RESEARCH ARTICLE

Genomic and proteomic characterization of

Staphylococcus aureus mastitis isolates of bovine origin

Carmen Wolf1, Harald Kusch1�, Stefan Monecke2, Dirk Albrecht1, Silva Holtfreter 3�, Christofvon Eiff 4�, Wolfram Petzl5, Pascal Rainard6, Barbara M. Broker3 and Susanne Engelmann1

1 Institute for Microbiology, University of Greifswald, Greifswald, Germany2 Institute of Medical Microbiology and Hygiene, Technical University of Dresden, Dresden, Germany3 Institute for Immunology and Transfusion Medicine, University of Greifswald, Greifswald, Germany4 Institute for Medical Microbiology, University Hospital M .unster, M .unster, Germany5 Clinic for Ruminants, Ludwig-Maximilians-University, Munich, Germany6 INRA, UR Infectiologie Animale et Sante Publique, Nouzilly, France

Received: November 1, 2010

Revised: March 11, 2011

Accepted: March 14, 2011

Staphylococcus aureus colonizes and infects humans as well as animals. In the present study,

17 S. aureus strains isolated from cows suffering from mastitis were characterized. The well-

established multilocus sequence typing (MLST) technique and a diagnostic microarray

covering 185 S. aureus virulence and resistance genes were used for genetic and epidemio-

logical analyses. Virulence gene expression studies were performed by analyzing the

extracellular protein pattern of each isolate on 2-D gels. By this way, a pronounced hetero-

geneity of the extracellular proteome between the bovine isolates has been observed

which was attributed to genome plasticity and variation of gene expression. Merely 12

proteins were expressed in at least 80% of the isolates, i.e. Atl, Aur, GlpQ, Hla, LtaS, Nuc,

PdhB, SAB0846, SAB2176, SAB0566, SspA, and SspB forming the core exoproteome. Fifteen

extracellular proteins were highly variably expressed and only present in less than 20% of the

isolates. This includes the serine proteases SplB, C, and F, and the superantigens SEC-bov,

SEL and TSST-1. Compared to human isolates we identified at least six proteins with

significantly different expression frequencies. While SAB0846 was expressed more frequently

in bovine isolates, LytM, EbpS, Spa, Geh, and LukL1 were seen less frequently in these

isolates.

Keywords:

Bovine mastitis / Exoproteome / Microbiology / Staphylococcus aureus

1 Introduction

Staphylococcus aureus is an important pathogenic bacterium

with a broad host range. It colonizes and infects humans as

well as farm and wild animals. In farm animals, S. aureus is

among the most common etiologic agents of bovine mastitis

and one of the pathogens most frequently isolated from raw

milk [1]. Mastitis is characterized by an inflammation of the

mammary gland that can be induced in all mammalians in

response to (intramammary) bacterial infections. As an

endemic disease it causes big financial/economic loss to the

dairy industry worldwide, mainly due to reduced milk

production and the need to discard contaminated milk [2, 3].

Abbreviations: CV, core-variable; MLST, multilocus sequence

typing; MSCRAMM, microbial surface components recognizing

adhesive matrix molecule; PBMC, peripheral blood mononuclear

cell; SAg, superantigen; ST, sequence type; TSB, tryptic soy

broth

�Current addresses: Harald Kusch, Institute for Microbiology and,

Genetics, University of Gottingen, Gottingen, Germany,

Silva Holtfreter, Department of Molecular Medicine and Pathology,

University of Auckland, Auckland, New Zealand,

Christof von Eiff, Pfizer Pharma, Berlin, Germany

Correspondence: Dr. Susanne Engelmann, Institut f .ur Mikro-

biologie, Ernst-Moritz-Arndt-Universit .at, F.-L.-Jahn-Str. 15,

D-17487 Greifswald, Germany

E-mail: Susanne.Engelmann@uni-greifswald.de

Fax: 149-3834-864202

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Proteomics 2011, 11, 2491–2502 2491DOI 10.1002/pmic.201000698

In contrast to mastitis caused by environmental patho-

gens such as Escherichia coli, S. aureus mastitis is contagious.

Bacteria are transmitted mainly during the milking process

[4]. Most of these infections take a subclinical course with

mild symptoms or they persist unnoticed, often resulting in

a long-lasting chronic form, which is particularly difficult to

treat [5].

The severity and clinical outcome of S. aureus infections

strongly depends on host factors such as the immune status

of the infected host. In addition, the virulence potential of

S. aureus that is characterized by its virulence gene equip-

ment may contribute to the course of infection [6]. S. aureushas the capacity to produce a large variety of virulence

factors, such as toxins, cell-surface-associated adhesins and

secreted exoproteins. It has been proposed that any S. aureusgenotype possesses the potential to transform into a life-

threatening pathogen, although strains from some clonal

lineages are more virulent than others [7].

Numerous typing methods have been applied to investi-

gate the genetic heterogeneity of S. aureus and revealed the

clonal population structure of the species [8]. They enable

epidemiological investigations to ascertain the source

and chain of infection in disease outbreaks [9, 10].

