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CTSL-2 upon specifically recognizing Vibrio splendidus directly cleaves complement C3 to promote the bacterial phagocytosis and degradation in oyster

Cell Communication and Signaling volume 23, Article number: 198 (2025) Cite this article

Abstract

Cathepsin L (CTSL) as a cysteine cathepsin protease mediates complement C3 cleavage and pathogen degradation. In the present study, a CTSL homolog was identified from Crassostrea gigas (designated as CgCTSL-2). Its mRNA expression increased significantly in hemocytes after Vibrio splendidus stimulation. The activity of rCgCTSL-2 was induced after incubation with LPS or V. splendidus in Ca2+-dependent manner. rCgCTSL-2 could specifically bound V. splendidus in Ca2+-dependent manner. The co-localization of rCgCTSL-2 and V. splendidus was observed in cell-free hemolymph. Upon binding V. splendidus, CgCTSL-2 interacted with CgC3 in cell-free hemolymph and hemocytes. CgC3 fragments in CgCTSL-2-RNAi oysters and full length CgC3 in rCgCTSL-2-treated oysters were both reduced in cell-free hemolymph, respectively. CgC3 fragments were accumulated in CgCTSL-2-RNAi or rCgCTSL-2-treated oysters. The co-localizations of V. splendidus, CgC3, CgCD18, CgCTSL-2 and lysosomes were observed in hemocytes. These results suggested that CgCTSL-2 upon binding V. splendidus directly interacted with CgC3 to lead to CgC3 cleavage and then CgC3 fragments coated on V. splendidus were mediated by CgCD18 into CTSL-2-lysosome pathway.

Introduction

Cathepsin L (CTSL) is an important member of the cysteine proteases to be involved in a variety of biological functions such as antigen processing [1], extracellular matrix remodeling [2], autophagy [3], and protein degradation [4]. CTSL is composed of a signal peptide, a pro-peptide and a mature enzyme with four active residues: Gln, Cys, His, and Asn [5]. Its primary role is the proteolysis of protein antigens from pathogen endocytosis [6]. There was one report that CTSL expressed in human CD4+ T cells processed C3 into C3a and C3b [7].

CTSL as an acidic protease is mainly localized in lysosomes and is the executor of non-oxidative killing during lysosomal clearance of bacteria [8]. In human and mouse macrophages, phagocytosed pathogens were encapsulated in phagosomes that transported them to lysosomes [9], where CTSL participated in their lysosome-mediated degradation [10]. In bony fish, such as Anguilla japonica [11] and Cynoglossus semilaevis [12], CTSL played a key function in the mucosal immune system in catabolizing pathogen. In crustaceans, such as Macrobrachium rosenbergii [5] and Sinonovacula constricta [13], CTSL was demonstrated to have the ability to lyse pathogen in the innate immune response.

CTSL plays crucial roles in mediating the activation of complement C3 in immune cells [14]. In mammals, C3 is primarily synthesized in liver [15]. Recently, C3 is also found to be expressed in intestinal epithelial cells [16] and some types of immune cells. In human intestinal epithelial cells [16] and CD4+ T cells [7], the activation of C3 was not dependent on the traditional C3 convertase, but the lysosomal CTSL. CTSL could directly cleave C3, and the specific location of its cleavage was at the arginine in the end of C3a fragment [17]. Extracellular C3b was able to bind to CR3 on the surface of macrophages, which in turn promoted intracellular degradation and clearance of pathogens [18]. In invertebrates, there are still no reports about the function of CTSL for regulating C3 activation. Only in some species such as Procambarus clarkii [19] and Cristaria plicata [20], CTSL was reported to be involved in the immune responses [21].

Molluscs play a crucial role in coastal environments and are known as “ecological engineers” and “keystone species” in evolution [22]. However, in recent years, the molluscan aquaculture industry has encountered severe disease problems, especially those caused by pathogenic microbes [23]. Vibrio splendidus is a Gram-negative pathogenic bacterium that causes high mortality in aquatic farmed animals, such as oysters [24]. Molluscs mainly rely on innate immunity to defend against pathogen invasion, in which the complement system outside the cell and the lysosomal process inside the cell constitute the two major immune barriers [25]. In the present study, CgCTSL-2 was identified from Crassostrea gigas with the objectives to (1) analyze its molecular characterization and immune recognition, (2) investigate its association with the activation of CgC3, and (3) confirm the degradation process of C3-coated Vibrio splendidus in lysosome pathway.

Materials and methods

Animals and sample collection

The oysters C. gigas (2-year-old adult, about 10 cm in shell length, about 120–160 g in weight) were obtained from a local farm in Dalian, Liaoning Province, China. All oysters were kept in a continuously aerated sea water tank at about 10℃ for more than one week before subsequent experiments. Six-week-old female Kunming mice were purchased from Dalian Institute of Drug Control. All the experiments were performed following the animal ethics guidelines approved by the Ethics Committee of Dalian Ocean University.

Bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Micrococcus luteus were obtained from Microbial Culture Collection Center (China), which were cultured in Luria-Bertani (LB) medium at 37℃. Vibrio alginolyticus, Pseudoalteromonas arctica and Pseudoalteromonas aliena were prepared and preserved in our laboratory [26], which were cultured in 2216E medium at 16℃. V. splendidus strain JZ6 was previously isolated from the lesioned area of a moribund scallop Patinopecten yessoensis and preserved in our laboratory [27], which was cultured in 2216E medium at 16℃.

Different tissues (gills, mantle, adductor muscle, gonad, hepatopancreas, labial palp, and hemolymph) of nine oysters were collected for RNA extraction, and the distribution of CgCTSL-2 mRNA was detected in different tissues. For the V. splendidus stimulation experiment, seventy oysters were divided into two groups: control group (ten oysters) and V. splendidus group (sixty oysters). The oysters in V. splendidus group received an intramuscular injection of 100 µL V. splendidus at 1 × 107 CFU/mL dissolved in sterile sea water. At the T0 h (control group, which did not receive V. splendidus stimulation), nine oysters were randomly chosen for collecting hemocytes. Then, nine oysters were randomly sampled at 3, 6, 12, 24, 48 and 72 h after V. splendidus stimulation (V. splendidus group) to collect hemocytes. The hemolymph from three oysters was put together as one sample and there were three samples for each group. The hemocytes were harvested from hemolymph by centrifugation at 800 g, 4℃ for 10 min. Total RNAs were extracted to analyze the mRNA expressions of CgCTSL-2.

