Veterinary Microbiology 119 (2007) 240247

www.elsevier.com/locate/vetmic

Characterization of cell wall associated proteins of

a Staphylococcus aureus isolated from bovine
mastitis case by a proteomic approach
Francesca Taverna, Armando Negri, Renata Piccinini, Alfonso Zecconi *,
Simona Nonnis, Severino Ronchi, Gabriella Tedeschi
Department of Animal Pathology, Hygiene and Health (DIPAV),
University of Milano, Via Celoria 10, 20133 Milano, Italy
Received 13 April 2006; received in revised form 11 September 2006; accepted 14 September 2006

Abstract
Staphylococcus aureus causes different pathologies in humans and animals. In particular, it is involved in intramammary
infections in cows, causing economic losses and milk-safety problems. Although it is well-known that surface components
(proteins and capsular polysaccharides) and exotoxins are virulence factors involved in the pathogenesis of bovine mastitis, less
is known about the precise biochemical identity of such molecules. Therefore, mapping of surface proteins using specific
disease- and environment-isolates provides a benchmark for strain comparison of pathogens with different pathogenic
characteristics and antibiotic resistance mechanism and can aid in defining specific vaccine and therapeutic targets. In this
study, we used a proteomic approach on protein extracts of lysostaphin-treated S. aureus in isotonic conditions, to produce a
reproducible and well resolved 2-D electrophoresis (2-DE) reference map of surface associated proteins of isolated S. aureus
from a case of bovine mastitis. The most abundant protein components were identified by Matrix assisted laser desorption
ionisation-time of flight (MALDI-TOF) mass spectrometry.
# 2006 Elsevier B.V. All rights reserved.
Keywords: Proteomics; Staphylococcus aureus; Mastitis; Cell wall proteins

1. Introduction
Staphylococcus aureus is a Gram-positive, facultative anaerobic, catalase positive coccus that causes
* Corresponding author at: DIPAVMalattie Infettive, Via Celoria
10, 20100 Milano, Italy. Tel.: +39 02 5031 8069;
fax: +39 02 5031 8079.
E-mail address: alfonso.zecconi@unimi.it (A. Zecconi).

many serious diseases in humans and animals, and it is

the most common aetiologic agent of contagious bovine
mastitis, with relevant losses in the dairy industry
(Zecconi et al., 2003). S. aureus isolates produce several
virulence factors including surface-associated secretory products, leukotoxins and enterotoxins (Kerro
Dego et al., 2002; Peacock et al., 2002), representing
potential target for vaccine development (Aarestrup
et al., 1995; Fitzgerald et al., 1997; Schuberth et al.,

0378-1135/$ see front matter # 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2006.09.007

F. Taverna et al. / Veterinary Microbiology 119 (2007) 240247

2001; Zecconi et al., 2006). However, the importance of

evaluating the combination of S. aureus virulence
factors has been recently emphasized both in human
and veterinary medicine as an indispensable step to
develop effective vaccines (Haslinger-Loffler et al.,
2005; Jarraud et al., 2002; Peacock et al., 2002; von Eiff
et al., 2004). The importance of surface components
(proteins and capsular polysaccharides) and exotoxins
in the pathogenesis of bovine mastitis is well-known.
Indeed, during S. aureus intramammary infections
surface components take part in bacterial adhesion to
host mammary tissues and to resistance to phagocytosis
by milk cells (Sutra and Poutrel, 1994). Therefore,
mapping of surface proteins provides a benchmark for
strain comparison of pathogens with different combination of pathogenic characteristics and antibiotic
resistance mechanism and can aid in defining vaccine
and therapeutic targets. In recent years, 2-D electrophoresis (2-DE) reference maps of the total or the
extracellular proteins from various S. aureus laboratory-adapted reference strains have been published
(Cordwell et al., 2001, 2002; Kohler et al., 2005; Scherl
et al., 2005; Ziebandt et al., 2001). On the contrary, a
single report on the subproteome of S. aureus surface
proteins, selectively obtained under carefully controlled lysis procedure, has been published recently
using a human pathogen strain isolated from a patient
diagnosed with osteomyelitis (Nandakumar et al.,
2005). Since recent development in genomic analysis
have revealed significant differences between various
strains of pathogens, including S. aureus, it has been
recently emphasized the importance of analysing
clinical isolates to detect specific virulence factors or
antigens (Fux et al., 2005). Therefore, the present
research is focused on obtaining a 2-DE reference map
of surface proteins isolated following lysostaphin
treatment from a S. aureus strain selected for its
virulence characteristics and for promoting mastitis in
cows.

