Streptococcus pneumoniae secretes a glyceraldehyde-3-phosphate dehydrogenase, which binds haemoglobinand haem

Zelene Edith Vazquez-Zamorano • Marco Antonio Gonzalez-Lopez •

Marıa Elena Romero-Espejel • Elisa Irene Azuara-Liceaga •

Mavil Lopez-Casamichana • Jose de Jesus Olivares-Trejo

Received: 2 December 2013 / Accepted: 2 June 2014

� Springer Science+Business Media New York 2014

Abstract Streptococcus pneumoniae is a gram posi-

tive encapsulated bacterium responsible of septicaemia

and upper respiratory infections in children. This

pathogen requires iron to survive in the host, which it

can obtain of haemoglobin (Hb) or haem. Only two Hb-

binding membrane proteins have been identified up to

now. However it is unknown whether this pathogen

secretes proteins in order to scavenge iron from the Hb or

haem. Therefore, in order to explore these possibilities,

cellular growth of S. pneumoniae was tested with several

alternative iron supplies. The bacterial growth was

supported with iron, Hb and haem. Additionally, S.

pneumoniae expressed and secreted a protein of 38 kDa

which was purified and characterized as Hb and haem-

binding protein. This protein was also identified by mass

spectrometry as glyceraldehyde-3-phosphate dehydro-

genase. Our overall results suggest that S. pneumoniae

secretes a protein capable of binding two usefull iron

sources for this bacterium (Hb and haem). This protein

could be playing a dynamic role in the success of the

invasive and infective processes of this pathogen.

Keywords Streptococcus pneumoniae � Haem �Haemoglobin � Glyceraldehyde-3-phospate

dehydrogenase � Secreted

Introduction

The capacity of microbial pathogens to obtain iron from

humans is essential for the infection establishment. Iron

can be acquired from lactoferrin (Lf), transferrin (Tf)

and ferritin (Ft) (Andrews et al. 2003). In addition, there

are other iron sources called haemoproteins such as

haemoglobin (Hb), which binds iron via haem (Wan-

dersman and Delepelaire 2004). Hb is a human iron

source found in the microenvironment of many tissues

and accessible to almost all pathogens (Wandersman

and Stojiljkovic 2000). Several mechanisms describing

these processes have been described in different bacte-

ria. In one of these processes, proteins called haemo-

phores are released to bind Hb or haem from the

extracellular media and deliver it to membrane recep-

tors for its subsequent internalisation (Brown et al.

2004). Although the amino acid sequence of haem-

binding proteins varies significantly between patho-

gens, it has been proposed that some of these receptor

Zelene Edith Vazquez-Zamorano and Marco Antonio

Gonzalez-Lopez have contributed equally to this work.

Z. E. Vazquez-Zamorano � M. A. Gonzalez-Lopez �M. E. Romero-Espejel � E. I. Azuara-Liceaga �M. Lopez-Casamichana � J. J. Olivares-Trejo (&)

Posgrado en Ciencias Genomicas, Universidad Autonoma

de la Ciudad de Mexico, San Lorenzo 290, Del Valle,

C.P. 03100 Ciudad de Mexico, D.F., Mexico

e-mail: olivarestrejo@yahoo.com

M. E. Romero-Espejel

Departamento de Infectomica y Patogenesis Molecular,

Centro de investigacion y de estudios avanzados del IPN,

Ciudad de Mexico, Mexico

123

Biometals

DOI 10.1007/s10534-014-9757-0

proteins bind the iron source via the two motifs FRAP

and NPNL (Simpson et al. 2000) or the motif KVAFDH,

which is found in Haemophilus influenzae (Reidl and

Mekalanos 1996). These mechanisms have been

described in more detail in gram negative bacteria than

in gram positive bacteria. In Staphylococcus aureus, at

least four proteins have been suggested to be involved in

haem binding (IsdA, B, C and H) (Torres et al. 2006).