Sequence feature analysis of CgCTSL-2

The full-length amino acid sequence of CgCTSL-2 (XP_034318852.1) was obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/). The sequence characteristics of CgCTSL-2 were predicted using ProtParam (http://web.expasy.org/cgi-bin/protparam/protparam). Multiple sequence alignment of CgCTSL-2 and CTSLs from other species were constructed using Clustal X v2.0 program and GeneDoc software and their phylogenetic tree was constructed by MEGA X software.

The tertiary structure of CgCTSL-2 was predicted by SWISS-MODEL (http://swissmodel.expasy.org.libyc.nudt.edu.cn:80). The tertiary structure of LPS (BCP09107) was predicted by pubchem (https://pubchem.ncbi.nlm.nih.gov/). For molecule docking, the structures of CgCTSL-2 and LPS were prepared by the AutoDockTools-1.5.7. And all water molecules were eliminated, and polar hydrogen was added in CgCTSL-2. LPS was manually added with water molecules and polar hydrogen bonds. The protein-small-molecule interactions between CgCTSL-2 and LPS were predicted and visualized using PyMOL. All the parameters for the protein and ligand preparation as well as docking run were set to their default values [28].

Real-time quantitative PCR (RT-qPCR) analysis of CgCTSL-2

RT-qPCR with the primers CgCTSL-2-RT-F/-R (Supplemental Table 1) was performed to analyze the tissue distribution of CgCTSL-2 mRNA and its temporal expression in hemocytes after V. splendidus stimulation. The fragments of elongation factor from C. gigas (CgEF, NM_001305313) amplified with primers CgEF-RT-F/-R (Supplemental Table 1) were employed as the internal reference. RT-qPCR was programmed at 95℃ for 10 min, followed by 40 cycles at 95℃ for 10 s and 60℃ for 45 s. The final product was analyzed via melting analysis from 65 to 95℃. The mRNA expressions of CgCTSL-2 were analyzed by 2−△△Ct method [26] with their corresponding primers (Supplemental Table 1). The data were analyzed by one-way analysis of variance (ANOVA), and multiple comparisons were performed by Duncan’s test, with statistically significant differences (p < 0.05) for comparisons between groups with different letters (a, b, and c).

The distribution of CgCTSL-2 in sub-populations of hemocytes

Flow cytometry (FCM) was used to analyze the protein distribution of CgCTSL-2 in three sub-populations of hemocytes. The hemocytes were collected according to previous description [28]. CgCTSL-2 (diluted in 3% bovine serum albumin (BSA) (Hyclone, 31840.0)) was used as the primary antibody, and Alexa Fluor 488-conjugated goat anti-mouse IgG (Beyotime, A0428) as the secondary antibody. The positive signals of heamocytes were analyzed by using FCM.

The expression and purification of recombinant CgCTSL-2 (rCgCTSL-2)

The nucleotide sequence of Inhibitor_I29 domain and Pept_C1 domain in CgCTSL-2 was amplified by using primers CgCTSL-2-Ex-F/R (Supplemental Table 1). The recombinant CgCTSL-2 was expressed and purified according to the previous description [26]. The PCR product, treated with restriction endonucleases BamH I and Xho I, was ligated into the pET-30a (+) vector containing a 6 × His tag (tag protein containing N-6×His, N-S, N-Thrombin, N-EK and C-6×His in expression vector pET-30a (+) is approximately 5.0 kDa [29]) (Sangong, B540185) and transformed into E. coli Transetta (DE3) (TransGen Biotech, CD801-02). The expression of rCgCTSL-2 were induced with isopropyl-β-D-thiogalactoside (IPTG), and its purification was performed using Ni2+ affinity chromatography.

Polyclonal antibody preparation and western blotting analysis of CgCTSL-2

The purified rCgCTSL-2 was used to treat the six-weeks female mice to acquire polyclonal antibody [28]. Mice were treated with an emulsion of rCgCTSL-2 and Freund’s adjuvant, once a week for a total of four times. After the fourth injection, mouse blood was collected, and serum was separated. The specificity of anti-CgCTSL-2 was confirmed using cell-free hemolymph and haemocyte lysates with anti-CgCTSL-2 as the primary antibody and HRP-conjugated goat anti-mouse IgG as the secondary antibody.

Proteolytic activity assay of rCgCTSL-2

The proteolytic activity of rCgCTSL-2 was measured using CTSL activity assay kit (Haling Biological technology, HL50115.1.1) according to the instruction of manufacturer. The hemocytes and cell-free hemolymph were collected according to previous description [28]. V. splendidus was also cultured and collected according to previous description [30]. Twenty microliter V. splendidus (1 × 105 CFU/mL) or 50 µL LPS (0.5 mg/mL) were mixed with 100 µL rCgCTSL-2 (with or without 10 mM CaCl2), respectively, and incubated at room temperature. The activity of CTSL was evaluated by detecting the release of yellow p-Nitrophenol through cleaving its substrate Ac-FR-pNA. The activity was calculated using the following formula: ((sample reading-background reading) * 0.20 (system capacity, mL)) / (0.095(sample capacity, mL) * 10.5 (millimolar absorption coefficient) * 90 (reaction time, minutes) * 0.3 (light path distance, cm)).

