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Iron regulated surface determinant (Isd) proteins of Staphylococcus lugdunensis. 1
Marta Zapotoczna1, Simon Heilbronner1, Pietro Speziale2 and Timothy J. Foster1. 2
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1Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College 4
Dublin, Dublin 2, Ireland. 5
2Department of Molecular Medicine, Viale Taramelli 3/b,27100 Pavia, Italy. 6
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Running title: Isd proteins of Staphylococcus lugdunensis. 8
Keywords: Staphylococcus lugdunensis, haemoglobin, haem, haem-uptake, Isd proteins, 9
protein sorting, sortase, cell wall, fibrinogen, cytokeratin 10, loricrin, linoleic acid, resistance 10
to fatty acid. 11
Corresponding author: Foster T.J., e-mail: [email protected]. 12
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Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.01195-12 JB Accepts, published online ahead of print on 21 September 2012
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ABSTRACT 14
Staphylococcus lugdunensis is the only species of coagulase negative staphylococcus with a 15
locus encoding iron regulated surface determinant (Isd) proteins. In S. aureus the Isd proteins 16
capture haem from haemoglobin, transfer it across the wall to a membrane-bound transporter 17
which delivers it into the cytoplasm where haem oxygenases release iron. The Isd proteins of 18
S. lugdunensis are expressed under iron-restricted conditions. We propose that S. lugdunensis 19
IsdB and IsdC proteins perform the same functions as those of S. aureus. S. lugdunensis IsdB 20
is the only haemoglobin receptor within the isd locus. It specifically binds human 21
haemoglobin with a KD of 23 nM and transfers haem on IsdC. IsdB expression promotes 22
bacterial growth in an iron-limited medium containing human haemoglobin but not mouse 23
haemoglobin. This correlates with weak binding of IsdB to mouse haemoglobin in vitro. 24
Unlike IsdB and IsdC, IsdJ and IsdK are not sorted to the cell wall in S. lugdunensis. In 25
contrast, IsdJ expressed in S. aureus and Lactococcus lactis is anchored to peptidoglycan, 26
suggesting that S. lugdunensis sortases may differ in signal recognition or could be defective. 27
IsdJ and IsdK are present in the culture supernatant suggesting they could acquire haem from 28
the external milieu. The IsdA protein of S. aureus protects bacteria from bactericidal lipids 29
due to its hydrophilic C-terminal domain. IsdJ has a similar region and protected S. aureus 30
and L. lactis as efficiently as IsdA but, possibly due to its location, was less effective in its 31
natural host. 32
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INTRODUCTION 38
Staphylococcus lugdunensis is a species of coagulase negative staphylococcus (CoNS) first 39
described by Freney et al. in 1988 (23). The bacterium is part of the normal flora of skin of 40
humans. It colonizes several distinct niches primarily in the lower part of the body (7). 41
Standard identification procedures used in clinical microbiology laboratories can allow 42
misidentification of S. lugdunensis as S. aureus (58). The bacteria have similar colony 43
morphology, and many strains of S. lugdunensis are haemolytic and express clumping factor 44
activity (38). S. lugdunensis can cause infections similar to those caused by S. aureus ranging 45
from localized to systemic, including skin and soft tissue infections (SSTI), breast abscesses, 46
osteomyelitis, prosthetic joint infections, pneumonia or meningitis (4, 8, 33, 35, 73). Most 47
notably S. lugdunensis can cause acute infective endocarditis (IE) which has a high mortality 48
rate of ca. 50% (2, 41, 68). S. lugdunensis infections occur at much lower frequency than 49
those caused by S. aureus, although the numbers may be underestimated. The majority of 50
infections caused by S. lugdunensis are community acquired, occurring in healthy adults, 51
which contrasts with the opportunistic character of other CoNS infections (3, 10, 75, 77). 52
Understanding the molecular determinants of virulence has been facilitated by the 53
availability of the genome sequences of two strains, N920143 and HKU09-01 (30, 67). S. 54
lugdunensis N920143 coding sequences have 77.8% matches with S. aureus MRSA252, 55
74.7% with S. epidermidis RP62a and 95.4% with HKU09-01 (30). Uniquely for CoNS, S. 56
lugdunensis possesses a cluster of genes with similarity both in terms of sequence and 57
organization to the iron regulated surface determinant (isd) locus of S. aureus (Fig. 1A). The 58
isd locus occurs in both sequenced and annotated genomes of S. lugdunensis and is tandemly 59
duplicated in the HKU09-01 (30). In S. aureus the isd genes are induced by iron limitation 60
with promoters that are controlled by the ferric uptake regulator (Fur). The S. aureus Isd 61
system is up-regulated in vivo during infection due to iron sequestration in the host (1, 6, 29). 62
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The system comprises nine proteins (IsdA-IsdI) whose main task is to bind haemoglobin and 63
to extract haem, which is transported into the cytoplasm to provide a source of iron. Four of 64
the proteins are anchored to cell wall peptidoglycan by either sortase A or sortase B. IsdA, 65
IsdB and IsdH each contain sortase A recognition sequences (LPXTG). IsdC has a distinct 66
recognition sequence NPQTN and is sorted by a type B sortase encoded by srtB which is 67
itself part of the isd regulon (44). These proteins each contain one or more near iron 68
transporter (NEAT) motifs, which bind to haemoglobin or haem. The NEAT domains are 69
conserved in several Gram-positive bacteria. They consist of ~125 amino acids forming an 70
IgG-like beta sandwich fold of 7 or more β-strands in 2 β-sheets (20, 25-27, 36). 71
The N-terminal NEAT motifs of IsdH (NEAT1 and NEAT2) and IsdB (NEAT1) bind 72
to the haptoglobin-haemoglobin complex and to haemoglobin but cannot bind haem (19, 55, 73
66). They share 47-65% identity and bind to a site on the α-chain of human Hb using 74
conserved aromatic residues within loop 1 (β1-β2) (36). Haem is then removed from Hb and 75
transported to the haem-binding NEAT domains that coordinate haem via a conserved Tyr in 76
β8 (25). The haem binding NEAT domains of IsdH NEAT3 and IsdB NEAT2 transfer haem 77
to those of IsdA and IsdC and then to the membrane located haem transporter (IsdEF) (26, 78
40, 49, 76). In the cytoplasm the porphyrin ring is broken by haem oxygenases (IsdG and 79
IsdI) and free iron is released (57). 80
There is evidence that surface-exposed Isd proteins may have a broader role in S. 81
aureus colonisation and pathogenesis (9, 14, 55, 66). The IsdA protein is multifunctional and 82
as well as transporting haem it can promote adhesion to the extracellular matrix and to the 83
surface of desquamated nasal epithelial cells, possibly by binding loricrin and keratin 10 in 84
the cornified envelope of squames (16). It promotes nasal colonization in the cotton rat, 85
survival on human skin by conferring resistance to bactericidal lipids in sebum and survival 86
within phagocytes by contributing to resistance to the oxidative burst (11-15, 17). IsdH was 87
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shown to facilitate enhanced conversion of complement opsonin protein C3b to iC3b and C3d 88
enabling S. aureus to avoid neutrophil uptake (70). The IsdB protein promotes S. aureus 89
adhesion to platelets resulting in platelet aggregation due to its ability to bind to the platelet 90
integrin (45). 91
The objective of this study was to investigate four putative cell wall anchored Isd 92
proteins of S. lugdunensis, IsdB, IsdC, IsdJ and IsdK with respect to their expression, 93
localization, haemoglobin and haem binding capabilities and their role in bacterial growth 94
under iron limitation. Finally the IsdJ protein, which has similarity to IsdA of S. aureus, was 95
tested for its ability to bind to fibrinogen, keratin 10 and loricrin, and to confer resistance to 96
killing by linoleic acid. 97
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MATERIALS AND METHODS 109
Growth conditions, bacterial strains and plasmids. The bacterial strains and plasmids used 110
in this study are listed in Table 1. S. lugdunensis and S. aureus strains were grown in tryptic 111