Molecular epidemiological analysis and comparative geno-

mics of bovine S. aureus demonstrated that a small number

of clonal types with a broad geographical distribution

caused the majority of mastitis cases [11, 12] and that a

limited number of genes or gene combinations confer host

specificity [13].

The genome of S. aureus consists of conserved and variable

regions [14]. The ‘‘core genome’’ is present in more than 95%

of the S. aureus strains and comprises approximately 75% of

all genes that are mainly associated with housekeeping (or

growth) and survival functions. It is subdivided into the so-

called ‘‘core-stable’’ genome and the ‘‘core-variable’’ (CV)

genome. CV genes make up approximately 10% of the

genome and show substantial variation between different

S. aureus strains, but they are typically stable and transferred

vertically [15]. Some of the CV genes encode virulence factors

involved in pathogenesis, e.g. toxins, superantigens (SAgs),

exoenzymes, and regulatory elements.

The remaining 25% of S. aureus genes encode non-

essential functions, ranging from virulence, resistance and

miscellaneous metabolic functions. Most of these ‘‘variable

or accessory’’ genes are carried on mobile (or once mobile)

genetic elements (MGEs). These elements, including

bacteriophages, S. aureus pathogenicity islands (SaPI),

staphylococcal cassette chromosomes (SCC), plasmids, and

transposons, are likely to transfer horizontally among

staphylococci [14] and contribute to the considerable genetic

heterogeneity of S. aureus and consequently to its enhanced

niche adaptation and virulence [16].

Whole genome sequencing of the common bovine

S. aureus strain RF122 identified a set of genetic features

that distinguishes this successful bovine clone from the

numerous sequenced human S. aureus strains. It was

proposed that specialized bovine clones diverged from

common human-associated ancestors through acquisition

and loss of DNA [17]. Kozytska et al. showed some genes are

specific for bovine isolates, but none of them was present in

all bovine isolates [18].

In the present study, 17 S. aureus isolates were subjected

to comprehensive genetic and proteomic analysis to reveal

virulence factors characteristic for bovine mastitis. Genetic

variability was determined by multilocus sequence typing

(MLST) and DNA microarrays. In addition we performed

2-D protein gel analyses, since we have previously shown

that differences in protein expression greatly contribute to

the diversity of the species S. aureus beyond the variability

that is conferred at the gene level. Finally, we analyzed

RNAIII transcription and studied T-cell-stimulating activ-

ities of the strains using both human and bovine peripheral

blood mononuclear cells (PBMC).

2 Materials and methods

2.1 Bacterial strains

Sixteen S. aureus isolates were derived from cows suffering

from clinical or subclinical mastitis in different geographical

regions (Table 1). All strains were identified as S. aureus by

plating and detection of genes encoding coagulase (coa) and

thermonuclease (nuc). Antibiotic sensitivity was determined

by the automated Vitek 2 system (bioMerieux, N .urtingen,

Germany). The sequenced S. aureus mastitis strain RF122

was used as a reference for genomic and proteomic studies

(Table 1). We used S. aureus COL and RN6390 as reference

strains for RNAIII transcription analyses [19, 20]. Control

strains for detection of the agr-type included COL (agr-1),

N315 (agr-2), TY114 8 (agr-3), and A920210 (agr-4)

[19, 21–23].

2.2 MLST

Total DNA of S. aureus was isolated with the WizardTM

Genomic DNA purification kit (Promega, Madison, WI,

USA) according to the manufacturer’s instructions with

some modifications. Most importantly, the addition of

0.6 mg lysozyme and 0.1 mg lysostaphin per sample resulted

in more efficient cell wall degradation. Isolated chromoso-

mal DNA was resolved in distilled water and stored at

�201C.

The protocol published by Enright et al. [24] was used for

MLST analyses. The resulting DNA sequences were

submitted to the Internet database (www.mlst.net). For each

isolate an allelic profile was generated which results in a

particular sequence type (ST). To classify different STs into

clusters of clonal complexes, the eBURST (based on related

sequences) algorithm software was applied (http://saureus.

mlst.net/eburst/) [25].

2492 C. Wolf et al. Proteomics 2011, 11, 2491–2502

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

2.3 Microarray hybridization

The diagnostic DNA microarray (StaphyType; Alere Tech-

nologies, Jena, Germany) used for this study covers about

300 target sequences corresponding to 185 distinct genes

and their allelic variants. A complete list of targets, as well as

the principle of the assay and related procedures, have

previously been described in detail [10, 26].

Additionally, to distinguish between agr types I and IV in

some ambiguous cases we used a multiplex PCR approach

for agr-1 to -4 as described previously [22].

2.4 Split network tree construction

Adapted from Monecke et al. [26], a split network tree was used

to visualize similarities between hybridization patterns. The

results of all array hybridization experiments were arranged in

a matrix where the columns represent the target genes and the

rows represent the experiments. The hybridization results

were converted into ‘‘sequences’’ using ‘‘A’’ for positive, ‘‘T’’

for negative results and ‘‘C’’ for ambiguous. Afterwards, the

matrix was converted into a series of ‘‘sequences.’’ These were

used for tree construction using SPLITSTREE 4.1

(www.splitstree.org) software (parameters: NJ, convex hull).

