SS Research_Project16_dunnel7

An Investigation into the Biological
Function of Subdomain N1 of the
Staphylococcus aureus protein
Clumping Factor A
BA (Mod) Microbiology Thesis 2016
Leah Dunne
Supervisor: Dr. Joan Geoghegan

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DECLARATION
I, Leah Dunne, certify that the experimentation recorded herein represents my own work.
I further certify that:
1. I have completed the Online Tutorial on avoiding plagiarism ‘Ready, Steady, Write’
located at http://tcd-ie.libguided.com/plagiarism/ready-steady-write
2. I have read and understand the plagiarism provisions in the General Regulations of
the University Calendar for the current year, found at: http://www.tcd.ie/calendar
3. this thesis represents my own unaided work.
Signed ____________
Leah Dunne
Date ____________

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ABSTRACT
Clumping factor A is a member of the microbial surface component recognising adhesive
matrix molecules (MSCRAMM) family, and an important fibrinogen-binding protein and
virulence factor of Staphylococcus aureus. The A region of this protein is composed of three
separately folded subdomains, N1, N2 and N3. Subdomains N2 and N3 are composed of
immunoglobulin G-like folds and comprise the minimum ligand binding subdomains. A
small linker region of 10 residues (211-220) in the N1 subdomain is required for the surface
expression of clumping factor A. Other than this nothing is known about the function of this
subdomain. The creation of a Staphylococcus aureus mutant expressing a truncated form of
the clumping factor A protein, lacking the remainder of the N1 subdomain (residues 40-210)
is demonstrated here. This study has shown that the N1 subdomain is not required for the
binding of this protein to immobilised fibrinogen. This study also examined the role of the
N1 subdomain in the activation of platelet aggregation, however no conclusions could be
drawn. Clumping factor A was shown to protect the bacterial surface from the deposition of
opsonins in the presence of fibrinogen and the N1 subdomain was required for this
protection. It is therefore thought that this domain could be involved in the binding to
soluble fibrinogen, creating a protective capsule-like shield around the bacterium, inhibiting
the deposition of host proteins.

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INTRODUCTION
Staphylococcus aureus is a Gram-positive commensal bacterium that asymptomatically
colonises around 20% of the population (31). This bacterium is also an important pathogen,
capable of causing opportunistic infections. These infections can be both superficial such as
skin and soft tissue infections or more serious invasive infections such as sepsis or
endocarditis (21). Antibiotic treatment of S. aureus infections is becoming increasingly
difficult due to the emergence of antibiotic resistance strains such as methicillin resistant
Staphylococcus aureus (MRSA) (17). Understanding how this organism is successful at
promoting infection and avoiding host immune defences is important for the advancement of
treatment and prevention approaches against S. aureus infections.
The success of this organism as a pathogen can be in part attributed to its expression of a
broad range of virulence factors, such as its repertoire of cell wall anchored proteins (5).
The largest class of proteins found on the surface of S. aureus are the microbial surface
components recognising adhesive matrix molecules (MSCRAMM) family, defined by their
similarities in structure and common ligand binding mechanism. Proteins of this family are
comprised of an N terminal ligand-binding A domain containing two immunoglobulin G
(IgG)-like folded subdomains, N2 and N3, and the N terminal N1 subdomain. The A region
is projected from the cell wall by an unfolded flexible R region. MSCRAMM proteins have
a crucial role in the colonisation by S. aureus and in the success of S. aureus infections (5).
Clumping factor A (ClfA) is a 130 kDa MSCRAMM protein of S. aureus. It is an
important fibrinogen (Fg) binding protein and potent virulence factor (10, 6). The gene
encoding this surface protein is carried by nearly all S. aureus clinical strains (6). ClfA
protein structure is composed of an N-terminal signal sequence (S) required for secretion
through the Sec secretion apparatus (Fig. 1). Features at the C-terminal domain are required
for cell wall anchoring. A wall spanning domain (W) and sorting signal (SS) containing an
LPXTG motif are located at the C-terminus. The conserved LPXTG motif within the sorting
signal is recognised by sortase enzymes, involved in anchoring the protein to the
peptidoglycan cell wall (2). The R region is a flexible region composed of serine-aspartate
dipeptide repeats, required for the projection of the remainder of the protein away from the
cell surface (13). The ligand binding function of this protein have been localised to the A
region. This region is comprised of three separately folded subdomains, N1, N2 and N3
(12). The N2 and N3 subdomains are composed of IgG-like folds and comprise the