2. Materials and methods

241

observed in a previous molecular epidemiological study

(Zecconi et al., 2006). Identification as S. aureus was
performed according to the following scheme: catalase
positive, Gram-positive cocci, haemolytic on blood
agar, coagulase positive in 418 h. The presumptive
identification was confirmed by API ID32 Staph
(99.9%, T = 0.63) (BioMerieux, Marcy LEtoile,
France). The genetic characteristics of the isolate are:
coagulase positive, collagen binding protein negative,
fibrinogen binding protein positive, protein A positive
(nine repetitions) and enterotoxin G and I positive.
The isolates were stored at 20 8C in BHI broth
with 15% (v/v) glycerol. Before proteomic analysis,
isolate was thawed and cultured on blood agar plate
with 5% bovine blood for 18 h, washed, suspended in
physiological saline and harvested by centrifuging at
12,000  g for 30 min at 4 8C.
2.2. Sample preparation for 2-D electrophoresis
Surface associated proteins were prepared by
lysostaphin treatment under isotonic conditions as
described in Vytvytska et al. (2002) with minor
changes. After harvesting bacteria in post-exponential
phase of growth, cells were washed twice with icecold PBS and once with digestion buffer (10 mM Tris
HCl, pH 7.6, 1 mM EDTA, 5 mM MgCl2). About
3  109 bacteria were suspended in digestion buffer
containing 35% raffinose, 1 mM PMSF and protease
inhibitor cocktail (Roche, Monza, Italy). After
incubation at 37 8C for 40 min in the presence of
5 U of lysostaphin, protoplasts were sedimented by
low speed centrifugation (5000  g) for 30 min at
4 8C and the supernatant was dialyzed (cut-off 10 kDa,
StepBio, Bologna, Italy) overnight in 10 mM Tris
HCl, pH 7.6, 1 mM PMSF. Protein content was
obtained by measuring the absorbance at 280 nm
(A280 of 1 equals 1 mg ml 1 protein). The supernatant was further treated with 10% (v/v) TCA in ice
for 30 min and the precipitate was collected by
centrifugation at 12,000  g for 30 min at 4 8C and the
precipitated was washed with 5% TCA to decrease
TCA concentration and stored at 20 8C.

2.1. Bacterial strains and growth condition

2.3. 2-D electrophoresis
S. aureus strain 1673 was isolated from a case of
bovine subclinical mastitis. The strain is representative
of the most frequently isolated strains cluster as

2-DE was carried out on surface proteins by

performing the reduction and the alkylation prior to

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F. Taverna et al. / Veterinary Microbiology 119 (2007) 240247

the first dimension as suggested by Castagna et al.