Interestingly, in Streptococcus pneumoniae, which is a

gram positive encapsulated bacterium responsible for

septicaemia and upper respiratory tract infections in

children (Tuomanen et al. 2004), not all of the available

iron from the host can be acquired by this pathogen

because S. pneumoniae can use Hb and haem but not Lf

and Tf (Tai et al. 1993). In this pathogen, only two

membrane proteins have been identified as Hb and

haem-binding proteins thus far (Romero-Espejel et al.

2013). Unfortunately, the complete mechanism utilised

by this bacterium to acquire iron is poorly understood

because no iron scavenging proteins secreted by this

bacterium has been described to date. In this study, we

have isolated a protein that is secreted by S. pneumoniae

and binds both Hb and haem. Although this protein was

revealed to be glyceraldehyde-3-phosphate dehydroge-

nase (GAPDH), which is an enzyme with well-estab-

lished metabolic roles, our findings suggest an addition

role for this protein in iron-binding.

Materials and methods

Conditions for bacterial growth

The R6 strain of S. pneumoniae was grown under

microaerophilic conditions in 5 % CO2 for 24 h at

37 �C using agar supplemented with 5 % sheep blood.

The cultures were then inoculated in Todd Hewitt-

Broth (THB) supplemented with 0.5 % yeast extract

(THB-Y) under the same conditions until the optical

density (600 nm) reached 0.3. To test the effect of

alternative iron sources such as haem (Sigma

51280-5G), Hb (Sigma H7379-10G), FeCl3 or holo-

Tf (Sigma T-4132), a chelating agent (500 lM 2,

2-dipyridyl) (Sigma D216305-25G) was used, and the

bacteria were incubated for 16 h at 37 �C in 5 % CO2.

To synchronise cellular growth, the optical density

(measured at 600 nm) was adjusted to 0.1 and mon-

itored every hour. After 3 h under iron starvation the

culture medium was supplemented with haem, Hb,

FeCl3, Tf or fresh medium (THB), with the concentra-

tion adjusted to 10 lM of iron. E. coli O157:H7 strain

EDL 933 was grown in LB broth for 12 h at 37 �C.

Separation of secreted protein from total proteins

To prevent cellular lysis, the bacteria were cultivated for

7 h in THB under iron-starved conditions. Cells were

then collected by centrifugation at 6,0009g for 10 min,

and the supernatant was filtered through a 0.45 lm pore

size membrane filter to remove residual bacteria and

then concentrated by centrifugation using Amicon

Ultra-15 filtration units at 5,0009g for 40 min to obtain

the S. pneumoniae secretome. The cellular pellet was

washed three times with THB by centrifugation at

6,0009g for 3 min and resuspended in medium supple-

mented with 1 mM PMSF. The bacterial cells were

lysed by sonication using 30 s pulses for 6 min. Samples

were centrifuged at 12,0009g for 20 min to remove

unbroken cells, and the supernatant was taken as the

total protein fraction (Gonzalez-Lopez et al. 2013).

Determination of b-galactosidase enzymatic

activity

b-Galactosidase activity was determined following pro-

tocol of Miller (Miller 1972), the bacteria were incubated

overnight, cells were then diluted in fresh medium, grew

to mid-log, the cultures were incubated for 20 min on ice

to stop growth and washed, 2 ml cells were centrifuged at

6,0009g for 10 min to separate the supernatant, the cell

pellet was resuspended in the same volume of Z buffer

chilled (60 mM Na2HPO4.7H2O, 40 mM NaH2PO4.H2-

O, 10 mM KCl, 1 mM MgSO4 and 50 mM b-mercap-

toethanol) and measured the optical density at 600 nm.

1 ml of Z buffer containing bacteria was mixed with

200 ll (4 mg/ml) of ortho-nitrophenyl-b-galactoside

(ONPG used as substrate). The reaction was developed

adding 500 ll 1 M Na2CO3. The colorimetric signal was

monitored by spectrophotometry at 420 and 550 nm.

Activity units were calculated using the following

equation: Miller units = 1,000 9 [(OD420 -1.75 9

OD550)]/(T 9 V 9 OD600), OD420 and OD550. OD600;

reflects cell density, T; time of the reaction (min) and V;

volume used in the assay (ml). The experiments were

performed by triplicate in three occasions.