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minimum ligand binding subdomains (2). A small linker region of ten residues (211-220) in
the N1 subdomain has been shown to be required for export and cell wall localisation of
ClfA (24). However, other than this, the structure or function of the N1 subdomain of ClfA
has not been described.
FIG. 1. Schematic representation of the domain organisation of ClfA. The N terminal
signal sequence (S) is followed by the A region, composed of the three separately folded
subdomains N1, N2 and N3. A small linker region in the N1 subdomain is required for
export and cell wall localisation of ClfA. The A region is followed by the flexible region
composed of serine – aspartate (SD) dipeptide repeats, a cell wall spanning domain (W) and
a sorting signal (SS).
Fg is a 340 kDa glycoprotein, composed of six chains, two Aα, two Bβ and two γ chains,
coming together to form a ‘dimer of trimers’ (2). Fg is abundantly found in plasma and is an
important host protein involved in coagulation. S. aureus utilises this protein in multiple
ways for its own advantage. ClfA binds the extreme C-terminus of the Fg γ-chain,
promoting bacterial clumping and adherence. ClfA promotes adhesion of S. aureus to
plasma coated surfaces through its binding to immobilised Fg (5). Binding of soluble Fg
promotes agglutination of bacteria with increased resistance to antibiotics (1). ClfA is also
capable of activating and causing aggregation of platelets which can lead to the development
of cardiovascular infections such as endocarditis (4).
The success of S. aureus as a pathogen can also be attributed to its vast repertoire of
immune evasion mechanisms. ClfA is the most important surface virulence factor
promoting S. aureus sepsis, through its agglutination in the presence of Fg or fibrin (22), it is
a proven virulence factor in a rat model of infective endocarditis and in mouse model of
septic arthritis (6). The importance of ClfA to S. aureus virulence in animal models may be
due to its ability to prevent phagocytosis of bacteria by human polymorphonuclear
leukocytes (14). The prevention of phagocytosis by ClfA occurs in both Fg-dependent and
independent mechanisms (14). The Fg independent mechanism is due to the ability of ClfA

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to bind host complement regulation protein, factor I. Binding of complement factor I to the
bacterial cell promotes cleavage of complement protein C3b to inactive C3b (iC3b), an
inactive form of the protein, unable to function in complement functions other than as an
opsonin (10, 11). The Fg-dependent mechanism is not fully understood. However, it is
hypothesised that Fg may form a protective shield around the bacteria, protecting it from
recognition by receptors on host phagocytic cells (14). A similar mechanism was described
for the S. aureus secreted protein Extracellular fibrinogen binding protein (Efb). This
protein protects the bacterium from innate immune defences by creating a ‘capsule-like
shield’ around the bacterium (18).
Due to the lack of knowledge about the N1 subdomain of ClfA and other MSCRAMM
proteins, the aim of this project was to determine whether the N1 subdomain of ClfA is
required for any of the known biological functions of ClfA. Attempts at creating a mutant,
with a truncated form of ClfA lacking the N1 subdomain, in the past had been futile due to
the need for the 10 residues of the N1 subdomain that are required for the export of ClfA.
This project demonstrates the creation of a ClfAΔN1 mutant that retains these important
residues and examines the effect of this deletion in some of the biological functions of ClfA.
These functions include its binding to immobilised and soluble Fg and its ability to activate
platelets. This study also aimed to investigate further the antiphagocytic properties of ClfA
in the presence of Fg, to determine whether the inhibition of phagocytosis occurs by
reducing deposition of opsonins on the bacterial cell surface.

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MATERIALS AND METHODS
Bacterial strains and growth conditions. Bacterial strains used in this study are listed in
Table 1. Staphylococcus aureus strains were grown on tryptic soy agar (TSA, Becton
Dickson, Franklin lanes, NJ) at 37oC, or in tryptic soy broth (TSB, Becton Dickson) or brain
heart infusion broth (BHI, Oxoid, Hampshire, UK) at 37oC with shaking. Escherichia coli
strains were grown in Luria broth at 37oC with shaking. Chloramphenicol (Cm, 10μg/ml)
and anhydrotetracycline (ATc, 1μg/ml) were added to the media as required. All reagents
unless otherwise stated, were obtained from Sigma-Aldrich (Wicklow, Ireland).
TABLE 1 Bacterial strains and plasmids used in this study
Reference
Strain or plasmid Description or source
S. aureus
Newman spa Derivative of S. aureus strain Newman (14)
deficient in protein A, spa::Kanr
Newman spa clfA Derivative of Newman deficient in protein A (14)
and clumping factor A, spa::Kanr clfA::Ermr
Newman spa Derivative of Newman spa lacking the N1 This study
clfAΔN140-210 (#1) subdomain (residues 40-210) of clumping factor A
Newman spa Derivative of Newman spa lacking the N1 This study
clfAΔN140-210 (#2) subdomain (residues 40-210) of clumping factor A
Escherichia coli
SA08B Plasmid propagation strain that allows direct (25)
transformation into S. aureus strain Newman
Plasmids
pIMAY:: Temperature sensitive vector for allelic exchange (K. Lacey,
clfAΔN140-210 to create a ClfA N1 subdomain deletion mutant; J. Geoghegan,
carries 628 bp of DNA from upstream and 600 bp unpublished)
from downstream of the clfA N1 subdomain; Cmr
* Kanr, Ermr and Cmr; resistance to kanamycin, erythromycin and chloramphenicol,
respectively.