2.5 Preparation of extracellular protein extracts

Bacteria were cultivated in tryptic soy broth (TSB) under

vigorous agitation at 371C to an optical density (OD540) of 10.

One hundred millilitres of culture were harvested by centri-

fugation (10 min, 41C, 10 000� g). Extracellular proteins were

precipitated from the supernatant with 10% w/v trichloro-

acetic acid (TCA) at 41C overnight. After centrifugation

(45 min, 41C, 10 000� g), the precipitates were washed

several times with 96% v/v ethanol and dried. Protein extracts

were resuspended in an appropriate volume of 8 M urea and

2 M thiourea. The protein concentration was determined by

using Roti-Nanoquant according to the manufacturer’s

instructions (Carl Roth, Karlsruhe, Germany).

2.6 Analytic and preparative 2-D-PAGE

Protein extracts (80 mg) were separated according to their pIon commercially available linear immobilized pH gradient

(IPG) strips in a pI range of 3–10 (GE-Healthcare, Uppsala,

Sweden). 2-D PAGE using the IPG technique was

performed as described previously [27–29]. Protein gels were

stained with KryptonTM Protein Stain (Thermo Scientific,

USA) according to the manufacturer’s instructions (http://

www.piercenet.com/files/1829kc9.pdf), scanned with the

Typhoon 9400 Scanner with a 532 nm laser light source.

Additionally, protein extracts (350 mg) were separated by

preparative 2-D gels as described above and the proteins

were stained with colloidal Coomassie brilliant blue (CBB)

G-250 [30] and scanned with the light scanner. For protein

identification by matrix-assisted laser desorption/ionization-

time of flight mass spectrometry (MALDI-TOF-MS),

proteins were cut from CBB-stained gels by using the spot

cutter Proteome Work (GE-Healthcare), digested and

analyzed as described previously [31, 32].

2.7 Transcriptional analyses

S. aureus cells were cultivated in TSB medium to an OD540

of 10 and total RNA was isolated using the acid–phenol

method with modifications as described previously [33, 34].

Table 1. Characteristics of the S. aureus mastitis isolates used in this study

S. aureus – strain Year of isolation Origin; reference Course of infection agr-type MLST CC

RF122 1993 Ireland [11]; GenBank: AJ938182) Clinical 2 151 705377 n/a W .urzburg, Germany n/a 1 115 97A 2006 Upper Bavaria, Germany Subclinical 2 1274 705B 2006 Upper Bavaria, Germany Subclinical 2 504 705C 2006 Upper Bavaria, Germany Subclinical 2 504 705D 2006 Upper Bavaria, Germany Subclinical 2 1275E 2006 Upper Bavaria, Germany Subclinical 2 479 479F 2006 Upper Bavaria, Germany Subclinical 2 504 7051027 2003 Hannover, Germany [64] Clinical 1 133 133D4-106.06 1961 Yonne, Burgundy, France [65] Subclinical 1 97 97D4-126.29 1977 Aveyron, southern France Probably subclinical 1 71 97D8-628.24 1992 Pays de Loire, western France Probably subclinical 1 389 20D8-644.15 1992 Savoie, Alps, France Probably subclinical 1 389 20D8-684.18 1993 Indre et Loire, Touraine, France Probably subclinical 2 1276D9-780.07 1997 Savoie, eastern France Probably subclinical 1 97 97D9-798.19 2004 Mayenne, Pays de Loir, France Clinical 1 352 97N305 1958 Ontario, Canada [66] Clinical 1 115 97

n/a, not available.

Proteomics 2011, 11, 2491–2502 2493

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Digoxygenin-labeled RNA probes for RNA III were prepared

by in vitro transcription with T7 RNA polymerase by using

PCR-generated fragments as templates. The PCR fragments

were generated by using chromosomal DNA of S. aureusCOL isolated with the chromosomal DNA isolation kit

(Promega) according to the manufacturer’s instructions and

the respective oligonucleotides as described by Ziebandt

et al. [32].

Northern blot analyses were carried out as previously

described [35]. The digoxygenin-labeled RNA marker I

(Roche) was used as a standard to calculate the sizes of the

transcripts.

2.8 T-cell proliferation assay

For preparation of S. aureus supernatants, S. aureus cultures

at OD540 of 10 were centrifuged (21 000� g, 5 min, 41C). Cell

debris was removed from the culture supernatants using

filters with a pore size of 2mm. The cell-free supernatants

were stored at �201C.

Human and bovine PBMC were isolated by density

centrifugation over Ficoll (Biocoll Separating Solution,

Biochrom, Berlin). Human blood was obtained from healthy

blood donors from the Department of Transfusion Medicine

in Greifswald. Bovine blood samples were taken from a

healthy cow. Human and bovine cells were cultured in

96-well flat-bottomed plates as described previously [36]. For

bovine cells Iscove’s medium instead of RPMI was used. The

cells were stimulated for proliferation and harvested accord-

ing to the protocol described by Holtfreter et al. [36]. Incor-

porated [3H]-thymidine was quantified. Two biological

replicates each in technical triplicates and with two different

blood donors were quantified. Standard errors of the means

(SEM) of technical triplicates and SEM of biological replicates

were below 20% except for values of below 1000 cpm.

