All animal procedures were reviewed and approved by the National Institute of Allergy and Infectious Diseases (NIAID) Animal Care and Use Committee under protocol LMVR4E and handled in accordance to the Guide for the Care and Use of Laboratory Animals and with the NIH OACU ARAC guidelines and also approved by Ethics Committee in Animal Experimentation (CETEA) of Universidade Federal de Minas Gerais (UFMG) under Protocol no. 87/2011.

Sand fly transcripts coding for Lufaxin and 11 other salivary proteins were cloned into the VR2001-TOPO vector as described before (21, 22). Recombinant proteins were produced in Leidos Biomedical Research PEL facility by transfecting HEK 293 F cells with the VR2001-TOPO DNA plasmids coding for the different sand fly salivary proteins and incubated for 72 h. The supernatant was concentrated and further purified by HPLC (NGC Chromatography system, Bio-Rad Laboratories, Inc.) using a HiTrap chelating HP column (GE Healthcare) charged with Ni₂SO₄. Imidazole was removed from fractions containing Lufaxin by washing with PBS using a 5,000, 10,000, or 30,000 MWCO Amicon filter (Millipore). Purified Lufaxin was analyzed by NuPage 4–12% gels (4) and stored at −80°C until use. Aliquots did not undergo more than three freeze-thaw cycles.

Twelve recombinant salivary proteins from L. longipalpis were tested on standardized AP-mediated hemolysis assays in order to detect anti-complement activity as previously described (23). The rabbit erythrocytes were acquired from CompTech or collected by venous puncture from a rabbit kept on the animal facility of UFMG. Before the experiments, 500 µl of rabbit blood were washed three times in 5 ml of Mg-EGTA solution (1 mM HEPES, 30 mM NaCl, 10 mM EGTA, 7 mM MgCl2, 3% glucose, and 0.02% gelatin, pH 7.4) as described in Ref. (16). The erythrocyte concentration was adjusted to 1 × 10⁸ cells/ml. All the experiments were performed at pH 7.4, unless specified. Briefly, in 1.5 ml microcentrifuge tubes, 25 µl of normal human serum (NHS, CompTech) diluted 1:20 in Mg-EGTA buffer (1 mM HEPES, 30 mM NaCl, 10 mM EGTA, 7 mM MgCl₂, 3% glucose, and 0.02% gelatin, pH 7.4) were mixed with 12.5 µl of PBS containing 1 µg of each recombinant protein. Then, 25 µl of Mg-EGTA containing 2.5 × 10⁶ rabbit red blood cells were mixed and the tubes incubated for 30 min at 37°C for complement activation. Final concentration of NHS in 62.5 µl of buffer was 2%. After incubation, 250 µl of cold PBS were added and the tubes rapidly centrifuged. Two hundred microliters of the supernatants were transferred to microplates and read at 415 nm. Tubes incubated without any recombinant protein were used as positive controls and tubes without NHS were used as negative controls. Tubes containing red blood cells but without NHS and recombinant proteins were combined with 250 µl of distilled water and were used to obtain total hemolysis. The assays were performed in duplicate, and in every test the mean of negative control was subtracted from the mean of the other results. The results were then transformed in percentage of lysis, considering tubes treated with distilled water as 100% of hemolysis.

In some experiments, AP-mediated hemolytic assays were performed with different concentrations of recombinant Lufaxin or LJS169 using Mg-EGTA buffer at pH 7.4 and pH 8.15 [pH in the intestinal lumen of L. longipalpis right after blood meal (24)]. Similar assays were also carried out using properdin-depleted serum (CompTech) diluted 1:2 in Mg-EGTA buffer (pH 7.4) and final concentrations of Lufaxin ranging from 0 to 200 nM.

In order to investigate if Lufaxin inhibits classical pathway, CP-mediated hemolytic assays were performed using IgG-sensitized sheep red blood cells (CompTech) as described by Ferreira et al. (6). Possible inhibition of LP by Lufaxin and LJS169 was investigated as described in the study of Mendes-Sousa et al. (23) through detection of the deposition of C4b component on Saccharomyces cerevisiae’s mannan-coated microplates using anti-C4 polyclonal antibodies (CompTech).