soya broth (TSB), or RPMI-1640 medium (Sigma) to create iron restricted conditions (47). E. 112
coli strains XL - 1 Blue and DC10B used for cloning were grown in Luria - Bertani (LB) 113
broth. E. coli TOPP10 used for recombinant protein expression was grown in LB broth, or 114
RPMI to purify proteins with low haem content. L. lactis was grown in M17 medium 115
supplemented with 0.5% glucose (GM17) (63). The following antibiotics (Sigma) were added 116
to the media as required: ampicillin (Amp) at 100 μg ml-1, chloramphenicol (Chm) at 10 μg 117
ml-1, tetracycline (Tet) at 2 μg ml-1, erythromycin (Erm) at 10 μg ml-1. 118
Expression and purification of recombinant proteins. DNA fragments encoding Isd 119
proteins were amplified using specific primers (Table S1) and cloned into pQE30 or pGE4t-2 120
vectors (Table 1). His-tagged and GST-tagged proteins were purified by nickel-affinity 121
chromatography using HisTrap HP (GE Healthcare) columns or glutathione-affinity 122
chromatography using GSTrap FF columns (GE Healthcare), as described previously (51). 123
Proteins were dialysed against PBS, concentrated by ultrafiltration (Amicon Ultra, EMD 124
Millipore) and stored at -70oC. 125
Anti-Isd sera. Antibodies were raised in rabbits to recombinant IsdB, IsdC, IsdJ (45-610) 126
and IsdK (Fig. S1) and the immunoglobulin fraction was purified. The potency and 127
specificity of the sera was determined by ELISA. Antisera were used to probe whole cells by 128
dot immunoblotting and cell fractions by Western immunoblotting. 129
Recombinant IsdB mutants. Mutations creating amino acid substitutions were introduced in 130
pQE30isdB by sequential primer overlap mutagenesis with Phusion High-Fidelity 131
Polymerase. Primers used for each step are listed as 13-18 in Supplementary Table 1. 132
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Complementary forward and reverse primers incorporating a mutation were extended by PCR 133
to produce a mutated plasmid. PCR products were digested with DpnI for 1.5 h at 37°C to 134
eliminate methylated parental DNA and then transformed into E. coli strain XL-1 Blue. 135
Inducible expression of IsdJ and IsdA. The isdA and isdJ genes were amplified by PCR 136
(Table S1) and cloned into pRMC2 using primers listed as 19 - 22 in Supplementary Table 1. 137
Plasmids were cloned in E. coli DC10B and subsequently electroporated into S. aureus 138
Newman clfA clfB isdA and S. aureus Newman spa (Table 1) using protocol of Lofblom et al. 139
(42). Plasmids of pRMC2isdA and pRMCisdJ allow anhydrotetracycline (aTc) inducible 140
expression of the full length IsdJ and IsdA proteins in S. aureus. To induce expression of the 141
proteins, aTc (1 μg ml-1) was added to exponentially growing cultures. 142
The isdJ gene was amplified (primers 23-24; Table S1) and cloned into pNZ8048, a 143
nisin-inducible expression vector. Plasmid pNZ8048isdJ was cloned into electrocompetent L. 144
lactis NZ9000. Induction of IsdJ expression was carried out by adding nisin (300 μg ml-1) to 145
exponentially growing cultures and incubating for 4 h. 146
Isolation of isd mutants by allelic replacement. DNA fragments comprising 500-1000 bp 147
upstream from the start codon of each gene and 500-1000 bp downstream from the stop 148
codon (see primers, Table S1) were cloned into either pKOR1 or pIMAY in E. coli DC10B 149
(Table 1) (5, 46). Plasmids were electroporated into S. lugdunensis N920143 (42). Allelic 150
exchange was performed by homologous recombination initiated by switching the bacterial 151
growth temperature from that optimal for plasmid replication (28oC) to the non-permissive 152
temperature of 37oC (pIMAY) or 42oC (pKOR1). Detailed protocols were described 153
previously by Bae et al. (pKOR1) and Monk et al. (pIMAY) (5, 46). 154
Cell fractionation. Cultures of S. lugdunensis were harvested by centrifugation (4,000 × g, 155
10 min), washed in phosphate buffer saline (PBS) and adjusted to an OD600 of 20. 1 ml of the 156
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bacterial suspension was pelleted and resuspended in 200 μl of digestion buffer (50 mM Tris-157
HCl, 20 mM MgCl2, 30 % (w/v) raffinose, pH 7.5) containing complete mini EDTA - free 158
protease inhibitors (Roche). Cell wall proteins were solubilized by digestion with lysostaphin 159
(500 μg ml-1) at 37°C for 30 min. Protoplasts were harvested by centrifugation (5,000 × g, 15 160
min) and the supernatant was retained as the cell wall fraction. Protoplast pellets were 161
washed once in digestion buffer, sedimented (5,000 × g, 15 min) and resuspended in ice-cold 162
lysis buffer (50 mM Tris-HCl, pH 7.5) containing protease inhibitors and DNase (80 μg ml-1). 163
Protoplasts were lysed on ice by vortexing. Complete lysis was confirmed by phase contrast 164
microscopy. The membrane fraction was obtained by centrifugation at 18,500 × g for 1 h at 165
4°C. The pellet (membrane fraction) was washed once and resuspended in ice-cold lysis 166
buffer. Both fractions were centrifuged again under the same conditions, the pellets 167
resuspended in 200 μl of lysis buffer and retained as the membrane fraction. Proportional 168
volumes of culture supernatants were concentrated using Amicon Ultra Centrifugal Filter 169
Units 10,000 K (Merck Millipore) and resuspended in final concentration of 200 μl to 170
maintain proportionally concentrated fractions. Fractions were mixed with sample buffer 1:1 171
(Laemmli) and separated by SDS-PAGE. Cellular fractionation of S. aureus and L. lactis was 172
performed as described (18, 60). 173
Western immunoblotting and fibrinogen - affinity blotting. Proteins or cellular fractions 174
were separated by SDS-PAGE and electroblotted onto PVDF membranes (Roche) for 1 h at 175
100 V using a wet transfer cell (Bio-Rad). Membranes were incubated for 1 h at 37°C, or 176
overnight at 4°C in TS buffer (10 mM Tris - HCl, pH 7.4, 150 mM NaCl) containing 10 % 177
skimmed milk (Marvel). Primary antibodies or fibrinogen were diluted in 10% Marvel TS 178
and incubated with the membranes for 1 h at room temperature with shaking. Unbound 179
antibody was removed by three 15 min washes with TS buffer. The HRP - conjugated 180
secondary reagents were incubated with the membranes for 1 h at room temperature with 181
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shaking. Unbound secondary reagent was removed by washing three times with TS buffer 182
and bound HRP developed with chemiluminescent substrate (LumiGlo; New England 183
Biolabs) in ImageQuant LAS imager (GE Healthcare). 184
Whole cell and protein dot immunoblotting. Bacterial cells were washed twice in PBS and 185
adjusted to an appropriate optical density. Each doubling dilution (5 µl) of the bacterial 186
suspensions or soluble protein (5 µl) was dotted onto a nitrocellulose membrane (Protran). 187
The membranes were blocked for 1 h with Marvel TS buffer followed by detection performed 188
as in Western blotting described above. 189
Haem content. The absorbance spectra of recombinant proteins were recorded on a 190
Shimadzu UV-160PC spectrophotometer. The haem content of proteins was estimated from 191
the reduced pyridine haemochrome spectra (61). Protein concentrations were estimated by 192
BCA Protein Assay Reagent (Thermo Scientific Pierce). 193
Haemin transfer from IsdB to IsdC. Recombinant IsdB, purified from E. coli grown in 194
RPMI, was gradually supplemented with porcine haemin (Sigma) dissolved in DMSO at 10 195
mM. Insoluble material was removed by centrifugation. The mixture was loaded onto 196
calibrated Sephadex G-25 (GE healthcare) and chromatography was performed to separate 197
the saturated IsdB from unbound haemin. IsdB (haemin donor) and IsdC - GST (haemin 198
acceptor) were mixed at 10 μM and 20 μM, respectively. After 4h incubation at 22oC the 199
mixture was loaded onto a GST column to separate GST-tagged IsdC from IsdB. Separation 200
of the two proteins was monitored by SDS-PAGE. The spectra of the separated donor and 201
acceptor were recorded to assess the haemin transfer. 202
Peroxidase (TMBZ) staining. Detection of haem - protein complexes was accomplished by 203
staining gels with 3,3',5,5'-tetramethylbenzidine (TMBZ; Sigma) a chromogen that turns blue 204
in areas of haem - associated peroxidase activity (64). 205
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Enzyme-linked immunosorbent assay (ELISA). Haemoglobin-haptoglobin (Hb-Hpt) 206