Pathogen-associated molecular pattern (PAMP) and microbial binding assay of rCgCTSL-2

The PAMP binding activity of rCgCTSL-2 was measured by enzyme-linked immune sorbent assay (ELISA) according to the previous report with minor modification [26]. Briefly, 96-well microliter plates were coated with LPS (from E. coli 0111: B4; Sigma, L2630) dissolved in Na2CO3-NaHCO3 buffer (50 mmol/L, pH 9.6) at a final concentration of 0.1 mg/mL, at 4 °C overnight. The wells were washed three times with TBST for 15 min and then blocked with 200 µL of 3% BSA in TBST at 37 °C for 1 h to prevent non-specific adsorption. One hundred microliter of rCgCTSL-2 (126.70 µg/mL, with or without 10 mmol/L CaCl2) with gradient dilution (126.70 µg/mL, 63.35 µg/mL, 31.68 µg/mL, 15.84 µg/mL, and 7.92 µg/mL), 6 × His Tag mouse IgG (Sangong, D191001; diluted to 1:1000 in 3% BSA) and HRP-conjugated goat anti-mouse IgG (Beyotime, A0473; diluted to 1:1000 in 3% BSA) were added to the wells successively and incubated at 37 °C for 1 h after TBST washes. The same concentration of rTrx was used as negative control (N). The TBS buffer was used as blank control (B). One hundred microliter of tetramethylbenzidine (Beyotime, P0206) was added and incubated at room temperature in dark for 15 min. The reaction was stopped by 50 µL ELISA Stop Solution (Beyotime, P0215), and the absorbance was measured at 405 nm by Tecan Infinite M1000 PRO (Tecan, Switzerland). P (sample)-B (blank)/N (negative)-B (blank) ratio > 2.1 was considered positive. The experiment was repeated three times for each sample, and the data are represented as mean ± SD (N = 3).

Gram-positive bacteria (S. aureus and B. subtilis) and Gram-negative bacteria (P. arctica, P. aliena, V. alginolyticus, E. coli, M. luteus, and V. splendidus) were cultured and collected according to previous description [30]. The 20 µL collected bacteria (OD600 = 1.0) were incubated with 100 µL rCgCTSL-2 (1.128 mg/mL). The samples were analyzed by western blotting with 6 × His Tag mouse IgG (Sangong, D191001; 1:1000). In the control rTrx group, the same procedure was performed as described above.

V. splendidus was cultured and collected according to previous description [30]. Fifty microliters rCgCTSL-2 and an equal amount of V. splendidus were mixed (with or without 10 mM CaCl2) and incubated at room temperature. Subsequent steps were performed as previously described with rCgCTSL-2 and V. splendidus [30]. The 96-well microliter plates with cultures were placed in Tecan Infinite M1000 PRO absorbance microplate reader (Tecan, Switzerland), and their OD600 values were measured every 30 min for investigating the growth of the tested microbes for 12.5 h. Each group was repeated three times.

Co-immunoprecipitation (Co-IP) assay of CgCTSL-2 and CgC3

The haemocyte lysates were incubated with 100 µL of Protein G (Beyotime, P2009) and 10 µL of pre-serum from mouse (to remove non-specific binding proteins). Then, the collected supernatant was added with 100 µL of Protein G and 10 µL of anti-CgC3 and anti-CgCTSL-2 and incubated overnight at 4 °C. The mixture was washed with PBS. The collected samples were analyzed by western blotting with anti-CgC3 and anti-CgCTSL-2 as primary antibodies, goat-anti-mouse IgG (Beyotime, A0473) and goat-anti-rabbit IgG (Beyotime, A0468) as the secondary antibody. The signal bands were imaged by Amersham Imager 600 (Thermo Fisher Scientific).

RNA interference (RNAi) of CgCTSL-2

Specific siRNAs targeting CgCTSL-2 were designed and synthesized to inhibit its expression. Eighteen oysters were divided into two groups, including negative control group (NC-RNAi group) and experimental group (CgCTSL-2-RNAi group). The oysters received an injection of 100 µL siRNAs of NC and 100 µL siRNAs of CgCTSL-2 (26 µg siRNAs dissolved in 100 µL sterile seawater), respectively. At 12 h after the injection of siRNAs, the oysters received another injection of V. splendidus (1 × 107 CFU/mL, 100 µL). Hemolymph was collected as description above and centrifuged to obtain hemocytes and cell-free hemolymph, respectively. There were three replicates in each group. The partial-hemocytes were used for RNA extraction to analyze the RNAi efficiency of CgCTSL-2. The remaining hemocytes and cell-free hemolymph were used for western blotting with anti-CgC3 as primary antibody, goat-anti-rabbit IgG (Beyotime, A0468) as the secondary antibody. The immunoblot protein bands were imaged by Amersham Imager 600 (Thermo Fisher Scientific, USA).

Immunocytochemical assay of CgCTSL-2

Subcellular localization of CgCTSL-2 in hemocytes was analyzed by immunocytochemistry assay with anti-CgCTSL-2 as the primary antibody and Alexa Fluor 488-labeled goat-anti-mouse IgG as the secondary antibody. Hemocytes were collected and processed as previously described [30]. Positive signals of hemocytes were observed by using an inverted fluorescence microscope (Axio Imager A2; Zeiss).

The co-localizations of V. splendidus with CgC3, CgCD18, lysosome, CgCTSL-2, and rCgCTSL-2

V. splendidus was labeled with FITC (FITC-V. splendidus) as the previously described method [25]. The collected hemocytes were obtained from 15 untreated oysters, mixed with FITC-labeled V. splendidus (1 × 107 CFU/mL), and then incubated at room temperature in the dark while rotating for 0.5 h. Anti-CgC3 (5.37 mg/mL; diluted 1: 1000 in 3% BSA), anti-CgCD18 (9.03 mg/mL; diluted 1: 1000 in 3% BSA) and anti-CgCTSL-2 (84.63 mg/mL; diluted 1: 1000 in 3% BSA) were used as the primary antibodies, respectively, and Alexa Fluor 647-labeled goat-anti-mouse IgG was used as the secondary antibody (showing red fluorescence signal). Nucleus stained with DAPI was in blue, lysosome stained with Lyso-Tracker Red (red) and FITC-V. splendidus was in green. The co-localizations of FITC-V. splendidus with CgC3, CgCD18, lysosome and CgCTSL-2 were observed using under inversion fluorescence microscope (Axio Imager A2; ZEISS), respectively.

Twenty microliters of FITC-V. splendidus was incubated with 1 mL of rCgCTSL-2 for 1 h at room temperature in dark with slight rotation. The primary antibody was 6 × His Tag mouse IgG (Sangong, D191001; 1:1000) and the secondary antibody was Alexa Fluor 647-labeled goat-anti-mouse IgG (Beyotime, A0473). DAPI-stained nucleus was in blue, rCgCTSL-2-conjugated-6 × His Tag mouse IgG was in red, and FITC-V. splendidus was in green. The co-localization signals of rCgCTSL-2 and FITC-V. splendidus were observed under inversion fluorescence microscope (Axio Imager A2; ZEISS).