Balb/c mice were injected three times every 2 weeks, intradermally in the ear with 5 µg of recombinant Lufaxin mixed (1:1 volume) with Magic™ Mouse Adjuvant (Creative Diagnostics) as recommended by the manufacturer. Fifteen days after the last inoculation, blood was collected to obtain the Lufaxin antiserum. The ability of Lufaxin antiserum to recognize the native and recombinant Lufaxin was checked by Western blot as previously described (4). Lufaxin (500 ng) and SGH (five pairs of L. longipalpis glands) were run in a NuPAGE 4–12% gel (ThermoFisher Scientific) and transferred to nitrocellulose membranes (ThermoFisher Scientific). Membranes were blocked with 5% non-fat milk solution overnight, incubated with Lufaxin antiserum (1:1,000 dilution) or mice pre-immune serum (control; 1:1,000 dilution), followed by anti-mouse secondary antibodies (Promega), and developed by addition of Western Blue^® stabilized substrate for alkaline phosphatase (Promega).

Rabbit erythrocytes (5 × 10⁶) with NHS (1:20) and samples were incubated at 37°C for 30 min, and the percentage of hemolysis was quantified. Erythrocytes incubated with distilled water were considered as 100% of hemolysis. Results were expressed as mean ± SEM of at least three independent experiments. Lufaxin (100 nM) or L. longipalpis salivary gland homogenate (SGH) was incubated at 37°C for 10 min with varying dilutions of anti-Lufaxin serum prior to the AP-hemolytic assays. The Lufaxin-serum or SGH/serum mixture was then used in hemolytic assay as described above.

ELISA plate (Corning Inc.) wells were filled with 100 µl of 0.1% agarose solution and dried completely by overnight incubation at 37°C. Then, 100 µl of HMEBN buffer (5 mM HEPES, 7 mM MgCl₂, 10 mM EGTA, 5 mg/ml BSA, and 140 mM NaCl, pH 7.4) containing 20% NHS and different concentrations of Lufaxin or LJS169 were added to the wells and the plate incubated at 37°C for 30 min under agitation. After two washes, the wells were incubated for 30 min with antibodies against each specific complement components produced in goat [C3, factor B, properdin, C5, or C9 (CompTech)] diluted in 10 mM HEPES and 140 mM NaCl solution. After two more washes, the wells were treated with anti-goat peroxidase-conjugated antibodies (Sigma-Aldrich) and then developed with 200 µl of developing buffer (50 mM Na₃C₆H₅O₇, 50 mM Na₂HPO₄, 1 mg/ml o-phenylenediamine (Sigma-Aldrich), and 0.075% H₂O₂, pH 5.0). The plate was read at 450 nm at kinetic mode (one read every 30 s) at 37°C for 10 min. Wells without serum and without recombinant protein were used as negative and positive control, respectively. The assays were performed in triplicate. The mean of negative controls was subtracted from the mean of other results and then transformed in percentage of deposition considering the positive control as 100% deposition.

To detect activation of complement components in the presence of Lufaxin, Western blotting assays were performed as described in the study of Mendes-Sousa et al. (23). First, AP-mediated hemolytic assays were done as described above in the presence of Lufaxin, LJS169, or PBS. After different times of incubation (0, 30, and 60 min), an aliquot of the supernatant of each tube was collected, mixed with denaturing sample buffer, and loaded onto SDS gels. Purified factor B (10 ng), Bb (10 ng), and C3a (5 ng) (CompTech) were loaded on the SDS gels as controls.

