THP1 cells, a human monocytic macrophage-like cell line was obtained from National Centre for Cell Science (Pune, Maharashtra, India). Mannan from Saccharomyces cerevisiae, penicillin G-sodium, streptomycin, human C4 molecule, anti-human C3b antibody, anti-human C4b antibody, NE from human leukocytes, substrate -N-α-Cbz-l-lysine thiobenzyl ester (Z-l-Lys-SBzl), VPR–7-amino-4 methyl coumarin, N-methoxy succinyl–Ala–Ala–Pro–Val-7-amino-4-methyl curamin, and DTNB [5,5-di thio-bis-(2 nitro-benzoic acid)] were purchased from Sigma-Aldrich (USA). Ficoll-Plaque Plus was purchased from GE Healthcare (Piscataway, NJ, USA). Human IgM and C1 complex were purchased from Merk-Millipore (US). M199 media and fetal bovine serum (FBS) were purchased from Invitrogen (USA) and Gibco (USA), respectively. C3c and C5c ELISA kits were purchased from Life Science, Inc. (USCN). Antibody against human C5aR, C5b-C9, PI3K, AKT, p-AKT, and p-PI3K were purchased from Santa Cruz Biotechnology (USA). Complement C1q antibody, anti-human MBL antibody, Factor B antibody against human were purchased from Abcam (Cambridge, UK). Secondary antibodies (HRP conjugated) were purchased from Jackson Laboratory (USA).

L. donovani promastigotes (AG83:MHOM/IN/1983/AG83) were used in our experiments. ISP2KD and ISP2 overexpressed (ISP2OE) Leishmania parasites were prepared in our lab as described previously (24). All the cell lines promastigotes were maintained according to Mandal et al. (25) at 25°C in 25 cm² flasks in fresh Medium-199 supplemented with 10% FBS. The ISP2KD and ISP2OE cells were maintained in M199 media supplemented with neomycin (G418) at 200 µg/ml concentration (24).

THP1 cells, a human monocytic macrophage-like cell line was cultured in RPMI-1640 medium containing 10 mM HEPES, 1 mM sodium pyruvate supplemented with 4 mM NaHCO₃, penicillin G-sodium (100 U/ml), streptomycin (100 mg/ml), and 10% (v/v) FBS. The cells were maintained in 5% CO₂ incubator at 37°C in tissue culture flask (Nunc A/S Roskilde, Denmark). The cells were subcultured at every 3–4 days to maintain the confluency of cells. For experimental infection, THP1cells (~10⁶) were treated with 20 nM phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis, MO, USA) and incubated for 24 h to become adherent and matured. Prior to infection, cells were washed with RPMI media without FBS to remove non-adherent cells.

Human mononuclear cells were isolated by the method as described by Singh et al. (26). Briefly, the peripheral blood of the healthy donor was collected in a heparinized tube. Isolated blood was processed immediately for mononuclear cell isolation by Ficoll-Paque density gradient method according to the manufacture instructions. The isolated monocytes were cultured (~1 × 10⁶ cells) in RPMI medium with 10% FBS in the presence of macrophage colony-stimulating factor (300 ng/ml) according to Mandal et al. (25). Washing was performed repeatedly to remove non- adherent cells and the cells were allowed to differentiate into human monocyte-derived macrophages (hMDM). Differentiated hMDM were harvested after 96 h for further infection study.

In our previous report, the robust homology models of LdISP1 and LdISP2 was used to monitor its interaction with trypsin and chymotrypsin (24). Here, we considered those putative models of ISP1 and ISP2 for further interaction study. First, the protein models of ISP1 and ISP2 were refined by Galaxy WEB server (27) and we have evaluated their reliability by SAVES server (ERRAT) (28). The refined models of ISP1 and ISP2 were further validated by Verify 3D. Finally ProSA-web server was used to evaluate the generated 3D structure of ISP1 and ISP2 for potential stability (29). The final refined protein model of LdISP1 and LdISP2 were used for the interaction study with the complement proteins C1r, C1s, MASP1, and MASP2. The PDB structure of the complement proteins of C1r (PDB: 1MD8_A), C1s (PDB: 1ELV_A), MASP1 (PDB: 3GOV_B), and MASP2 (PDB: 1Q3X_A) was taken from PDB database (3D structures) (30–33). The protein preparation of the selected protein structure was performed in Discovery Studio 2.5 (DSv2.5) with an application of a CHARMm force field (34). GRAMM (Global Range Molecular Matching)-X (Web Server v.1.2.0 program) was used for protein–protein docking (35). The generated complex model of proteins were further analyzed and visualized in the DSv2.5 for protein–protein interaction (24, 36).