The co-localizations of CgCTSL-2, CgC3, and lysosome

Hemocytes were incubated with Lyso-Tracker Green. Anti-CgC3 and anti-CgCTSL-2 were used as the primary antibody, respectively. Alexa Fluor 647-labeled goat-anti-rabbit IgG (Beyotime, A0468) and Alexa Fluor 488-labeled goat-anti-mouse IgG (Beyotime, A0428) were used as the secondary antibodies, respectively. DAPI-stained nucleus was in blue, anti-CgC3-conjugated Alexa Fluor 647-labeled goat-anti-rabbit IgG was in red, and anti-CgCTSL-2-conjugated Alexa Fluor 488-labeled goat-anti-mouse IgG was in green. Lysosome-conjugated Lyso-Tracker Green was in green and lysosome-conjugated Lyso-Tracker Red was in red. The co-localization signals in hemocytes were observed under inversion fluorescence microscope (Axio Imager A2; ZEISS).

Results

CgCTSL-2 with Ca2+ binding sites was identified from C. gigas

The full-length cDNA of CgCTSL-2 cloned from oyster was of 1433 bp with an open reading frame of 996 bp that encoded a polypeptide of 331 amino acids (aa) with a predicted molecular mass of 37.05 kDa. CgCTSL-2 consisted of a signal peptide, an Inhibitor_I29 domain, and a Pept_C1 domain. CgCTSL-2 shared high similarities with CTSLs from vertebrates and other invertebrates, such as 52% similarity with Homo sapiens, 54% similarity with Cyprinus carpio, 53% similarity with Xenopus laevis, 51% similarity with Danio rerio, 64% similarity with Mizuhopecten yessoensis, 33% similarity with Mytilus edulis, and 89% similarity with C. virginica. In the phylogenetic tree of CTSLs (Supplemental Fig. 1A), the molluscan CTSLs were clustered together and among which, CgCTSL-2 was closer to that of C. virginica. Multiple sequence alignments showed that the four sites (Q, C, H, and N) of CTSLs were conserved in various species (marked by red asterisk in Fig. 1B). The corresponding sites in CgCTSL-2 were Q133, C165, H255, and N298 (Supplemental Fig. 1B). A Ca2+ binding sites “EPA (Glu-Pro-Ala)” were identified in CgCTSL-2 (marked by blue box in Supplemental Fig. 1B).

Fig. 1
figure 1

The mRNA expression s and protein distribution features of CgCTSL-2. A The mRNA expressions of CgCTSL-2 in different tissues. B The mRNA expressions of CgCTSL-2 in hemocytes after V. splendidus stimulation. C SDS-PAGE analysis of rCgCTSL-2. Lane M: protein marker, Lane 1: negative control (without induction), Lane 2: induced rCgCTSL-2, Lane 3: purified rCgCTSL-2. D The specific Ab detection of CgCTSL-2 in hemocytes and cell-free hemolymph. Lane M: protein marker, Lane 1: hemocytes, Lane 2: cell-free hemolymph. E Three sub-populations of hemocytes separated by FCM. P3: agranulocytes, P4: semi-granulocytes, P5: granulocytes. FCgCTSL-2 protein levels in the sub-populations of hemocytes. G The subcellular localization of CgCTSL-2 (green). Nucleus was stained with DAPI (blue). Scale bar, 10 μm. The data were represented as the mean ± SD from three independent replicates. Different letters (a and b): p < 0.05 (one-way ANOVA)

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The mRNA expressions of CgCTSL-2 increased significantly after V. splendidus stimulation

The mRNA expressions of CgCTSL-2 in different tissues were investigated by RT-qPCR with CgEF as internal control. CgCTSL-2 mRNA was expressed in all the tested tissues, including gills, mantle, adductor muscle, gonad, hepatopancreas, labial palp, and hemolymph. The highest expression of CgCTSL-2 mRNA was found in hepatopancreas, which was 67.98-fold (p < 0.05) of that in mantle (Fig. 1A). The temporal expressions of CgCTSL-2 mRNA were also examined in hemocytes after V. splendidus stimulation. After V. splendidus stimulation, the mRNA expressions of CgCTSL-2 increased significantly at 3 h (4.76-fold of that at 0 h, p < 0.05) (Fig. 1B).

CgCTSL-2 was distributed in the cytoplasm of hemocytes

rCgCTSL-2 was purified by Ni-NTA affinity chromatography and characterized by 12.5% SDS-PAGE. A clear band was observed, corresponding to the predicted molecular weight of rCgCTSL-2 of approximately 40 kDa (Fig. 1C). The specificity of CgCTSL-2 antibody was confirmed in cell-free hemolymph and hemocytes by western blotting. A band of 30 kDa was observed, corresponding to the activated peptide of CgCTSL-2 (Fig. 1D).

The distribution of CgCTSL-2 in hemocytes was analyzed using FCM and immunocytochemistry. Anti-CgCTSL-2 conjugated with Alexa Fluor 488 was observed in green. The green fluorescence signals of CgCTSL-2 were distributed in three sub-populations of hemocytes, predominantly in granulocytes. The fluorescence intensity in granulocytes was 2.54-fold (p < 0.01) of semi-granulocytes and 16.19-fold (p < 0.01) of agranulocytes (Fig. 1E-F). Moreover, the positive signals of CgCTSL-2 were mainly distributed in the cytoplasm of the three sub-populations (Fig. 1G).

The activity of CgCTSL-2 was induced after it bound to V. splendidus and LPS in Ca2+-dependent manner

rCgCTSL-2 with pro-peptide Inhibitor_I29 domain and mature peptide Pept_C1 domain was expressed and purified from E. coli. And its activity was detected by using CTSL activity assay kit. rCgCTSL-2 had CTSL activity (Fig. 2C and H). The structure of CgCTSL-2 was constructed by SWISS-MODEL, and its EPA sites (Ca2+ binding sites) and interaction sites with LPS were both in the catalytic region (Fig. 2A). The binding activity of rCgCTSL-2 to LPS was examined by ELISA, with a P/N value > 2.1 considered positive for binding. rCgCTSL-2 bound polysaccharides in a concentration dependent manner. When the concentration of rCgCTSL-2 was below 15.84 µg/mL, there was no binding activity to LPS regardless of the presence of Ca2+. With the concentration of rCgCTSL-2 increased, its binding activity to LPS was enhanced. When the concentration of rCgCTSL-2 reached 126.70 µg/mL, its binding activity to LPS was the highest. The addition of Ca2+ promoted the binding activity of rCgCTSL-2 to LPS (Fig. 2B). There was no significant difference in CTSL enzymatic activity between rCgCTSL-2 group and rCgCTSL-2 + LPS group (Fig. 2C). In the presence of Ca2+, the activity of rCgCTSL-2 + LPS group increased significantly, which was 2.57-fold (p < 0.05) of that in rCgCTSL-2 group and 2.85-fold (p < 0.05) of that in rCgCTSL-2 + LPS group (Fig. 2C).