3 Results

3.1 Clonal relationship of the bovine S. aureus

isolates

First, the 17 bovine S. aureus isolates were assessed by MLST

to ascertain their clonal relationship. They belonged to 12

different STs (Fig. 1). Three of them were new (ST1274,

ST1275, and ST1276) and were submitted to the MLST

database (http://www.mlst.net). The new STs resulted from

new combinations of known alleles (ST1275 and ST1276) or

from the presence of a new allele (tpi-157 in ST1274). The

isolates clustered into five clonal complexes with six isolates

assigned to CC97, five to CC705, two to CC20, and one

strain to CC479 and CC133, respectively. The remaining two

isolates are singletons.

Of the 16 isolates three belong to lineages of known

prevalence in cattle: ST133 (n 5 1), and ST97 (n 5 2)

[13, 37–41]. ST133 represents one of the most common

animal-associated MLST types, and it has been rarely found

in humans so far [41]. A further seven isolates were assigned

to STs closely related to common intercontinental bovine

mastitis lineages: ST115 (CC97), ST71 (CC97), ST352

(CC97), and ST504 (CC705 (formerly CC151)). The two

ST389 isolates (CC20) have so far only been described in

humans albeit at low frequency [42]. Nine isolates were

classified as belonging to agr-type I (CC97, CC133, and

CC20) and six isolates to agr-type II (CC 151, CC479, and the

three singletons) (Table 1).

3.2 Detection of virulence-associated genes using

a diagnostic DNA microarray

The prevalence of 185 genes was determined using a diag-

nostic DNA microarray [26], which covered regulatory genes,

genes encoding virulence factors and microbial surface

components recognizing adhesive matrix molecules

(MSCRAMMs), capsule type-specific genes, as well as

resistance determinants in the different staphylococcal

strains including RF122.

The results are presented in the Supporting Information

Table S1. Altogether, 43 of the virulence genes were

conserved among the bovine S. aureus isolates. These are the

hemolysin-encoding genes hla, hlb, and hld, the two genes hl(SACOL0921) and hlIII (SACOL2160) with homologies to

hemolysin-encoding genes, the protease-encoding genes

aur, sspA, sspB, sspP, splA, and splB, the ssl genes setB, setB2,

50.00

26.79

42.86

24.29

35.71

0.71

10.71

11.43

3.57

7.14

7.14

N305

377

D4-106.06

D9-780.07

D4-126.29

D9-798.19

D9-628.24

D8-644.15

E

D

1027

F

C

B

A

D8-684.18

7.14

7.14

14.29

14.29

14.29

35.71

7.14

1276

705

97

115

115

1274

504

504

504

133 133

1275

479

97

97

97

97

705

705

71

352

97

389

389 20

20

strain ST CC

97

97

RF12216.07

8.93

151 705

479

705

Figure 1. Dendogram of multilocus STs detected among

the bovine S. aureus isolates of this study. The tree was

computed by using concatenated nucleotide sequences of seven

housekeeping genes and was constructed using the tool

provided by the MLST database (http://saureus.mlst.net/sql/

uniquetree.asp?]). Bootstrap values are indicated above the

branches. Clonal complexes were calculated by using the

eBURST algorithm.

2494 C. Wolf et al. Proteomics 2011, 11, 2491–2502

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

setB3, setC, ssl1, ssl5, and ssl3, the leukocidin genes hlgA and

hlgB ( 5 lukF) and hlgC ( 5 lukS) and lukX/Y and other

virulence genes as coa, sbi, spa, nuc, hysA1, hysA2, isaB, and

isdA. Moreover, we found that all isolates harbored the

MSCRAMM genes clfA, clfB, ebh, ebpS, eno, fib, fnbA, map,

sdrC, and vwb. Genes encoding capsular polysaccharide

biosynthesis proteins of serotype 5 (capH5/J5/K5) and

serotype 8 (capH8/I8/J8/K8) were similarly distributed

among the strains (eight and seven strains, respectively).

Other virulence genes were more variable. In nine

isolates we detected SAg-encoding genes, the enterotoxin

gene cluster (egc containing seg, sei, sem, sen, seo, and seu)

being most frequent (eight isolates). Two strains addition-

ally contained the bovine variants of tst1 and sec, and sel,indicating that they harbor the pathogenicity island SaPIbov

which is specific for bovine isolates [43]. Moreover, an

enterotoxin-like gene ORF CM14 was detected in five

strains.

Genes encoding the bovine specific virulence genes lukMand lukF’-PV were detected in 50% of the strains.

As expected, specific human S. aureus virulence genes

such as those encoding the Pantone Valentine Leukocidin

(PVL) (lukS-PV1lukF-PV), the exfoliative toxins (eta, etb, and

etd) or the epidermal cell differentiation inhibitors (edinA-C)

were absent from the bovine S. aureus isolates (Supporting

Information Table S1). Also, genes located on hlb-integrat-

ing phages, sak, sea, chp, and scn were not detected [44].

Accordingly, hlb was not disrupted in all isolates.