Biometals

123

Protein quantification

The concentration of proteins was determined by

spectrophotometry at 595 nm using the Bradford

method; a standard curve was generated using Brad-

ford reagent and bovine serum albumin at concentra-

tions of 1, 5, 10, 25 and 50 lg/ml, the concentration of

samples was determined by interpolation.

Densitometry

The proteins separated by SDS-PAGE were submitted

to Chemi Doc (BioRad) equip and the protein bands

were quantified using the Quantity One program

version 4.6.3 with the tool volume analysis report to

determine the concentration of a protein by their

intensity in the gel.

Coomassie brilliant blue and hydrogen peroxide

staining

Proteins were resolved in gels and used for subsequent

mass spectrometry analysis were visualised by a

Coomassie brilliant blue R-250 method; the gel was

immersed in a staining solution (0.1 % Coomassie

brilliant blue R-250, 50 % methanol and 10 % glacial

acetic acid) for 15 min with gentle agitation and then a

destaining solution was added (40 % methanol and

10 % glacial acetic acid) until the gel background was

fully clear. To identify haem interaction, native gels

were revealed with hydrogen peroxide (3.5 %).

SDS-PAGE and native gels

50 lg proteins were prepared in loading buffer (1 %

SDS, 10 % glycerol, 10 mM Tris–Cl, pH 6.8, 0.5 M

dithiothreitol, 1 % Bromophenol blue) and loading

onto SDS-PAGE gel (30 % acrylamide/bisacrylamide

(29:1), 1.5 M Tris–HCl buffer (pH 8.8), 10 % ammo-

nium persulfate solution, 0.08 % TEMED, 0.1 % SDS,

in the case of native gel the sample was mixed with

binding buffer (250 mM Tris/HCl, pH 8.0, 5 mM

EDTA and 10 % glycerol) for 30 min at 25 �C, after

that the sample was loaded in the native gel (7 %

acrylamide/bisacrylamide, 1.5 M Tris–HCl buffer (pH

8.8), 10 % ammonium persulfate solution and 0.08 %

TEMED, the sample was resolving at 35 mA.

Western blotting

A Western blot was performed using anti-b-galacto-

sidase antibodies as an internal control to verify that no

intracellular proteins were released under our cellular

culture conditions. Proteins were transferred to nitro-

cellulose membranes in a semi-dry trans-blot cell for

1 h at 100 mA in a solution of 30 mM Tris, 1.4 %

glycine and 20 % methanol. The membranes were

soaked in PBST buffer (137 mM NaCl, 2.7 mM KCl,

10 mM Na2HPO4, 2 mM KH2PO4 and 0.1 % Tween

20) containing 5 % non-fat milk overnight to saturate

all remaining active binding sites. The membrane was

washed three times with PBST and then was incubated

with anti-b-galactosidase antibody (Millipore)

(1:40,000) for 1 h. Five washes were performed with

PBST, and the secondary antibody was added (anti

rabbit coupled to horseradish peroxidase; 1:10,000).

The antibody was specific for E. coli protein but cross-

react with homologous protein from S. pneumoniae.

Purification of secreted proteins by haem-affinity

chromatography

The secretome of bacteria grown in THB media under

iron-starved supplemented with Hb was loaded onto

haem-affinity chromatography resin (Sigma H6390)