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TABLE 2 Primers
Primer Sequence Source
pIMAY MCS F 5’-TACATGTCAAGAATAAACTGCCAAAGC- 3’ (26)
pIMAY MCS R 5’-AATACCTGTGACGGAAGATCACTTCG- 3’ (26)
ClfA OUT FWD 5’ -GTAGGGCACGGTTTACTAAG- 3’ (K. Lacey,
ClfA OUT REV 5’ -CGCACTTTAATTGCTCCTCTTC- 3’ J. Geoghegan,
ClfA D REV 5’ -CGGCGCAATAACGTTATC- 3’ unpublished,
ClfA A FWD 5’ -GGTATTGGGAAGCGATTGATTC - 3’ IDT®)
Electroporation. Electroporation was performed as described by Löfblom et al. (19) with
slight modifications. An overnight culture of S. aureus was grown in 10 ml BHI medium (in
50 ml tubes). The culture was then adjusted to an optical density at 578 nm (OD578) of 0.5 in
50 ml prewarmed TSB. The culture was reincubated for 30 min and then placed on ice for
10 min. All of the following steps were carried out at 4oC. The cells were harvested in a
swinging bucket centrifuge at 3,900 x g for 10 min. The pellet was washed, centrifuged
again and resuspended in 50 ml sterile ice cold water. The centrifugation and resuspension
steps were repeated. The cells were first resuspended in 5 ml, then 1 ml and then 210 μl of
sterile ice cold 10% (w/v) glycerol. 50 μl aliquots were frozen at -70oC. Before
electroporation, cells were placed on ice for 5 min and then at room temperature for 5 min.
Cells were centrifuged at 5000 x g for 1 min and resuspended in 50 μl of 10% glycerol/500
mM sucrose. Plasmid DNA was precipitated by Pellet Paint Co-Precipitant (Novagen, Cork,
Ireland) and 5 μg of plasmid was added to the cells. The cells were transferred to a 0.1 cm
electroporation cuvette (MBP, Dublin, Ireland) and pulsed using a Bio-Rad Gene Pulser at
21 kV/cm, 100 Ω and 25 μF, time constant 2.4 ms. Following electroporation, cells were
suspended in 1 ml BHI + 500 mM sucrose and incubated at 28oC for 2 h with shaking before
being plated onto TSA + Cm plates and incubated at 28oC for 48 h.
PCR. Colony PCR was carried out by resuspending half a toothpicked colony in 40 μl
sterile TE buffer (1 mM EDTA/ 10 mM Tris-HCl, pH 7.8). This solution was then heated for
10 min at 100oC and then centrifuged for 10 min at 4000 x g. 2 μl of the supernatant was then
used as template for the PCR reaction. Each 25 μl reaction also included, 14 μl DNase free
water (ThermoFisher Scientific, Dublin, Ireland), 5 μl Phire Buffer (ThermoFisher), 250 μM
deoxynucleoside triphosphates (dNTPs) (Bioline, London, UK), 4 μM MgCl2 (Bioline), 0.5
μM forward and reverse primers and 0.5 μl Phire Hot Start II polymerase (Finnzymes, Dublin,

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Ireland). Reactions were carried out in a Piko Thermal Cycler (ThermoFisher). Reactions
began with a 30 s denaturation step at 98oC, followed by 30 cycles of 98oC (5 s), 52oC (5 s),
72oC (20 s / kb product). The final extension step was carried out at 72oC for 1 min. PCR
amplification of genomic DNA was carried out using Velocity DNA polymerase (Bioline).
Each 50 μl reaction contained nuclease free water (made up to 50 μl), 10 μl 5 x Hi-Fi Buffer
(Bioline), 250 mM dNTPs, 0.4 μM forward and reverse primers, 150 ng genomic DNA
template and 0.5 μl Velocity DNA polymerase. Initial denaturation was at 98oC for 2 min,
and followed by 30 cycles of 98oC (30 s), 54oC (30 s), 72oC (30 s / kb genomic DNA) and a
final extension of 72oC for 1 min. Sizes of PCR products were estimated by agarose gel
electrophoresis. Samples were run through a 0.8% agarose gel in 1 x Tris-acetate-EDTA
(TAE) buffer (ThermoFisher) and DNA was visualised under UV light following ethidium
bromide staining. Sizes of bands were compared against a 1 kb DNA ladder (HyperLadder I,
Bioline).
Mutant strain production. All strains and plasmids used are listed in Table 1. Deletion
of the N1 subdomain of the clfA gene was performed by allelic exchange using the plasmid
pIMAY (26). Plasmid pIMAY::clfAΔN140-210 was isolated from E. coli SA08B using the
Plasmid Plus Midi Kit from QIAGEN (Manchester, UK). The plasmid was then
transformed into competent S. aureus strain Newman spa by electroporation, as described
above (19). Colonies were screened by PCR (as above) with primers against the MCS of
pIMAY (pIMAY MCS F and pIMAY MCS R) (Table 2) to identify colonies positive for the
presence of pIMAY. Deletion of DNA encoding residues 40-210 of the N1 subdomain of
clfA was achieved by allelic exchange as previously described (26). Briefly, integration of
the plasmid was accomplished by taking a single colony positive for replicating plasmid and
emulsifying it in 200 μl TSB. This was then diluted to 10-3 and dilutions were plated on TSA
+ Cm and incubated 37oC overnight. Colony PCR analysis was then performed again using
MCS primers to determine the absence of replicating plasmid. Clones that gave a negative
result, were then screened by colony PCR to ascertain the side of integration using (1) ClfA
OUT FWD and ClfA D REV primers for the left side or (2) ClfA OUT REV and ClfA A
FWD primers for the right side (Table 2). Overnight cultures of colonies of both left and
right side of integration were diluted to 10-6 and plated on TSA + ATc and incubated for 48
h at 28oC. Large colonies were streaked onto TSA + ATc and TSA + Cm plates and
incubated at 37oC overnight. Cm sensitive colonies screened by PCR with OUT primers
(ClfA OUT FWD, ClfA OUT REV) to identify clones with the desired deletion. Genomic