(2002). The sample was solubilised in 7 M urea, 2 M
thiourea, 4% CHAPS, 40 mM Tris and incubated at
room temperature for 90 min with 5 mM tributylphosphine (TBP). After this step, the sample was
incubated with 20 mM iodoacetamide (IAA) at room
temperature for 90 min in the dark. Following
precipitation in an anhydrous solution of acetone
and methanol (8:1, v/v) at 20 8C, the precipitate was
suspended in the 2-D PAGE sample buffer (7 M urea,
2 M thiourea, 4% CHAPS, 0.5% Resolyte 3.510 NL,
Bromophenol Blue) and sonicated for 1 min. For the
first dimension, 1000 mg (preparative 2-DE) or
250 mg (analytical 2-DE) of protein were applied to
rehydrated IPG strips (immobiline dry strip pH 310
NL, 180 mm; Amersham Pharmacia, Cologno Monzese, Italy) by sample cups. IEF was carried out at
14 8C, 74,650 V total voltage, for 23 h. The first
dimension IEF was performed with the Multiphor II
system (Amersham Pharmacia, Milano, Italy).
Before the second dimension, the strips were rinsed
with buffer (6 M urea in 0.375 M TrisHCl pH 8.8, 2%
SDS, 20% glycerol, Bromophenol Blue). The second
dimension was performed on home-made 916%
polyacrylamide linear gradient gels (170 mm  190
mm  1.5 mm), at 40 mA/gel constant current until the
bromophenol band had reached the end of the gel
(Protean II, BioRad, Segrate, Milano, Italy). Finally, the
gels were stained with colloidal blue CBB G (Sigma,
Milano, Italy) (preparative 2-DE, 4 gels) or ammoniacal
silver (analytical 2-DE, 4 gels).
The gels were scanned in an Epson 1660
densitometer (Bruker Daltonics, Milano, Italy).
2.4. In situ digestion and protein identification by
MALDI-TOF analysis
Matrix assisted laser desorption ionisation-time of
flight (MALDI-TOF) analysis was carried out on the
spot to be identified following 2-DE, in gel digestion
and peptide extraction as described in Tedeschi et al.
(2005). Briefly, each spot was excised, destained with
50% acetonitrile in ammonium bicarbonate 0.1 M
(40 min at 25 8C), dried in a Speed Vac, soaked with
ammonium bicarbonate 0.1 M and digested overnight
with trypsin sequencing grade (Roche, Monza, Italy) at
37 8C. The in gel tryptic digest was extracted with 30 ml
50% acetonitrile in 0.1% trifluoroacetic acid and the

peptide mixture was subjected to MALDI-TOF analysis

by using a Bruker Daltonics Reflex IV instrument
(Bruker Daltonics, Milano, Italy) equipped with a
nitrogen laser (337 nm) and operated in reflector mode
with a matrix of a-ciano-4-hydroxy-cinnamic acid. The
peptide mixture was loaded on an AnchorChip plate
(Bruker Daltonics, Milano, Italy). External standards
were used for calibration (Bruker peptide calibration
standard). Each spectrum was accumulated for at least
600 laser shots. Measured peptide masses were used to
search the Swiss-Prot, MSDB, TrEMBL and NCBI
sequence databases for protein identifications by the
Mascot program (http://www.matrixscience.com).
The presence of predicted signal peptides was
determined by the program SignalP available as a tool
in Expert Protein Analysis System (ExPASy) proteomics server of the Swiss Institute of Bioinformatics
(SIB). Theoretical molecular weight and hydropathy
(Grand Average of Hydropathicity, GRAVY) values
were calculated by the ProtParam tool in ExPASy. The
predicted transmembrane regions for proteins were
identified by the program TMPred in ExPASy.

3. Results
3.1. Surface proteins preparation and 2-DE PAGE
The aim of the present research is to obtain a 2-DE
reference map of the subproteome of surface
associated proteins of a S. aureus strain selected for
promoting intramammary infections in cows. Fig. 1
shows a representative silver-stained analytical gel
(out of four highly reproducible gels) loaded with S.
aureus cell wall associated proteins following
lysostaphin treatment. Similarly to what was previously reported (Nandakumar et al., 2005; Vytvytska
et al., 2002), most proteins were found in the acidic
range and many of them, in the 30150 kDa range,
were represented by a pattern indicative of multiple
forms of the same polypeptide, suggesting possible
post-translational processing or experimental artefact
such as deamidation.
3.2. Protein identification by mass spectrometry
For identification of proteins, a preparative 2-DE
gel was stained with colloidal blue and the spots were

F. Taverna et al. / Veterinary Microbiology 119 (2007) 240247

Fig. 1. Analytical 2-D gel electrophoresis of surface associated

proteins from S. aureus after lysostaphin treatment. A 250 mg of
protein were applied to a rehydrated IPG stip (180 mm, pH 310 NL).
The second dimension was carried out on an homemade 916%
polyacrylamide linear gradient gel (170 mm  190 mm  1.5 mm).
After 2-D electrophoresis the gel was stained by silver. The numbers
refer to the spots reported in Table 1.

digested by trypsin and analysed by MALDI-TOF.