Genes associated with antibiotic resistance in human

isolates were rarely detected in the bovine isolates

(Supporting Information Table S1). For example, genes

conferring resistance to b-lactam antibiotics (blaZ, blaI, and

blaR) were only found in strain D4-126.29, the vanB gene in

strain D9-780.07 and the fosB gene in strains D8-628.24 and

D8-644.15. Other genes involved in antibiotic resistance

present on the DNA microarray have not been detected in

any of the isolates.

By split network tree construction [26] we compared the

overall hybridization patterns of the DNA-arrays of the

different isolates (Fig. 2). Some isolates cluster together

indicating a very similar virulence and resistance gene

pattern (Fig. 2). In general, these isolates belong to the same

STs (D8-628.24 and D8-644.15) or to closely related STs

(N305, 377, and D4-106.06; D9-780.07 and D9-798.19; A, B,

C, and F, E and D) (Fig. 1).

3.3 The exoproteome of the different bovine

isolates

From previous studies we knew that many virulence factors

in S. aureus are regulated by the quorum sensing system agrand are induced under high ODs [32, 45]. An agr mutant was

impaired in virulence in different mouse models indicating

that the expression of at least some of these proteins was

relevant for infection [46–49]. To get a first impression of

virulence gene expression of different clinical S. aureusisolates, a detailed comparison of the virulence factor patterns

under highly standardized in vitro growth conditions was

required, preferably at high ODs. Since many virulence

factors are known to be secreted, we focused on the exopro-

teome of these isolates. Extracellular proteins of the 17 bovine

S. aureus isolates (including RF122) were prepared from the

supernatants of bacterial cell cultures grown in TSB medium

to OD540 of 10 and separated on 2-D gels.

Altogether 232 different proteins were identified by

MALDI-TOF MS/MS (Supporting Information Table S2).

Seventy-four of them possessed signal sequences specific for

the Sec-dependent translocation machinery. Using PSORTb

(http://www.psort.org/psortb), 44 of these proteins were

predicted to be extracellular and eight cell wall-associated.

The localization of a further 37 proteins has not been exactly

predicted: 18 contained a signal peptide and 19 did not.

Similar to other studies [32, 50], we identified possible

membrane-associated (six proteins) and a considerable

number of cytoplasmic proteins (139 proteins) in the

exoproteomes of the bovine mastitis isolates.

As expected from our genomic studies, the extracellular

protein patterns varied considerably between the different

isolates (Fig. 3). Interestingly, only one protein (LtaS) was

found in the exoproteome of all isolates (Supporting Infor-

mation Table S2). LtaS (formerly YfnI) is a polytopic

membrane protein, which is processed by signal peptidase I

(SspI) as was previously shown for the homologue of

N305, 377D4-106.06

D9-780.07D9-798.19

D4-126.29

D8-644.15D8-628.24

D8-684.18

1027

ED

FC B

ARF122

0.01

Figure 2. Split network tree based on hybridization results for

every single gene on the diagnostic DNA microarray. The tree

was generated by Splitstree version 4.1 (http://www.splitstree.

org/) and shows genetic relationships between different bovine

S. aureus isolates.

Proteomics 2011, 11, 2491–2502 2495

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

BA DC

FE N305 377

1027 D4-106.06 D4-126.29 D8-628.24

D8-644.15 D8-684.18 D9-780.07 D9-789.19

10

pI[pH]

10 3

MW[kDa]

150

B

ApH 10 pH 3

Figure 3. Extracellular proteomes

of different S. aureus isolates

from bovine mastitis infections.

Cells were grown in TSB medium

to the stationary phase. Proteins

of the culture supernatants were

collected by trichloroacetic acid

precipitation and separated by 2-

D protein gel analysis. Protein

spots were detected by staining

with KryptonTM Protein Stain

(Thermo Scientific). Labeled

proteins were identified by MS.

(A) 2-D protein map of secreted

proteins of S. aureus RF122.

Protein spots are labeled with

protein names as listed in the

Supporting Information Table S2

and colored according to their

localization as predicted by

PSortb: extracellular (red),

unknown with signal peptide

(black), cell wall associated

(green), membrane bound

(violet), cytoplasmatic (blue),

unknown (gray). Superscript ‘‘F’’

indicates protein fragments. (B)

2-D protein gels of extracellular

proteins of different mastitis

isolates. The respective isolate is

indicated in the upper left corner

of each gel.

2496 C. Wolf et al. Proteomics 2011, 11, 2491–2502

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

B. subtilis [51]. Depletion of ltaS impaired LTA synthesis

and thus caused defects in cell division in S. aureus [52].

Eleven proteins (i.e. Atl, Nuc, SAB0566, GlpQ, Hla, SAB2176,

SspA, SspB, Aur, SAB0846, and PdhB) were expressed

in at least 80% of the isolates, with eight of them being

predicted as secreted proteins (Supporting Information

Table S2). Together with LtaS they represent the core

exoproteome of the bovine S. aureus mastitis isolates.

According to PSORTb prediction, two proteins (SAB0566 and

SAB0846) are of unknown subcellular localization but

possess signal sequences, and only one cytoplasmic protein

(PdhB) belongs to the core exoproteome. Strikingly, the

protein amounts of the eleven members of the core exopro-

teome varied significantly between isolates (Supporting

Information Fig. S1).

Fifteen extracellular proteins were almost unique, i.e.

they were found secreted by at most three of the 17 isolates.