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DNA of putative mutants was isolated using the Bacterial Genomic DNA Purification Kit
(EdgeBio, Gaithersburg, MD) and PCR (as above) using OUT primers was used to amplify
across the deletion. The PCR product was purified using the High Pure PCR product
purification Kit (Roche, Dublin, Ireland), verified by agarose gel electrophoresis and was
subsequently sent for DNA sequencing (GATC Biotech, Constance, Germany).
Growth curve. 5 ml overnight cultures in TSB were centrifuged at 4000 x g for 5 min to
harvest cells. The cells were washed with prewarmed TSB and the optical density at 600 nm
(OD600) was adjusted to 0.2 in 2 ml TSB. The culture was then diluted 10-1 in 1 ml and 200
μl was added in triplicate for each strain, to the wells of a 96 well plate. A broth only control
was also added to three wells. The OD600 was read at 30 min intervals at 37oCwith shaking,
in a Synergy H1 Multi-Mode plate reader (BioTek, Bad Friedrichshall, Germany).
Toxin activity testing. Haemolytic activities of the strains were tested by streaking the
strains on a Columbia sheep blood agar plate (Oxoid), as described previously (30). β-
haemolysin producing strain RN4220 was streaked across the centre of the plate,
perpendicularly to this the test strains were streaked. Production of δ-toxin and α-toxin were
tested by this method.
Immobilised fibrinogen adherence assay. This assay was performed as previously
described by Hartford et al. with some modifications (13). 96 well flat bottom plates
(Sarstedt, Nümbrecht, Germany) were coated with doubling dilutions of Fg (Enzyme
Reasearch Laboratories, South Bend, IN) in 1 x phosphate-buffered saline (PBS)
(ThermoFisher) beginning at a concentration of 20 μg/ml. The plate was then incubated
overnight at 4oC. The following day the Fg dilutions were removed from the plate and the
plate was washed three times with PBS, the plate was then blocked with 5% (w/v) bovine
serum albumin (BSA) (ThermoFisher) for 2 h at 37oC. Overnight cultures of S. aureus were
washed and diluted to an OD600 of 1.0 using PBS and 100 μl of bacterial culture was added
to each well of the 96 well plate. The plate was incubated with the bacteria at 37oC for 2 h.
The plate was then washed with PBS and 100 μl of 25% (w/v) formaldehyde was added to
each well to fix adherent cells. The cells were then stained with crystal violet for 1 min and
absorbance was measured at the wavelength of 570nm (A570) on a Thermo Scientific
Multiskan EX plate reader. Each Fg concentration was performed in triplicate for each
strain.