Table 1 reports the proteins identified grouped in
functional categories, the coverage percentage, the
molecular weight and the corresponding accession
number in the protein sequence databases as well as
the GRAVY values, the length of the signal peptide
and the number of the predicted transmembrane
helices (TM). The position of the spots corresponding
to the proteins listed in Table 1 and indicated in Fig. 1.
Most of the proteins migrated according to their
predicted pI and molecular weights. The identification
confirmed the sample obtained after lysostaphin
treatment is enriched in proteins with a predictable
signal sequence and TM motifs, including known cell
wall associated proteins, or predicted to be at least
temporarily associated to the membrane in analogy
with their localization in various microorganisms. In
particular, signal peptide sequences which targets are
protein for secretion were present in 11 spots (spots 7
10, 23, 24, 36, 38, 39, 49, 50). Spots 710 and 48 were
identified as well-known adhesins. Twenty-five spots

243

were identified as proteins with predicted transmembrane helices (spots 2, 710, 1424, 2628, 36, 38, 39,
41, 49, 50). On the contrary, 16 proteins (spots 1, 36,
1113, 25, 2935, 37, 40, 4248) had no predicted
signal sequence or TM motifs. Among them, there are
four proteins which are clearly described as membrane
associated or named as putative membrane proteins in
data banks due to their primary structure features
(spots 32, 35, 4648) and five proteins which present
hydrophobic properties (as indicated by the GRAVY
value) that may allow them to associate with the lipid
bilayer (spots 1, 1113, 37, 4245). Only 7 proteins
out of 33 (50 spots) reported in Table 1 are not
predicted to be potentially associated to the cell wall
or the membrane based on their primary structure
features (spots 36, 25, 2931, 33, 34, 40). However,
as detailed in Section 4, several observations suggest
that also EF-TU (spots 36), alkyl hydroperoxide
reductase C (spots 30, 31) and hypothetical protein
ansA (spot 34) can be temporarily associated to the
membrane. As far as the last three proteins are
concerned (spots 25, 29, 33), two are either
hypothetical or putative, thus no experimental
observations on their potential localization are
available so far from any source.

4. Discussion
It is well-known that S. aureus expresses cell wall
associated surface proteins, which play an important
role in virulence and in biofilm formation. In
particular, in bovine intramammary infections, surface
components play a major role in bacterial adhesion to
host mammary tissues and to resistance to phagocytosis by milk cells.
Therefore, the identification of surface proteins
produced by S. aureus and the availability of
reproducible proteomic procedures to evaluate the
pattern of antibody expressed by infected versus
healthy subjects will provide new information for
developing vaccines and diagnostic tools (Etz et al.,
2002; Sellman et al., 2005; Vytvytska et al., 2002;
Weichhart et al., 2003). Moreover, cell wall constituents may hold some of the answers to resistance in
different strains (Hecker et al., 2003).
The actual polypeptide content of protein extracts
is highly dependent on the preparation procedure;

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F. Taverna et al. / Veterinary Microbiology 119 (2007) 240247

Table 1
Proteins identified by mass spectrometry
Spot no.

Identified
protein

Coverage
(%)

Accession
number

1.131
0.303
0.904

45
32
60

Q53779
SA0774
Q6GKG5

//

0.764

29

Q7A606

//

0.625

40

Q7A5R3

68,654
48,706
22,357

4
3
//

2223
3435
//

0.001
0.277
0.330

36
27
30

Q6GCX4
JC4555
Q6GFK2

51,400
36,047
41,382

1
1
1

//
//
//

0.514
0.046
0.484

44
44
45

P99112
Q6GBM4
P60089

32,910

//

//

0.484

47

P99117

59,855
59,871
52,850
34,674

2
2
2
//

//
//
//
//

0.115
0.105
0.173
0.127

34
40
31
42

Q6G8J5
Q99TD2
P99106
Q6G716

35,552
30,411

//
1

//
//

0.165
0.359

31
36

Q99U11
Q99V48

Detoxification
1113
Catalase
30, 31
Alkyl hydroperoxide reductase C
42, 43
Superoxide dismutase SodA