Fig. 2
figure 2

The activation of CgCTSL-2 upon binding to LPS and bacteria. A The molecular docking of CgCTSL-2 and LPS. B The binding activity of rCgCTSL-2 to LPS. P/N > 2.1 was considered positive. C The proteolytic activity of rCgCTSL-2 with LPS. D-E The bacteria binding activities of rCgCTSL-2 without Ca2+ or with Ca2+. F-G The growth of V. splendidus after incubation with rCgCTSL-2 without Ca2+ or with Ca2+. H The proteolytic activity of rCgCTSL-2 with V. splendidus. I-J The proteolytic activity of CgCTSL-2 in cell-free hemolymph and hemocytes. K The subcellular localization of rCgCTSL-2 (red) and FITC-V. splendidus (green) in cell-free hemolymph. Nucleus was stained with DAPI (blue). proteolytic activity: (µmol p-Nitrophenol / min) * mg. Vertical bars represented the mean ± S.D. (N = 3). Different letters: p < 0.05 (one-way ANOVA). ***: p < 0.001 (Student t-test)

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The microbial binding assay was conducted to analyze the binding activity of rCgCTSL-2 to microbes. In the absence of Ca2+, there was no binding obvious band observed after the incubation of rCgCTSL-2 and V. splendidus. Gram-positive bacteria (S. aureus and B. subtilis) and Gram-negative bacteria (P. arctica, P. aliena, V. alginolyticus, E. coli, M. luteus, and V. splendidus) were selected for analyzing the microbial binding activity of rCgCTSL-2 in the presence of Ca2+. The binding band was only observed after V. splendidus was incubated with rCgCTSL-2. As control, there was no band observed in rTrx groups (Fig. 2D-E).

The ability of rCgCTSL-2 to inhibit the growth of V. splendidus was evaluated by detecting the microbe growth curve. The OD600 value of V. splendidus was recorded every 30 min. Compared with the rTrx group, there were no significant changes about the growth of V. splendidus after the bacteria were incubated with rCgCTSL-2 and rTrx for 1–12 h (with or without Ca2+) (Fig. 2F-G).

The enzymatic activity of rCgCTSL-2 significantly increased in the group treated with rCgCTSL-2 and V. splendidus, which was 2.49-fold (p < 0.05) of that in rCgCTSL-2 group (Fig. 2H). In the presence of Ca2+, the enzymatic activity of rCgCTSL-2 in the rCgCTSL-2 + V. splendidus group increased significantly, which was 4.84-fold (p < 0.05) of that in rCgCTSL-2 group and 1.94-fold (p < 0.05) of that in rCgCTSL-2 + V. splendidus (Fig. 2H). In cell-free hemolymph and hemocytes, the activities of CTSL both increased significantly, which was 1.20-fold (p < 0.05) and 1.45-fold (p < 0.05) of that in seawater group (Fig. 2I-J).

Immunocytochemistry assay was used to observe the co-localization of rCgCTSL-2 and FITC-labeled V. splendidus. DAPI-stained nucleus was observed in blue, rCgCTSL-2 conjugated-6 × His Tag mouse IgG was in red, and FITC-labeled V. splendidus was in green. After incubation of FITC-labeled V. splendidus and rCgCTSL-2 in hemolymph, the red signals of rCgCTSL-2 were colocalized with the green signals of FITC-labeled V. splendidus in cell-free hemolymph (Fig. 2K).

CgCTSL-2 interacted with CgC3 to lead to CgC3 cleavage in cell-free hemolymph and hemocytes

The interaction between CgCTSL-2 and CgC3 in cell-free hemolymph and hemocytes was confirmed by Co-IP assay. When cell-free hemolymph lysates were coimmunoprecipitated with anti-CgC3 and anti-CgCTSL-2, respectively (Fig. 3A and C), the band of CgC3 fragments (alpha chain fragments of iCgC3b and CgC3γ) and CgCTSL (p-CgCTSL and CgCTSL (active)) were observed (Fig. 3B and D). When hemocytes were coimmunoprecipitated with anti-CgC3 and anti-CgCTSL-2, respectively (Fig. 3E and G), the band of CgC3 fragments (CgC3b, alpha chain fragments of iCgC3b and CgC3γ) and CgCTSL (p-CgCTSL and CgCTSL (active)) were observed (Fig. 3F and H). In rCgCTSL-2-treated oysters, the band intensity of full-length CgC3 was reduced, and those of CgC3b and alpha chain fragments of iCgC3b were induced in cell-free hemolymph after V. splendidus stimulation (Fig. 4A-B). The band intensities of CgC3b and CgC3γ were also enhanced in hemocytes after V. splendidus stimulation (Fig. 4C).