ELISA was performed to study the interaction of rLdISP1 and rLdISP2 with C1 complex and MASPs according to Ferreira et al. (13) with minor modification. Briefly, ELISA plate was coated with either human IgM (10 µg/ml) or mannan from S. cerevisiae (5 µg/ml) in coating buffer (15 mM of NaCO₃, 35 mM of NaHCO₃, pH 9.6). Purified C1 complex (0–100 µg/ml) was diluted in PBS/1% w/v bovine serum albumin (BSA)/0.05% tween 20, whereas normal human serum (NHS) was diluted in veronal buffer saline (VBS) (145 mM/L NaCl, 1.8 m M/L sodium barbiturate, 2.8 mM barbiturate acid) in different dilution (1:100; 1:50; 1:10). Different concentration of C1 complex and serum was added to the antibody-coated plates or mannan-coated plates, respectively, incubate at 37°C for 2 h. Washing was performed throughout the assay with washing buffer, TBS (10 mM of NaCl)/0.05% Tween. Subsequently, after washing different concentration (0–50 µM) of rLdISP1 and rLdISP2 were added to the coated plates and incubated at 37°C for 2 h. Bound rLdISP1 and rLdISP2 was detected with the anti-ISP1 or anti-ISP2 antibody (1:500 dilutions) followed by addition of HRP conjugated anti-goat IgG-Ab and TMB substrate (3,3′,5,5′-tetramethylbenzidine).

The enzymatic assay was performed to observe the inhibitory property of rLdISP1 and rLdISP2 on C1 complex, MASPs and NE activities. ELISA plate was coated either with human IgM or mannan. Purified C1 complex (100 µg/ml) was bound to the antibody-coated plate as discussed previously. Mannan-coated wells were bound with 100 µl of serum (1:10 v/v) diluted in serum dilution buffer (40 mM HEPES, 2 M NaCl, 10 mM CaCl₂, pH 7.4) and incubated for 2 h at 37°C. C1 and serum bound wells were washed with washing buffer according to Presanis et al. (37). After washing, both C1 complex and MASPs-bound plate was incubated with or without rLdISP1/LdISP2 (0–50 µM) or C1-inhibitor (C1-inh) (10 nM) for 1 h. The C1 complex or mannan-coated plates without inhibitor were used as a control. Relative activity of C1 complex and MASP2 was tested by using the chromogenic substrate (100 µM) such as-N-α-Cbz-l-lysine thiobenzyl ester (Z-l-Lys-SBzl) and DTNB [5,5-di thio-bis-(2 nitro-benzoic acid)] according to Keizer et al. (38). The absorbance was recorded at 405 nm for every 10 min for 1 h. Simultaneously, MASP1 activity was analyzed by addition of MASP1 specific substrate VPR–7-amino-4 methyl to the MASPs-bound plate and incubated for 1 h at 37°C. Approximately, 100 µM of substrate diluted in 20 mM HEPES, 5 mM CaCl2, pH 8.5 (VB2 + B/T) buffer was used in the experiments. The fluorescent AMC group is released upon cleavage by active protease and the relative release of AMC was monitored over a time course using an excitation wavelength of 355 nm and an emission wavelength of 450 nm by spectroflurometer.

Additionally, the enzymatic assay was performed according to Morrisons et al. (39) to see the inhibitory effect of rLdISPs (rLdISP1 and rLdISP2) on NE. Briefly, NE (50 nM) was either left untreated or treated with inhibitor ecotin (1 µM) or rLdISP1/rLdISP2 (0.1–10 µM) in 100 mM Tris–HCl, pH 8.0, 2.5% (v/v) dimethyl sulfoxide and incubated in ice for 30 min. Subsequently, the appropriate chromogenic substrate, i.e., N-methoxy succinyl–Ala-Ala-Pro-Val-7-amino-4-methyl curamin (200 µM) was added to treated and untreated NE. Enzymatic hydrolysis of the substrate was monitored by spectrofluorometer by measuring the release of fluorescence (excitation 380 nm, emission at 460 nm). All the experiments were performed in triplicate, and the data expressed as means ± SD from three independent experiments.