58,379
21,133
22,711

//
//
//

//
//
//

0.846
0.331
0.576

60
74
66

Q8NWV5
BAB41593
Q9Z5W5

Transcription
32
33
40

36,060
28,755
23,909

//
//
//

//
//
//

0.230
0.179
0.280

60
49
45

P99175
P63844
Q7A4V0

Protein synthesis and folding

1
DnaK protein
2
GroEL
36
Traslation elongation factor Tu
44, 45
50S ribosomal protein L4

66,361
56,297
43,103
22,451

//
1
//
//

//
//
//
//

0.523
0.150
0.251
0.348

41
30
65
62

P99110
Q8VTM8
P99152
P61059

Other
21
25
38, 39
49
50

67,182
34,702
39,973
26,733
56,274

1
//
1
1
1

//
//
2324
3031
2837

0.425
0.120
0.504
0.706
0.708

35
31
34
31
40

Q6GKP0
Q7A417
Q8CQ38
Q6GC59
Q6GCY0

Transport and binding

710
Immunoglobulin G binding protein A
35
ABC transporter ATP-binding protein
36
Binding protein dependent
transport systems membrane
48
Fibronectin binding protein
Cell wall metabolism
20
Factor essential for expression of
methicillin resistance (FemA)
23
Capsular polysaccharide synthesis enzyme
24
Serine-Type D-Ala-D-Ala Carboxypeptidase
46, 47
Putative membrane protein (putative
glucosamine 6-P dehydrogenase)
Energy metabolism
22
ATP synthase beta chain
26
Alcohol dehydrogenase
27, 28
Piruvate dehydrogenase
E1 component, a-subunit
37
Fructose-bisphosphate aldolase class I
Amino acid,
14
15, 16
1719
29
34
41

cofactor and purine metabolism

Formate-tetrahydrofolate ligase
Formyltetrahydrofolate synthetase
Inosine-5-monophosphate dehydrogenase
Putative D-isomer specific
2-hydroxyacid dehydrogenase
Hypothetical protein ansA
Naphthoate synthase

regulation
Catabolite control protein A
Trascription pleiotropic repressor codY
Hypothetical protein SA1666

Putative membrane protein

Hypothetical protein SA2098
Lipase A/Esterase
Exotoxin
Putative exported protein

Mw

TM

Signal P

51,320
28,275
29,388

2
//
6

136
//
3839

65,810

//

49,139

GRAVY
value

F. Taverna et al. / Veterinary Microbiology 119 (2007) 240247

preliminary experiments confirmed the protocol based

on controlled lysis by lysostaphin under isotonic
conditions originally reported in Vytvytska et al.
(2002) was suitable to obtain reproducible starting
material (not shown). While our investigation was in
progress, this protocol was confirmed as the best
method for obtaining a preparation enriched in surface
proteins, in terms of spot number and image quality, by
Nandakumar et al. (2005). These authors compared
different cell lysis methods to obtain membrane and cell
wall associated proteins from S. aureus (strain Phillips)
isolated from a human patient diagnosed with
osteomyelitis. The protocol used in our study, described
in Section 2, differs from the one reported in
Nandakumar et al. (2005) mainly for the presence of
a dialysis step to remove raffinose. Adding this step
resulted in better recoveries of proteins in the following
precipitation step with TCA, and for the use of larger
gels in 2-DE, which allowed better spot resolution and
reduction in streaking compared with the results
reported in Nandakumar et al. (2005) using mini-gels.
As the first step toward the complete proteomic
characterization of cell wall components in pathogenic isolates, we set up a 2-DE reference map of
surface proteins from a strain isolated from cow
mastitis and selected for its pathogenic activity
(Zecconi et al., 2006). The importance of a diseaseand environment-specific analysis of isolates has
already been emphasized (Fux et al., 2005; Lammers
et al., 2000; Scherl et al., 2005) and confirmed by the
comparison between the results reported in the present
research and in Nandakumar et al. (2005). Although
both investigations were conducted using basically the
same protocols and the number of identified spots is
nearly the same in the two studies, only 5 out of the 33
proteins identified in the present research were listed
among the proteins identified in Nandakumar et al.
(2005): DnaK (spot 1), protein A (spots 710), ATPsynthase (spot 22), alkyl hydroxiperoxide reductase C
(spots 30, 31) and ABC transporter ATP-binding
protein (spot 35). Such observation confirms a highly
selective expression of most abundant surface proteins
by the two different clinical isolates.
As detailed in Section 3, most of the proteins
identified in the present study are predicted as either cell
wall or membrane associated proteins based on analysis
of their primary structure, confirming the procedure is
suited for the analysis of this subproteome of S. aureus.

245

However, the presence in our extracts of polypeptides

usually considered as strictly cytoplasmic needs further
comments. Although abundant cytoplasmic proteins
are often found in the enriched cell surface fractions
(Cordwell et al., 2001; Nandakumar et al., 2005) and are
often considered as contaminant components, it should
be pointed out that for most of such proteins listed in
Table 1, a possible partial association with membrane
and cell wall has been proposed in previous studies
using various microorganisms. In particular, the stress
proteins GroEL, DnaK and the cytosolic elongation
factor TU (EF-TU) are very abundant proteins often
identified in the insoluble fraction of different bacteria
despite they are not usually considered cell surface
exposed proteins. However, very recently, GroEL was
shown to be present predominantly in the structurebound fraction in Helicobacter pylori (Backert et al.,
2005). Moreover, enhanced expression of GroEL was
observed after exposure to oxacillin, suggesting that
this component could play a role in the antibiotic-stress
response (Singh et al., 2001).
DnaK is a major surface-exposed antigen in H.
pylori (Backert et al., 2005). The authors suggest that in
such organism DnaK may be a stress-induced surface
adhesin capable of mediating the recognition of
receptors in the epithelial cells and a possible target
for the development of effective vaccines. A similar role
for DnaK and EF-TU, as plasminogen binding proteins,
has been recently suggested in Listeria monocytogenes
(Schaumburg et al., 2004), while EF-TU has been
characterised as a plasminogen binding protein also in
Candida albicans (Crowe et al., 2003). Moreover, the
data in Backert et al. (2005) show that this protein in H.
pylori is 95% structure-bound distributed. This confirms the observations of Dallo et al. (2002) in
Mycoplasma influenzae where EF-TU, in addition to
its major cytoplasmic biosynthetic role, was also
present in a subpopulation of a surface translocated
molecule showing fibronectin binding ability. Therefore, it would be of interest to examine whether these
three proteins (GroEL, DnaK and EF-TU) might posses
similar roles in S. aureus as suggested by their presence
in surface associated protein preparations.
A similar role as plasminogen binding proteins has
been proposed by a proteomic analysis of cell wall
proteins of C. albicans for alcohol dehydrogenase,
catalase, fructose-bisphosphate aldolase (Crowe et al.,
2003) and as a species-specific antigen of pathogens

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F. Taverna et al. / Veterinary Microbiology 119 (2007) 240247

for alkyl hydroperoxide reductase C (Rhee et al.,

2005), whose partial surface localization has been
confirmed also in Saccharomyces cerevisiae (Ogita
et al., 2005). It has been suggested that hydroxiperoxide reductase, catalase and superoxide dismutase at
the bacterial surface may act as defence enzymes
against oxidants produced by macrophages and
neutrophils (Crowe et al., 2003; Rhee et al., 2005).
Finally, cell wall localization of hypothetical protein
asnA has been reported in S. cerevisiae (Ogita et al.,
2005; Sinclair et al., 1994).

5. Conclusions
In our knowledge, this is the first report of a
proteomic characterization of a strain of S. aureus
isolated from a case of bovine mastitis. As confirmed
by the selected examples reported above, the recent
development of proteomic approaches allows uncovering possible multiple localizations and roles for
many proteins, thus requiring re-evaluation of the
significance of the presence of cytoplasmic proteins
in membrane and surface associated preparations.
The data reported in this paper increase the
knowledge on this field since they meet the need to
set up reproducible and well resolved 2-DE reference
map of surface associated proteins suitable. These maps
could be used in future studies aimed at comparing
different strains selected for their pathogenic activity
against cows and at identifying vaccine candidate
antigens by a serological proteome analysis (SERPA).

Acknowledgements
We thank Francesco Corniola for the assistance in
figures preparation and Dr. Dario J. Roncelli for
technical assistance. This work was supported by
grants from FIRST 20032004 (University of Milano)
and by grant from PRIN 2003-2005 (MIUR-Italy).

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