International Journal of Biological Macromolecules 120 (2018) 135–143 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac Streptococcus pneumoniae surface adhesin PfbA and its interaction with erythrocytes and hemoglobin Deepthi Radhakrishnan a, Masaya Yamaguchi b, Shigetada Kawabata b, Karthe Ponnuraj a,⁎ a b Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025, India Department of Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, Suita, Osaka 565-0871, Japan a r t i c l e i n f o Article history: Received 18 June 2018 Received in revised form 14 August 2018 Accepted 14 August 2018 Available online 17 August 2018 Keywords: Surface adhesin PfbA Streptococcus pneumoniae Adhesin-RBC interaction Erythrocyte invasion a b s t r a c t Streptococcus pneumoniae is one of the major colonizers of human nasopharynx and its surface protein PfbA interacts with host molecules like plasmin(ogen), ﬁbrinogen and ﬁbronectin for colonization. Most of the binding partners of PfbA are glycoproteins. Recently we found that PfbA exhibited high afﬁnity towards carbohydrates. It was reported that S. pneumoniae invades erythrocytes and utilizes them to evade human innate immunity. The results of this study suggested that LPXTG motif containing pneumococcal surface proteins, erythrocyte lipid rafts and erythrocyte actin remodeling are all involved in the invasion mechanism. The erythrocyte cell membrane contains different glycoproteins and glycolipids. Therefore, to ﬁnd out if PfbA plays any role in erythrocyte binding, we carried out the binding studies of rPfbA49–684 with human red blood cells (RBCs) especially with its surface molecules employing ELISA and Bio Layer Interferometry. The results from these experiments show that rPfbA49–684 has a broad speciﬁcity for carbohydrates and remarkable afﬁnity towards RBCs and in particular with extracted surface glycolipids. Further rPfbA49–684 also exhibited moderate afﬁnity towards hemoglobin. Thus the results of the present study provide clear evidence that PfbA can interact with RBCs and this could be one of the important factors in erythrocyte invasion of S. pneumoniae. © 2018 Published by Elsevier B.V. 1. Introduction The bipolar lipid layer of human Red Blood Cells (RBCs) membrane is occupied by a vast family of proteins like ankyrin, spectrin, actin and many surface antigens acting as the skeleton of the cell . The role of surface antigens is played mainly by the characteristic carbohydrate moieties attached onto the surface through protein and lipid anchoring thus forming conjugate as glycoproteins and glycolipids . These antigens are recognition factors for the blood group identiﬁcation in humans . The main carbohydrates that build up these self antigens are galactose, fucose, N-acetyl glucosamine and N-acetyl galactosamine. The terminal sugars of ABO blood group antigens are utilized by many bacteria to bind to RBC through protein-carbohydrate interactions . Earlier studies reported that many Gram-positive bacterial species interact with the RBC surface through the RBC surface sugars [5–7]. Apart from ABO blood group antigens, there is a massive family of neuraminic acids (sialic acids) and its variants bound to the terminal of RBC surface glycolipids and glycoproteins, which due to its negative charge attaches many components on to it . Studies on bacterialRBC interactions have shown the importance of the surface antigens or molecules for the host invasion, where sialic acids are playing the ⁎ Corresponding author. E-mail address: firstname.lastname@example.org (K. Ponnuraj). https://doi.org/10.1016/j.ijbiomac.2018.08.080 0141-8130/© 2018 Published by Elsevier B.V. major role for pumping more bacteria into the attachment . As already established, S. pneumoniae utilizes sialylated oligosaccharides for its adherence on the respiratory epithelial cells . Apart from these, pneumococcal cholesterol dependent cytolysins pneumolysin and streptolysin O is found to have hemolytic activity against RBC and this activity is dependent on the interaction of these proteins with RBC glycans [11,12]. Adhesins and toxins from Prevotella loescheii and Bordetella pertussis also have shown binding capacity with RBCs [13,14]. Many bacteria – RBC interactions are dependent on carbohydrate chains of the glycolipids and glycoproteins present on the surface of RBCs [15–17]. PfbA is a plasmin-and ﬁbronectin-binding adhesin of S. pneumoniae and this protein has been characterized as an important factor in the development of pneumococcal infections [18,19]. We have recently determined the crystal structure of rPfbA  (Fig. 1) and characterized its interaction with various host molecules like ﬁbronectin, plasminogen, ﬁbrinogen, collagen, laminin and human serum albumin. In addition, we also demonstrated that rPfbA binds to different carbohydrate moieties with high afﬁnity . In a recent study, S. pneumoniae was shown to invade erythrocytes and utilizes them to evade human innate immunity . The results of this study suggested that the LPXTG motif containing pneumococcal proteins, erythrocyte lipid rafts, and erythrocyte actin remodeling are all involved in the invasion mechanism. In this context, the overall aim of this study is to investigate the interaction 136 D. Radhakrishnan et al. / International Journal of Biological Macromolecules 120 (2018) 135–143 Fig. 1. Structural organization of PfbA. (A). Schematic representation showing the different regions of PfbA. ‘S' indicates signal peptide which is followed by the ligand binding region (residues 49–684). The C-terminal end consists of the cell wall-anchoring region (W), the membrane-spanning region (M) and the cytoplasmic tail (C) (B). Ribbon representation of the crystal structure of rPfbA150–607 monomer. Since the crystal structure is not known for N (aa 49–149) and C (aa 608–684) terminal segments, ab initio models were obtained using I-TASSER server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) which is represented in the diagram (C). SDS-PAGE gel shows the highly puriﬁed rPfbA49–684 (Lane 1) and rPfbA150–570 (Lane 2) proteins used in the present study. Molecular Weight Ladder (kDa) is shown in lane 3. of rPfbA with the surface molecules of RBCs and to understand the role of carbohydrates in the same by performing different assays. The results obtained here suggest that rPfbA has a broad speciﬁcity for carbohydrates and it can directly interact with the surface molecules of RBCs through the carbohydrate mediated interactions. Further, this study revealed that rPfbA has moderate afﬁnity towards hemoglobin. 2. Materials and methods 2.1. Cells and reagents Human blood was obtained from the Blood Bank (Voluntary Health Services, Chennai), N-acetyl neuraminic acid, hemoglobin, monoclonal anti-polyhistidine antibody and monoclonal anti-polyhistidine−alkaaline phosphatase were obtained from Sigma-Aldrich, India. Para Nitro Phenyl Phosphate (PNPP) and glucose were obtained from Sisco Research Laboratories (SRL), India. 2.2. Puriﬁcation of full length (rPfbA49–684) and beta fragment (rPfbA150–570) of PfbA The expression and puriﬁcation of rPfbA49–684 and rPfbA150–570 were carried out as reported earlier by us . Brieﬂy, the full length rPfbA (aa 49–684) and PfbA β construct (aa 150–570) were transformed into E. coli BL21 (DE3). A 10 ml of overnight culture was prepared and transferred to 1 l of Luria Bertani (LB) medium supplemented with 100 μg/ml ampicillin. The culture was grown at 37 °C and when an optimum optical density (A600 of 0.6–0.8) was reached, the culture was induced with 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG). After induction, the culture was allowed to grow for an additional 4 h and following this the cells were collected by centrifugation at 5011g for 20 min at 4 °C. The pellet was resuspended in a 10 ml of lysis buffer containing 20 mM Tris pH 8.0, 200 mM NaCl, 5 mM β mercaptoethanol, 10% glycerol and 1 mM Phenyl Methyl Sulfonyl Fluoride (PMSF). The cells were lysed by sonication and the cell debris in the lysate was removed by centrifugation at 6876g for 30 min at 4 °C. The clear supernatant containing the soluble protein as well as the pellet was analyzed on a 12% SDS-PAGE gel. The supernatant which contains rPfbA49–684 was directly subjected to Ni-NTA afﬁnity chromatography and eluted with different concentrations of imidazole. Fractions collected were analyzed by SDSPAGE gel and pure fractions were taken for further binding studies. Concentration of the protein was measured at 280 nm using UV–Vis spectrophotometer. 2.3. Collection and puriﬁcation of human RBC Human blood samples of blood groups A, B, AB and O (all Rhpositive) of healthy individuals were collected fresh from Voluntary Health Service Hospital (Chennai, India) blood bank. The samples were collected each time with 10% EDTA and stored under 4 °C. Processing and separation of Red Blood Cells (RBCs) were carried out using the standard procedure which is brieﬂy mentioned here. Initially 1 ml of cells were spun down at 5474 g for 30 min at 4 °C. Recovered pellets were washed 3–4 times with 2 volumes of 0.9% NaCl, which is the isotonic solution for RBCs. During each spin upper yellow colour layer containing plasma, the middle white puffy coat layer containing platelets and white blood cells (WBCs) were removed to obtain ﬁnal pure RBCs without any contamination. Final pellet obtained was again resuspended in 500 μl of 0.9% NaCl. The cells were preserved at 4 °C for a maximum of 2 weeks. Obtained cells were checked under microscope each time before the experiment to make sure RBCs are alive and not mixed with platelets and WBCs. 2.4. rPfbA-RBC binding: ELISA analysis As a preliminary experiment to ﬁnd out whether rPfbA can bind to RBCs, an ELISA experiment was performed using rPfbA49–684 and type O RBCs. Puriﬁed RBCs (0.02% suspension) were coated onto 96 well ﬂat bottom microtiter plate (NEST, Tarsons, India) and kept for incubation at room temperature for 1 h. Later the plate was washed with phosphate buffered saline (PBS) for three times. After blocking with 5% bovine serum albumin (BSA) for 1 h and washing with PBS for three times, varying concentrations (0–2.5 μM) of rPfbA49–684 along with a positive control were added to the wells. Human ﬁbrinogen was taken as a positive control  for the assay where the binding was detected using anti-ﬁbrinogen antibody. After washing with PBS to remove unbound protein, mouse monoclonal anti-polyhistidine antibody was added to the wells (1:5000 dilution, 3% BSA in PBS) and incubated overnight at 4 °C. Plates were then incubated with alkaline phosphataseconjugated goat anti mouse IgG (Calbiochem) at room temperature for 1 h (1:10000, 3% BSA in PBS). Later, after treating the wells with 0.1% of p-nitrophenyl phosphate salt solution, the plate was kept for reaction for 20 min. Following this the absorbance was measured at D. Radhakrishnan et al. / International Journal of Biological Macromolecules 120 (2018) 135–143 405 nm using an ELISA plate reader and a graph was plotted by taking concentration of the protein in X axis and absorbance in Y axis. 2.5. rPfbA-RBC interaction: bio-layer interferometry (BLI) analysis The rPfbA49–684 - RBC interaction was analyzed using bio-layer interferometry with BLItz system (FortéBio PALL Life Sciences). The data acquisition for a single run was about 9 min 30 s. The initial baseline correction was carried out with PBS for 30 s, followed by loading of rPfbA49–684 protein into Ni-NTA biosensor tip at a concentration of 1 mg/ml for 5 min. The ﬁnal baseline correction was carried out with PBS for 30 s. Association of RBCs (A+, B+, AB+, O+) against rPfbA49–684 was done for 2 min followed by dissociation of 60 s. 2.6. Extraction of RBC surface glycolipid and glycoprotein Puriﬁcation and separation of glycolipid and glycoprotein fraction of A+, B+, AB+ and O+ RBCs were done as previously described . For the extraction, fresh human blood with EDTA was taken from which the RBCs were puriﬁed and stored at 4 °C. The cell membrane of the puriﬁed RBCs was extracted by rupturing the cells by hemolysis. Excess salts and hemoglobin was removed by washing the cells thrice by ice cold water. As a ﬁrst level of organic extraction, membrane was mixed with methanol/chloroform mixture (2:1 v/v) vigorously under room temperature for 30 min followed by centrifugation at 14454g. For a second level of extraction, pellet from the ﬁrst extraction was taken and subjected to methanol/chloroform mixture (1:2 v/v) followed by centrifugation. Supernatant from both the extractions were pooled and air dried to obtain the glycolipid fraction whereas the pellet from the ﬁnal extraction was retained which contains the glycoprotein fraction. The presence of lipids in the supernatant was further conﬁrmed by performing calcium chloride precipitation test  and Salkowski's test . Calcium chloride precipitation test was performed by the addition of 1 M calcium chloride to 10 μl of the extracted glycolipid solution there by noting down the presence of undissolved white precipitate produced. For Salkowski's test, about 10–20 μl of the extracted glycolipid solution was taken and concentrated sulfuric acid was added drop by drop and the subsequent colour change was observed. The presence of proteins in the glycoprotein fraction was conﬁrmed by SDS-PAGE analysis. 137 fractions of each group sequentially, the association and disassociation with rPfbA49–684 was studied. The above experiment was also carried out for PfbA β fragment (rPfbA150–570). Similar to glycolipid fractions, the interaction between glycoprotein fractions of the four blood groups with rPfbA49–684 was studied. Based on the binding curves the association rate constant (ka), the dissociation rate constant (kd) and the afﬁnity constant (KD) for the complexes were calculated using Forte Bio Data analysis package with the global ﬁtting function. 2.9. Role of surface antigens and its interaction with rPfbA: BLI analysis and blood group testing To analyze the interaction between RBC surface antigen and rPfbA, a BLI study was carried out. For this, 100 μl of 1 mg/ml of rPfbA49–684 was incubated with 50 μl of 10 mM of galactose overnight. The excess of galactose was removed by passing the incubated rPfbA-galactose mixture through Sephadex G-25 medium. The rPfbA-galactose pre-incubated complex was taken for binding studies with RBCs of type B using BLI. Similarly rPfbA49–684 was incubated with N-Acetyl Galactosamine (GalNAc) and this complex was subjected to BLI interaction analysis with glycolipid fraction of RBCs type A. In another study, 20 μl of 15–20 mg/ml of rPfbA49–684 was incubated with 1 μl of A+, B+, AB+ and O+ RBCs overnight and taken for blood group testing using a kit as described in the manufacturer's instruction. RBCs devoid of rPfbA49–684 were taken for a positive control. Blood group testing was done using Anti-A, Anti-B and Anti-D monoclonal antibodies provided in the kit. 2.10. Role of sialic acid in RBC-rPfbA49–684 interaction: BLI analysis 250 μl of 1 mg/ml of rPfbA49–684 was incubated with 50 μl of 50 mM of sialic acid overnight following which the excess sialic acid was removed by passing the incubated rPfbA-sialic acid mixture through Sephadex G-25 medium. This pre-incubated complex was taken for further binding studies with RBC's type B and O using BLI. To analyze the binding of sialic acid and rPfbA49–684, a gel shift assay was done with 10% native PAGE gel with rPfbA49–684 and rPfbA49–684-sialic acid incubated samples. 2.11. Hemolysis assay of rPfbA49–684 2.7. Estimation of total carbohydrates in glycolipid fraction Different volumes of working standards of glucose starting from 20 to 100 μg/ml were taken in test tubes and it was made up to a ﬁnal volume of 1 ml by the addition of water. As a blank, 1 ml of double distilled water was taken. The conversion of glucose to furfural derivatives was obtained by the addition of 1 ml of 5% phenol and 5 ml of 96% sulfuric acid to each test tube including blank. The tubes were incubated at room temperature for 10 min, followed by 20 min of incubation in a water bath at 25–30 °C. At the end of incubation a colour change from pale yellow to brown was observed and subsequently the readings were taken at an absorbance of 490 nm using UV–Vis spectrophotometer . The experiment was performed three times. Amount of total carbohydrate in each fraction was calculated by plotting the readings in a standard glucose graph by taking the concentration of glucose in X axis and absorbance in Y axis. Average values of the three readings were taken for further study. 2.8. rPfbA49–684/rPfbA150–570 – RBC glycolipid/glycoprotein interaction: BLI analysis The extracted glycolipid fractions of the four blood groups were subjected to interaction studies with rPfbA49–684. Based on the estimation of total carbohydrates, a constant sugar concentration of 730.3 nM was used for all the four groups. The protein was immobilized to Ni-NTA biosensor tip at a concentration of 1 mg/ml and by taking the glycolipid To analyze whether or not rPfbA49–684 exhibits the hemolysis property, tube assay was carried out by incubating the protein (at a concentration of 5 mg/ml in PBS) and RBCs (10 μl) for 30 min at 37 °C. Tubes were taken out and spun at 3079 g for 10 min and observed the range of lysis. RBCs with 0.1% Triton X-100 and 0.1% DMSO were taken as positive and negative controls respectively [27,28]. 2.12. rPfbA - hemoglobin interaction: ELISA and BLI analysis In order to study the interaction between rPfbA49–684 and human hemoglobin, initially an ELISA was performed. For this, hemoglobin of varied concentration (1–15 μM) was coated on to the 96 well ﬂat bottom microtiter plate (NEST, Tarsons, India) for 1 h. The plate was then washed with PBS for three times. rPfbA49–684 of 0–2.5 μM then added to the wells after being blocked with 5% BSA for 1 h and washing with PBS for three times. The plate was then stored under 4 °C overnight for the protein to get bound sufﬁciently. Next day, the plate was washed again with PBS to remove the unbound protein and after the wash the mouse monoclonal anti-polyhistidine antibody was added to the wells (1:5000 dilution, 3% BSA in PBS) and incubated for 1 h at RT. After adding the alkaline phosphatase-conjugated goat anti mouse IgG (Calbiochem), the reaction mixture was kept at room temperature for 1 h (1:10000, 3% BSA in PBS). Later, the substrate, 0.1% of pnitrophenyl phosphate salt solution was added and kept for reaction for 20 min. Following this the absorbance was measured at 405 nm 138 D. Radhakrishnan et al. / International Journal of Biological Macromolecules 120 (2018) 135–143 Fig. 2. rPfbA49–684 – RBC interaction. (A). Dose-dependent response of rPfbA49–684 interacting with type O RBC as quantiﬁed by ELISA and plotted on a linear scale, compared with the positive control ﬁbrinogen. Bars represent the standard deviation for each value. (B). Interaction of rPfbA49–684 with human RBCs of different blood groups. The BLI response curves obtained during the association and dissociation of RBCs of different blood groups with rPfbA49–684. The results show that O+ group exhibits more afﬁnity compared to other groups. using an ELISA plate reader and a graph was plotted by taking concentration of the protein in X axis and absorbance in Y axis. Following this the BLI studies were employed to analyze the interaction of hemoglobin with rPfbA49–684 and rPfbA150–570. The rPfbA49–684 and rPfbA150–570 of 1 mg/ml was loaded onto the Ni-NTA biosensor tip and the association and disassociation were carried for four concentrations of hemoglobin. Complement factor H (CFH) binding HcpA protein of Borrelia recurrentis was taken as a negative control for the binding studies. 3. Results 3.1. RBC-rPfbA49–684 interaction: ELISA and BLI analysis To analyze the interaction between rPfbA49–684 and RBCs, initially an ELISA was carried out. The RBCs of blood group O+ were coated on the microtiter plate and examined the binding of rPfbA49–684. From this study, it was prominently seen that rPfbA49–684 binds to RBC in a similar manner to that of ﬁbrinogen. This experiment, thus establish the binding capacity of rPfbA49–684 towards RBCs (Fig. 2A). Further, the interaction between rPfbA49–684 and the RBCs of different blood groups were analyzed by BLI technique (Fig. 2B). From this study, it was found that the RBCs of all the four blood groups exhibit an apparent binding with rPfbA49–684, but variation in the binding afﬁnity was noticed. The binding curves depicted that type O+ RBCs show more afﬁnity followed by A+, AB+ and B+. 3.2. Glycolipid and glycoprotein extraction To determine the speciﬁcity of the interaction between glycolipid/ glycoprotein fractions of RBCs and rPfbA49–684, these two fractions were extracted separately. After the extraction, the presence of lipids in the glycolipid fraction was conﬁrmed by Salkowski's test . This test provided a positive result as the colour of the solution changes from a yellow to mild cherry red (Fig. 3A). Degree of colour intensity varied based on the amount of glycolipids extracted from the surface of RBCs of different blood groups. Among the four blood groups tested, A+ and AB+ RBCs showed higher concentrations of lipids whereas in O+ and B+ RBCs it is found relatively lesser. The presence of lipid was further conﬁrmed by the calcium chloride precipitation test Fig. 3. Extraction and identiﬁcation of glycolipid and glycoprotein fractions of RBC. (A). Salkowski's test for identiﬁcation of lipids. The bright pink colour indicates relatively higher concentration of lipids present in AB+ and A+ RBCs. (B). A 7–15% gradient reducing SDS-PAGE gel of glycoprotein fraction shows the presence of proteins (indicated by arrows) of different molecular weights. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) D. Radhakrishnan et al. / International Journal of Biological Macromolecules 120 (2018) 135–143 139 3.4. PfbA-RBC interaction analysis using BLI Table 1 Concentration of carbohydrates present in RBC glycolipid fractions. Glycolipid fraction obtained from RBC type Concentration of carbohydrates (mg/ml) Standard deviation A+ B+ AB+ O+ 3.1 1.8 4.1 1.35 0.06 0.05 0.05 0.17 which has resulted in the formation of an undissolved white precipitate (Data not shown), thus proving the presence of lipid. Similarly the presence of proteins in the glycoprotein fraction was conﬁrmed by running a 7–15% gradient reducing SDS-PAGE gel. The observed bands indicate the presence of proteins of different molecular weights (Fig. 3B). 3.3. Total carbohydrate estimation The total carbohydrates in the glycolipid fraction of the RBCs from four blood groups were estimated using phenol sulfuric acid method as described previously . In this analysis a yellow-orange coloured product (Data not shown) was observed in all four fractions indicating the presence of carbohydrates. To quantify the amount of carbohydrates present in the solution, absorbance was taken at 490 nm and the concentration was calculated . The experiment was performed three times and the average values were taken for each reading. From this calculation, it was found that A+ and AB+ RBCs showed higher concentrations of carbohydrates and among these two groups, AB+ got higher concentration of carbohydrates. The other two fractions from blood group O+ and B+ showed lesser carbohydrate concentration (Table 1). From this a ﬁxed concentration of carbohydrates was used for further experiments. Based on the estimation of total carbohydrates in the extracted glycolipid fraction of the four blood groups, binding studies between the extracted glycolipid fractions and rPfbA49–684/rPfbA150–570 were carried out in triplicates. Both proteins showed remarkable and variable binding towards glycolipid fractions (Fig. 4) The afﬁnity constant (KD) was also calculated for the interaction (Table 2) and the carbohydrates extracted from A+ showed higher afﬁnity followed by O+ N B+ N AB+. The binding pattern seen here is slightly different from the results obtained before when RBC cells as such were used in the interaction study with rPfbA49–684 (Fig. 2B). This slight change in the binding pattern could be due to the difference in the accessibility of carbohydrate molecules of extracted fractions of surface glycolipid molecules in comparison to the glycolipid molecules bound to the surface of RBCs. In order to compare the afﬁnities of glycoprotein and glycolipid fractions of RBCs, the binding of rPfbA49–684 with glycoprotein fraction was also analyzed. From this analysis, it was observed that the glycoprotein fraction exhibited a different binding pattern and afﬁnity towards rPfbA49–684 in comparison with glycolipid fraction. Comparing with the glycolipid fraction, the glycoprotein fraction showed a signiﬁcantly less KD value towards rPfbA49–684 with AB+ showing higher afﬁnity followed by B+, A+ and O+ (Fig. 4C, Table 2). Since rPfbA49–684 exhibited very less afﬁnity towards glycoprotein fraction, similar BLI analysis was not carried out for rPfbA150–570. 3.5. PfbA partially recognizes the surface antigens of RBC Binding studies of rPfbA with glycolipid and glycoprotein fractions revealed that rPfbA preferentially binds to glycolipids. To ﬁnd out if rPfbA binds to surface antigens of RBC, rPfbA49–684 was incubated with galactose and this complex was subjected to the interaction analysis Fig. 4. The BLI response curves obtained during the association and dissociation of glycolipid/glycoprotein fractions extracted from RBCs of different blood groups with rPfbA49–684/ rPfbA150–570. Interaction of (A) rPfbA49–684 and (B) rPfbA150–570 with glycolipid fractions. (C). Interaction of rPfbA49–684 with glycoprotein fractions extracted from RBCs of different blood groups. 140 D. Radhakrishnan et al. / International Journal of Biological Macromolecules 120 (2018) 135–143 Table 2 Binding afﬁnity constant (KD) of rPfbA49–684 - glycolipid/glycoprotein and rPfbA150–570 glycolipid interaction as determined by Bio-Layer Interferometry. Type of RBC Interaction between rPfba49–684 and glycolipid fraction Interaction between rPfba150–570 and glycolipid fraction Interaction between rPfba49–684 and glycoprotein fraction A+ B+ AB+ O+ 5.97 μM 0.10 mM 1.4 mM 2.6 μM 3.87 μM 0.85 μM 1.6 μM 1.97 μM 3.9 mM 4.6 mM 5.6 mM 3.8 mM with B-RBC using BLI. The incubated rPfbA49–684 exhibit decreased binding afﬁnity towards B-RBC when compared with a normal rPfbA49–684 (Fig. 5A). In RBC B antigen, the terminal sugar is a galactose. The observed decrease in binding afﬁnity towards B-RBC by galactose bound rPfbA49–684 suggests that, in this case, rPfbA binds to the terminal galactose of the surface antigen. Based on the same principle, another experiment was carried out to understand the interaction of rPfbA49–684 and surface antigen of A-RBC glycolipid fraction. In this study, rPfbA49–684 was incubated with N-Acetyl Galactosamine (GalNAc) and the interaction analysis of this complex with glycolipid fraction of RBC type A was carried out using BLI. As observed in the interaction of B-RBC with rPfbA-galactose complex mentioned above, a signiﬁcant decrease in the binding afﬁnity was observed for rPfbA-GalNAc complex in comparison with untreated rPfbA (Fig. 5B). GalNAc is a terminal sugar in the surface antigen of A-RBC. The decrease in binding afﬁnity for a GalNAc treated rPfbA suggests that rPfbA is preferentially binds to GalNAc. Similarly to ﬁnd out the role of surface antigens of glycoproteins in recognizing rPfbA, the RBCs pre-incubated with rPfbA49–684 were subjected to blood group testing in order to ﬁnd out whether rPfbA49–684 can interact with the blood group identifying antigens on the RBC surface. If rPfbA interacts with the surface antigens which are involved in blood group identiﬁcation this interaction would inhibit the antibodies to access and interact with the antigens. However, rPfbA49–684 was unable to mask the blood group identifying antigens, as the monoclonal antibodies identiﬁed the exact blood group antigens even after ﬂooding more rPfbA49–684 to interact with the RBCs (Supplementary ﬁg. S1). This may be due to either lack of interaction or poor interaction with the sugar chains of RBC glycoproteins and rPfbA49–684. Even though the characteristic oligosaccharide units of glycolipids and glycoproteins together function as blood group antigens, the contribution from glycoproteins is predominant as previously described . This has lead to a conclusion that the interaction of rPfbA49–684 with surface antigens may be associated to the glycolipids rather than glycoproteins of the RBC as normal agglutination was observed in the blood group identiﬁcation. This observation highly correlates with the results obtained in the binding studies with the extracted glycolipid and glycoprotein fractions of RBC where glycolipid fractions exhibit high afﬁnity towards rPfbA49–684 in comparison with glycoprotein fractions. 3.6. Sialic acid enhancing the interaction between PfbA and RBCs Previous studies have shown that sialic acid plays important roles in cell-cell signaling pathway by mediating carbohydrate-carbohydrate interactions. It also has proven that sialic acid mediated pathogen-ABO blood group interaction is a prominent mechanism in the downstream signaling . As already mentioned, rPfbA could interact with different carbohydrates including sialic acid . Again to analyze the binding of sialic acid with rPfbA49–684, a native PAGE was carried out. A slight upward shift in the band corresponding to the molecular weight of sialic acid incubated rPfbA49–684 when compared with the native rPfbA49–684 indicates the binding of sialic acid with the protein (Supplementary ﬁg. S2). Next, to ﬁnd out the afﬁnity of sialic acid bound rPfbA49–684 towards RBCs, the BLI experiment was conducted. Based on the results obtained from the BLI analysis of rPfbA49–684 interaction with four different glycolipid fractions (Fig. 2B), B+ (low binding afﬁnity) and O+ (high binding afﬁnity) RBCs were chosen for binding studies with sialic acid incubated rPfbA49–684. The sialic acid bound rPfbA49–684 showed increased binding afﬁnity towards RBC when compared with native rPfbA49–684. This could be due to the interaction between rPfbA bound sialic acid with sialic acid or other carbohydrates of the RBC cell surface (Fig. 6A&B). In a recent study it was demonstrated that host–glycan:bacterial–glycan interactions are of high afﬁnity in nature and suggested that these interactions may play a critical role in host:pathogen biology . In this context, the sialic acid mediated interaction found in this study might help the bacteria in host cell recognition and thereby adherence to the host cell surface . However, there is a possibility for PfbA bound sialic acid to interact with RBC protein which might also result in increased binding afﬁnity. 3.7. Hemolysis test PfbA is an adhesin. Therefore, it is expected that PfbA do not show any activity or characters for hemolytic activity. However to conﬁrm its non-hemolytic property, hemolysis assay was carried out by incubating rPfbA49–684 with RBCs for 30 min at 37 °C followed by centrifugation. This analysis revealed a negative hemolysis of rPfbA49–684. Thus this result is relevant to the previous ﬁnding of S. pneumoniae invasion of erythrocytes . 3.8. rPfbA49–684/rPfbA150–570 – hemoglobin interaction Many bacteria require iron for several cellular processes and they have developed strategies for scavenging it from host proteins . Apart from lactoferrin, transferrin and ferritin, hemoglobin is also one of the iron sources for bacteria. Since it was demonstrated that S. pneumoniae can invade erythrocytes, our interest is to analyze whether or not rPfbA can interact with hemoglobin. For this, as an initial experiment, an ELISA was performed with rPfbA49–684 and hemoglobin. Fig. 5. Interaction of rPfbA49–684 with terminal sugars of the surface antigens. (A). Comparison of BLI response curve of rPfbA49–684 interaction with RBC type B and the pre incubated complex of rPfbA49–684 and galactose with RBC type B. The pre incubated complex exhibits reduced binding. (B). BLI interaction analysis of rPfbA49–684 – GalNAc incubated complex with glycolipid fraction of RBC type A and the comparison of the same interaction in the absence of GalNAc. Reduced binding was observed in the presence of GalNAc. D. Radhakrishnan et al. / International Journal of Biological Macromolecules 120 (2018) 135–143 141 Fig. 6. Sialic acid incubated rPfbA49–684 - RBC interaction analyzed by BLI. After the incubation with sialic acid, rPfbA49–684 shows increased binding towards RBCs of (A) blood group B and (B) O. In the diagrams, ‘SIAL’ refers to Sialic acid. From this study it was observed that rPfbA49–684 is binding to hemoglobin in a dose dependent manner (Fig. 7A). From the plot of absorbance against concentration of the hemoglobin the dissociation constant of the interaction was calculated as 3.5–3.7 × 10−6 M. This shows that rPfbA49–684 is binding to hemoglobin with a moderate afﬁnity. Subsequently we carried out BLI interaction analysis of rPfbA49–684 and rPfbA150–570 with hemoglobin. As obtained in ELISA, a moderate binding afﬁnity between rPfbA49–684 and hemoglobin was observed with a binding constant of 3.63 × 10−6 M (Fig. 7B). To analyze the role of beta fragment of PfbA alone in the interaction with hemoglobin, BLI was performed (Fig. 7C). The afﬁnity of the interaction was measured as 3.7 × 10−5 M which is lower than that of between full length PfbA (rPfbA49–684) and hemoglobin. This suggests, apart from the central beta helical region, the other regions of PfbA (N and C-terminal regions) are also involved in the interaction with hemoglobin. To get a glimpse of mode of binding of PfbA and hemoglobin, the molecular docking was carried out with the crystal structures of these two molecules (rPfbA150–607, PDB 4MR0 and Human hemoglobin, PDB 4HHB) using GRAMM-X server . From this it was observed that hemoglobin preferentially binds to C-terminal end of rPfbA150–607 (Supplementary ﬁg. S3). The binding afﬁnity of rPfbA49–684 is higher than of rPfbA150–570 on their interaction with hemoglobin as calculated from the BLI studies mentioned above. This result correlates well with the rPfbA150–607 – hemoglobin binding mode as obtained from the docking where rPfbA150–607 lacks the C-terminal region consisting of residues 571 to 684 and probably due to this reduced binding afﬁnity was observed for rPfbA150–570. 4. Discussion Towards the functional characterization of PfbA, we initially determined the crystal structure of rPfbA150–607 and the structural analysis revealed that the molecule exhibits a parallel beta-helical structure which is strikingly similar to that of carbohydrate-active enzymes (CAZymes) like polysaccharide lyases and glycoside hydrolases . In our recent study, we found that rPfbA49–684 has a remarkable afﬁnity towards various host glycoproteins and different saccharides . It was proposed that the channel-like groove or cleft found on the surface of rPfbA150–607 as a carbohydrate recognizing region  (Fig. 8A&B). The current work is focused on studying the interaction of rPfbA49–684 Fig. 7. The interaction of rPfbA49–684/rPfbA150–570 with hemoglobin (Hb) (A). Quantiﬁcation of rPfbA49–684 binding to hemoglobin by ELISA. The hemoglobin of varied concentration was immobilized on microtitre plates and probed with rPfbA49–684 followed by incubation with anti-His tag and secondary antibodies. Points representing the means of the triplicates and standard deviations are indicated. The BLI response curves obtained for (B) rPfbA49–684 – hemoglobin and (C) rPfbA150–570 - hemoglobin interaction. 142 D. Radhakrishnan et al. / International Journal of Biological Macromolecules 120 (2018) 135–143 Fig. 8. Putative carbohydrate binding region of PfbA. (A) Crystal structure of rPfbA150–607 monomer shown in ribbon representation with a view along the beta helical axis (B) Electrostatic potential surface of rPfbA150–607 with a view perpendicular to the helical axis. The basic and acidic regions are shown in blue and red, respectively. The putative carbohydrate binding cleft on the surface of the molecule is shown with a yellow lining. The green sphere represents the metal atom. (C). Residues of the cleft region. Charged residues are scattered throughout the cleft. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) with the human RBC cell surface molecules such as the glycolipids and the glycoproteins as carbohydrates present in it and in addition it was proposed that LPXTG motif contain surface adhesins like PfbA are involved in the pneumococcal invasion of RBC. In the present study, it was observed that rPfbA49–684 binds to the human RBCs in a variable manner. When the importance is given to the surface molecules, the glycolipids showed higher afﬁnity than glycoproteins. Further, variations in the afﬁnity between the different glycolipid fractions of the ABO groups were also observed which could be due to different types of carbohydrate moieties present in these molecules. In order to study whether rPfbA49–684 binds to surface antigens or not, interaction analysis between B-RBC and pre incubated rPfbA-Galactose complex was carried out. From this analysis, it was found that rPfbA recognizes the terminal sugar (Galactose) of the surface antigen of blood type B. In a similar study, where the interaction between pre incubated rPfbAGalNAc complex and glycolipid fraction of A-type RBC was carried out. The results obtained from this study suggest that rPfbA interacts with the surface antigen of blood type A by recognizing its terminal sugar GalNAc. In the same line of understanding the interaction of rPfbA and surface antigen, another experiment with pre incubated rPfbA49–684 RBCs were carried out where the RBCs of different types were subjected to agglutination test with antibodies. In this study it was found that the speciﬁc antibodies recognize the surface antigens and agglutinates RBC as in a standard blood group identiﬁcation test. This result suggests that rPfbA49–684 is unable to mask the surface antigens, especially of surface glycoproteins, since the surface antigens of glycoproteins rather than glycolipids play a dominant role in ABO activity of human erythrocytes . The sialic acids of RBC membrane, primarily from the sialic acid-rich glycophorins that span the membrane of the RBC, are considered to play an important role in the physiology of the RBC. Many pathogenic bacteria bind to sialic acid residues on human and animal RBCs for colonization and infection [7,34]. In our previous work we found that rPfbA49–684 binds to sialic acid . Since RBC surface contains high amount of sialic acid, it is possible that these surface sialic acids could interact with rPfbA49–684 bound sialic acid. It has been shown that interactions between carbohydrates of host and pathogen are important in bacterial pathogenesis  and in this context we tested the interaction of preincubated rPfbA49–684 – sialic acid complex against the RBCs. When compared with a normal rPfbA49–684, in the presence of sialic acid the afﬁnity between rPfbA and RBCs was increased. This could be due to carbohydrate:carbohydrate/protein:carbohydrate or both of host (RBC): pathogen (sialic acid bound to rPfbA) interaction. The present study thus clearly establishes the role of carbohydrates in the binding of rPfbA49–684 with RBCs. Although rPfbA49–684 shows the ability to interact with RBC through the RBC's surface glycolipids, hemolysis assay suggests that rPfbA49–684 has no role in lysis RBC. This property is important and probably relevant to the invasion of erythrocytes by S. pneumoniae. Taken together, the interaction of rPfbA with carbohydrate moieties of glycolipids including its surface antigens and other carbohydrate molecules like sialic acid suggests that PfbA has broad speciﬁcity for carbohydrates and this is perhaps essential for the colonization of the bacterium under different physiological conditions based on the availability/accessibility of the different substrates. The putative carbohydrate binding cleft of PfbA also supports the broad speciﬁcity where the charged residues are distributed throughout the long cleft (Fig. 8C) Further, in this study it was demonstrated that rPfbA49–684 could also bind to hemoglobin. Although the biological signiﬁcance of this interaction is not known now, it is possible that S. pneumoniae can utilize this interaction for iron scavenging. 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