To observe the inhibitory effect of rLdISPs on MASPs functional activity, C4 cleavage assay was performed in presence or absence of rLdISP1/rLdISP2 according to Ferreira et al. (13) with minor modification. Briefly, the microtitration plate was firstly coated with mannan as discussed previously. NHS was diluted (1:10 v/v) in BVB⁺ buffer (VBS containing 0.5 mM MgCl2·6H₂O, 1.5 mM CaCl₂, 0.05% tween-20, 1% BSA, pH 7.5). NHS pretreated with rLdISP1/rLdISP2 (0–50 µM) at 4°C for 1 h, or untreated NHS was added to the mannan-coated plates and incubated for 2 h at 37°C. The ELISA plate bound with serum-derived MASPs was used as a control. NHS pre-incubated with C1-inh (10 nM), the known inhibitor of classical and LP was used as the control inhibitor. C4 molecule (0.2 µg/ml) containing 20 mM HEPES, 140 mM NaCl, 5 mM CaCl₂, pH 7.4, was added to the serum bound plate and incubated at 37°C for 2 h. Simultaneously, another plate was coated with C4b antibody (diluted in coating buffer) and incubated for 2 h. The cleaved C4 product was added to C4b antibody bound plate and further incubated for 2 h. The C4b antibody was added to the plate and incubated for another 2 h. The plates were washed in each step with TBS/0.05% Tween/5 mM of CaCl₂, according to Presanis et al. (37). The bound C4b antibody was quantified by addition of HRP-conjugated secondary antibody followed by TMB substrate. Absorbance was recorded at 450 nm in an ELISA plate reader. Each experiment was done in triplicate and data are means ± SD from three independent experiments.

Functional convertase assay was performed to monitor the C3c and C5c formation via all active complement pathway and LP individually. Serum was prepared according to the different experimental setup. Briefly, to study the involvement of all three complement pathways, NHS was firstly diluted (1:10 v/v) in modified veronal buffer (VB²⁺—4 mM of 5,5-diethylbarbituric acid sodium salt, 10 mM of NaCl, 2 mM of CaCl2, and 1 mM of MgCl2, pH 7.4). To determine the contribution of LP in C3c and C5c formation, lectin pathway-specific serum (LPSS) was prepared by incubating NHS (10% v/v) with “C1q antibody + Factor B antibody” (40). To block the formation of C3 and C5c formation via all pathways and individually by LP NHS or LPSS was incubated with “C1-inh + Factor B antibody” and “C1-inh” (10 nM), respectively, kept at 37°C for 2 h. The appropriate dilution of the antibody used in the experiment was standardized experimentally.

Further, to assess the inhibitory role of rLdISP1/rLdISP2 on convertase formation, the different group of serum was incubated with rLdISP1/rLdISP2 (0–50 µM) at 37°C for 2 h. Untreated NHS and LPSS were used as a control. Leishmania promastigotes (~2 × 10⁶) were processed according to Okroj et al. (41) for the convertase formation. Processed cells were added in the different group of untreated serum or serum pre-incubated with different inhibitors and kept in an incubator shaker at 30°C with shaking at 300 rpm for 5–10 min for C3 and C5c formation. Simultaneously, for the assessment of C3c, C5c formation C3 and C5c ELISA kits were used, respectively, to perform ELISA by using the processed serum sample. The experiments were performed in triplicate and data are means ± SD from three separate experiments.

The comparative study of the complement pathway activation by Leishmania parasites was detected by the deposition of C3b molecule on parasite surface and formation of MAC (Detail in SI) (11, 42). Additionally, LP activation assay was performed using rLdISP2 to analyze the relative MAC (C5b-9) formation and parasite lysis. For the LP activation, LPSS was used. Heat inactive LPSS, active LPSS, LPSS pretreated with C1-inh (10 nM), or LPSS pretreated with different concentration of rLdISP2 (5–50 µM) were used for this study. Briefly, for LP activation, Leishmania cells were first washed three times with ice-cold Gelatin HEPES buffer saline containing 2 mM CaCl₂ and 0.5 mM MgCl₂ (GHB²⁺). Two experimental set up was made, in which an equal number of Leishmania promastigotes (~1 × 10⁷) were taken. In one set, washed parasites were fixed in 4% paraformaldehyde and coated in ELISA plate for overnight at 4°C (12). The wells were washed with phosphate buffered saline (PBS-137 mM NaCl, 1.47 mM KH₂PO₄, 4.3 mM Na₂HPO₄, 2.7 mM KCl)/0.05% tween 20 (3 times) then blocked with 3% BSA in PBS for 2 h at room temperature. For LP activation, different group of LPSS were added into the well and incubate for 1 h. Active formation of MAC via each group of serum was detected by using anti-human C5b-9 complex antibody (1: 1,000 dilution), followed by incubation with HRP conjugated secondary antibody (1:5,000). In another set, parasites were either treated with heat inactive LPSS, active LPSS, LPSS pretreated with C1-inh (10 nM), LPSS pretreated with different concentration of rLdISP2 (5–50 µM). The treated parasites were used to analyze the complement-mediated lysis by counting the number of viable parasites in hemocytometer by trypan blue method. The parasites were stained with Giemsa for the microscopic examination of the different group of serum treated parasites.