In the assays for detection of C3 activation, an aliquot with diluted NHS without inhibitor and red blood cells was also loaded onto the gel. After transfer to nitrocelullose membranes (Bio-Rad Laboratories, Inc.), the membranes were blocked for 2 h with 0.05% Tween 20 and 10% dried non-fat milk in PBS under agitation and then incubated with goat anti-factor B or rabbit anti-C3a antibodies (CompTech) diluted 1:1,000 in 0.05% Tween 20 and 1% BSA in PBS for 1 h. After washing three times with 0.05% Tween 20 in PBS, the membranes were treated with anti-goat or anti-rabbit peroxidase-conjugated antibodies (Sigma-Aldrich) diluted 1:3,000 in the same buffer of primary antibodies for 1 h. Visualization of the proteins was possible after treating the membranes with the peroxidase substrate diaminobenzidine kit (Vector Laboratories).

To determine if Lufaxin acts at MAC formation, AP-mediated hemolytic assays were performed with some modifications. Primarily, in 1.5 ml microcentrifuge tubes, 25 µl of C6-depleted serum (CompTech) diluted 1:20 in Mg-EGTA buffer were mixed with 12.5 µl of PBS containing recombinant Lufaxin or LJS169 and 25 µl of rabbit red blood cells and incubated at 37°C for 15 min. The final concentration of the recombinant proteins was 75 nM. Tubes without recombinant proteins (only PBS) were used as negative controls. After incubation, the tubes were centrifuged (1 min, 1,700 g) and the supernatant discarded. Erythrocytes were resuspended in a mixture of 12.5 µl PBS plus 50 µl of NHS diluted 1:40 in GHB-EDTA buffer (5 mM HEPES, 145 mM NaCl, 10 mM EDTA, and 0.1% gelatin, pH 7.4) and incubated at 37°C for 30 min. After this step, the assay followed as described previously.

For detection of inhibition after the C6 component (MAC assembly), the assay was performed without recombinant protein during the first incubation, and the erythrocytes were resuspended in a mixture of 12.5 µl of PBS containing Lufaxin or LJS169 and 50 µl of NHS diluted 1:40 in GHB-EDTA buffer. Detection of hemolysis was evaluated as described above.

In order to check if Lufaxin affects factor D activity, enzymatic assays based on Kam et al. (25) were performed. Purified factor D (6 µg/ml) (CompTech), Lufaxin (0.1 µg), and 0.1 mM aldrithiol (Sigma-Aldrich) were combined in 0.1 M HEPES, 0.05 M NaCl, and 10% DMSO (pH 7.4) buffer (100 µl final volume) in 1.5 µl microcentrifuge tubes. After homogenization, the content of the tubes was transferred to ELISA microplates and 100 µl of 2 mM Z-1-Lys-SBzL substrate (Sigma-Aldrich) diluted in the same buffer was added to the wells. The plate was immediately read at the kinetic mode at 37°C for 30 min at 324 nm. Tubes without Lufaxin and factor D were used as positive and negative controls, respectively. Tubes with only Lufaxin, aldrithiol, and the substrate were also used to check if Lufaxin have any effect over the substrate.

ELISA plates (Corning Inc.) were incubated overnight at 4°C with 50 µl of coating buffer (35 mM Na₂CO₃, 15 mM NaHCO₃, pH 9.6) containing 0.5 µM of purified C3b, properdin, factor B, Bb, or factor D (CompTech). Wells incubated with 1% BSA in coating buffer were used as negative controls. Following a 1 h blocking with 1% BSA in PBS, the wells were incubated with 120 nM of Lufaxin in 50 µl of PBS for 30 min under agitation. The plate was washed twice with 0.05% Tween 20 in PBS and then treated with mouse anti-Lufaxin serum diluted 1:500 in blocking buffer (1% BSA in PBS). After two more washes, the wells received 50 µl of blocking buffer containing alkaline phosphatase-conjugated anti-mouse antibody (Sigma-Aldrich) diluted 1:10,000 for 30 min. The plate was washed and the reaction was completed by adding 100 µl of p-nitrophenyl phosphate substrate for alkaline phosphatase (Sigma-Aldrich). After 20 min of incubation at 37°C, the plate was read at 450 nm.