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Platelet aggregation. Platelets were prepared and aggregation was tested as previously
described by Loughman et al. (20). Breifly, platelet-rich plasma (PRP) was prepared by
drawing nine parts blood into one part 3.8% (w/v) Na citrate. Blood was centrifuged for 10
min at 170 x g and PRP was carefully removed. The remaining blood was centrifuged again
at 2000 x g for 10 min to prepare platelet-poor plasma (PPP). PPP was used as a reference
of 100% light transmission in the aggregation experiments. S. aureus overnight cultures
were washed and adjusted to an OD600 of 1.6 in PBS. 5 μl bacterial culture was added to 245
μl PRP in a glass cuvette. The cuvettes were incubated at 37oC with stirring for 15 min in a
PAP-8 aggregometer (Bio/Data) and light transmission was monitored. Blood was donated
from healthy volunteers. Ethical approval for the use of human blood was obtained from the
Trinity College Dublin (TCD) Faculty of Health Sciences Ethics Committee and the Royal
College of Surgeons in Ireland (RCSI) Research Ethics Committee.
Opsonisation assay. 10 x HEPES buffered saline (HBS) was prepared with 100 mM
HEPES and 1.5 mM NaCl (pH 7.4). Overnight cultures of S. aureus were centrifuged at
20,000 x g for 10 min, washed once in 1 x HBS with 5 mM CaCl2 + 2.5 mM MgCl2 (called
HEPES++) and adjusted to an OD600 of 1.0 in HEPES++ with 0.1% (w/v) BSA. 50 μl of the
bacterial culture was incubated with 50 μl Fg at a final concentration of 10 μg/ml (or PBS),
at 37oC and 200 rpm for 30 min. The bacteria were then centrifuged again (as above),
washed with HEPES++ and resuspended in 100 μl 1% (v/v) normal human serum (NHS)
(Complement Technology Inc, Tyler, TX) (or PBS). The bacteria were incubated again at
37oC, 200 rpm for 30 min. Bacteria were centrifuged and washed with PBS + 0.1% BSA,
before being resuspended in 50 μl sheep polyclonal anti-C3 antibody conjugated to
fluorescein isothiocyanate (FITC) (Abcam, Cambridge, UK). The antibody was a 1:50
dilution in PBS + 0.1% BSA. The antibody was incubated with bacteria on ice for 30 min,
the bacteria were then centrifuged and washed with PBS. The bacteria were resuspended in
100 μl PBS, transferred to the wells of a black 96 well plate (Falcon) and fluorescence was
analysed in a Synergy H1 Multi-Mode plate reader (BioTek). Each condition was performed
in duplicate for each strain.
Statistical analysis. The values from replicate experiments were normalised and the
average and standard errors of the means were calculated. Statistical analysis was performed
using the Prism GraphPad program. P values were calculated using paired t tests. P < 0.05 -
*, P < 0.01 - **, P < 0.001 - ***, n.s.- not significant (P > 0.05).

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RESULTS
Construction and verification of two Newman spa clfAΔN1 mutants. To examine the
role of the N1 subdomain of ClfA, S. aureus mutants expressing a truncated form of this
protein, lacking the N1 subdomain were generated. The mutants were created by deleting
DNA encoding residues 40-210 of the ClfA protein in the S. aureus strain Newman spa.
This strain was chosen as the background in which to create the deletion as it lacks cell wall
anchored fibronectin binding proteins, which are also capable of binding to Fg (9). The
strain is also deficient for protein A, this allowed the use of blood and serum later in the
study, without the complication of protein A binding Fc region of IgG (3). The presence of
Fg binding protein clumping factor B (ClfB) in this strain does not affect the experiments
conducted as ClfB does not contribute to Fg binding in stationary phase cultures, due to
break down of the protein on the surface and a halt in its transcription (28, 23). Therefore
the study of ClfA in this project was not affected by the presence of Fg-binding proteins,
ClfB and the fibronectin-binding proteins.
The deletion was achieved by performing allelic exchange using the plasmid pIMAY (26),
as described in the Materials and Methods. Following completion of the protocol for allelic
exchange, genomic DNA was isolated from putative mutants. PCR amplification of the clfA
gene was carried out using OUT primers (Table 2) and the sizes of PCR products were
estimated by agarose gel electrophoresis (Fig. 2A). From this gel image it can be seen that
the Newman spa (wild-type) product (lane 6) is about 4 kb in size, whereas the two Newman
spa clfAΔN1 mutant products (lanes 1 and 2) are approximately 3.5 kb in size. The reduced
size of these bands corresponds to the 513 bp size of the deleted fragment of this gene.
Subsequently, the PCR products of the two mutants were sequenced and alignment to the
wild-type ClfA amino acid sequence (GenBank accession no. BAF67028) is shown (Fig.
2B). The same sequence was generated for the two mutants and therefore only one is shown.
The sequence alignment confirmed that DNA encoding residues 40-210 had been deleted in
the Newman spa clfAΔN1 mutant strains. This was the desired deletion, retaining the linker
region of the N1 subdomain, which is required for export and cell wall localisation of ClfA
(24).
Phenotypic studies of the ClfAΔN1 mutants. Following verification of the two
ClfAΔN1 mutants, the growth rate of these strains were tested and compared to the wild-

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type strain- Newman spa. This was carried out to ensure that the ClfAΔN1 mutants had not
developed any growth defect during the mutation process. The growth curves of these
strains, referred to as Newman spa clfAΔN1 #1 and Newman spa clfAΔN1 #2 and the wild-
type strain, Newman spa, are shown (Fig. 3A). The ClfAΔN1 mutants had a similar growth
rate to the wild-type
FIG. 2. Verification of two ClfAΔN1 mutants. (A) Agarose gel electrophoresis of PCR
products, amplified from genomic DNA using OUT primers upstream and downstream of
the clfA gene. Products from the two ClfAΔN1 mutants (lanes 2 & 3) show smaller bands
than the wild-type product (lane 6). Length of amplified fragments were estimated by
comparison with a 1 kb DNA ladder (lane 1). Lanes 4 and 5 are negative controls
representing unamplified genomic DNA and lanes 7 and 8 represent no template and no
polymerase controls respectively. (B) Alignment of amino acid sequences from PCR
products from the ClfAΔN1 mutants with the wild-type ClfA sequence (GenBank accession
no. BAF67028). Conserved amino acid residues are indicated by an asterisk (*). Alignment
was carried out using the Clustal Omega program.