For infection experiment, equal number of THP1 and hMDM (~10⁶) were used for two experimental setups. In one set, the adhered THP1 cells and hMDM were infected with either wild-type (WT), ISP2KD, or ISP2OE Leishmania parasites at parasite/macrophage multiplicities of 10:1. Cultures were kept at 37°C in 5% CO₂ for 12 h. The unbound parasites were removed by washing with RPMI without FBS. The cells were harvested (12 h after infection) from each group, washed with PBS and kept at −80°C for RNA and protein isolation. In other set, macrophages (THP1 and hMDM) were pretreated with wortmannin (200 nM) for 3 h and then infected with WT parasites. Untreated macrophages were infected with WT, ISP2KD, and ISP2OE parasites. Infected cells were kept in incubator and cells were harvested (after 12 h) for protein isolation and the supernatant was collected for ELISA and kept at −80°C. The parasite load was measured both in THP1 cells and hMDM at 24 h after infection by counting the number of intracellular amastigotes per 100 macrophages and the rate of infection was also analyzed in both cells (THP1 and hMDM) by counting the percent infected macrophages after Giemsa staining.

THP1cells (~10⁷) were treated with 20 nM PMA to obtain a macrophage-like cell line. Further to observe the effect of NE on the expression of C5aR, macrophages (~10⁷) were treated with purified NE (10 µg/ml) and kept at 37°C for 2 h. Simultaneously, to evaluate the effect of the inhibitor on NE, NE was pre-incubated with ecotin (1 µM) or rLdISP1/rLdISP2 (10 µM) for 30 min at 4°C. These pretreated NE was used to treat the macrophages and kept at 37°C for 2 h. The macrophages were harvested for protein isolation and the protein samples were stored at −80°C for further study.

UI macrophages (THP1 and hMDM) and macrophages infected with WT, ISP2OE and ISP2KD Ld parasites (for 12 h) were harvested and used for RNA isolation, using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Simultaneously, RNA concentration was measured and cDNA was prepared by using 20 µg of RNA. cDNA was quantified and semi-quantitative PCR was performed to amplify C5aR (F-GAGGAGTACTTTCCACCAAAGG and R-AAATCGTGAGCGTGAGTAGAG). GAPDH was used as an endogenous control. The PCR was performed in thermocycler machine using thermocycling conditions -denaturation at 94°C for 5 min and 28 amplification cycles (94°C for 45 s, 56°C for 45 s, and 72°C for 1 min) followed by a final extension at 72°C for 5 min. The PCR products were run on the agarose gel (1.5%) and stained with ethidium bromide prior to analysis.

The protein samples were prepared from NE treated macrophages to evaluate the C5aR expression. 12% SDS-PAGE was used for the separation of protein samples. Western blot was performed by transferring the gel onto PVDF membrane, followed by blocking with 3% BSA. The expression of C5aR was analyzed by using anti-human C5aR antibody (1:1,000 dilution). Alkaline phosphatase conjugated secondary antibody (1:5,000 dilution), followed by NBT/BCIP was used to detect the band. Additionally, macrophages (THP1 and hMDM) infected with WT, ISP2KD and ISP2OE Ld parasites were harvested and used for protein isolation and Western blot analysis using antibody against the C5aR. Furthermore, Western blot was performed to observe the expression of AKT, p-AKT, PI3K, and p-PI3K proteins in UI macrophages (THP1) or wortmannin pretreated Ld infected macrophages, macrophages infected with ISP2KD, ISP2OE, and WT parasites using anti -AKT, p-AKT, PI3K, p-PI3K primary antibody, respectively. Alkaline phosphatase conjugated secondary antibody (1:5,000) followed by addition of NBT/BCIP solution was used for detection. GAPDH was taken as an endogenous control.

The culture supernatant of the different group of infected macrophages (THP1 and hMDM) was used to measure cytokine production of IL-12, IL-10. The cytokines level was measured by sandwich ELISA using commercially available BD OptEIA ELISA kits (BD, San Jose, CA, USA). The experiments were performed in triplicate and the data are means ± SD from three separate experiments. An asterisk (*) denotes P ≤ 0.05 and a double asterisk (**) denotes P ≤ 0.001 when compared to control.