and was incubated overnight at 4 �C. The chromato-

graphic fraction was centrifuged at 1,0009g for

Table 1 List of the bacterial species and UniProt access

numbers of GAPDH protein used to perform the alignment

illustrated in Fig. 6

Bacteria GAPDH Access

number UniProt

Streptococcus pneumoniae Q8CWN6

Bacillus anthracis Q81X74

Bacillus subtilis P09124

Clostridium botulinium C1FQW2

Escherichia coli P58072

Haemophilus influenzae E1X8N0

Haemophilus influenzae e-P4 C9MJZ6

Helicobacter pylori Q9ZJP0

Neisseria maningitidis E7BH93

Staphylococcus aureus Q6GIL8

Streptococcus pneumoniae 24 Q97P40

Streptococcus pneumoniae 37 Q97RH40

Biometals

123

1 min, the flow-through was collected, and the resin

was washed three times with wash buffer (50 mM

Tris–HCl pH 7.0, 300 mM NaCl) to eliminate non-

specific interactions. Secreted proteins bound to haem

were eluted using a 6 M guanidine hydrochloride

solution. The samples were then cleaned of salts, lipids

and nucleic acids by precipitation using a kit (Cleanup

kit, Bio-Rad), and the proteins were recovered in the

pellet. Protein concentration was determined by the

Bradford method. A total of 50 ll of protein was

resolved by SDS-PAGE and visualised by Coomassie

blue staining to evaluate the integrity and resolution of

the proteins in the mixture (Asuthkar 2007).

Hb-binding experiments

The secreted fraction purified by haem-affinity was

used to evaluate the Hb-binding capability of the

38 kDa protein. First, 30 lg of purified proteins was

suspended in binding buffer (250 mM Tris/HCl,

5 mM EDTA and 10 % glycerol, pH 6.8) and was

incubated with 20 lM Hb at 37 �C for 30 min. The

complex was then separated by native PAGE (7 %

gel). To identify the interaction of the 38 kDa protein

with the iron source (Hb or Tf) the native gels were

treated with hydrogen peroxide (3.5 %) and protein

was visualised by Coomassie blue staining (Cruz-

Castaneda et al. 2011).

Competition assays

Competition experiments were performed in the same

way as haem-affinity chromatography, but the bound

proteins were eluted using several possible competi-

tors such as haem (180 lmol), Hb (180 lmol) or Tf

(180 lmol) instead of guanidine hydrochloride. Com-

petitors were used at an iron concentration ten-fold

higher than that used in haem-affinity chromatogra-

phy. Finally, 30 ll of each sample was resolved by

SDS-PAGE and visualised by Coomassie blue stain-

ing (Romero-Espejel et al. 2013).

Mass spectrometry analysis

The 38 kDa protein band was excised from the gel and

was digested with trypsin. LC–MS/MS was performed

on a Micromass QTof I equipped with a LC Packings

nanoflow LC. A total of 5 ll of digested protein

solution was injected onto a LC Packings C18 PepMap

column (0.75 lm 9 15 cm) and was eluted with a

linear acetonitrile gradient at a flow rate of 200 nL/

min. The peptides eluted from the column were

introduced into the mass spectrometer through a New

Objective PicoTip held by a New Objective adapter.

The experimental conditions were as follows: capil-

lary voltage of 1.8 kV, cone voltage of 32 V and

collision energy according to the mass and charge of

the ion, ranging from 14 eV to 50 eV. Raw data files

were processed using the MassLynx ProteinLynx

software and pkl files were analysed by www.

matrixscience.com using the Mascot algorithm (Pro-

tein Core Facility, Columbia University Medical

Center).

Multiple amino acid sequence alignment

The amino acid sequence of the S. pneumoniae

GAPDH (Q8CWN6) protein was compared with the

Fig. 1 Alternative iron sources such as Hb and haem rescue the

cellular growth of S. pneumoniae. After maintaining S.

pneumoniae under iron starvation conditions (500 lM dipyri-

dyl) for 16 h, the optical density was adjusted to 0.1 and the

bacteria were cultured further for 3 h using alternative iron

sources (Hb, filled circle; haem, filled square; Tf, asterisk and

FeCl3 filled triangle). The cultures were incubated for a total of

7 h, and culture samples were harvested every hour. Cellular

growth was determined by spectrophotometry (600 nm). Data

represent the mean ± SD of three independent experiments.

Bacterial growth after the addition of THB fresh medium was

used as control (open circle)

Biometals

123

protein sequences of GAPDH from Bacillus anthracis,

Bacillus subtilis, Clostridium botulinum, Escherichia

coli, Helicobacter pylori, Neisseria meningitidis, S.

aureus and two membrane proteins of S. pneumoniae

of 22 and 37 kDa (Romero-Espejel et al. 2013) and the

protein e-P4 of H. influenza, which had the haem-

binding motif (Reidl and Mekalanos 1996) (Table 1).