complexes were prepared by mixing human plasma haptoglobin (Molecular Innovations) 207
with haemoglobin (Pointe Scientific) at a 1:1 molar ratio for 1 h at 22 oC. Haemoprotein 208
binding assays were performed in two ways. (I) Microtitre plates (Nunc) were coated with 209
recombinant Isd proteins at 2 μM in 100 μl of 50 mM sodium carbonate buffer, pH 9.5, 210
overnight at 4oC. Coated wells were washed three times with PBS and blocked with 5% 211
Marvel in PBS for 2 h at 37oC. Following three washes with PBS, various concentrations of 212
methaemoglobin (MetHb), haemoglobin (Hb), Hb-Hpt complexes or haptoglobin (Hpt) were 213
added to the wells in 2% Marvel PBS and incubated for 2 h at 37oC. Wells were washed with 214
PBS and bound proteins detected by incubation with rabbit anti-human Hb serum (Dako) or 215
rabbit anti – human Hpt serum (Sigma) in 2% Marvel PBS for 1 h at 37oC. (II) Reciprocal 216
binding experiments were performed by coating microtitre (Nunc) wells with MetHb, Hb, 217
Hb-Hpt, Hpt or myoglobin (Mb) at 1 μM in 100 μl of 50 mM sodium carbonate buffer, pH 218
9.5 overnight at 4oC. After blocking with 5% Marvel PBS, the wells were incubated with 219
increasing amounts of recombinant Isd proteins in 2% skimmed milk in PBS for 2 h at 37oC. 220
Bound proteins were detected by incubation with appropriate polyclonal rabbit anti-Isd IgG 221
in 2% Marvel PBS for 1 h at 37oC and washed three times with PBS. To detect specific 222
interactions in both types of assays, HRP-conjugated goat anti-rabbit IgG (Dako) in 2% 223
Marvel PBS was added for 1 h at 37oC. After three washes, 100 μl of TMBZ (Sigma) at 0.1 224
mg ml-1 prepared in 0.05M phosphate citrate buffer containing 0.006% (v/v) hydrogen 225
peroxide was added, and plates were developed for 5 min. The reaction was stopped by the 226
addition of 50 μl of 2M H2SO4 to each well. Plates were read at 450 nm using a Multiscan 227
ELISA plate reader. Half-maximum binding values reported for all ELISA-type binding 228
assays were predicted using GraphPad Prism software. 229
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Human GST tagged - loricrin (1 μM), GST-cytokeratin 10 (1 μM) (71), GST (1 μM) 230
and human fibrinogen (10 μg ml -1) (Calbiochem) in 50 mM sodium carbonate buffer, pH 9.5 231
were coated onto microtiter wells (Nunc) overnight at 4oC. All plates were blocked with 5% 232
BSA in PBS for 2h at 37°C. Following three washes with PBS, various concentrations of 233
recombinant IsdJ in 1% Marvel PBS were added and incubated at 37°C for 2 h. After three 234
washes with PBS, bound protein was detected by incubation with rabbit anti-IsdJ IgG in 1% 235
Marvel PBS at 37°C. After 1 h incubation the wells were washed three times with PBS 236
followed by incubation with protein A- peroxidase conjugated to HRP (Dako) in 1% Marvel. 237
After incubation for 1 h at 37°C wells were washed three times with PBS, and bound HRP 238
detected with TMBZ as described above. Purified human GST-loricrin and GST-cytokeratin 239
10 were kindly provided by Michelle Mulcahy. 240
Bacterial adherence to immobilized ligands. Microtiter plates were coated with doubling 241
dilutions of fibrinogen, GST-cytokeratin 10 and or GST in PBS overnight at 4oC. Control 242
wells contained PBS only. After washing (PBS), the plates were blocked for 2 h at 37oC with 243
5% (w/v) BSA in PBS. After washing three times, 100 μl of a cell suspension (OD600 of 1 in 244
PBS) was added to each well, and the plates incubated for 2 h at 37oC. After washing three 245
times, adherent cells were fixed by adding 100 μl of 25% formaldehyde, and incubating at 246
room temperature for at least 20 min. The plates were then washed and stained with crystal 247
violet for 2 min. After several washes 100 μl of 5% (v/v) acetic acid was added to the wells to 248
solubilise the crystal violet. Absorbances were read in Multiscan ELISA plate reader at 570 249
nm. 250
Surface Plasmon Resonance (SPR). SPR analysis of IsdB - haemoglobin binding was 251
performed using the BIAcore X100 system (GE Healthcare). Recombinant IsdB was captured 252
and amine-coupled to monoclonal anti-His antibodies (R&D Systems) on a flow cell. The 253
antibodies at 50 μg ml-1 in sodium acetate buffer, pH 5.0 had been previously covalently 254
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immobilized on both flow cells of a CM5 chip, using amine coupling. This was performed 255
according to manufacturer’s protocol, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide 256
hydrochloride, followed by N-hydroxysuccinimide (NHS) and ethanolamine. Increasing 257
concentrations of analytes, such as methaemoglobin (34 kDa) or haemoglobins (68 kDa) in 258
running buffer (PBS, pH 7.4, 0.005% P20) were flowed over immobilized IsdB and a 259
reference flow cell (coated only with anti-His antibody) at a rate of 5 μl min-1. The 260
sensorgram data presented were subtracted from the corresponding data on the reference flow 261
cell and from the response generated by injection of buffer over the chip. We used affinity 262
analysis (BIAevaluation software) to determine the dissociation constants (KD). Purified 263
human and mouse haemoglobins were kindly provided by Dr Gleb Pishchany and Dr Eric 264
Skaar. 265
Growth in modified RPMI supplemented with Hb. The protocol by Pishchany et al. was 266
used with minor modifications (55). A single colony of S. lugdunensis was inoculated into 267
RPMI and grown overnight. The concentration of cells was normalized to OD600 of 3.0, 268
bacteria were sedimented (4,000 × g, 10 min) and resuspended in 1 ml NRPMI containing 269
0.25 mM of the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid (EDDHA, LGC 270
Standards GmbH). NRPMI is RPMI containing 1% casamino acids, treated with Chelex 100 271
(Sigma) at 20 g l-1 and supplemented with 25 μM ZnCl2, 25 μM MnCl2, 100 μM CaCl2, and 1 272
mM MgCl2. The S. lugdunensis suspension was subcultured (1:50) into 5 ml NRPMI + 0.25 273
mM EDDHA with haemoglobin at 10 μg ml-1. Cultures were incubated at 37°C in 15 ml 274
conical tubes with shaking at 40 r.p.m. OD600 measurements were taken at indicated time 275
points. 276
Bacterial killing by linoleic acid. Bacteria were harvested by centrifugation and washed 277
twice in sterile dH2O. Cell suspensions were adjusted to 1 × 108 cfu ml-1 in dH2O and 278
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incubated at 37oC with linoleic acid (Sigma) at 10 μg ml-1. The cfu was determined at 0, 30 279
and 60 minutes by viable counting. 280
In silico structural modelling. Structural models were obtained using the PHYRE website 281
(34) (Imperial College London, Structural Bioinformatics Group 282
[http://www.sbg.bio.ic.ac.uk/~phyre/]) and analyzed using the molecular modelling UCSF 283
Chimera software (University of California, Resource for Biocomputing Visualisation and 284
Informatics [http://www.cgl.ucsf.edu/chimera/]). 285
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RESULTS 299
Sequence analysis of S. lugdunensis proteins with NEAT domains. The predicted amino 300
acid sequences of putative cell wall anchored Isd proteins of S. lugdunensis N920143 and 301
HKU01-09 were compared to homologous Isd proteins of S. aureus. The IsdB and IsdJ 302
proteins of S. lugdunensis are each predicted to have two NEAT domains, while IsdC and 303
IsdK have one (Fig. 1B). IsdB and IsdJ have sortase A recognition sequences (LPATG and 304
LPNTG, respectively) while IsdC and IsdK have sortase B - like recognition sequences 305
(NPQTS and NKQPN, respectively) (Fig. 1B). S. lugdunensis IsdB and IsdC proteins are 306
orthologues of S. aureus proteins with 37% and 57% identities, respectively (Fig. 1A). The 307
identities occur mostly within the NEAT domains. The S. lugdunensis IsdB NEAT1 domain 308
is 60% identical whereas the NEAT2 domain is 56% identical to S. aureus IsdB NEAT1 and 309
NEAT2 domains, respectively (Fig. 1D). Both IsdJ and IsdK have segments with similarities 310
to IsdA of S. aureus (Fig. 1CD). The NEAT1 and NEAT2 domains of IsdJ have 48% and 311
55% identity, respectively, to the single NEAT domain of IsdA, while the IsdJ NEAT2 and 312
C-terminal residues 457-646 have 39% identity to IsdA 63-350 (Fig. 1CD). The IsdK NEAT 313
domain is 36% identical and 60% similar to the IsdA NEAT domain (Fig. 1D). 314