Fig. 3
figure 3

The interaction between CgCTSL-2 and CgC3 in cell-free hemolymph and hemocytes. A-D Co-IP assay of CgCTSL-2 and CgC3 in cell-free hemolymph. (A) Input samples analyzed by anti-CgC3. (B) CgC3 and CgCTSL-2 IP samples analyzed by anti-CgC3. (C) Input samples analyzed by anti-CgCTSL-2. (D) CgC3 and CgCTSL-2 IP samples analyzed by anti-CgCTSL-2. E-H Co-IP assay of CgCTSL-2 and CgC3 in hemocytes. (E) Input samples analyzed by anti-CgC3. (F) CgC3 and CgCTSL-2 IP samples analyzed by anti-CgC3. (G) Input samples analyzed by anti-CgCTSL-2. (H) CgC3 and CgCTSL-2 IP samples analyzed by anti-CgCTSL-2. p-CTSL: pro-CgCTSL-2, CTSL (active): activated peptide of CgCTSL-2

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Fig. 4
figure 4

CgC3 cleavage in rCgCTSL-2-treated or CgCTSL-2-RNAi oysters after V. splendidus stimulation. A Quantification of total protein in cell-free hemolymph of rCgCTSL-2-treated oysters. BCgC3 full-length and its fragments in cell-free hemolymph of rCgCTSL-2-treated oysters. CCgC3 full-length and its fragments in hemocytes of rCgCTSL-2-treated oysters. D RNAi efficiency of CgCTSL-2 after treated with CgCTSL-2 siRNAs. *p < 0.05, Student t-test. E Quantification of total protein in cell-free hemolymph of CgCTSL-2-RNAi oysters. FCgC3 full-length and its fragments in cell-free hemolymph of CgCTSL-2-RNAi oysters. GCgC3 full-length and its fragments in hemocytes of CgCTSL-2-RNAi oysters

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In CgCTSL-2-RNAi oysters, the band intensities of full-length CgC3 and CgC3b were enhanced, while those of alpha chain fragments of iCgC3b and CgC3γ were reduced in cell-free hemolymph after V. splendidus stimulation (Fig. 4E-F). The mRNA expression of CgCTSL-2 in hemocytes of CgCTSL-2-RNAi oysters decreased significantly, which was 0.09-fold (p < 0.05) of that in the NC group (Fig. 4D). In CgCTSL-2-RNAi oysters, the band intensities of the full-length CgC3 and CgC3b were enhanced in hemocytes after V. splendidus stimulation. While the band intensities of alpha chain fragments of iCgC3b and CgC3γ were also enhanced (Fig. 4G).

CgC3-coated V. splendidus was degraded through the CD18-CTSL-2-C3-lysosome pathway

The co-localizations of V. splendidus with CgC3 and CgCD18 in hemocytes were observed by immunocytochemistry. Anti-CgC3 and anti-CgCD18 conjugated with Alexa Fluor 647 were observed in red, respectively and nucleus stained by DAPI was in blue. After the incubation of hemocytes with FITC-labeled V. splendidus for 1 h, the red signals of CgC3 and CgCD18 were found to be co-localized with the green signals of FITC-labeled V. splendidus, respectively (Fig. 5A-B). After V. splendidus stimulation, the green signals of CgCTSL-2 were co-localized with the red signals of CgC3 in haemocyte cytoplasm (Fig. 5C). The co-localization value was 3.81-fold (p < 0.01) compared with that in sea water group (Fig. 5D). After V. splendidus stimulation, the red signals of lysosomes labeled with Lyso-Tracker Red were co-localized with the green signals of CgCTSL-2 in haemocyte cytoplasm (Fig. 6A). The co-localization value was 3.75-fold (p < 0.05) compared with that in sea water group (Fig. 6B). After V. splendidus stimulation, the green signals of lysosomes labeled with Lyso-Tracker Green were co-localized with the red signals of CgC3 in haemocyte cytoplasm (Fig. 6D). The co-localization value was 2.22-fold (p < 0.01) compared with that in sea water group (Fig. 6C). After the incubation of hemocytes with FITC-labeled V. splendidus for 1 h, the red signals of CgCTSL-2 and lysosomes were found to be co-localized with the green signals of V. splendidus in the cytoplasm, respectively (Fig. 6E-F).

Fig. 5
figure 5

The co-localization of FITC-V. splendidus with CgC3 and CgCD18 in hemocytes, and that of CgCTSL-2 with CgC3. A The co-localization of FITC-V. splendidus (green) and CgC3 (red). B The co-localization of FITC-V. splendidus (green) and CgCD18 (red). C The co-localization of CgCTSL-2 (green) and CgC3 (red) in hemocytes after V. splendidus stimulation. D The statistical analysis of co-localization of CgCTSL-2 and CgC3. The histogram was the statistical analysis about the co-localization of CgCTSL-2 and CgC3 in 150 hemocytes. Nucleus was stained with DAPI (blue). Scale bar, 10 μm. Vertical bars represented the mean ± S.D. (N = 3). Different letters (a, b): p < 0.05 (one-way ANOVA)

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Fig. 6
figure 6

The co-localization of lysosomes with CgCTSL-2 and CgC3, and co-localization of FITC-V. splendidus with CgCTSL-2 and lysosomes. A The co-localization of CgCTSL-2 (green) with lysosomes (red). B The statistical analysis of co-localization of CgCTSL-2 and lysosomes. C The statistical analysis of co-localization of CgC3 and lysosomes. D The co-localization of CgC3 (red) with lysosomes (green). E The co-localization of CgCTSL-2 (red) and FITC-V. splendidus (green). F The co-localization of FITC-V. splendidus (green) and lysosomes (red). The histogram was the statistical analysis about the co-localization of V. splendidus with CgCTSL-2 and V. splendidus with CgC3 in 150 hemocytes. Nucleus was stained with DAPI (blue). Scale bar, 10 μm. Vertical bars represented the mean ± S.D. (N = 3). Different letters (a, b): p < 0.05 (one-way ANOVA)

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Discussion

CTSLs as lysosomal enzymes are reported to be involved in the activation of complement C3 [31]. CTSL zymogen is processed into active mature enzymes in lysosomes [32], then cleaves intracellular C3 into biologically active C3a and C3b [33]. Until now, the role of CTSL in activating C3 had been reported in mammalian T cells, while there are still no reports in other species [7]. C3 is an ancient and evolutionary conserved molecule in metazoan and C3 homologs had been identified in lower species [34], such as fish [35], shrimp [36], oyster, etc [22]. Among which, in oyster, C3 could be activated by Vibrio to participate in multiple antibacterial immune processes [25]. In the present study, CgCTSL-2 identified in oyster could specifically recognize V. splendidus and bind to CgC3 in cell-free hemolymph and hemocytes to promote the activation of CgC3. The activated CgC3 upon binding bacteria mediated bacterial degradation into the CTSL2-lysosome pathway.