The amino acid sequences were submitted to the http://

www.ebi.ac.uk/Tools/msa/clustalw2/ server to obtain

an alignment. The residues were highlighted in bold-

face using the JalView program 2.8. The amino acid

sequence of S. pneumoniae GAPDH (Q8CWN6)

protein was submitted to the CPH models 3.2 server

(http://www.cbs.dtu.dk/services/CPHmodels/) to

obtain the PDB file, which was used to construct a 3-D

model using PyMol program 0.9.

Results

Hb and haem support the cellular growth of

S. pneumoniae but transferrin does not

It has been proposed that S. pneumoniae uses Hb or

haem as iron sources, and under these conditions two

membrane proteins are expressed by a direct mecha-

nism (Romero-Espejel et al. 2013). However, an

indirect mechanism explaining the role of haemophore

secretion in iron acquisition by the bacterium has not

been documented (Brown et al. 2001). To investigate

whether S. pneumoniae secretes proteins capable of

scavenging iron from Hb or haem, this bacterium was

grown using different iron sources Hb (filled circle),

haem (filled square), FeCl3 (filled triangle), Tf

Fig. 2 Secretome is not contaminated with cytoplasmic pro-

teins. In order to demonstrate that the secretome is not

contaminated with cellular proteins (total proteins), secretome

and total protein fractions were analysed by Western Blotting

using anti-b-galactosidase (b-Gal) antibodies (a). The protein

bands were visualized by chemiluminescence. Arrows indicate a

116 kDa protein band in E. coli and a 69 kDa band in S.

pneumoniae. Secretome and total proteins were visualized by

SDS-PAGE and Coomassie blue staining (b). Secretome and

total protein fractions were determinated for specific biological

activity of b-Galactosidase (measured by absorbance). Lane 1,

E. coli secretome; lane 2, E. coli total proteins; lane 3, S.

pneumoniae secretome; lane 4, S. pneumoniae total proteins.

Data represent the mean ± SD of three independent experi-

ments. The P value, obtained by a t test, is 0.120 (c)

Biometals

123

(asterisk) and free iron (open circle) after previous

iron depletion using dipyridyl addition (iron chelator).

Our results showed that this pathogen uses selective

iron sources for its cellular growth, and while iron, Hb

and haem support the cellular growth of S. pneumo-

niae, Tf does not (Fig. 1).

S. pneumoniae expresses a 38 kDa haem-binding

protein

To determine whether cytoplasmic proteins released

upon lysis of S. pneumoniae contaminate the secreted

proteins, the total and secreted protein fractions from S.

pneumoniae were resolved by SDS–PAGE (Fig. 2b),

and the presence of b-galactosidase (b-Gal), an exclu-

sive cytoplasmic protein, was detected by Western blot

analysis. b-Gal was detected in total protein samples

(Fig. 2a, lane 4), but not in the secreted protein fraction

(Fig. 2a, lane 3). These results indicated that under our

conditions of bacterial culture, the cytoplasmic proteins

do not contaminate the secreted proteins. As an

experimental control, this protocol was also performed

using E. coli (Fig. 2a and b, lanes 1 and 2). To

corroborate that the secretome is not contaminated with

Fig. 3 S. pneumoniae

secretes a 38 kDa haem-

binding protein and its

amount is not modified by

the presence of iron.

Proteins of the S.

pneumoniae secretome were

purified by haem-affinity

chromatography. Samples

of each fraction were loaded

onto SDS-PAGE and

staining by Coomassie blue

(a). Uninoculated Todd

Hewitt medium was used as

a negative control (b). Lane

1, secretome; lane 2, flow-

through; lanes 3–5, washes;

lane 6, elution with

guanidine hydrochloride.

Molecular weight markers

are indicated on the left.

Secretome proteins

separated of cellular cultures

and cultivated under iron

replete or iron starve

conditions, were loaded onto

SDS-PAGE staining by

Coomassie blue (c) thus, the

gel was submitted to

densitometry in order to

quantified the 38 kDa bands

(d). Arrow indicates the

estimated size of the haem-

binding protein

Biometals

123

cellular proteins (total proteins), secretome and total

protein fractions were determined for b-galactosidase

activity following protocol of Miller (Fig. 2c), the b-

galactosidase activity was detected only in total protein

samples, the values obtained were 83.93 Miller units to

E. coli and 97.03 Miller units to S. pneumoniae (Fig. 2c,

lanes 2 y 4) and 0 Miller units in the secreted proteins

(Fig. 2c, lanes 1 y 3). Our results clearly showed that

under these culture conditions the secreted and cyto-

plasmic proteins do not mix.