Nowadays, bioinformatics play a vital role in elucidating the possible protein–protein or protein–ligand interacting residues and their mode of binding (24, 43–45). In our study, the final models of ISP1 and ISP2 was validated with Galaxy WEB server. To monitor the reliability of the model of ISP1 and ISP2, diverse statistical parameters were considered (Table 1). Significant favoured region in the Ramachandran plot was observed for both ISP1 (96.53%) and ISP2 (98.52%). Simultaneously, ERRAT gives a measure of the structural error for each residue in the protein (ISP1-83.178% and ISP2-71.875%). Precision of the model was checked using the ProSA web server by analysing Z-score for ISP1 (−3.76) and ISP2 (−4.68) respectively (Table 1). In this notion, in silico protein–protein interaction was performed between LdISP1/LdISP2 and the peptidase involved in classical or lectin pathway of complement channels, such as C1r, C1s, MASP1, and MASP2. Our preliminary analysis revealed that predictive model of both LdISP1 and LdISP2 formed a stable complex with C1r, C1s, MASP1, and MASP2 via non-covalent hydrogen bonds (H-bonds). The different interacting amino acids of protein–protein interaction have been shown in Figure 1 (Tables S1 and S2 in Supplementary Material). Notably, during interaction of C1r with ISP1/ISP2, three H-bonds were formed in both cases, i.e., between C1r–ISP1 complex and between C1r–ISP2 complex. Specifically, during C1r and ISP1 interaction the strong H-bond between C1r:ARG650:HH12 and ISP1:GLN57: O plays a crucial role in the formation of a stable ISP1–C1r complex (Figure 1Ai). During the interaction of C1s with ISP1 and ISP2, nine and eight H-bonds were formed, respectively (Figure 1Bi,ii). Moreover, CYS136 of ISP1 formed 4H-bonds with CYS534 and LYS631 of C1s (Figure 1Bi). This implies the possible central role of CYS136 of ISP1 in the formation of a stable ISP1-C1s complex. Similarly, the interface analysis also depicted that four and two H-bonds was formed during MASP1 interaction with ISP1and ISP2, respectively (Figures 1C). GLN142 of ISP1 was found to be participated most in H-bond formation (4H-bond) (Figure 1Ci). Interestingly, the formation of two strong H-bonds (distance <2Ǻ) between MASP2 and ISP1 as well as MASP2 and ISP2 implies that both ISP1 and ISP2 are capable to form a stable ISPs–MASP2 complex (Figure 1D). Additionally, the key residue, MASP2:LEU575 formed two H-bond with ISP2:ASN55 and enhances its interaction stability (Figure 1Dii). Therefore, from the in silico analysis, it can be speculated that both LdISP1 and LdISP2 strongly interact with the complement serine peptidases C1r, C1s MASP1, and MASP2, where H-bond profile act as one of the most driving forces for protein–protein interaction stability.

It was reported previously that the strength of the protein–protein interaction could be indirectly estimated by some measured output signals that is directly proportinal to the receptor–ligand complex formation (46). Therefore, to further assess the interaction of rLdISP1 and rLdISP2 with C1 complex and MASPs, direct binding experiment, i.e., ELISA was conducted (Figure 2). In order to quantify the interactions between the biological molecules, the determination of dissociation constants (Kd) or association constant (Ka) is one of the most important factor (46, 47). Kd represent the concentration of free ligand that is responsible for the half of the maximum saturation of receptor. In our study, Kd and Ka was determined according to Wilkinson et al. (46) during rLdISP1/rLdISP2 interaction with C1 complex (Table 2i) and MASPs (Table 2ii). At different concentration of C1 complex the dissociation constant for rLdISP1/rLdISP2 was measured and it was found inbetween the range (3.8–1.6 μM) for rLdISP1 and (3.5–1.6 µM) for rLdISP2 (Table 2i). The maximum binding affinity of rLdISP1/rLdISP2 was observed at 100 µg/ml concentration of C1 complex and assocition constant (Ka) was found to be 0.62 and 0.76 µM⁻¹ for rLdISP1and rLdISP2, respectively. This result showed that low range of rLdISPs (3.8–1.6 µM) was required for half of the saturation of C1 complex. A dose-dependent saturation curve was observed by increasing concentration of rLdISP1/rLdISP2 up to 50 µM (Figures 2A,B). Similarly, a dose-dependent and saturation binding of rLdISP1/rLdISP2 was also observed with MASPs at different dilution of serum (Figures 2C,D). In different dilutions of serum, the half of the saturation was observed between 1.2–3.2 µM concentration of rLdISP1 or rLdISP2 (Table 2ii). These findings suggest that low range of rLdISP1 and rLdISP2 (1.2–3.2 µM) was required for half of the MASPs saturation. It was found that the binding capacity of rLdISPs increases with decreasing dilution of serum. Moreover, the maximum binding of rLdISPs was observed at 1:10 dilution of serum and Kd was found to be 1.4 and 1.2 µM for rLdISP1 and rLdISP2, respectively. This result showed that rLdISP2 has higher binding affinity (Ka-0.83 μM⁻¹) for MASPs than rLdISP1 (Ka-0.71 μM⁻¹). The control protein sample containing only C1 complex or MASPs (no ISP), taken as the negative control did not show significant optical density. These results indicated that rLdISP1 and rLdISP2 have an efficient binding affinity for both C1 complex and serum-derived MASPs.