CTSL is an evolutionary highly conserved molecule that consists of a signal peptide, Inhibitor_I29 domain, and Pept_C1 domain [37]. Its enzymatically active catalytic domain also contains four highly conserved amino acid residues: Gln, Cys, His, and Asn [38]. In our previous study, CgCTSL1 with a single Pept_C1 domain and three amino acid residues (Q25, H135, and N178) identified from C. gigas [39]. In the present study, CgCTSL-2 with a signal peptide, Inhibitor_I29 domain and Pept_C1 domain and four amino acid residues (Q133, C165, H255, and N298) were identified. The structural domains of CgCTSL-2 were the same as those of CTSLs in mammals. In the phylogenetic tree of CTSLs, CgCTSL-2 and CTSLs from molluscs (except for CgCTSL1) were clustered together and they were dropped into the invertebrate branch. The results indicated that CTSL was conserved in different species of metazoan. In C-type lectins, the conserved motif “EPA/N” is the calcium-binding sites [40]. In the present study, the EPA sites were found in CgCTSL-2 and CvCTSL from C. virginica and EPN sites were found in MmCTSL from Mus musculus, suggesting that CTSL from oyster and mouse had the ability to bind Ca2+.

CTSLs are widely found in a variety of cells and tissues, and particularly abundant in macrophages and liver, respectively [41]. The expressions of CTSL in various tissues suggest its potential diverse functions [5]. In our previous study, CgCTSL1 mRNA was highly expressed in gills and hemolymph [39]. In the present study, the mRNA transcripts of CgCTSL-2 could be detected all the tested tissues with the highest level in the hepatopancreas. Hepatopancreas is a major source of immune-related proteins that can be secreted into the serum to perform immune functions [42]. The hemolymph containing a large number of hemocytes in oysters, is considered to be a key immune component that plays a significant role in mediating cellular and humoral immunity [43]. The expressions of CTSLs were up-regulated after viral or bacterial stimulation, which has been reported in both fish [44] and molluscs [5]. In the present study, the mRNA transcripts of CgCTSL-2 were also found in hemocytes and its expressions increased significantly after V. splendidus stimulation. There are three sub-populations of hemocytes, including agranulocytes, semi-granulocytes, and granulocytes [45]. CgCTSL-2 proteins were distributed in all of them with the highest distribution in granulocytes. These results suggested that CgCTSL-2 might play vital roles in defending against bacterial infection.

CTSLs mainly utilizes protein hydrolysis function to degrade protein antigens produced by pathogens [46]. In mammals, LPS triggers the maturation of CTSL and enhances its lysosomal activity [47]. LPS is the major constituents of the outer layer of Gram-negative bacteria’s outer membrane [48]. In Japanese eel epidermis, CTSL was found to participate in degrading proteoglycans, a major component of bacterial cell walls [11]. In the present study, rCgCTSL-2 with pro-peptide domain and mature domain was demonstrated to bind to LPS (in or not Ca2+). The binding sites of CgCTSL-2 with LPS were in the catalytic region. In the presence of Ca2+, LPS could enhance the activity of rCgCTSL-2. The immature pro-CTSL is translocated through the endoplasmic reticulum-golgi apparatus-lysosome axis, and then the pro-region was removed in lysosome, resulting in mature CTSL with proteolytic activity [49]. In rat macrophages, CTSL was firstly synthesized as a precursor polypeptide, then processed to an active single-chain form for stable existence [50]. In Epinephelus coioides, rEcCTSL with pro-peptide domains still performed lysis activity [44]. In our previous study, rCgCTSL1 from oyster (which lacked the signal peptide and pro-peptide domains) had enzymatic activity [39]. In the present study, rCgCTSL-2 containing pro-peptide (Inhibitor_I29) domain and Pept_C1 domain could also cleave the synthetic substrate Ac-FR-pNA to release yellow p-Nitrophenol indicating that it had the proteolytic activity. CTSL is secreted into the extracellular matrix and can also be localized within extracellular vesicle [51, 52]. In the present study, CgCTSL-2 (active) was observed in hemocytes and cell-free hemolymph, suggest that CgCTSL-2 existed as mature form with proteolytic activity in oysters. CTSL as a major member of the lysosomal cysteine protease family has the ability to lyse bacteria. CTSL was also responsible for nonoxidative killing of S. aureus within rat macrophages [53]. In E. coioides, CTSL had the bacteriolytic activity of Gram-negative bacteria (Vibrio campbellii and E. coli) [44]. In the present study, rCgCTSL-2 had no bacteriolytic activity to V. splendidus (in or not Ca2+). Interestingly, rCgCTSL-2 was demonstrated to co-localize with V. splendidus in cell-free hemolymph and had specific binding activity to V. splendidus in the presence of Ca2+. V. splendidus also promoted the activity of rCgCTSL-2 (in or not Ca2+). CgCTSL-2 specifically sensed V. splendidus and the binding of CgCTSL-2 to LPS might promote the activation of CgCTSL-2.

CTSL can cleave the intracellular complement C3 into C3a and C3b [54]. So far, the mechanism had only been reported in human CD4+ T cells [7] and Jurkat T cells [55]. Among which, CTSL in Jurkat T cells could promote iC3b production [55]. The function of CTSL in cleaving C3 is still largely unknown in other species. In invertebrates, the complement activation system had been widely studied in different species, such as shellfish [25], ascidians [56], and echinoderms [57]. Among which in oysters, the lectin pathway was outlined, which was activated by CgCLec-CCP-CgMASPL-1 axis, leading to the cleavage of CgC3 in cell-free hemolymph [25]. In the present study, CgCTSL-2 was found to interact with alpha chain fragments of iCgC3b and CgC3γ in cell-free hemolymph, as well as with CgC3b, alpha chain fragments of iCgC3b and CgC3γ in hemocytes. In rCgCTSL-2-treated oysters, the full-length CgC3 was reduced and that of CgC3b and alpha chain fragments of iCgC3b were induced in cell-free hemolymph. In addition, C3 fragments CgC3b and CgC3γ were induced in hemocytes. These results indicated that CTSL had the ability to cleave the full-length C3 and its fragments to promote C3 fragments production in oysters. In mammals, a CTSL-specific inhibitor could inhibit CTSL-mediated C3 fragments production [7, 55]. In the present study, in CgCTSL-2-RNAi oysters, the full-length CgC3 was enhanced both in cell-free hemolymph and hemocytes. While interestingly, the band intensities of alpha chain fragments of iCgC3b and CgC3γ were also enhanced in hemocytes. It was speculated that CgCTSL-2 possibly participated in the C3 fragment-mediated bacteria degradation and the inhibition of CgCTSL-2 might lead to the accumulation of C3 fragments in hemocytes.