To investigate whether S. pneumoniae secretes

proteins with an affinity for haem, S. pneumoniae was

cultivated in THB. The supernatant (secretome) was

separated and loaded onto a haem-affinity chromatog-

raphy column (Fig. 3a, lane 1). The flow-through

containing unbound proteins of the bacterial secre-

tome (Fig. 3a, lane 2) and three subsequent wash

fractions (Fig. 3a, lanes 3–5) were collected, and

finally, proteins bound to the resin were eluted by

increasing the stringency of the elution buffer (Fig. 3a,

lane 6). These results showed the presence of a major

protein of 38 kDa (obtained by RF calculation). To

exclude the possibility that this protein could be a

contaminating component from the Todd Hewitt

Broth, a negative control was performed with this

medium. As expected, no protein was eluted from the

control sample (Fig. 3b, lane 6). Our results clearly

showed that an S. pneumoniae 38 kDa protein was

secreted, but we did not know if the growing

conditions modified the amount of 38 kDa protein

secreted, in order to investigate this assumption S.

pneumoniae was cultivated under iron starvation or

iron replete condition, after that, the secreted proteins

were separated and loaded onto SDS-PAGE. Gels

were analysed by densitometry (Fig. 3c). However,

we did not find difference between both conditions, it

seems to be, that the amount of 38 kDa protein

secreted remains without changes (Fig. 3d). There-

fore, we think that the iron source and growth

condition do not modify the amount of 38 kDa protein.

Characterization of the secreted 38 kDa protein

Because the 38 kDa protein secreted by S. pneumo-

niae was found to have an affinity for haem, we

characterized this interaction in more detail, eluting

this haem-binding protein fraction using different

types of potential competitors. As expected, the

38 kDa protein was eluted with Hb (Fig. 4, lane 2)

and haem (Fig. 4, lane 3), but not with Tf (Fig. 4, lane

4). Guanidine hydrochloride elution was used as a

control (Fig. 4, line 1). This result suggested that the

38 kDa protein bound to both Hb and haem.

To corroborate this observation further, Hb-binding

experiments were performed. Under native conditions

the 38 kDa protein was incubated with Hb, and the

complex was separated on 7 % native gels. The results

clearly showed that the 38 kDa protein bound Hb

(Fig. 5a, lane 3) and was unable to bind Tf (used as a

negative control) (Fig. 5a, lane 4). The distinct

migration profiles of the 38 kDa protein (Fig. 5a, lane

1), Hb (Fig. 5a, lane 2) and the Hb-38 kDa protein

complex (Fig. 5a, lane 3) were observed, but no

complex with Tf (Fig. 5a, lane 4) was detected.

Consistent with this observation, the presence of iron

was revealed by hydrogen peroxide reaction (Fig. 5b,

lanes 2, 3, 4 and 5). Our overall results clearly indicate

that the 38 kDa protein specifically binds Hb and

haem. We think that S. pneumoniae secretes this

protein to scavenge iron from the haem present in

human Hb.

Fig. 4 The 38 kDa protein of S. pneumoniae was eluted with

different compounds. The haem-binding protein in the secre-

tome fraction bound to the affinity chromatography resin was

eluted using different compounds. 6 M guanidine chloride (lane

1), Hb (lane 2), haem (lane 3) and Tf (lane 4) fractions.