In order to investigate the effect of rLdISP1 and rLdISP2 on C1 complex and MASP1/MASP2 activity, specific enzyme assay was carried out (Figure 3). No significant inhibition of the enzyme activity of C1 complex and MASP1 was observed in presence of rLdISP1 and rLdISP2 (concentration up to 50 µM) (Figures 3A,B) when compared to control. However, the enzyme activity of serum-derived MASP2 was reduced by ~30, ~44, and ~60% in the presence of 5, 10, and 50 µM of rLdISP2, respectively, when compared to the untreated serum (control) (Figure 3C). C1-inh (C1-inh), significantly inhibited the proteolytic activity of both enzyme C1 complex and MASP1/MASP2.

It was previously reported that MASP2 has the ability to cleave C4 molecule to produce C4a and C4b (4). Therefore, in this study, we aimed to investigate the effect of rLdISP1 and rLdISP2 on the MASPs-mediated cleavage of C4 molecule that might affect C4b formation. The ELISA plate coated with serum-derived MASPs was used as a control. No significant inhibition in C4b formation was observed in presence rLdISP1 even at higher concentration (50 µM), when compared to control (Figure 4A). However, a significant inhibition in C4b formation was observed in presence of rLdISP2 and C1-inh. C4b formation was found to be decreased by ~2.1 and ~2.9-fold in presence of 10 and 50 µM rLdISP2, respectively, when compared to control (Figure 4B). Since we have observed previously that rLdISP2 was unable to inhibit the enzyme activity of MASP1, it is evident that the reduction in C4 cleavage occurred solely due to reduced activity of MASP2 by rLdISP2.

Addition of Leishmania parasites to the active human serum lead to activate complement pathways resulting in the formation of C3 and C5c. The effect of the rLdISPs on C3 and C5c formation was assessed by using human serum or human serum pre-incubated with different concentration (0–50 µM) of rLdISP1 or rLdISP2. C3c and C5c formation via all complement pathways or individually via lectin pathway were accessed by sandwich ELISA. ~1.5- and ~2.5-fold reduction in C3c by all pathways was observed in presence of 10 and 50 µM of rLdISP2, respectively, when compared to control (Figure 5A). Simultaneously, by lectin pathway a ~2- and ~4-fold reduction in C3c was observed in presence of 10 and 50 µM of rLdISP2, respectively, when compared to control (Figure 5B). Similarly, the C5c formation by all three pathways was reduced by ~1.5- and ~2.9-fold at 10 and 50 µM rLdISP2, respectively, when compared to control (Figure 5C). Simultaneously, a ~1.4- and ~3.2-fold reduction in C5c by lectin pathway was observed at 10 and 50 µM of rLdISP2, respectively, when compared to control (Figure 5D). Serum pre-incubated with “C1-inh + Factor B antibody” and C1-inh was used as a negative control that significantly reduced the convertase formation by all contributing pathways and lectin pathway, respectively. No significant change in C3c, C5c formation was observed in presence of rLdISP1 when compared to the untreated NHS or LPSS (control).

Altered level of C3c and C5c in presence of rLdISP2 would ultimately lead to affect C3b formation, MAC (C5b-9 complex) formation and pathogen killing by complement-mediated lysis. Therefore, C3b deposition and MAC formation individually by the CP, LP, AP was analyzed in case of Leishmania donovani infection, and it was observed that the lectin pathway is the most activated pathway among all the three pathways (CP, LP, AP) (SI). As previous findings suggested that LdISP2 inhibits LP associated MASP2; therefore, MAC formation and complement-mediated lysis by LP was analyzed in presence of rLdISP2. ~1.5- and ~2.3-fold decrease in MAC formation was observed via LP in presence of 10 and 50 µM of rLdISP2 when compared to active LPSS (control) (Figure 6A). Simultaneously, a ~1.8- and ~2.5-fold reduction in the parasites lysis via LP was also observed in presence of 10 and 50 µM of rLdISP2, respectively, when compared to control (Figure 6B). Microscopic observation of the parasite also revealed that the parasite lysis via lectin pathway was significantly reduced in presence of rLdISP2 (5–50 µM) (Figure 6C). However, no significant changes in MAC formation and parasite lysis was observed in presence of heat inactive serum and C1-inh treated serum.