C3b was diluted in 10 mM sodium acetate buffer (pH 5.0) and immobilized on CM5 sensor chips (GE Healthcare) by the amine-coupling method. Assays were conducted at 25°C on a Biacore T100 instrument (GE Healthcare) by passing different combinations of complement factors and Lufaxin over the C3b immobilized surface. The buffer for these experiments (HBS-N) contained 10 mM HEPES, pH 7.4, 150 mM NaCl. In most cases, either MgCl₂ or NiCl₂ was added to the buffer to a concentration of 2 mM.

To assay the ability of Lufaxin to inhibit the activation of C3, the C3b-Bb complex was formed in solution by incubating 200 nM C3b, 100 nM factor B, and 50 nM factor D in HBS-N buffer containing 2 mM MgCl₂ for 2 min at room temperature. After stopping the formation of activated complex by the addition of EDTA (5 mM), purified C3 was added at various concentrations in the presence or absence of Lufaxin (1 µM), and the mixtures were incubated for 20 min at room temperature before the addition of SDS-PAGE sample preparation buffer. Inhibition of factor B activation was evaluated by incubating 200 nM C3b, 100 nM factor B, and 50 nM factor D in the presence or absence of 1 µM Lufaxin in HBS-N containing 2 mM MgCl₂ for 30 min. The reactions were stopped by adding EDTA to a concentration of 5 mM. In both types of experiments, the reaction products were separated by SDS-PAGE and blotted to nitrocellulose. To detect C3 cleavage, blots were incubated with rabbit anti-C3a, and to detect factor B cleavage they were incubated with goat anti-factor B, followed by incubation with the appropriate alkaline phosphatase conjugate antibody. The blots were then developed using Western Blue alkaline phosphatase substrate reagent (Promega).

Inhibition of samples was analyzed using GraphPad Prism 5.0 software. All assays were carried out in duplicate (hemolytic assays) or triplicate (deposition assays) with at least three biological repetitions. Mean of groups was statistically compared using ANOVA followed by Tukey’s test using 95% as significant level (p < 0.05).

To identify the AP-complement inhibitor in L. longipalpis saliva, 12 recombinant salivary proteins, chosen from among the most abundant molecules (26), were tested in AP hemolytic assays. Only one sand fly salivary recombinant protein of 32.5 kDa, Lufaxin, produced a complete reduction in AP erythrocyte lysis (Figure 1A). Lufaxin inhibited AP erythrocyte lysis in a dose-dependent manner at host physiological pH (7.4) and sand fly midgut pH (8.15) (24) in comparison to the control protein LJS169, another sand fly recombinant salivary protein (Figure 1B). Statistical significance was observed at Lufaxin concentrations of 40 nM or above (p < 0.0001), confirming its inhibitory effect on the AP.

The possible activity of Lufaxin against other complement pathways was also examined. The CP-mediated hemolytic assay (Figure 1C) and LP-mediated activation (Figure 1D) were not inhibited by Lufaxin or the control protein LJS169.

We tested whether neutralization of Lufaxin by antibodies would prevent blockage of AP-mediated rabbit erythrocyte hemolysis by Lufaxin or sand fly saliva. Anti-Lufaxin serum produced in mice was able to recognize both the recombinant and native Lufaxin (Figure 2A). Recombinant Lufaxin inhibited AP activation in a dose-dependent manner (Figure 2B), and addition of anti-Lufaxin serum to recombinant Lufaxin partially rescued complement activation at a dilution of 1:25, and completely rescued activation at a 1:10 dilution (Figure 2C). In order to investigate if Lufaxin is the only AP inhibitor in L. longipalpis saliva, we tested the effect of anti-Lufaxin serum on L. longipalpis SGH in the AP hemolysis assay. First, we confirmed that SGH also inhibited AP activation in a dose-dependent manner (Figure 2D) and next that addition of anti-Lufaxin serum at a dilution of 1:10 completely rescued AP complement activation (Figure 2E). These results indicate that Lufaxin accounts for most or all AP-inhibitory activity in L. longipalpis saliva.