Molecular weight markers are indicated on the left. The arrow

shows the estimated size of the purified secreted haem-binding

protein

Biometals

123

The secreted 38 kDa protein is identified

as GAPDH

To investigate the identity of the 38 kDa protein, the

corresponding band was excised from the gel and digested

with trypsin. The resulting peptides were purified using

cartridges and analysed by mass spectrometry, 8 peptides

were identified (VGINGFGR, YDTTQGRFDGTVEVK,

KVVITAPGGNDVK, AGAANIVPNSTGAAK, AI-

GLVIPELNGK, TLEYFAK, INDLTDPVMLAHLLK

and NVTVDEVNAAMK), this represented 28 % of

coverage, the Mascot score was 149, the method allowed

us to identify this protein as S. pneumoniae glyceraldehyde

3-phosphate dehydrogenase (GAPDH) with a molecular

weight of 38.76 kDa and an isoelectric point (pI) of 5.37

(Access Number Q8CWN6) (Table 2) in the UniProt

database. This result clearly shows that GAPDH protein is

a Hb and haem-binding protein.

S. pneumoniae GAPDH has the motifs necessary

for haem-binding

It has been proposed that some haem-binding proteins

use a critical motif (KVAFDH) to bind haem (Sirover

Fig. 5 The 38 kDa protein

binds Hb. Native gel

electrophoresis and

Coomassie blue staining

were performed to

determine whether the

38 kDa binds Hb in addition

to haem (a). The native gel

was developed with

hydrogen peroxide (b). Lane

1, 38 kDa protein; lane 2,

Hb; lane 3, complex 38 kDa

protein-Hb; lane 4, 38 kDa

protein and Tf; lane 5, Tf.

Arrows indicate bands

corresponding to the 38 kDa

protein-Hb complex, Hb, Tf

and the 38 kDa protein

alone

Biometals

123

2011). To explore if S. pneumoniae GAPDH has this

motif, amino acid sequence multiple alignment was

performed. The S. pneumoniae GAPDH sequence was

compared with sequences of other proteins including

the S. pneumoniae 22 kDa protein, S. pneumoniae

37 kDa protein and GAPDH proteins from B. anthracis,

B. subtilis, C. botulinum, E. coli, H. influenzae, H.

pylori, N. meningitidis and S. aureus (Table 1). This

analysis revealed the existence of the essential haem-

binding motif (KVAFDH) in the S. pneumoniae GAP-

DH sequence (Fig. 6). To determine the localization of

this motif in the 3D structure of the protein, 3D

modelling was performed using PyMol 0.9. Our

analysis revealed that this motif (KVAFDH) is exposed

in the GAPDH 3D structure (Fig. 7).

Discussion

S. pneumoniae is a pathogenic bacterium capable of

utilising Hb or haem as its only iron source (Tai et al.

1993, Tuomanen et al. 2004). Unfortunately, very

little is known about the mechanisms involved in its

iron acquisition from Hb or haem. Here, we present the

first evidence suggesting that this pathogen secretes an

Table 2 The 38 kDa protein was identified as GAPDH

Access

number

Protein name Coverage Mass

(kDa/

pl)

Mascot

score

Peptides Function

Q8CWN6 Glyceraldehyde-3-

phosphate

dehydrogenase

GAPDH

28 % 38.76/

5.38

149 VGIGFGR, YDTTQGRFDGTVENK,

KVVITAPGGNDVK,

AGAANUVPNSTGAAK,

AIGLVIPELINGK, TLEYFAK,

INDLTDPVMLAHLLK,

NVTVDEVNAAMK

Glucose metabolic

process NAD

binding NADP

binding

The purified protein was resolved by SDS-PAGE. The band corresponding to the protein was then excised from the gel, digested with

trypsin and the purified peptides were analysed and identified by mass spectrometry

Fig. 6 The 38 kDa protein of S. pneumoniae has the motif

necessary for haem-binding. Multiple amino acid sequence

alignment was performed to compare the sequence of S.

pneumoniae GAPDH with that of GAPDH from B. anthracis,

B. subtilis, C. botulinum, E. coli, Haemophilus influenzae, H.

pylori, N. meningitidis, S. aureus and two membrane proteins

containing the haem-binding motif (S. pneumoniae 22 kDa and

S. pneumoniae 37 kDa). The KVAFDH motif is essential for

haem-binding (shown as dark boxes). Val (V) is the most

conserved residue followed by Lys (K) and His (H). Multi-

alignment was performed using Clustal W server and marked

residues were obtained using Jalview program 2.8

Fig. 7 The motif necessary for haem-binding is exposed in

GAPDH as observed from the 3D analysis of the spatial

structure of the protein. The 3D GAPDH structure was

determined using PyMol 0.9 program. The haem-binding motif

is indicated in black

Biometals

123

38 kDa protein which binds Hb and haem and its

amount was not modified under iron starvation

condition. This protein was identified as GAPDH.