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Toxin activity of the strains was also tested. The activity of α-toxin and δ-toxin are tested
to ensure functionality of the SaeRS and Agr two component systems of S. aureus (30, 8,
15). These systems regulate many virulence genes in S. aureus and it is therefore important
to test their functionality (Fig. 3B) (30, 16).
The activity of α-toxin is seen around the test strains steaks on the sheep blood agar plate,
while a halo of complete lysis corresponding to δ-toxin activity is seen at the junction where
the test strains meet the RN4220 β-haemolysin producing strain. Complete lysis is seen
because the activity of δ-toxin is enhanced by the presence of β-haemolysin from RN4220
(30). The ClfAΔN1 mutants are phenotypically identical to the wild-type with regard to
haemolysis of sheep blood agar.
FIG. 3. Phenotypic studies of the ClfAΔN1 mutants. (A) Growth curve of Newman spa
(wild-type) and the two Newman spa clfAΔN1 mutants (#20 & #21). Absorbance at 600nm
(A600) was read at 30 min intervals until stationary phase of growth was reached. (B) Toxin
activity test of the ClfAΔN1 strains and the wild-type. The haemolytic activity of the strains
were tested on sheep blood agar. β-haemolysin producing strain RN4220 was streaked down
the plate, at right angles to this, the test strains were streaked. δ-toxin activity is indicated by
the black arrow and α-toxin activity is indicated by the white arrow.
ClfAΔN1 mutants adhere to immobilised fibrinogen. The N2 and N3 subdomains have
been identified as the minimum Fg binding domain of ClfA (12). To test whether the N1
subdomain is involved in the binding of immobilised Fg by ClfA and to ensure that the ClfA
protein is being expressed by the ClfAΔN1 mutants, adherence assays were performed (Fig.
4). A ClfA deficient mutant was used as a negative control. It can be seen from this graph,
that the ClfAΔN1 mutants bind strongly to Fg, at a level similar to that of the wild-type. The

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interaction between ClfA and Fg is specific, dose-dependent and saturable. Whereas the
interaction seen by the ClfA deficient mutant- Newman spa clfA is weak and represents non-
specific binding. This assay indicates that the N1 subdomain is not involved in the binding
to immobilised Fg by ClfA. This assay also ensures that the ClfA protein is being expressed
by the ClfAΔN1 mutant strains. Quantification of the levels of ClfA on the surface of the
ClfAΔN1 strains and wild-type strain was attempted by flow cytometry using a monoclonal
antibody against an epitope in the N3 domain (Aurexis), followed by a secondary antibody
against the primary antibody, which was conjugated to FITC. However, this experiment was
unsuccessful.
FIG. 4. Adherence of Newman spa, Newman spa clfA and the two Newman spa clfAΔN1
strains to immobilised Fg. A range of Fg concentrations from 0.625 – 20 μg/ml were tested
and absorbance at 570 nm (A570) was measured. The data represents the mean of three
independent experiments. Error bars indicate the standard errors of the means.
ClfAΔN1 mutant causes aggregation of platelets. S. aureus is capable of binding to and
activating platelets by a range of mechanisms. ClfA has been shown to be an important
factor in the stimulation of platelets (29). In order to cause aggregation of platelets by ClfA,
in a Fg-dependent manner, two interactions are required. First Fg bound to ClfA on the
surface of S. aureus is recognised by the platelet integrin glycoprotein (GP) IIb/IIIa. ClfA

16
specific antibodies are also required to cause activation of platelets. IgG bound to ClfA links
the bacterial cell to the platelet via the platelet FcγRIIa receptor (20).
To test whether the N1 subdomain is involved in the activation of platelets, the ability of
one of the ClfAΔN1 mutants to cause platelet aggregation was tested (Fig. 5). Bacteria were
incubated with PRP and aggregation was monitored by light transmission. Results are
expressed as percentage aggregation. As is well documented in literature, a significant
difference was seen in the percentage aggregation between the wild-type strain and the ClfA
deficient strain (29). Aggregation was stimulated by the Newman spa clfAΔN1 (#2) mutant
in all three experiments. However, there was high variation in the percentage aggregation by
this strain on each occasion. Therefore there is no significant difference between the
ClfAΔN1 mutant and either of the other two strains. For this reason it is unknown whether
the N1 subdomain is playing a role in the activation of platelets. There does seem to be a
trend in the data, showing the ClfAΔN1 mutant to be capable of causing aggregation, similar
to the wild-type. More investigation is required here to get conclusive results.
FIG. 5. Testing the ability of the ClfAΔN1 mutant to aggregate platelets. Bacteria were
injected into platelet-rich plasma and percentage aggregation was recorded over a 15 min
period. The data presented represents the mean of three independent experiments with three
different donors, error bars indicate standard errors of the means, ** p < 0.01, n.s. – not
significant.