Agarose-coated microplates were used to measure the deposition of complement components after activation of the AP. In the presence of Lufaxin, deposition of C3b, the first activated component of the AP, was strongly inhibited in a dose-dependent manner (Figure 3A). This correlates with the inhibition of subsequent components of the complement cascade (Bb, properdin, C5b, and C9) by Lufaxin (Figures 3B–E). The negative control (LJS169) showed no effect on the deposition of the tested components (Figures 3A–E).

Inhibition of complement activation by Lufaxin was further confirmed by Western blotting of supernatants from the AP-hemolysis assay using anti-factor B and anti-C3a polyclonal antibodies in the presence of Lufaxin or LJS169. Lufaxin blocked factor B cleavage, since no bands (or very weak bands) corresponding to factor Bb or Ba were detected on immunoblots at 30 or 60 min after AP complement initiation as compared to treatment with LJS169 (Figure 3F). Similarly, when Lufaxin was added to tubes with serum and red blood cells, a band corresponding to C3a was not observed, indicating that it inhibited the activation of C3, even after 60 min at 37°C (Figure 3G). These results strongly suggest that Lufaxin prevents the formation of C3b and Bb, the components of the AP C3-convertase complex.

Hemolysis assays were then performed to determine if Lufaxin acts exclusively at the early steps of the activation cascade or may act on any MAC component. Hemolytic assays were carried out using C6-depleted serum and red blood cells, and, after removal of the supernatant, MAC formation was elicited by addition of NHS diluted in buffer containing EDTA to prevent the formation of additional C3 convertase complexes. When Lufaxin was added with the C6-depleted serum, hemolysis was significantly reduced (p < 0.0001), suggesting that Lufaxin impairs C3 convertase formation (Figure 3H). The strong hemolysis seen in tubes without inhibitor, or with the control recombinant LJS169, indicates that the C6-depleted serum was active (Figure 3H). When Lufaxin was added together with the NHS, after C3 convertase assembly, no effect on MAC formation was observed (p = 0.7657) (Figure 3I), providing further evidence that Lufaxin acts exclusively on the early steps of the AP, where C3b and Bb are produced.

To determine how Lufaxin impairs C3-convertase formation, we first tested whether it is a direct inhibitor of factor D, the serine protease that cleaves factor B in the AP pathway. Lufaxin, purified factor D, and aldrithiol were mixed and added to factor D substrate Z-1-Lys-SBzL. Similar levels of substrate cleavage were seen in tubes containing factor D with or without Lufaxin, indicating that Lufaxin does not inhibit the enzymatic activity of factor D (Figure 4A).

As Lufaxin does not act on factor D, we then checked which AP complement components may be affected by Lufaxin. ELISA plates were coated with different AP complement components followed by addition of Lufaxin which was able to bind significantly to properdin (p < 0.001). Lufaxin was not able to bind significantly (p > 0.05) to C3b, factor B, Bb, and factor D when they were alone on the plate surface (Figure 4B).

The role of Lufaxin binding to properdin was assessed by hemolytic assays using properdin-depleted sera. Lufaxin maintained its AP inhibitory activity even in the absence of properdin (Figure 4C), suggesting properdin binding is not essential for AP inhibition.

The ability of Lufaxin to bind the active C3 convertase C3b-Bb, or the proconvertase C3b-B, was evaluated using SPR. In these experiments, factor D, factor B, and Lufaxin were run in different combinations over surfaces of immobilized C3b in the presence of Mg²⁺. Factor B bound detectably to immobilized C3b, and stability of the complex was significantly increased by adding factor D to the mobile phase (Figures 5A,B), indicating the formation of C3b-Bb. Lufaxin alone did not bind C3b (Figures 5A,B), but when it was added to factor B, or to the factor B-factor D mixture prior to injection, a large increase in the stability of the complex formed on immobilized C3b was observed (Figure 5A). Under conditions of constant factor B concentration, complex formation was found to depend on the concentration of Lufaxin (Figure 5C). These results indicate that Lufaxin binds to the C3b-B complex and possibly to the C3b-Bb complex. To distinguish these possibilities, the C3b-Bb complex was formed by injection of a mixture of factor B and factor D over a C3b surface (Figure 5B) and, after formation of the complex, Lufaxin was injected alone while the complex slowly dissociated. No significant Lufaxin binding was observed in this case, suggesting that the inhibitor binds only to the C3b-B complex and not to C3b-Bb (Figure 5B). The fact that complex stabilization by Lufaxin was essentially equivalent in the presence and absence of factor D suggests that the inhibitor stabilizes the C3b-B complex, while preventing activation of factor B by factor D (Figure 5A).

Magnesium ion binds at the metal ion-dependent adhesion site (MIDAS) of the VWA domain of factor B and is coordinated by the C-terminus of C3b (27, 28). Replacement of Mg²⁺ with Ni²⁺ stabilizes an open form of C3b-bound factor B that is susceptible to cleavage by factor D (29, 30). To gain insight into the mechanism of complex formation we examined the ion specificity of C3b-B-Lufaxin formation using SPR. Binding of factor B to a C3b surface in the presence of Ni²⁺ was enhanced, and the addition of Lufaxin led to a further increase in the amplitude of the sensorgram (Figure 5D). However, fitting the dissociation phase data to a single exponential decay function indicated that the half-time for C3b-B-Lufaxin dissociation (~400 s) was of similar magnitude to that for C3b-B (~600 s) suggesting that Lufaxin binds to C3b-B, but does not provide the degree of stabilization in the presence of Ni²⁺ seen in the presence of Mg²⁺ (Figures 5A,F, t1/2 ~ 135 s). Injection of Lufaxin on the same surface after formation of the C3b-B complex (during the dissociation phase) showed an increase in binding to produce a total sensorgram amplitude that was similar to that seen with coincident injection of factor B and Lufaxin (Figure 5E, B + Lufaxin), again suggesting that Lufaxin binds to the complex but does not further stabilize the interaction of factor B with C3b in the presence of Ni²⁺. Comparison of assembly on the same C3b surface in Ni²⁺ and Mg²⁺ buffers revealed a higher level of C3b-B-Lufaxin complex formation in the presence of Ni²⁺ than Mg²⁺ (Figure 5F), but comparison of Figures 5A,D,E suggests that Lufaxin enhances the binding of factor B to the C3b surface in the presence of Mg²⁺, but not Ni²⁺. Perhaps, Lufaxin binds preferentially to the open form of C3b-bound factor B, thereby increasing its abundance in the presence of Mg²⁺ but not in the presence of Ni²⁺ where the open form is already favored. When divalent cations were removed completely (HBS-N buffer alone) neither factor B nor Lufaxin bound detectably to the C3b surface, while binding of the factor B-Lufaxin mixture was similar to that seen in the presence of Mg²⁺ (Figure 5G). This indicates that the presence of Mg²⁺ or Ni²⁺ in the MIDAS of the VDW domain of factor B is not essential for formation of a C3b-B-Lufaxin complex and suggests that Lufaxin may stabilize the open form of factor B independently of metal ions.

The effect of Lufaxin on the enzymatic activity of the proconvertase and convertase complexes was tested using reconstituted systems. The C3b-Bb complex was assembled from soluble purified components by activating C3b-bound factor B with factor D in the absence of Lufaxin. The cleavage of C3 by the complex was then evaluated in the presence and absence of Lufaxin using Western blots to detect the release of C3a (Figure 6A). Under these conditions, Lufaxin did not inhibit C3 cleavage, which is consistent with SPR results showing a lack of binding to the C3b-Bb complex (Figure 6A). However, when C3b was mixed with factor B in the presence and absence of Lufaxin prior to the addition of factor D, cleavage of factor B, as indicated by the appearance of Bb on Western blots, was inhibited in the presence of Lufaxin, but not in its absence (Figure 6B). This result is also consistent with SPR data and supports the idea that Lufaxin binds to the C3b-B complex and inhibits the cleavage of factor B by factor D.