The activity of NE in presence of rLdISP1or rLdISP2 was analyzed by enzymatic assay using chromogenic substrate. ~49, ~60, and ~82% reductions in enzyme activity was observed in presence of 0.1, 1, and 10 µM of rLdISP2, respectively (Figure 7A). However, only ~27% inhibition of enzyme activity was observed in presence of rLdISP1 (10 µM). Additionally, the effect of NE and NE specific inhibitor including rLdISP1/rLdISP2 on the expression of macrophage C5aR was analysed (Figure 7B). An approximately 1.6-fold decrease in C5aR expression was observed in NE (10 µg/ml) treated macrophages. Furthermore, a ~2.5-fold increase in C5aR expression was observed in macrophages treated with NE pretreated with rLdISP2 (10 µM), when compared to control. In presence of NE pretreated with ecotin (1 µM) the C5aR expression was increased by ~2-fold when compared to control. However, no significant change in expression of C5aR was observed in macrophages treated with NE pre-incubated with rLdISP1 when compared to control. Our results demonstrated that NE activity leads to decrease in C5aR expression whereas inhibition of NE activity by ecotin or rLdISP2 significantly induces the expression of C5aR.

The expression of C5aR was analyzed at the transcript and protein level in macrophages (THP1 and hMDM) infected with WT, ISP2OE, and ISP2KD Ld parasites. Interestingly, at transcript level in case of THP1 cells, a ~5-fold increase in C5aR expression was observed in WT infected macrophages when compared to UI macrophages. However, a ~1.8-fold increase and a ~2.3-fold decrease in expression of C5aR was observed in ISP2OE and ISP2KD infected macrophages, respectively, when compared to WT infected macrophages (Figure 8A). However, in hMDM cells, a significant ~2.2-fold increase in C5aR expression was observed in WT infected macrophages when compared to UI macrophages whereas, a significant ~2.7-fold increase and a ~1.3-fold decrease in expression of C5aR was observed in ISP2OE and ISP2KD infected macrophages, respectively, when compared to WT infected macrophages (Figure 8B).

Simultaneously, at protein level in THP1 cells, a ~5-fold increase in the expression of C5aR was also observed in WT infected macrophages when compared to UI macrophages. Moreover, a ~1.6-fold increase in expression of C5aR in ISP2OE infected macrophages and a ~1.7-fold decrease in C5aR expression in ISP2KD infected macrophages was observed when compared to WT Ld infected macrophages (Figure 8C). Similarly, in case of hMDM cells, the expression of C5aR was found to be significantly increased by ~2.2-fold in WT infected macrophages when compared to UI macrophages. Simultaneously, we have observed a significant ~2.5-fold increase in expression of C5aR in ISP2OE infected macrophages and a ~2-fold decrease in C5aR expression in ISP2KD infected macrophages when compared to WT Ld infected macrophages (Figure 8D). All these results demonstrated that knocked down of LdISP2 significantly downregulate the expression of host macrophages C5aR expression.

The expression of PI3K, p-PI3K, AKT and p-AKT was analyzed in case of WT, ISP2KD, and ISP2OE Leishmania infection to macrophages (THP1 cells) (Figure 9A). Interestingly, a ~2- and ~1.5-fold increase in phosphorylation of PI3K and AKT was observed in WT infected macrophages when compared to UI. However, a ~3.3- and ~3.2-fold decrease in p-PI3K and p-AKT expression was observed in wortmannin-treated macrophages infected with WT Ld when compared to untreated macrophages infected with WT Ld. Additionally, a ~2-fold increase in p-PI3K and ~1.6-fold increase p-AKT expression was observed in macrophage infected with ISP2OE parasites when compared to WT infected macrophages. Moreover, a ~2- and ~1.6-fold decrease in expression of p-PI3K and p-AKT were observed, respectively, in macrophages infected with ISP2KD parasites when compared to WT Ld-infected macrophages. These results demonstrated that Leishmania infection induces PI3K and AKT activation and their phosphorylation is significantly increased in the presence of LdISP2.

Simultaneously, the involvement of PI3K and AKT in Th1/Th2 balance was further assessed by measuring the cytokines, i.e., IL-12 and IL-10 at the released level in macrophages (THP1 and hMDM) infected with Leishmania parasites (Figures 9B,C). In case of THP1 cells, in wortmannin pretreated macrophages infected with WT parasites, IL-12 level increased ~2.7-fold. Additionally, a ~1.6-fold decrease and ~2.6-fold increase in IL-12 was observed in macrophages infected with ISP2OE and ISP2KD parasites, respectively (Figure 9Bi). Similarly, in case of hMDM cells, in wortmannin pretreated macrophages infected with WT parasites, IL-12 level increased ~2.5-fold. However, a ~2.3-fold decrease and ~1.5-fold increase in IL-12 was observed in macrophages infected with ISP2OE and ISP2KD parasites, respectively (Figure 9Bii). Moreover, the IL-10 level was also found to be significantly altered in macrophages (THP1 and hMDM) infected with ISP2KD and ISP2OE parasites. Interestingly, in THP1 cells we observed a ~2-fold increase in IL-10 in WT infected macrophages when compared to noninfected macrophages and a ~1.8-fold decrease in IL-10 level in wortmannin pretreated WT infected macrophages when compared to untreated macrophages infected with WT Ld. We have also found significant ~1.6-fold decrease and ~1.3-fold increase in IL-10 production in macrophages infected with ISP2KD and ISP2OE parasites, respectively (Figure 9Ci). Similarly, in case of hMDM cells we have found a ~1.8-fold increase in IL-10 in WT infected macrophages when compared to noninfected macrophages and a ~1.6-fold decrease in IL-10 level in wortmannin pretreated WT infected macrophages when compared to untreated macrophages infected with WT Ld. Simultaneously, we have found significant ~1.5-fold decrease and ~1.4-fold increase in IL-10 production in macrophages infected with ISP2KD and ISP2OE parasites, respectively (Figure 9Cii).

To evaluate the effect of LdISP2 on the survival of Leishmania inside macrophages (THP1 and hMDM), parasite load and percentage of infectivity were measured in wortmannin pretreated macrophages infected with WT Ld and macrophages infected with WT, ISP2KD, ISP2OE Leishmania parasites for 24 h. Microscopic picture after Giemsa staining displaying different parasite load inside infected macrophages (THP1) (Figure 10A). In case of THP1 cells, an approximately ~2.5-fold decrease and a ~1.7-fold increase in parasite load were observed in macrophages infected with ISP2KD and ISP2OE Ld parasites, respectively, when compared macrophages infected with WT parasites (Figure 10B). Parasite load was found to be decreased by ~2.8-fold in wortmannin pretreated infected macrophages when compared to WT Ld infected macrophages (Figure 10B). Moreover, in case of hMDM cells, the parasite load was found to be ~2.3-fold decreased and ~1.75-fold increased in macrophages infected with ISP2KD and ISP2OE Ld parasites, respectively, when compared macrophages infected with WT parasites. Parasite load was severely affected in wortmannin pretreated infected macrophages and found to be ~2.6-fold decreased when compared to WT Ld infected macrophages (Figure 10C). Simultaneously, the percentage of infectivity was also severely affected in macrophages (THP1 and hMDM) infected with ISP2KD and ISPEOE parasites respectively when compared WT infected macrophages. In case of THP1 cells the percentage of infectivity was reduced by ~1.6-fold in wortmannin pretreated Ld infected macrophages when compared to WT Ld infected macrophages. The percentage of infectivity, in case of macrophages infected with ISP2KD parasites was ~1.4-fold less than WT Ld infected macrophages whereas, in case of ISP2OE Ld infected macrophages the percent infectivity was found to be ~1.3-fold higher than the WT Ld infected macrophages (Figure 10D). Moreover, in case of hMDM cells, the percentage of infectivity was decreased by ~1.6-fold in wortmannin pretreated Ld infected macrophages when compared to WT Ld infected macrophages. However, the percentage of infectivity was reduced by ~1.3-fold and increased by ~1.3-fold in case of macrophages infected with ISP2KD and ISP2OE Ld, respectively, when compared to WT Ld infected macrophages (Figure 10E). Therefore, our data demonstrated that LdISP2 activates PI3K/AKT pathway and increased IL-10 level that promotes parasite survival inside the host, whereas downregulation of ISP2 led to decreased activation of PI3K/AKT pathway and increased IL-12 level that severely affects parasite survival inside the host.