17
Binding of Fg by ClfA protects the bacteria from opsonin deposition. Phagocytosis
by neutrophils is a key element of the host immune defence against infection (27). The
engulfment of bacteria by these immune cells is greatly increased by the presence of
opsonins such as complement proteins and antibodies on the bacterial cell surface.
Complement-mediated opsonisation is very important to the clearance of pathogenic
bacteria, as plasma proteins such as C3b label the bacteria for uptake by phagocytic cells (7).
Higgins et al. have shown that ClfA is capable of inhibiting phagocytosis of S. aureus by
human polymorphonuclear leucocytes, in the presence of Fg. It was hypothesised that ClfA
binding Fg formed a protective layer around the bacterium, perhaps preventing recognition
of opsonins by receptors on phagocytic cells (14). Here it was tested whether the binding of
Fg by ClfA could influence the deposition of opsonins such as C3b on the bacterial cell
surface, which would then result in reduced phagocytosis by host cells.
Bacteria were incubated with Fg and then with human serum. An anti-C3 antibody
conjugated to FITC, recognising C3, C3b, iC3b and C3dg, was used to detect opsonins
associated with the bacteria. Flow cytometry was first used to detect opsonins on the
bacterial cell surface. Unusual results were seen with this method, in which higher amounts
of opsonins were associated with the bacteria in the presence of Fg and double peaks of
fluorescence were observed (data not shown). This led to questioning as to whether the
results were truly representative and if perhaps the bacteria could be clumping in the
presence of Fg. Clumping of bacteria could have resulted in the flow cytometer analysing
clumps of bacteria, rather than a single cell at a time, giving higher fluorescence values in
the presence of Fg.
To overcome this problem, a plate reader method was employed, as described in the
Materials and Methods. This method was more successful, as the plate reader read the
fluorescence value of an entire well, rather than cell by cell as was the case with the flow
cytometer. Relative fluorescence units (RFU) were used as a measure of opsonins associated
with the bacteria. A significant (P < 0.05) decrease in the amount of opsonins associated to
the bacteria was seen with the wild-type strain in the presence of Fg, compared to in its
absence (Fig. 6). This shows that in the presence of Fg, the bacteria are protecting
themselves from deposition of opsonins. This protection was shown to be dependent on
ClfA, as no effect was seen with the ClfA deficient mutant in the presence of Fg. The ClfA
deficient mutant, which is unable to bind Fg showed a similar level of opsonin deposition to

18
native ClfA in the absence of Fg. This shows that this protection is occurring by an Fg-
dependent mechanism. Controls in the absence of serum showed very low RFU values of
less than 10, ensuring the results were serum dependent (data not shown).
The ability of the ClfAΔN1 strains to protect themselves from opsonin deposition was also
tested. No significant decrease in opsonins associated with the bacteria was seen in the
presence of Fg with these strains. The contrasting results observed between the wild-type
and ClfAΔN1 strains, suggests that the N1 subdomain may be involved in this Fg dependent
protection mechanism. The observation of the same result in two independent ClfAΔN1
mutants, further consolidates the results shown here.
FIG. 6. The ability of ClfA to inhibit deposition of opsonins on the bacterial cell surface in
the presence of Fg (10 μg/ml). Bacteria were incubated with/without Fg and then with
serum. C3 binding the bacterial surface was detected with an anti-C3 antibody conjugated to
FITC. Relative fluorescence units (RFU) were used as a measure of C3 association with the
bacteria. The data presented represents the mean three independent experiments for
Newman spa clfAΔN1 #1, and four independent experiments for the remainder of the strains.
Each experiment was performed in duplicate for each strain/condition. Error bars indicate
standard errors of the means, * p < 0.05, n.s – not significant.

