12, 20 μg/mL). (b) The bound recombinant proteins were detected by anti-His monoclonal antibody. Data are expressed as mean of OD450 ± SDs of three independent experiments (*** p < 0.0005, ns, not significantly different compared to plates coated with BSA). (B) Far western blot was performed to detect rEmCRT and its fragments (15 μg/mL) bound onto MBL (1 μg) transferred on membrane, visualized by anti-His antibody (1:1000). SDS-PAGE of MBL and control BSA was shown on the right panel. (C) Densitometry reading of recognized bands. The binding ability of rEmCRT-S and rEmCRT-NP was similar to rEmCRT. (* p < 0.005, *** p < 0.0005, ns, no significant difference compared to bands incubated with rEmCRT). (Original Western blot and SDS-PAGE images of subfigures (B) can be found in Figure S3).
Pathogens 14 00354 g004
Pathogens 14 00354 g005
Figure 5. rEmCRT and its MBL binding regions inhibited binding of MBL to mannan. (A) Pre-incubation of different amounts of rEmCRT, rEmCRT-S, and rEmCRT-NP (0, 0.5, 1.0 µM) with MBL (10 μg/mL) before adding to plates coated with mannan (50 μg/mL), detected with anti-MBL monoclonal antibody. (B) Under the same ELISA condition, different amounts of rEmCRT, rEmCRT-S, or rEmCRT-NP (0, 1, 2 μM) were pre-incubated with C1qD (1:50) before adding into plates coated with mannan. Data are expressed as mean of OD450 ± SDs of three independent experiments (* p < 0.005; ** p < 0.001, *** p < 0.0005, ns, not significantly different compared to the reading from MBL without incubation with recombinant EmCRT).
Figure 5. rEmCRT and its MBL binding regions inhibited binding of MBL to mannan. (A) Pre-incubation of different amounts of rEmCRT, rEmCRT-S, and rEmCRT-NP (0, 0.5, 1.0 µM) with MBL (10 μg/mL) before adding to plates coated with mannan (50 μg/mL), detected with anti-MBL monoclonal antibody. (B) Under the same ELISA condition, different amounts of rEmCRT, rEmCRT-S, or rEmCRT-NP (0, 1, 2 μM) were pre-incubated with C1qD (1:50) before adding into plates coated with mannan. Data are expressed as mean of OD450 ± SDs of three independent experiments (* p < 0.005; ** p < 0.001, *** p < 0.0005, ns, not significantly different compared to the reading from MBL without incubation with recombinant EmCRT).
Pathogens 14 00354 g005
Pathogens 14 00354 g006
Figure 6. Inhibition of C3b and C4b deposition by rEmCRT and its functional binding regions measured by ELISA. (A) Pre-incubation of 100 μL C1qD (1:50) as source of natural MBL with different amounts of rEmCRT, rEmCRT-S, or rEmCRT-NP (0, 2, 4 μM) before adding into mannan-coated plates (50 μg/mL). After being washed, C1qD (1:150) was added as source of complement components to initiate lectin pathway of complement activation. Deposition of C3b (a) and C4b (b) was detected with anti-C3 or C4 monoclonal antibodies. (B) The similar ELISA procedure was performed using NHS as source of physiological MBL instead of C1qD serum to detect the generation of C3b (a) and C4b (b). Data are expressed as mean of OD450 ± SDs of three independent experiments (* p < 0.005, ** p < 0.001, *** p < 0.0005, ns, no significant difference compared to plates without added rEmCRT).
Figure 6. Inhibition of C3b and C4b deposition by rEmCRT and its functional binding regions measured by ELISA. (A) Pre-incubation of 100 μL C1qD (1:50) as source of natural MBL with different amounts of rEmCRT, rEmCRT-S, or rEmCRT-NP (0, 2, 4 μM) before adding into mannan-coated plates (50 μg/mL). After being washed, C1qD (1:150) was added as source of complement components to initiate lectin pathway of complement activation. Deposition of C3b (a) and C4b (b) was detected with anti-C3 or C4 monoclonal antibodies. (B) The similar ELISA procedure was performed using NHS as source of physiological MBL instead of C1qD serum to detect the generation of C3b (a) and C4b (b). Data are expressed as mean of OD450 ± SDs of three independent experiments (* p < 0.005, ** p < 0.001, *** p < 0.0005, ns, no significant difference compared to plates without added rEmCRT).
Pathogens 14 00354 g006
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Shao, Y.; Xia, M.; Song, Y.; Yan, Y.; Dong, X.; Zong, H.; Zhan, B.; Wang, Y.; Zhao, L. Echinococcus multilocularis Calreticulin Inhibits Lectin Pathway of Complement Activation by Directly Binding to Mannose-Binding Lectin. Pathogens 2025, 14, 354. https://doi.org/10.3390/pathogens14040354

AMA Style

Shao Y, Xia M, Song Y, Yan Y, Dong X, Zong H, Zhan B, Wang Y, Zhao L. Echinococcus multilocularis Calreticulin Inhibits Lectin Pathway of Complement Activation by Directly Binding to Mannose-Binding Lectin. Pathogens. 2025; 14(4):354. https://doi.org/10.3390/pathogens14040354

Chicago/Turabian Style

Shao, Yuxiao, Meng Xia, Yinghui Song, Yan Yan, Xiaofang Dong, Haoran Zong, Bin Zhan, Yanhai Wang, and Limei Zhao. 2025. "Echinococcus multilocularis Calreticulin Inhibits Lectin Pathway of Complement Activation by Directly Binding to Mannose-Binding Lectin" Pathogens 14, no. 4: 354. https://doi.org/10.3390/pathogens14040354

APA Style

Shao, Y., Xia, M., Song, Y., Yan, Y., Dong, X., Zong, H., Zhan, B., Wang, Y., & Zhao, L. (2025). Echinococcus multilocularis Calreticulin Inhibits Lectin Pathway of Complement Activation by Directly Binding to Mannose-Binding Lectin. Pathogens, 14(4), 354. https://doi.org/10.3390/pathogens14040354

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop