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International Journal of Biological Macromolecules 120 (2018) 135–143
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International Journal of Biological Macromolecules
journal homepage:
Streptococcus pneumoniae surface adhesin PfbA and its interaction with
erythrocytes and hemoglobin
Deepthi Radhakrishnan a, Masaya Yamaguchi b, Shigetada Kawabata b, Karthe Ponnuraj a,⁎
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
Surface adhesin
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 [1]. 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 [2]. These antigens are recognition factors for the blood group identification in
humans [3]. 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 [4].
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 [8]. 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: (K. Ponnuraj).
0141-8130/© 2018 Published by Elsevier B.V.
major role for pumping more bacteria into the attachment [9]. As already established, S. pneumoniae utilizes sialylated oligosaccharides
for its adherence on the respiratory epithelial cells [10]. 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 [20] (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 [21]. In a recent study, S. pneumoniae was shown
to invade erythrocytes and utilizes them to evade human innate immunity [22]. 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
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 ( 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 [20]. 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
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 [23] 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 [8]. 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 [24] and Salkowski's test [25]. 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.
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 [26]. 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
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
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
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 [25]. 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
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)
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 [26]. 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 [26]. 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.
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.
rPfba49–684 and
glycolipid fraction
rPfba150–570 and
glycolipid fraction
Interaction between
rPfba49–684 and
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 [29]. 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 [30]. As already mentioned, rPfbA could interact with different
carbohydrates including sialic acid [21]. 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 [31]. 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 [30]. However, there is a possibility for PfbA bound sialic acid to
interact with RBC protein which might also result in increased binding
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 [22].
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 [32].
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
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 [33]. 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 [20]. In
our recent study, we found that rPfbA49–684 has a remarkable affinity towards various host glycoproteins and different saccharides [21]. It was
proposed that the channel-like groove or cleft found on the surface of
rPfbA150–607 as a carbohydrate recognizing region [21] (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.
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 [21]. 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 [31] 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.
KP gratefully acknowledges the Department of Science and
Technology-Science and Engineering Research Board (DST-SERB), Government of India for the financial support in the form of a grant. DR
thanks DST, India for the Fellowship.
Conflict of interest
The authors have declared no conflict of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
[1] T.L. Steck, Organization of proteins in the human red blood cell membrane, J. Cell
Biol. 62 (1974) 1–19.
[2] P. Zahler, Blood group antigens in relation to chemical and structural properties of
the red cellmembrane, Vox Sang. 15 (1968) 81–101.
[3] W.L. Marsh, Biological roles of blood group antigens, Yale J. Biol. Med. 63 (1990)
[4] L.E. Balanzino, J.L. Barra, C.G. Monferran, F.A. Cumar, Differential interaction of
Escherichia coli heat-labile toxin and cholera toxin with pig intestinal brush border
D. Radhakrishnan et al. / International Journal of Biological Macromolecules 120 (2018) 135–143
glycoproteins depending on their ABH and related blood group antigenic determinants, Infect. Immun. 62 (1994) 1460–1464.
T. Boren, P. Falk, K.A. Roth, G. Larson, S. Normark, Attachment of Helicobacter pylori
to human gastric epithelium mediated by blood group antigens, Science 262 (1993)
M.J. Haverkorn, W.R. Goslings, Streptococci, ABO blood groups, and secretor status,
Am. J. Hum. Genet. 21 (1969) 360–375.
P.K. Shin, P. Pawar, K. Konstantopoulos, J.M. Ross, Characteristics of new Staphylococcus aureus-RBC adhesion mechanism independent of fibrinogen and IgG under
hydrodynamic shear conditions, Am. J. Phys. Cell Phys. 289 (2005) C727–C734.
T. Bulai, D. Bratosin, A. Pons, J. Montreuil, J.P. Zanetta, Diversity of the human erythrocyte membrane sialic acids in relation with blood groups, FEBS Lett. 534 (2003)
B.A. Bensing, J.A. Lopez, P.M. Sullam, The Streptococcus gordonii surface proteins
GspB and Hsa mediate binding to sialylated carbohydrate epitopes on the platelet
membrane glycoprotein Ibα, Infect. Immun. 72 (2004) 6528–6537.
R. Barthelson, A. Mobasseri, D. Zopf, P. Simon, Adherence of Streptococcus
pneumoniae to respiratory epithelial cells is inhibited by sialylated oligosaccharides,
Infect. Immun. 66 (1998) 1439–1444.
S.D. Taylor, M.E. Sanders, N.A. Tullos, et al., The cholesterol-dependent cytolysin
pneumolysin from Streptococcus pneumoniae binds to lipid raft microdomains in
human corneal epithelial cells, PLoS One 8 (2013), e61300.
L.K. Shewell, R.M. Harvey, M.A. Higgins, et al., The cholesterol-dependent cytolysins
pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity, Proc. Natl. Acad. Sci. U. S. A. 111 (2014) E5312–E5320.