Among them were well-characterized virulence factors such

as SAgs toxic shock syndrome toxin-1, SEC-bov and SEL,

serine proteases SplB, C, and F, FPRL1 inhibitory protein

FLIPr, HysA1and HysA2, Pbp2, Ssl11, and Ssl9 but also

proteins with unknown function (SAB0739, SAB1980c, and

SAB2421c) (Supporting Information Table S2).

As already mentioned, we found four groups of isolates

whose genetic composition was very similar: (i) D8-628.24

and D8-644.15, (ii) N305, 377, and D4-106.06, (iii) D9-780.07

and D9-798.19, and (iv) A, B, C, F, and RF122. Sometimes,

this genetic similarity was reflected by similar protein

expression patterns as exemplified by N305 and 377 of

group II. More frequently, however, even strains with very

similar virulence gene equipment, differed strongly in their

exoproteome patterns. This became impressively clear

when we compared expression of those proteins whose

genes were conserved among the isolates belonging

to the same group as exemplified for isolates A, B, C, and F.

Of the 82 genes that have been shown to be conserved in

these isolates, the gene products of 20 genes were identified

on the extracellular proteome of at least one of the four

isolates. Strikingly, only six proteins (Aur, Hlb, LukD,

LukF’-PV, LukM, and SspA) were secreted by all four

isolates while 14 proteins were found to be expressed

in two or less isolates. Isolates B and F shared the highest

number of proteins (16 proteins) followed by isolate A and C

(13 proteins).

3.4 Mitogenic activity of bovine S. aureus culture

supernatants

To analyze the mitogenic activity of the S. aureusisolates, supernatants from bacterial cultures at an OD at

540 nm (OD540) of 10 were incubated with human and

bovine PBMCs, respectively. T-cell activation was deter-

mined by [3H]-thymidine incorporation. Despite donor-

dependent variations, the titers of the supernatants with

maximal proliferation as well as the (relative) degree

of T-cell stimulation were reproducible between the experi-

ments. Data from one representative experiment are shown

in Fig. 4.

Accordingly, nine isolates (A, B, C, D, E, F, D8-628.24,

D8-644.15, and D8-684.18) showed mitogenic activity, which

correlated well with the presence of SAg genes (Fig. 4).

Isolate D8-684.18 was exceptional, since no SAg gene

was found but nevertheless the bacterial supernatant

was mitogenic. Point mutations interfering with oligonu-

cleotide binding on the DNA-array or an unknown

SAg could be the reason. In contrast, ORF entCM14 does

not appear to encode a functional SAg, since the super-

natant of strain D4-126.29 was devoid of mitogenic

activity. The isolates B and C, which contained the bovine

pathogenicity island, stimulated bovine PBMCs much

more strongly than human cells. The others, which

only encoded egc SAgs (sometimes together with ORF

entCM14) showed no significant differences in mitogenic

activity when incubated with blood cells from humans or

cattle (Fig. 4).

3.6 Transcription of RNAIII in bovine isolates

Variations in virulence gene expression may be caused

by differential activities of S. aureus regulators such

as RNAIII [32]. As shown previously, a deletion of

RNAIII affects the virulence factor pattern of S. aureusdrastically [32, 45]. RNAIII transcription was analyzed

by Northern blot experiments with RNA prepared from

the different isolates grown to an OD540 of 10 in TSB

medium. Accordingly, RNAIII was not detectable in four

isolates (A, C, 1027, and D4-106.06) (Fig. 5), which is

reflected by a low-level virulence factor expression in these

isolates.

4 Discussion

In the post-genomic era, the availability of the genome

sequences of several S. aureus strains including one bovine

isolate provides the basis for a better understanding of

staphylococcal infection biology. However, despite extensive

comparative genomic studies of bovine and human S. aureusisolates, the molecular basis of host specificity and virulence

of bovine S. aureus mastitis clones is still a matter of debate

[13, 17, 18, 38, 53, 54]. By dissecting the exoproteomes of 25

clinical human S. aureus isolates, we have recently demon-

strated that, within a single bacterial species, the exopro-

teome composition can vary to the extreme. The diversity

was induced not only by genome variations, but also by an

exceptionally high variability in gene expression [32]. These

findings suggest that the combination of genomics and

proteomics will result in a more comprehensive picture of

the virulence potential of a given bacterial strain. The same

may be true for host specialization [32, 45, 55–57].

Proteomics 2011, 11, 2491–2502 2497

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

In the present study we have characterized 17 S. aureusisolates from cows suffering from clinical or subclinical

mastitis in different geographical regions (Table 1). Most of

them belong to clonal lineages known to be prevalent in

cattle or they are closely related to one of these lineages.

Only two bovine isolates were assigned to an ST exclusively

described in humans where they occur at low frequency.

The genetic variability of these isolates was extensive;

therefore, differences in the extracellular proteome pattern

were expected. However, even highly similar virulence gene

patterns did not necessarily result in similar virulence factor

expression as impressively shown for isolates A, B, C, and F

(Figs. 2 and 3B and Supporting Information Table S1). In

contrast, loss of RNAIII, possibly due to mutations within

Figure 5. Transcription of

RNAIII in different bovine

mastitis isolates. Total RNA

was prepared from cells grown

in TSB to an OD540 of 10. Total

RNA (2 mg) was separated on a

denaturating agarose gel and

subsequently blotted and

crosslinked onto a positively

charged nylon membrane. The

membrane-bound RNA was

hybridized with the digox-

ygenin-labeled RNA probe

complementary to RNAIII. S.

aureus RN6390 served as a

RNAIII-positive control. The

arrow indicates the size of the

transcript.

titer

0

20

40

60

80

100

120

140

160A B

titer

10–7 10–6 10–5 10–4 10–3 10–2 10–110–8

10–7 10–6 10–5 10–4 10–3 10–2 10–110–80

20

40

60

80

100

120

140

160

T c

ell p

rolif

erat

ion

[cpm

x 1

03 ]

seg, sei, sem, sen, seo, seu, ORF entCM14

seg, sei, sem, sen, seo, seu, sec-bov*, sel*, tst*, ORF entCM14

seg, sei, sem, sen, seo, seu, sec-bov*, sel*, tst*, ORF entCM14

seg, sei, sem, sen, seo, seu

seg, sei, sem, sen, seo, seu

seg, sei, sem, sen, seo, seu, ORF entCM14

-

-

-

-

ORF entCM14

seg, sei, sem, sen, seo, seu

seg, sei, sem, sen, seo, seu

-

-

-

A

B

C

D

E

F

N305

377

1027

D4-106.06

D4-126.29

D8-628.24

D8-644.15

D8-684.18

D9-780.07

D9-798.19

bovine PBMC

human PBMC

symbol

T c

ell p

rolif

erat

ion

[cpm

x 1

03 ]

superantigen encoding genesS. aureusstrain

Figure 4. Mitogenic activity of S. aureus culture supernatants. (A) S. aureus strains were grown in TSB to an OD540 of 10 and supernatants

were obtained by centrifugation and subsequent filter sterilization. T-cell mitogenic activity was assessed by stimulating human and

bovine PBMC with serial dilutions (10�1 to 10�8) of the culture supernatants. T-cell proliferation was measured after 72 h by [3H]-thymidine

incorporation. Values greater than two times of the background value were considered as significant T-cell activation. One representative

data set of three biological replicates is depicted for the strain A (�), strain B (m), strain C (& ), strain D(~), strain E(3), strain F (D), strain

D8-628.24 (&), strain D8-644.15 (� ) and strain D8-684.18 (B). The remaining strains (377, N305, 1027, D4-106.06, D4-126.29, D9-780.07,

and D9-798.19) showed no mitogenic activity. (B) Occurrence of SAg-encoding genes in the different S. aureus isolates. Our data show

differences in the extracellular proteomes between bovine and human S. aureus isolates.

2498 C. Wolf et al. Proteomics 2011, 11, 2491–2502

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

the agr locus, predicted low expression levels of late viru-

lence factors (A and C) (Figs. 3B and 5) that might be

associated with a specific virulence behavior. Consequently,

to understand virulence and host specialization of a given

S. aureus isolate, virulence gene expression has to be

considered besides genomic information.

With regard to specific differences in virulence gene

expression between human and bovine strains, our data

show a core exoproteome of the bovine isolates consisting of

12 proteins. These were found in the supernatant of at least

80% of the bovine isolates (Supporting Information Table

S2). The human S. aureus core exoproteome of 15 clonally

different isolates comprises 26 proteins and is therefore

significantly larger [32]. Since the number of proteins in the

supernatants of the bovine isolates was not different from

that of human isolates, the smaller core exoproteome indi-

cates a higher complexity and diversity of the total exopro-

teome of bovine isolates. Eight proteins were conserved

between bovine and human S. aureus isolates. These are

LtaS (YfnI), Nuc, GlpQ, SspA, Hla, Aur, SAB0566 (SA0570),

and SAB2176. The four proteins SspB, Atl, PdhB, and

SAB0846 (SA0841) were more frequent in bovine isolates.

While SspB, Atl, and PdhB were found in 67–74% of the

human isolates, SAB0846 was expressed by human isolates

with strikingly lower frequency (20%). SAB0846 codes for a

protein highly similar to Map proteins. Typical for the core

exoproteome of the human isolates were LytM, Geh, Spa,

and LukL1, which were found in less than 50% of the bovine

isolates, although the genes encoding LytM, Geh, and Spa

are conserved among bovine isolates.

The use of a diagnostic DNA microarray [26] facilitated a

comprehensive genomic profiling (Supporting Information

Table S1). In total, we identified 27 virulence genes that

belong to the core genome of the 17 bovine as well as the 15

human isolates (Supporting Information Table S1) [32]. In

the secretomes of these isolates, the gene products of 15 of

these genes were observed, most of them with almost the

same frequency in human and bovine isolates (Supporting

Information Table S2). The sole exceptions were EbpS and

Spa. Both proteins EbpS and Spa were frequent in the

secretomes of human isolates (72 and 88%) but rare in the

secretomes of bovine isolates (0 and 11.7%). There are at

least two explanations for this phenomenon: either the

respective genes represent pseudogenes in bovine isolates (i)

or they are repressed (ii).