C3 covalently deposited onto the surfaces of pathogens then is taken into the intracellular environment through its membrane receptor, such as CR3, leading to their degradation in lysosomes [58]. CR3 belongs to the family of integrins, which is consisted of a beta subunit (CD18) and an alpha subunit (CD11b) [59]. CR3 binds to iC3b, facilitating the phagocytosis of C3-opsonized pathogens, which has been reported in both mammals [58] and fish [60]. Some of the integrin family members have been identified in molluscan species [61]. CD18 with a VWA domain (adhesion to C3 molecules) and an INB domain (N-terminal integrin β subunit) is conserved [62]. So far, CD18 had been reported mainly in vertebrates [63], and it is still largely unknown in invertebrates except for oysters [64]. In our previous study, CgC3 was found to be located on the cell surface of V. splendidus in cell-free hemolymph [25]. In the present study, it was observed that CgC3 and CgCD18 could colocalize with V. splendidus in hemocytes, respectively, suggesting the existence of pathogen-C3-CD18 axis. Co-localization of CTSL and C3 in the lysosomal compartment has been demonstrated in human CD4+ T cells [14], but it is not clear whether this phenomenon is in other species. In our previous study, CgCTSL1 were found to be co-localized with the lysosomes in hemocytes [39]. In the present study, it was observed that CgCTSL-2, CgC3 and lysosomes could colocalize with each other in haemocyte cytoplasm. CTSL as a lysosomal protease is involved in the degradation of pathogens in lysosomes [53]. In rat macrophages, CTSL was involved the degradation of S. aureus in lysosomes [65]. In the present study, both CgCTSL-2 and lysosomes co-localized with V. splendidus in hemocytes. With these results, it was speculated that C3 fragment-coated on V. splendidus mediated by CgCD18 into hemocytes was degraded by the CTSL-lysosomal pathway.

In conclusion, a CTSL (CgCTSL-2) with Ca2+ binding sites were identified from oyster, whose mRNA expressions increased significantly in hemocytes after V. splendidus stimulation. It exhibited binding activity to LPS and had specific binding activity to V. splendidus in the presence of Ca2+. CgCTSL-2 cleaved CgC3 into CgC3 fragments in cell-free hemolymph and hemocytes. CgC3 fragment-coated on V. splendidus was mediated by CgCD18 into lysosomes for degradation. In addition, C3 fragments were accumulated in hemocytes of CgCTSL-2-RNAi oysters (Fig. 7). Taken together, the results provided evidence that the activation of C3 by CTSL in oysters led to the opsonization of pathogens for degradation in lysosomes, which was important for understanding the activation mechanism of complement system and its roles in antibacterial immunity in invertebrates as well as the origin and evolution of complement system.

As is known, many pattern recognition receptors identified in molluscan species all had relatively broad-spectrum ligand recognition functions. In the present study, CgCTSL-2 could bind to LPS, while it had specific binding activity to V. splendidus. LPS is a central component of the outer membrane in Gram-negative bacteria, while CgCTSL-2 had only binding activity to V. splendidus, suggesting that there might be some other binding mechanisms between CTSL-2 and V. splendidus.

Fig. 7
figure 7

CgCTSL-2 upon binding V. splendidus cleaved CgC3 to produce CgC3 fragments in cell-free hemolymph, which then opsonized V. splendidus through CgCD18 into the lysosomal pathway for bacterial degradation in hemocytes. (Figdraw was used to assist in drawing patterns)

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Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

We were grateful to all the laboratory members for technical advice and helpful discussions.

Funding

This research was supported by National Science Foundation of China (32222086, 32173002), the fund for CARS-49 and for Outstanding Talents and Innovative Team of Agricultural Scientific Research in MARA, the innovation team of Aquaculture Environment Safety from Liaoning Province (LT202009), Liaoning Revitalization Talents Program (XLYC2203087), Dalian High Level Talent Innovation Support Program (2022RG14), and Dalian Outstanding Young Scientific and Technological Talent (2022RY01).

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Authors and Affiliations

  1. Liaoning Key Laboratory of Marine Animal Immunology & Disease Control, Dalian Ocean University, 52 Heishijiao Street, Dalian, 116023, China

    Qiuyan Guo, Wenwen Yang, Weishuai Shan, Hongsheng Yao, Xiangqi Shi, Lingling Wang, Jiejie Sun & Linsheng Song

  2. Laboratory of Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266235, China

    Lingling Wang & Linsheng Song

  3. Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China

    Qiuyan Guo, Wenwen Yang, Weishuai Shan, Hongsheng Yao, Xiangqi Shi, Lingling Wang, Jiejie Sun & Linsheng Song

  4. Dalian Key Laboratory of Aquatic Animal Disease Control, Dalian Ocean University, Dalian, 116023, China

    Qiuyan Guo, Wenwen Yang, Weishuai Shan, Hongsheng Yao, Xiangqi Shi, Lingling Wang, Jiejie Sun & Linsheng Song

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  1. Qiuyan Guo
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Contributions

Conceptualization: J.S., and L.S.; methodology: Q.G., and W.Y.; formal analysis: Q.G., J.S., and W.Y.; investigation: Q.G., W.Y., H.Y., W.S., and S.S.; resources: J.S., L.W., and L.S.; writing– original draft: Q.G., and W.Y.; writing– review and editing: J.S., and L.S.; visualization: Q.G. and J.S.; supervision: J.S., L.W., and L.S.; project administration: J.S., L.W., and L.S.; funding acquisition: J.S., L.W., and L.S.

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Correspondence to Jiejie Sun or Linsheng Song.

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Guo, Q., Yang, W., Shan, W. et al. CTSL-2 upon specifically recognizing Vibrio splendidus directly cleaves complement C3 to promote the bacterial phagocytosis and degradation in oyster. Cell Commun Signal 23, 198 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12964-025-02205-z

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Keywords

Cell Communication and Signaling

ISSN: 1478-811X