J. Zwickel, E.I. Weiss, A. Schejter, Degradation of native human hemoglobin following hemolysis by Prevotella loescheii, Infect. Immun. 60 (1992) 1721–1723.
M.C. Gray, W. Ross, K. Kim, E.L. Hewlett, Characterization of binding of adenylate cyclase toxin to target cells by flow cytometry, Infect. Immun. 67 (1999) 4393–4399.
K.A. Karlsson, Animal glycosphingolipids as membrane attachment sites for bacteria,
Annu. Rev. Biochem. 58 (1989) 309–350.
B.J. Cameron, L.J. Douglas, Blood group glycolipids as epithelial cell receptors for Candida albicans, Infect. Immun. 64 (1996) 891–896.
J. Baum, R.H. Ward, D.J. Conway, Natural selection on the erythrocyte surface, Mol.
Biol. Evol. 19 (2002) 223–229.
M. Yamaguchi, Y. Terao, Y. Mori, S. Hamada, S. Kawabata, PfbA, a novel plasminand fibronectin-binding protein of Streptococcus pneumoniae, contributes to
fibronectin-dependent adhesion and antiphagocytosis, J. Biol. Chem. 283
(2008) 36272–36279.
M.D. Suits, A.B. Boraston, Structure of the Streptococcus pneumoniae surface protein
and adhesin PfbA, PLoS One 8 (2013), e67190.
[20] D.S.J. Beulin, M. Yamaguchi, S. Kawabata, K. Ponnuraj, Crystal structure of PfbA, a
surface adhesin of Streptococcus pneumoniae, provides hints into its interaction
with fibronectin, Int. J. Biol. Macromol. 64 (2014) 168–173.
[21] D.S.J. Beulin, D. Radhakrishnan, S.C. Suresh, C. Sadasivan, M. Yamaguchi, S.
Kawabata, K. Ponnuraj, Streptococcus pneumoniae surface protein PfbA is a versatile
multi-domain and multi-ligand-binding adhesin employing different binding
mechanisms, FEBS Lett. 284 (2017) 3404–3421.
[22] M. Yamaguchi, Y. Terao, Y. Mori-Yamaguchi, H. Domon, Y. Sakaue, T. Yagi, K.
Nishino, A. Yamaguchi, V. Nizet, S. Kawabata, Streptococcus pneumoniae invades
erythrocytes and utilizes them to evade human innate immunity, PLoS One 8
(2013), e77282.
[23] D. Lominadze, W.L. Dean, Involvement of fibrinogen specific binding in erythrocyte
aggregation, FEBS Lett. 517 (2002) 41–44.
[24] Ranjana Chawla, Practical clinical biochemistry: methods and interpretations, in: R.
Chawla (Ed.), Test for Lipids, 4th ed.Jaypee Brothers Medical Publishers (P) Ltd,
New Delhi 2014, p. 60.
[25] D.C. Sharma, M. Riyat, Practical medical biochemistry, in: D.C. Sharma, M. Riyat
(Eds.), Qualitative Analysis: Lipids, BI Publications Pvt Ltd, New Delhi 2007, p. 17.
[26] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for
determination of sugars and related substances, Anal. Chem. 28 (1956) 350–356.
[27] S. Chakraborti, S. Bhattacharya, R. Chowdhury, P. Chakrabarti, The molecular basis of
inactivation of metronidazole-resistant Helicobacter pylori using polyethyleneimine
functionalized zinc oxide nanoparticles, PLoS One 8 (2013), e70776.
[28] J.S. Novais, V.R. Campos, A.C.J.A. Silva, et al., Synthesis and antimicrobial evaluation
of promising 7-arylamino-5, 8-dioxo-5, 8-dihydroisoquinoline-4-carboxylates and
their halogenated amino compounds for treating Gram-negative bacterial infections, RSC Adv. 7 (2017) 18311–18320.
[29] H. Schenkel-Brunner, Blood-group-ABH antigens of human erythrocytes, FEBS J. 104
(1980) 529–534.
[30] M. Cohen, N. Hurtado-Ziola, A. Varki, ABO blood group glycans modulate sialic acid
recognition on erythrocytes, Blood 114 (2009) 3668–3676.
[31] C.J. Day, E.N. Tran, E.A. Semchenko, et al., Glycan:glycan interactions: high affinity
biomolecular interactions that can mediate binding of pathogenic bacteria to host
cells, Proc. Natl. Acad. Sci. U. S. A. 112 (2015) E7266–E7275.
[32] J.R. Sheldon, H.A. Laakso, D.E. Heinrichs, Iron acquisition strategies of bacterial pathogens, Microbiol. Spectr. 4 (2016) 1–32.
[33] A. Tovchigrechko, I.A. Vakser, GRAMM-X public web server for protein-protein
docking, Nucleic Acids Res. 34 (2006) W310–W314.
[34] R. Del, C. Rocha-Gracia, E.I. Castañeda-Roldán, S. Giono-Cerezo, J.A. Girón, Brucella sp.
bind to sialic acid residues on human and animal red blood cells, FEMS Microbiol.
Lett. 213 (2002) 219–224.
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