19
DISCUSSION
Clumping factor A is the archetypal MSCRAMM Fg-binding protein of S. aureus. It is
composed of the defining features of a ligand-binding A region, and a flexible unfolded stalk
linking the A region to the wall spanning domain, which anchors the protein in the bacterial
cell envelope. The A domain is composed of two separately folded, IgG-like folded
subdomains, designated N2 and N3. ClfA also contains, in the A region, a subdomain,
termed N1 with very little known about its structure or function (5). A function has been
attributed to 10 residues (211-220) of this domain in export and cell wall localisation of
ClfA, however no function has been described for the remainder of this subdomain (24).
This project reports for the first time a possible role for the remaining residues of this
domain.
This study has demonstrated the creation of two independent S. aureus mutants expressing
a truncated form of the ClfA protein, lacking the N1 subdomain residues 40-210. Creation
of these mutants were verified by DNA sequencing. They were also shown to display no
defect in growth or in the functionality of the Agr and SaeRS two-component systems
through the testing of toxin activity. The expression of ClfA on the surface of the cells was
ensured by the adhesion of these mutants to immobilised fibrinogen. Quantification of the
levels of the protein on the surface of these strains would be merited, if further studies were
to be performed.
Adhesion assays conducted showed no difference in the binding of Newman spa wild-type
strain and Newman spa clfAΔN1 mutant strains to immobilised Fg. These results indicated
that the ClfA N1 subdomain is not required for binding of ClfA to immobilised Fg. Platelet
aggregation experiments were inconclusive. The ClfAΔN1 mutant #2 was capable of
aggregating platelets, however, the variation in percentage aggregation between donors was
too large for any statistically significant results to be generated. This variation between
donors could be in part due to the requirement of ClfA specific immunoglobulins for
activation of platelets by S. aureus (4). Donors could have different levels of these
antibodies, depending on their exposure to this organism. Proteolytic cleavage of the surface
proteins of S. aureus or cell lysis over time before conducting the experiments may also
explain the variation in these results. This could be optimised for further studies of this type.
More investigation, into the activation of platelets by the ClfAΔN1 mutants, with many more

20
donors is required to elucidate if the N1 subdomain does have any role in the activation of
platelet aggregation by S. aureus.
It was hypothesised that the antiphagocytic property of ClfA (14) was in part due to the
inhibition of opsonin deposition on the bacterial cell surface in the presence of Fg.
Accordingly, a protection from opsonin deposition was observed by the wild-type bacteria in
the presence of Fg. It should be noted, that no significant difference in opsonin deposition
was observed between the Newman spa wild-type and Newman spa clfA mutant in the
absence of Fg. As the antibody used (polyclonal anti-C3 antibody) recognises C3, C3b and
iC3b, the influence of ClfA recruiting factor I to the surface of the bacterium and its
degradation of C3b to iC3b is not seen here. Although iC3b is recognised by this antibody
and can still function as an opsonin, it cannot function in complement activities such as the
formation of C3 convertase, required for the generation of more C3b on the bacterial cell
surface.
The protection from opsonin deposition observed by the wild-type bacteria in the presence
of Fg, was not seen with the Newman spa clfAΔN1 mutants. This result was observed with
both of the ClfAΔN1 mutant strains, which consolidates the results generated. This shows
for the first time a possible role for the remainder of the N1 subdomain of ClfA. The N1
subdomain seems to be required for this Fg-mediated protection of the bacterium from
opsonin deposition. The mechanism of protection is Fg-dependent and therefore it is
thought that perhaps the bacterium could be creating a protective shield composed of Fg
around itself, preventing the opsonin proteins attaching to the cell surface. No difference
was observed in binding immobilised Fg by these mutants compared to the wild-type,
however, perhaps the N1 subdomain is involved in the binding of ClfA to soluble Fg.
The role of the N1 subdomain in binding to soluble Fg warrants further investigation. It
was attempted to test this binding by immobilising bacteria to a microtitre plate and
performing a type of enzyme-linked immunosorbent assay (ELISA). This was performed
with an antibody directed against Fg, bound to the immobilised bacteria and a secondary
antibody, directed against the primary antibody and conjugated to horse radish peroxidase,
that would allow for detection. This method however, proved unsuccessful as the
immobilisation of the bacteria was not occurring evenly. To study this interaction further, I
would suggest employing a method similar to that used to test opsonin deposition, as
described in the Materials and Methods. This method could test the binding of ClfA to

21
soluble Fg with the bacteria in solution, rather than immobilised to a plate. Fg bound to the
bacteria could be detected using an anti-fibrinogen antibody conjugated to FITC and
fluorescence recorded. This would also be more representative of what is occurring in the
blood during the establishment of infection.
In summary, this project demonstrates that the N1 subdomain of ClfA is not involved in
the binding to immobilised Fg, but may be required for the binding of soluble Fg and the
protection of S. aureus from opsonophagocytosis. It has also demonstrated a Fg-dependent
mechanism of inhibition of phagocytosis, through reduced opsonin deposition. This
supports the idea of a Fg composed protective capsule around the bacterium, preventing
interaction of innate immune responses (14, 18).

22
ACKNOWLEDGEMENTS
I would like to thank all those who assisted me in completing this project. Particularly my
supervisor Dr. Joan Geoghegan and the PhD students, Dara and Joana, who assisted in my
supervision. I would also like to thank all of the other members of the Geoghegan Lab,
Leanne, Aisling, Marta, Orla, Keenan and Deirdre, who were very helpful throughout the
project. I also thank Ana Lopez-Alonso and Ghada Alharbi from the Royal College of
Surgeons in Ireland, who welcomed me so warmly and assisted me in the completion of
aggregation experiments in their laboratory.

23
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