The NEAT domains of S. lugdunensis Isd proteins were modelled in 3D based on 315
known X-ray crystal structures of the highly conserved NEAT domains of S. aureus. IsdA 316
(PDB ID: 2ITE) was used to model the IsdJ NEAT1 and NEAT2 domains, and the IsdK 317
NEAT domain (27). S. aureus IsdH NEAT3 (PDB ID: 2E7D) and IsdB NEAT2 (PDB ID: 318
3RTL) were used to model S. lugdunensis IsdB NEAT2 (24, 72). The NEAT domain of IsdC 319
was modelled on its orthologue from S. aureus (PDB ID: 2O6P) (59, 69). 320
Each of the putative haem binding NEAT domains was predicted to be composed of 321
an IgG-like β-sandwich fold with 8 anti-parallel β-strands. Each has conserved residues 322
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within the predicted hydrophobic haem binding pocket (Fig. 2A). A key metal coordinating 323
Tyr is conserved in β8. Conserved Lys, Ser, and Met residues which have been shown to 324
form hydrogen bonds with the propionate groups of bound haem occur in a predicted helix 325
linking β1b and β2 (20, 25, 27, 54). Hydrophobic residues involved in capturing the 326
porphyrin occur also in β1-β2 (Tyr) and β7 (Val, Ile, Val or similar) (Fig. 2A) (24, 25). S. 327
aureus IsdC is unique in using Tyr and Ile in β4 to make van der Waal’s contacts with haem. 328
These residues are present in the S. lugdunensis orthologue (Fig. 2A) along with a 5 residue 329
insertion in loop β7-β8 that is identical to that in IsdC of S. aureus (Fig. 2A) (59, 69). 330
The NEAT1 domain of IsdB from S. lugdunensis is 56% identical to the IsdH NEAT1 331
domain of S. aureus (Fig.1 D). Therefore the available crystal structures of IsdH NEAT1 332
(PDB ID: 3SZK, 3OVU and 2H3K) were used to model the domain allowing the prediction 333
that it binds to haemoglobin (36, 54). The NEAT1 domain of IsdB from S. lugdunensis is 334
60% identical to the NEAT1 domain of S. aureus IsdB and 59% identical to the NEAT 2 of 335
IsdH (Fig. 1D). The structural model presented a 9 stranded distorted β-barrel with an 336
immunoglobulin-like fold. A characteristic helix-containing loop occurs between β1 and β2 337
which contains a string of aromatic residues which are known to be crucial for binding to α-338
chain of haemoglobin (Fig. 2B). 339
Cellular localization of Isd proteins in S. lugdunensis. Specific antibodies were used to 340
detect surface-exposed proteins of S. lugdunensis using a whole cell dot blotting method. S. 341
lugdunensis N920143 was grown to stationary phase in TSB containing iron (48) or iron-342
restricted RPMI with or without added FeCl3. Whole cells were dotted onto membranes 343
beginning at an OD600 of 1 for probing with anti-IsdC and anti-IsdB sera or an OD600 of 50 344
for IsdK and IsdJ. IsdB, IsdC and IsdK were detected on the surface of cells grown in RPMI 345
but not on TSB-grown cells indicating that they were only expressed in iron limited 346
conditions (Fig. 3ABC). Addition of increasing concentrations of FeCl3 to RPMI resulted in 347
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decreasing levels of the proteins being expressed. 1 mM FeCl3 completely abolished 348
expression of IsdC and IsdK whereas 2 mM FeCl3 inhibited production of IsdB (Fig. 3ABC). 349
Unexpectedly IsdJ was not detected on the cell surface. In order to determine if the 350
antisera recognized the N-terminal region (likely to be surface exposed) rIsdJ (45-295) and 351
rIsdJ (295-610) truncates were expressed and purified. Dot immunoblotting and ELISA 352
indicated that both regions were equally immunogenic (Fig. S2) so it can be concluded that 353
the N-terminal region is not exposed on the surface. Since the anti-IsdK serum was as potent 354
in recognizing the recombinant protein as anti-IsdB and anti-IsdC serum (data not shown) it 355
can also be concluded that IsdK is poorly expressed on the surface whereas IsdB is well 356
exposed (Fig. 3ABC). 357
Cells grown in TSB and RPMI were suspended in a buffer containing 30% raffinose 358
and protoplasts formed by treatment with lysostaphin. The protoplasts were removed by 359
centrifugation leaving solubilised cell wall proteins in the supernatant (wall fraction). The 360
protoplasts were lysed and the membranes separated by centrifugation to yield the membrane 361
fraction. Wall and membrane fractions were analyzed by Western immunoblotting (Fig. 3B). 362
Each protein was expressed in RPMI with only very slight expression of IsdB seen in TSB. 363
IsdB and IsdC were predominantly found in the wall fraction but a significant amount was 364
always found in the membrane fraction. Interestingly IsdC from the membrane fraction is 365
larger than that from the wall fraction which indicates that a proportion of IsdC has not been 366
sorted and is retained in the membrane with its hydrophobic and positively charged C-367
terminus (Fig. 3D and 4B). Surprisingly IsdJ and IsdK were only detected in the membrane 368
fraction and appeared not to be sorted at all (Fig. 3D and 4B). The same pattern of expression 369
of Isd proteins was seen in four other isolates of S. lugdunensis (Fig. S3). This suggests that 370
sortases are defective in anchoring the IsdJ and IsdK, perhaps due to their inability to 371
recognize the proteins’ sorting signals. 372
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We investigated the possibility that IsdJ and IsdK are secreted by S. lugdunensis. 373
Wild-type N920143 and the isdK and isdJ mutants were grown in RPMI, culture supernatants 374
were concentrated and proportional amounts of each fraction were probed with anti-Isd sera. 375
Similar amounts of IsdJ and IsdK were detected in the membrane and supernatant fractions 376
and were missing from cultures of appropriate mutants (Fig. S4). However, the cell wall 377
anchored IsdB protein was also detected in the supernatants possibly resulting from autolysis 378
during growth. It is therefore difficult to determine if IsdJ and IsdK occur in the supernatant 379
due to direct secretion because all three Isd proteins found in the supernatants were smaller in 380
size suggesting that proteolytic cleavage had occurred (Fig. S4). 381
Isd mutants. Mutations were isolated to delete the isdC, isdJ, isdK, isdB genes and the entire 382
isd locus (15.174 kb) by allelic exchange using the S. aureus vectors of pKOR1 or pIMAY 383
(Table 1). Each of the mutants was grown in RPMI and expression of Isd proteins measured 384
by whole cell dot immunoblotting with anti-IsdC and anti-IsdB sera. The data shows that the 385
proteins were expressed on the surface at the same level as wild-type and were absent from 386
the appropriate mutants (Fig. 4A). Western immunoblotting of fractionated cells revealed the 387
same pattern of expression as in Figure 3D with IsdB and IsdC partially sorted to the wall and 388
with IsdJ and IsdK being found only in the membrane fraction (Fig. 4B). The level of 389
expression of IsdB was the same in the wild-type and in the isdC, isdJ and isdK mutants and 390
was missing in the isdB mutant and the complete isd deletion. Similar patterns were obtained 391
with IsdC, IsdJ and IsdK indicating an absence of polarity (Fig. 4B). 392
In order to investigate expression of IsdJ in other Gram-positive bacterial hosts the 393
isdJ gene was cloned into the nisin-inducible Lactococcus lactis expression vector pNZ8048 394
and into the anhydrotetracycline-inducible staphylococcal expression vector pRMC2 (Table 395
1). Following induction, expression of IsdJ was detected on the surface of L. lactis and on 396
the surface of S. aureus Newman spa by whole cell dot immunoblotting (Fig. 5A). 397
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Interestingly the IsdJ protein was found in the cell wall fractions of L. lactis and S. aureus 398
indicating that it had been sorted in the heterologous host (Fig. 5B). This further suggests that 399
sortase A is defective in S. lugdunensis. 400
Absorption spectra of Isd proteins. The his-tagged recombinant Isd proteins purified from 401
E. coli grown in LB were red in colour indicating that haem was bound. When the absorption 402
spectra of each protein were measured, Soret peaks at 400 nm (IsdB), 406 nm (IsdC), 404 nm 403
(IsdK) and 403 (IsdJ) were detected (Fig. 6A). Both NEAT domains of IsdJ bound haem 404
with each having Soret peaks at 403 nm (data not shown). It was determined from the spectra 405