GAPDH protein is a cytoplasmic enzyme involved

in the glycolytic pathway, which is an essential

metabolic pathway utilised by a wide variety of

organisms ranging from prokaryotic to eukaryotic.

Nevertheless, in addition to metabolic role, the

eukaryotic GAPDH participates in several cellular

functions including transcription, oxidative stress,

apoptosis, autophagy (Sirover 1996, 2011) and haem

binding (Hannibal et al. 2012). Our results suggest that

S. pneumoniae also secretes GAPDH to bind Hb and

haem. We believe that S. pneumoniae GAPDH has

unique characteristics and they differ from rabbit

muscle GAPDH. Rabbit-GAPDH binds haem, but its

tetramer form has a higher haem affinity than the

monomer (Hannibal et al. 2012). In contrast, mono-

meric S. pneumoniae GAPDH binds Hb and haem

efficiently, as was demonstrated by overlay experi-

ments (data no showed). Because these experiments

were performed with high stringency and nevertheless

GAPDH still bound Hb (These conditions maintain the

monomeric form of GAPDH. We suggest that forma-

tion of its quaternary structure of GAPDH is not

necessary for Hb- or haem-binding.

Despite the lack of secretory signals in its amino

acid sequence, GAPDH has been localized on the

surface of several bacteria, fungi and even protozoans

(Pancholi and Chhatwal 2003). Although the mecha-

nism of secretion is not known, there is report in which

GAPDH is releases of the bacterium using a method

detergent-assisted lysis (Oliveira et al. 2012). In the

present study, the bacteria culturing method designed

allowed the growth of bacteria without lysis, our

results suggest that this protein could be secreted using

a different unknown pathway. This notion was further

corroborated by the b-galactosidase control experi-

ment in which the exclusively cytoplasmic protein b-

galactosidase with enzymatic activity was not detected

in the growth media. This observation was consistent

with experiments performed in H. Pylori and S.

pneumoniae (Vanet and Labigne 1998; Choi et al.

2012; Gonzalez-Lopez et al. 2013). Although the

experimentally obtained molecular size of 38 kDa for

GAPDH was higher than that of the other GAPDH

proteins described in the multiple alignment (Fig. 6),

we cannot exclude the possibility that all of these

proteins have this function (Hb and haem-binding).

Multiple alignment revealed the essential motif for

haem-binding (KVAFDH) previously described for

the e-P4 lipoprotein of H. influenzae (Reidl and

Mekalanos 1996). This result was corroborated by

3D modeling in which the spatial location of the

functional motif was elucidated. Our hypothesis is

supported by current evidence suggesting that GAP-

DH is a multifunctional protein displaying diverse

activities distinct from its conventional metabolic role.

In humans, new roles for GAPDH may be dependent

on its subcellular localization, oligomeric state (Han-

nibal et al. 2012) or the proliferative state of the cell

(Mazzola and Sirover 2001). Furthermore, the GAP-

DH of Chlamydomonas reinhardtii has been shown to

be a bifunctional protein (Erales et al. 2009).

In summary, S. pneumoniae GAPDH could play

another role distinct from its role in carbohydrate

metabolism. We propose that S. pneumoniae GAPDH

is an Hb- and haem-binding protein that participates in

iron acquisition and is essential for the successful

invasion of S. pneumoniae in several tissues during the

infection process.

Acknowledgments This work was supported by CONACyT

(Consejo Nacional de Ciencia y Tecnologıa) (Grant numbers

SALUD-2010-01-139945, SALUD-2012-01-181641). We thank

Ms. D. Alma Lidia Olivares from Hospital Infantil de Mexico

‘‘Federico Gomez’’ for providing the R6 strain used in this study.

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