of pyridine haemochromes that the occupancy level was 10-30%. In contrast, proteins 406
purified from E. coli cells grown in RPMI were not red in colour and had only 1-3% haem 407
occupancy. 408
Haem transfer. A conserved Tyr in β8 of the NEAT motif of haem-binding Isd proteins is 409
essential for ligand binding. A substitution Y467A was introduced into NEAT2 of rIsdB of S. 410
lugdunensis which, when purified from E. coli, resulted in a protein that lacked a Soret peak 411
(Fig. 6B) and did not gain it after incubation with porcine haemin (data not shown). This 412
strongly suggests that IsdB uses the same conserved haem binding mechanism as S. aureus. 413
In S. aureus haem is stripped from haemoglobin bound to NEAT1 and NEAT2 of IsdH 414
and/or NEAT1 of IsdB and transferred (via IsdA) to IsdC and then to the membrane 415
transporters (40, 49, 76). S. lugdunensis lacks an orthologue of IsdH and IsdB NEAT1 is the 416
only putative haemoglobin binding NEAT domain among its Isd proteins. As IsdB and IsdC 417
are the only wall-anchored surface-exposed Isd proteins, we propose that IsdB NEAT1 418
captures haemoglobin and the stripped-out haem is then transferred from NEAT2 to IsdC. To 419
test part of this hypothesis we grew E. coli pQE30isdB in RPMI, purified the apo form of the 420
protein and saturated it with haemin. This was then incubated with GST-tagged IsdC. The 421
latter was separated from the mixture by glutathione sepharose chromatography. The eluted 422
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GST-IsdC had an enhanced Soret peak at 406 nm indicating that the protein had accepted 423
haem transferred from IsdB (Fig. 6C). Equal amounts of the proteins were separated by SDS-424
PAGE and stained either with Coomassie blue or TMBZ peroxidase, a reagent that turns blue 425
as a result of the haemin-generated peroxides (Fig. 6D). 426
Isd proteins binding to haemoglobins. We used solid phase binding assays and surface 427
plasmon resonance to investigate the binding of recombinant Isd proteins to methaemoglobin 428
(MetHb; occurs mostly as an αβ dimer with haem groups that contain oxidated ferric iron that 429
are unable to bind oxygen), human haemoglobin (hHb, an α2β2 tetramer), the haemoglobin-430
haptoglobin complex (Hb-Hpt), human haptoglobin (Hpt) and human myoglobin (hMb). IsdB 431
is the only Isd protein of S. lugdunensis that bound to soluble haemoproteins (Fig. 7AB). 432
Human MetHb, Hb and Hp-Hpt bound to immobilized IsdB dose-dependently and saturably 433
with half maxima at 8 ± 1.6 nM, 12 ± 2 nM and 2 ± 0.5 nM, respectively (Fig. 7AB, data not 434
shown). Binding to haptoglobin or myoglobin was not detected (data not shown). Aromatic 435
residues in the NEAT1 and NEAT2 domains of IsdH (β1-β2 loop) were shown to be critical 436
for binding to α-chains of hHb (36, 54). A mutant of S. lugdunensis recombinant rIsdB 437
(Y189A/H190E/Y191A) was generated and found not to bind to hHb (Fig. 7C) or to Hb-Hpt 438
complexes (data not shown). This demonstrates a conserved mechanism of binding of 439
haemoglobin by IsdB of S. lugdunensis and IsdH of S. aureus. 440
Solid phase assays measured specific binding of rIsdB to hHb, MetHb and Hb-Hpt 441
with half-maximal binding concentrations in the low nanomolar range. To measure the 442
affinities more accurately surface plasmon resonance binding analysis was performed. rIsdB 443
was immobilized onto a dextran chip and the hHb was passed over the surface in 444
concentrations ranging from 9.766 nM to 6 μM (Fig. 8A). Human haemoglobin immediately 445
associated to recombinant IsdB reaching an equilibrium response for every concentration 446
(Fig. 8A). The steady-state responses against concentrations were fitted with a two-binding-447
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site affinity model, as there are two predicted binding sites (2 α-chains) on tetrameric Hb. An 448
accurate fitting was judged from the low χ2 value of 9 giving an affinity KD of 23.13 ± 4 nM. 449
A similar affinity was obtained for human MetHb (data not shown). 450
As demonstrated by Pishchany et al. S. aureus IsdB has a higher affinity for hHb than 451
mouse haemoglobin (mHb) (55). We measured S. lugdunensis IsdB binding by mHb using 452
SPR. Purified mHb was passed over the surface in concentrations ranging from 156.25 nM to 453
40 μM. In contrast to hHb, the mouse Hb binding levels were too low to be evaluated for an 454
accurate affinity KD as the response levels did not reach the required steady state equilibrium 455
(Fig. 8B). This demonstrated a low affinity of S. lugdunensis IsdB for mHb with a KD 456
predicted at approximately 177 μM. 457
Growth of iron-starved S. lugdunensis is enhanced by haemoglobin. To test the 458
hypothesis that the Isd system of S. lugdunensis is important for iron acquisition from 459
haemoglobin and that IsdB in particular has a crucial role, bacteria were cultured in a 460
modified RPMI medium that was pre-treated with Chelex 100 and contained a divalent metal 461
chelator. S. lugdunensis growth was significantly impaired when cultured in iron depleted 462
medium by comparison with rich medium (TSB) (data not shown). Addition of haemoglobin 463
resulted in significant growth enhancement starting at 16 h post inoculation and continuing 464
until 48 h post inoculation. Wild-type S. lugdunensis grew to an OD600 nm of 0.86 in 48 h in 465
modified RPMI containing human haemoglobin while in the absence of hHb the OD600 nm 466
was less than 0.4 (Fig. 9AE). Mutants of S. lugdunensis lacking IsdB or the entire Isd locus 467
grew to an OD600 nm of ca 0.6 in the presence of hHb and to less than 0.4 in its absence (Fig. 468
9BCD). This demonstrates that growth of S. lugdunensis is enhanced by hHb in an IsdB-469
dependent fashion. Nevertheless the presence of hHb did support growth to a limited extent, 470
perhaps due to similar unspecific binding of bacteria to haemoglobin that was previously 471
observed for S. aureus (66). To confirm the specificity of the role of hHb and IsdB, wild-type 472
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S. lugdunensis was grown in modified RPMI supplemented with mouse Hb which in in vitro 473
studies was showed to bind very weakly to IsdB (Fig. 8B). Wild-type S. lugdunensis grew 474
better when supplemented with human than mouse Hb (Fig. 9EF), demonstrating the IsdB-475
dependent preference towards the human haemoprotein. However, addition of mouse Hb 476
resulted in enhanced growth compared to growth in the medium without added Hb. This 477
growth enhancement initiated by mHb resulted in similar optical densities to those of isdB 478
mutant supplemented with hHb, suggesting it is caused by the same IsdB-independent 479
mechanism (Fig. 9EF). This indicates a role for IsdB in binding haemoglobin to support 480
bacterial growth when iron is strictly limited. 481
Binding of IsdJ to fibrinogen and cytokeratin 10. The IsdA protein of S. aureus can bind 482
to several ligands other than haem, including fibrinogen, loricrin and keratin 10, the last two 483
of which are found only in the cornified envelope of desquamated epithelial cells (11, 15). 484
Since significant similarities exist between IsdA and IsdJ (Fig. 1C) of S. lugdunensis, rIsdJ 485
was tested for its ability to bind fibrinogen, keratin 10 and loricrin in solid phase assays. rIsdJ 486
bound to each ligand with half maxima of approximately 200 nM for loricrin, 250 nM for 487
keratin 10 and 350 nM for fibrinogen, 50-100 fold lower than the affinity of rIsdB for hHb 488
(Fig. 10A). Ligand affinity blotting was performed with full length rIsdJ, and the N- and C-489
terminal truncates, probing with soluble fibrinogen (Fig. 10B). As with IsdA, the N-terminal 490
NEAT-containing segment supported fibrinogen binding. 491
In order to determine if IsdJ could support bacterial adherence to immobilized 492
fibrinogen, wild-type S. lugdunensis was compared to the fbl and isdJ mutants grown in 493
RPMI. There was no difference in binding by S. lugdunensis and the isogenic isdJ mutant. 494
Moreover the fbl mutant could not bind to fibrinogen at all indicating that IsdJ does not 495
contribute to bacterial adherence (Fig. S5AB). 496
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To test if heterologous expression of IsdJ in S. aureus or L. lactis, where the protein is 497
exposed on the cell surface, could support adhesion to immobilized ligands, the IsdJ protein 498
was expressed in S. aureus clfA clfB isdA, a mutant that is defective in all fibrinogen and 499
keratin 10 adhesins and in L. lactis which cannot bind these ligands. In neither case could 500
adhesion be detected (data not shown). Either the affinity of binding is too weak to support 501
adhesion or the protein is perhaps partially buried in the cell wall and not fully accessible to 502
the ligand. 503
IsdJ promotes resistance to killing by linoleic acid. The multifunctional IsdA protein of S. 504
aureus promotes resistance to killing by bactericidal lipids that occur in sebum due to the C-505
terminal region that confers decreased hydrophobicity on the bacterial cell (14). Since the C-506
terminus of IsdJ has similarity to IsdA it was decided to investigate if the S. lugdunensis 507
protein conferred the same property. Mutants of S. aureus and S. lugdunensis that were 508
defective in IsdA and IsdJ, respectively, were compared with the parental strains for survival 509
in a buffer containing linoleic acid for 30 min. The isdA mutant survived worse than S. 510
aureus wild-type, while there was a trend towards the same effect that was not statistically 511
significant for S. lugdunensis isdJ (Fig. 11A). To test if less effective protection by IsdJ could 512
be caused by its cellular localization we expressed IsdJ on the surface of S. aureus Newman 513
isdA to see if the cell wall anchored IsdJ could complement the isdA mutation in the killing 514
assay. IsdA and IsdJ were expressed from the anhydrotetracycline inducible pRMC2 vector 515
in S. aureus Newman isdA and bacterial survival was measured after 30 min. Expression of 516
IsdA and IsdJ both resulted in increased protection compared to the isdA mutant or the 517
mutant carrying the empty pRMC2 vector reaching survival levels of Newman wild-type 518
(Fig. 11B). This suggested that the protein could protect bacterium from killing by the fatty 519
acid when exposed on the bacterial surface. Similarly expression of IsdA or IsdJ in L. lactis 520
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promoted enhanced survival (Fig. 11C). Thus IsdJ appears to confer resistance to linoleic 521
acid and may help promote survival of S. lugdunensis in its natural habitat on the human skin. 522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
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DISCUSSION 540
Uncharacteristically for CoNS S. lugdunensis is capable of causing serious systemic diseases 541
in healthy adults. To do so it needs to overcome several challenges, among which is nutrient 542
accessibility. Iron is a critical component for bacterial growth but is sequestered in humans 543
due to its insolubility and as prevention from oxidative damage with approximately 90% 544
bound to haem, mostly within haemoglobin (52, 53). Additional iron-sequestration 545
mechanisms are employed by the human host during infection to counteract the ability of 546
pathogens to obtain iron (6, 29, 31, 39). The inevitable competition with the host has led to 547
both Gram-positive and Gram-negative bacterial pathogens developing several distinct 548
mechanisms for accessing iron from haem (21, 43, 50, 56, 65, 74). 549
Here we have characterized four proteins from an iron acquisition system of the 550
emerging pathogen S. lugdunensis being to date the sole example of a CoNS utilizing the Isd 551
system for iron acquisition. The Isd proteins share significant structural and functional 552
homology with the Isd receptors of S. aureus. Both systems are expressed under low-iron 553
conditions. The S. lugdunensis proteins IsdB, IsdC, IsdJ and IsdK were induced in cultures 554
grown in iron deficient medium. This is consistent with report by Haley et al. which studied 555
the role of S. lugdunensis haem monooxygenase (IsdG) (28). These findings suggest that 556
transcription of the isd locus in S. lugdunensis, like S. aureus, is regulated by the iron-557
dependent repressor Fur. Indeed Fur boxes were identified upstream from the start codons of 558
isdB, isdC and isdJ genes (Fig. 1A.). Interestingly, the isd locus is duplicated in S. 559
lugdunensis HKU09-01 which may increase the levels of transcription of the isd genes in that 560
strain (30). 561
In both systems the IsdB and IsdC orthologues are localized in the cell wall. IsdB is 562
likely to be processed by sortase A (SrtA), while IsdC linkage to peptidoglycan is most 563
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probably catalyzed by sortase B (SrtB). We observed that the S. lugdunensis IsdB and IsdC 564
proteins seem only to be partially wall-anchored, as a portion of those proteins could be 565
found in the membrane fraction. IsdK and IsdJ were detected only in the membrane fraction 566
and were either hardly detectable on the surface of the bacterium (IsdK) or could not be 567
detected at all (IsdJ). IsdJ and IsdK contain C-terminal sorting signals composed of sortase 568
recognition motifs (LPXTG or NKQPN, respectively) followed by a hydrophobic domain and 569
a tail of positively charged residues and were therefore expected to be anchored to the cell 570
wall and exposed on the cell surface. Despite SrtA and SrtB being capable of anchoring IsdB 571
and IsdC, it is unclear why IsdJ and IsdK remain in the membrane. IsdJ was sorted to cell 572
wall of the heterologous hosts S. aureus and L. lactis indicating that differences in anchoring 573
surface proteins to the cell wall must exist in S. lugdunensis. 574
The IsdJ and IsdK proteins were detected in S. lugdunensis culture supernatants 575
suggesting an additional role in iron acquisition. Haem acquisition by secreted proteins 576
containing NEAT-like domains has been well characterized in Bacillus anthracis (20, 32, 577
62). The bacterium secretes IsdX1 and IsdX2 haemophores (acquiring haem from host 578
haemoglobin), which deliver haem to IsdC exposed on bacterial surface, allowing transfer of 579
the iron-porphyrin into the cytoplasm. IsdX1 contains a single NEAT domain, whereas IsdX2 580
has five. NEAT1, 3, 4, and 5 bind haem while all motifs associate with haemoglobin. NEAT1 581
and NEAT5 extract haem which is then transferred by NEAT1, NEAT3 and NEAT4 to the 582
cell wall-anchored IsdC protein. S. lugdunensis IsdJ and IsdK cannot bind Hb but perhaps 583
could acquire haem directly from the extracellular environment during infection or from IsdB 584
which occurs abundantly in the external milieu. Perhaps two parallel haemoglobin-haem 585
acquisition pathways exist in S. lugdunensis, one utilizing the cell wall-anchored IsdB and 586
IsdC and the second employing secreted proteins which acquire haem from the environment 587
(IsdB, IsdJ and IsdK) and pass it onto IsdC before transferring the porphirin ring into the 588
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cytoplasm. However more studies are required to reveal the role of IsdJ and IsdK in the haem 589
acquisition mechanism. 590
The specific binding of haemoproteins by the Isd system of S. lugdunensis is 591
restricted to a single IsdB receptor which contrasts with S. aureus where IsdH and IsdB 592
express three haemoglobin binding NEAT domains (19, 66). In S. lugdunensis the IsdB 593
protein uses its NEAT1 domain to bind to haemoglobin and haemoglobin-haptoglobin 594
complexes but has no affinity for myoglobin or haptoglobin. Even though haemoglobin 595
binding has been reported for a number of bacterial proteins containing NEAT-like domains, 596
the characteristic aromatic loop co-ordinating haemoglobin is unique to S. lugdunensis and S. 597
aureus. Furthermore both species show selectivity in the source of haemoglobin. Here we 598
show that S. lugdunensis IsdB binds strongly to human Hb (KD of 23 nM) and barely 599
associates with mouse Hb. This correlates with the bacterial preference in utilizing the human 600
Hb over mouse Hb to support growth in iron restricted media. Therefore it could be difficult 601
to evaluate the importance of the Isd system in mouse models of colonization or infection. 602
Since the nutrient availability may have a critical effect on bacterial pathogenesis the 603
humanized animal model (mice expressing human haemoglobin) should be strongly 604
considered to study invasiveness and virulence of S. lugdunensis (55). A preference for hHb 605
over mHb is characteristic of bacteria associated with humans, like S. aureus and S. 606
lugdunensis but also S. simulans and Corynebacterium diphtheriae, whereas environmental 607
bacteria that infect numerous hosts like Acinetobacter baumannii, Pseudomonas aeruginosa, 608
B. anthracis and B. cereus grow at comparable levels on hHb and mHb (55, 56). 609
The S. lugdunensis IsdB protein was shown to be capable of binding haem by a 610
conserved Tyr (IsdB Y467) probably in a pentacoordinate complex (a hydrogen bond is 611
formed with the phenolate of another conserved Tyr in β8). This interaction is absolutely 612
conserved among haem binding NEAT domains (25). The S. aureus haem uptake pathway 613
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IsdH-IsdB-(IsdA)-IsdC-IsdEF transfers haem from the surface exposed haemoprotein 614
receptors (IsdH and IsdB) through the cell wall-bound IsdA or directly onto the IsdC (buried 615
in the cell wall) which passes it onto the membrane transporter (IsdEF) (40, 49, 76). The S. 616
lugdunensis Isd system may present a reduced set of receptors, considering that IsdJ and IsdK 617
may be redundant due to their cellular localization. We observed direct transfer of haemin 618
from recombinant holo-IsdB to apo-IsdC suggesting that these two abundant cell wall 619
receptors may be sufficient for the efficient transfer of haem onto the putative membrane 620
transporter system encoded by isdE and isdF. The likely reduction in the number of Hb and 621
haem receptors could be the consequence of the lower haemolytic capacity of S. lugdunensis, 622
which only expresses the delta toxin-like haemolysin called SLUSH (22, 68). In contrast S. 623
aureus expresses a range of haemolysins (α, β, γ ,δ) which lyse the erythrocytes and provide a 624
rapid access to a pool of haemoglobin. 625
Several different ligands were identified for IsdJ. Recombinant IsdJ binds human 626
loricrin and cytokeratin 10 (implicated in adherence to squamous epithelial cells and nasal 627
carriage), as well as human plasma fibrinogen. Each ligand was previously shown to interact 628
with IsdA and is therefore most probably recognized by conserved regions (11, 15). The 629
recombinant N-terminal domain of IsdJ containing NEAT1 was shown to bind to fibrinogen. 630
However, IsdJ expression did not promote bacterial adherence to immobilized ligands. Iron 631
starved S. lugdunensis adhered strongly to fibrinogen but this was not due to IsdJ, as there 632
was no difference in adherence between the wild-type strain and the isdJ deficient mutant. 633
Also the iron starved S. lugdunensis fbl mutant did not adhere to fibrinogen suggesting that 634
Fbl is the only fibrinogen receptor on S. lugdunensis. S. aureus and L. lactis strains 635
expressing IsdJ on their surfaces also failed to adhere to cytokeratin, fibrinogen and loricrin, 636
which demonstrates that the protein is not sufficient to promote bacterial adherence to those 637
ligands. IsdJ expression could confer some protection to S. lugdunensis from killing by 638
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linoleic acid, albeit not as efficiently as IsdA in S. aureus. However, IsdJ protected L. lactis 639
and S. aureus as well as IsdA. Either IsdJ is expressed at a higher level in the surrogate hosts 640
than in S. lugdunensis, or its location on the cell surface following sorting is responsible for 641
the greater efficacy. 642
To summarize, S. lugdunensis, like S. aureus utilizes the Isd system to acquire haem-643
iron. IsdB is an abundant cell wall receptor which preferentially recognizes human Hb. IsdC 644
is a haem receptor located in the cell wall which can accept haem from IsdB. Haem bound to 645
IsdC could then be transferred onto the putative membrane transporters IsdEF. IsdJ and IsdK 646
are haem binding proteins that occur in the cytoplasmic membrane and external milieu 647
despite their C-terminal motifs for anchorage to the cell wall peptidoglycan. Finally, IsdJ is 648
multifunctional as it recognizes several host ligands and can confer resistance to skin fatty 649
acids. 650
651
ACKNOWLEDGEMENTS 652
We thank Gleb Pishchany and Eric Skaar for providing human and mouse haemoglobins. We 653
are also grateful to Michelle Mulcahy for providing purified human loricrin and cytokeratin 654
10. We wish to acknowledge funding from the Health Research Board (RP/2008/20), from 655
IRCSET for an Embark Scholarship (RS2000192), from Science Foundation Ireland for a 656
Programme Investigator Grant (08/IN.1/B1854) and from Fondazione CARIPLO for a grant 657
(Vaccines 2009-3546). 658
659
660
661
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Figure legends. 902
Fig. 1. Schematic representations of the isd locus and Isd proteins of S. lugdunensis with their 903
comparison to conserved Isd proteins of S. aureus. (A) Comparison of the isd loci of S. 904
aureus and S. lugdunensis. Black lines connect orthologous genes. NEAT domains are 905
indicated by grey boxes. The isdH and isdI genes of S. aureus are located outside the locus as 906
indicated by circles. (B) Schematic representation of the putative cell wall anchored proteins 907
IsdB, IsdC, IsdJ and IsdK of S. lugdunensis and IsdA, IsdB, IsdC and IsdH of S. aureus. The 908
positions of the NEAT domains were determined from their predicted structures based on 909
homology to available crystal structures of Isd proteins of S. aureus. Signal sequences (S) and 910
putative cell wall anchoring motifs (W T) are indicated. (C) Scheme representing 911
conservation within specified regions of S. aureus IsdA and S. lugdunensis IsdJ. (D) Table 912
comparison of the amino acid conservation between NEAT domains of (vertical) S. aureus 913
and (horizontal) S. lugdunensis Isd proteins (as indicated). Fields highlighted in grey 914
represent Hb-binding NEAT domains while white fields show haem-binding NEAT domains. 915
(-) Represents homology lower than 25% of identity between the domain’s amino acid 916
chains. Most significant levels of homology are underlined. 917
918
Fig. 2. Multiple sequence alignments of the NEAT domains of S. lugdunensis and S. aureus. 919
(A) Alignment of the putative haem binding NEAT domains of S. lugdunensis (IsdB NEAT2, 920
IsdC, IsdJ NEAT1, IsdJ NEAT2 and IsdK) and their structural predictions based on 921
homology to available crystal structures of S. aureus NEAT domains of IsdA, IsdC, and 922
IsdH. Underlined residues represent fragments predicted to form β-strands as indicated above 923
the alignment. (B) Multiple sequence alignment of the putative haemoglobin binding NEAT 924
domain S. lugdunensis IsdB NEAT1 with haemoglobin binding NEAT domains of S. aureus 925
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(IsdB NEAT1, IsdH NEAT1 and NEAT2). Residues predicted to form β-strands are 926
underlined. Conserved residues are indicated by asterisks (*), ‘:’ marks at least two identical 927
residues across the alignment. Residues predicted to be involved in haem capture are 928
displayed in bold. 929
930
Fig. 3. Expression of the IsdB, IsdC, IsdJ and IsdK proteins by S. lugdunensis under iron-931
restricted and iron-rich conditions. (ABC) Whole cell dot immunoblot to detect surface 932
displayed of Isd proteins of S. lugdunensis grown in either RPMI, RPMI supplemented with 933
various concentrations of FeCl3 (as indicated) or TSB. (D) Western immunoblotting to detect 934
expression of Isd proteins in cellular fractions of S. lugdunensis N920143 grown in either 935
TSB or RPMI. Lanes marked as CW contain solubilised cell wall fraction. Lanes marked 936
with M contain membrane fractions. Dot and Western immunoblotting was performed using 937
rabbit serum recognizing (AD) IsdB (anti-IsdB), (BD) IsdC (anti-IsdC), (D) IsdJ (anti-IsdJ) 938
and (CD) IsdK (anti-IsdK). Bound IgG was detected by HRP-conjugated protein A. 939
940
Fig. 4. Validation of protein expression by S. lugdunensis isd deletion mutants. (A) 941
Expression of surface-exposed IsdB and IsdC by S. lugdunensis deletion mutants 942
demonstrated by whole-cell dot immunobloting of RPMI grown cells. (B) Western 943
immunoblot to detect expression of Isd proteins in the cell wall (CW) and the membrane (M) 944
fractions of S. lugdunensis N920143 grown in RPMI. Immunobloting was performed using 945
rabbit anti-Isd IgG (as indicated) followed by HRP-conjugated protein A. 946
947
Fig. 5. Cellular localization of IsdJ expressed in S. aureus and L. lactis from inducible 948
plasmids pRMC2isdJ and pNZ8048isdJ, respectively. (A) Whole-cell immunoblot to detect 949
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expression of surface-exposed IsdJ by S. aureus Newman spa and L. lactis NZ9000 and S. 950
lugdunensis. (B) Western immunoblot identifying cellular localization of IsdJ expressed by S. 951
aureus and L. lactis. Cell wall (CW) and membrane (M) fractions were separated by SDS-952
PAGE. Immunoblotting was performed using rabbit anti-IsdJ serum followed by HRP-953
conjugated goat anti-rabbit IgG. Experiments were performed three times with similar results. 954
955
Fig. 6. The absorption spectra of Isd proteins and spectral shifts following co-incubation of 956
haemin-saturated IsdB with GST - IsdC. (A) Recombinant His - tagged IsdB, IsdC, IsdJ, and 957
IsdK proteins (at 10 μM) purified from E. coli grown in LB broth. (B) Recombinant IsdB and 958
the Y467A mutant purified from E. coli grown in LB broth. (C) Visible spectra of His - 959
tagged IsdB and GST - tagged IsdC (5 μM) purified from E. coli grown in RPMI and their 960
spectral shifts following IsdB saturation with haemin and haemin transfer to GST - IsdC. (D) 961
Coomassie and TMBZ stained SDS-PAGE gels with equal amounts of following proteins: 962
apo - IsdB, IsdB saturated with haemin, IsdB post haemin transfer to IsdC, apo - IsdC and 963
IsdC upon haemin transfer from IsdB. These are representatives of three independent 964
experiments. 965
966
Fig. 7. IsdB binding to human haemoglobin (Hb) and haemoglobin-haptoglobin (Hb–Hpt) 967
complexes. 96-well plates were coated with (A and B) recombinant IsdB, IsdC, IsdJ and IsdK 968
or (C) recombinant IsdB wild-type and IsdB Y189A/H190E/Y191A following incubation 969
with various concentrations of (A and C) Hb, (B) Hb-Hpt complexes. Bound proteins were 970
detected with polyclonal rabbit anti - human haemoglobin serum followed by HRP-971
conjugated goat anti - rabbit IgG. Values represent the mean of triplicate wells. Binding 972
assays was preformed three times with similar results. 973
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Fig. 8. Surface Plasmon Resonance (SPR) binding analysis of (A) human Hb (hHb) and (B) 974
mouse Hb (mHb) to recombinant IsdB. Data were generated using Biacore X-100. IsdB was 975
immobilized on the surface of a CM5 sensor chip, followed by passing increasing 976
concentrations of hHb or mHb over the surface. Injections began at 0 s and ended at 180 s. 977
The data are presented as real-time graphs of (left) response units (RU) against time and 978
(right) as SPR signals against ligands concentration in the late dissociation phase. 979
980
Fig. 9. Growth of S. lugdunensis N920143 strains in iron restrictive medium supplemented 981
with haemoglobins. (ABCD) Growth of wild-type S. lugdunensis, ΔisdB and Δisd locus 982
mutants in modified RPMI medium either supplemented with human Hb (grey columns) or 983
without added Hb (white columns). Changes in optical densities at 600 nm (OD600) were 984
measured over 48 h. Values are means representing three independent experiments. (D) 985
Represent a mean of yield of growth (after 48h) of three independent experiments. (EF) 986
Growth of S. lugdunensis wild-type in iron depleted medium supplemented with human Hb 987
(dark grey), mouse Hb (light grey) or grown without added Hb (white). Values are means 988
representing three independent experiments. (F) Represent a mean of yield of growth (after 989
48h) of three independent experiments. Error bars represent standard deviation of the OD600. 990
Statistically significant differences are indicated (Student’s two tailed t test, * P<0.05). 991
992
Fig. 10. Recombinant IsdJ binding to loricrin, cytokeratin 10 and fibrinogen. (A) 96-well 993
plates were coated with GST-loricrin, GST-cytokeratin 10, GST or fibrinogen and incubated 994
with increasing concentrations of recombinant IsdJ (45-610). Bound IsdJ was detected with 995
rabbit anti-IsdJ serum followed by HRP-conjugated protein A. Results are the means of 996
triplicate wells. (B) Fibrinogen (Fg) affinity immunoblot to detect Fg - binding domain of 997
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IsdJ. Lanes contain equal amounts of recombinant IsdJ variants, as indicated. Bound Fg was 998
detected with HRP-conjugated monoclonal anti-human Fg IgG. These are representatives of 999
three independent experiments. 1000
1001
Fig. 11. Bacterial killing by human skin fatty acid – linoleic acid. (A) S. lugdunensis 1002
N920143 wild type and the ΔisdJ mutant, S. aureus Newman wt and isdA mutant were grown 1003
in RPMI overnight. (B) S. aureus Newman and Newman isdA were grown in RPMI 1004
overnight. Newman isdA mutant carrying pRMC2, pRMC2isdA and pRMC2isdJ were grown 1005
to exponential phase and induced with anhydrotetracycline at 1 μg ml-1 overnight. (C) L. 1006
lactis NZ9000 (pNZ8048), (pNZ8048isdA) and (pNZ8048isdJ) were grown in GM17 to mid-1007
exponential phase and induced for 4 h with nisin. (A-C) Bacterial suspensions at 1 x 108 cfu 1008
ml-1 in dH2O were treated with 10 μg ml-1 of linoleic acid for 30 minutes or as indicated. 1009
Survival was measured by viable counting. Values are the means of three independent 1010
experiments. 1011
1012
1013
1014
1015
1016
1017
1018
1019
1020
1021
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Table 1. Strains and plasmids used in the study. 1022
Relevant characteristics Reference
Strains
Staphylococcus lugdunensis
S. lugdunensis N920143 Human breast abscess isolate. (30)
S. lugdunensis N910319 Human endocarditis isolate. Vandenesch, F.
(unpublished).
S. lugdunensis N910320 Human endocarditis isolate.
S. lugdunensis N930432 Human endocarditis isolate.
S. lugdunensis ΔisdB isdB null mutation in N920143.
This study.
S. lugdunensis ΔisdC isdC null mutation in N920143.
S. lugdunensis ΔisdJ isdJ null mutation in N920143.
S. lugdunensis ΔisdK isdK null mutation in N920143.
S. lugdunensis Δisd locus isd locus null mutation in N920143.
S. lugdunensis Δfbl fbl null mutation in N920143.
Staphylococcus aureus
S. aureus Newman Widely used laboratory strain.
S. aureus Newman isdA Human clinical isolate, Newman isdA::Tn917 ErmR. (14)
S. aureus Newman isdA
clfA clfB Frameshift mutation in clfA, clfB::TetR, isdA. (45)
Lactococcus lactis
L. lactis NZ9000 L. lactis subsp. Cremoris MG1363 carrying nisin
resistance cassette.
(37)
Escherichia coli
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E. coli XL-1Blue E. coli cloning host, TetR. Stratagene
E. coli DC10B A dcm deficient E. coli DH10B strain. (46)
E. coli TOPP10 E. coli cloning and protein purification host. Invitrogen
Plasmids
pQE30 E. coli cloning and expression vector, AmpR. Stratagene
pQE30isdB pQE30 encoding residues 45 to 655 of IsdB.
This study.
pQE30isdC pQE30 encoding residues 30 to 190 of IsdC.
pQE30isdJ (45-610) pQE30 encoding residues 45 to 610 of IsdJ.
pQE30isdJ (45-295) pQE30 encoding residues 45 to 295 of IsdJ.
pQE30isdJ (290-610) pQE30 encoding residues 290 to 610 of IsdJ.
pQE30isdK pQE30 encoding residues 35 to 426 of IsdK.
pGEX-4t-2 E. coli cloning and expression vector, AmpR. Amersham
pGEX-4t-2isdC pGEX4t-2 encoding residues 30-190 of IsdC. This study.
pRMC2 ChmR vector allowing aTc - inducible expression. (16)
pRMC2isdA ChmR, plasmid for inducible expression of IsdA. This study.
pRMC2isdJ ChmR, plasmid for inducible expression of IsdJ.
pNZ8048
L. lactis shuttle vector containing PnisA promoter and
start codon in NcoI site, ChmR . Allowing nisin-
inducible expression of insert.
(37)
pNZ8048isdJ pNZ8048 encoding isdJ, for controlled expression of
IsdJ in L. lactis. This study.
pNZ8048isdA
pNZ8048 encoding isdA insert, for controlled
expression of IsdJ in L. lactis.
pKOR1 E. coli/ Staphylococcus shuttle vector allowing allelic
replacement in staphylococci. (5)
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pKOR1ΔisdC pKORI plasmid for in frame deletion of isdC. This study.
pKOR1ΔisdK pKORI plasmid for in frame deletion of isdK.
pIMAY E. coli/ Staphylococcus shuttle vector allowing allelic
replacement in staphylococci. (46)
pIMAYΔisdJ pIMAY plasmid for in frame deletion of isdJ. This study.
pIMAYΔisdB pIMAY plasmid for in frame deletion of isdB.
pIMAYΔisd pIMAY plasmid for in frame deletion of isd locus.
1023
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