RF122 is the only sequenced bovine isolate thus far. This

strain also lacks the ability to express protein A, EbpS, Geh,

and LytM. Notably, the spa gene of RF122 contains premature

stop codons and thus is a pseudogene. The same is true for

spa in other successful bovine isolates such as ET3 isolates

different from RF122 and non-ET3 isolates [17]. The lack of

protein A in the supernatant of bovine isolates observed in

the present study supports the idea that premature trunca-

tions of the spa gene might be very common in origin S.aureus strains isolated from cattle. In contrast the ebpS, geh,

and lytM genes are intact in RF122 but obviously not

expressed. This might be induced by mutations in the regu-

latory regions of these genes and/or varying activities of

regulatory molecules. A very detailed comparison of the

upstream regions of lytM and geh, however, revealed no

significant sequence differences between strain RF122 and

strain COL which that abundantly express both genes [55].

The present data clearly show that virulence gene expres-

sion studies add an important dimension to our under-

standing of host specificity of S. aureus isolates which is not

covered by genomic studies. This has now been shown for the

extracellular as well as surface-associated proteins. In view of

the pronounced specific differences in the expression of

surface-associated proteins such as Spa, LytM, and EbpS

between bovine and human isolates, new techniques are now

required to analyze surface-associated virulence factors in

more detail. The method applied in this work to investigate

the extracellular proteome does not efficiently cover surface-

associated proteins such as MSCRAMMs, SERAMs and

lipoproteins. MS-based approaches, which rely on the

separation of complex protein or peptide mixtures by liquid

chromatography or 1-D SDS gel electrophoresis, allow the

identification of proteins in complex protein mixtures and

circumvent the obstacle of separation of proteins by 2-D gels

[58]. With specific targeting of the surface-associated

proteome, an even more comprehensive picture of the

features determining host specificity can be expected.

Despite extensive efforts in functional characterization of

virulence factors in S. aureus, the overall understanding of

the pathogenesis of S. aureus infection is limited. The

functions of at least 60% of the putative secreted proteins, as

derived from the genome sequence, are completely

unknown. In the secretome of the 17 bovine S. aureusisolates, at least 25 of these proteins have been identified

indicating that they are expressed. Functional characteriza-

tion of these particular proteins will be a challenging task for

future studies.

Previous studies suggest that SAgs are important during

mastitis pathogenesis due to their immunmodulatory effects

[59, 60]. SAg-encoding genes were identified in ten of the

isolates with three of them encoding the bovine variants of

SEC and TSST. Only three strains (RF122, B, and C) produced

the SAgs SEC and TSST in amounts detectable on 2-D gels.

However, T cells proved to be a more sensitive read out since

all ten SAg-positive strains induced T-cell proliferation. To test

whether these SAgs induce T-cell proliferation in humans and

cows with comparable intensities, human and bovine PBMCs

were compared. Two strains (B and C) stimulated bovine cells

much more efficiently than human cells suggesting some

degree of species specificity in SAg action. This is in line with

the previous observation that three amino acid differences

between SEC variants from bovine and ovine origin result in a

host-dependent superantigenic activity [61]. To ascertain

whether the SEC and TSST variants of bovine origin are

indeed more active in bovine than in human T cells, detailed

analyses have to be performed with recombinant SAg variants.

The egc SAgs appeared to be comparably active in bovine or

Proteomics 2011, 11, 2491–2502 2499

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

human PBMCs. ORF entCM14 probably does not encode a

functional SAg.

In summary, our data show differences in the extracellular

proteomes between bovine and human S. aureus isolates.

While EbpS, Geh, LytM, Spa, and LukL1 were more

frequently expressed in human isolates, for SAB0846 the

opposite was true. Most probably, the varying extracellular

protein patterns observed under highly standardized in vitro

conditions reflect a similar degree of variability in vivo.

Comparative genome sequencing of ruminant S. aureusisolates belonging to CC133 provided first evidence that

mechanisms of host adaptation involve gene decay and

diversification of proteins important in host pathogen inter-

action [17, 62, 63]. We propose that variability in the regula-

tion of virulence gene expression (in particular the loss of

expression of defined virulence genes) is also linked to the

host specificity of S. aureus.

We are indebted to Anita Harang and Thomas Meier forexcellent technical assistance. We thank Elke Lange (Friedrich-Loffler Institute, Riems, Germany) for providing bovine bloodsamples. The study was supported by research grants from theDFG (FOR 585, GRK 840, and SFB-TRR34), BMBF (ERA-NET Pathogenomics Network: sncRNAomics project) and EU(StaphDynamics: LSHM-CT-2006-019064).

The authors have declared no conflict of interest.

5 References

[1] Barkema, H. W., Green, M. J., Bradley, A. J., Zadoks, R. N.,

The role of contagious disease in udder health. J. Dairy Sci.

2009, 92, 4717–4729.

[2] Miller, G. Y., Bartlett, P. C., Lance, S. E., Anderson, J., Heider,

L. E., Costs of clinical mastitis and mastitis prevention in

dairy herds. J. Am. Vet. Med. Assoc. 1993, 202, 1230–1236.

[3] Seegers, H., Fourichon, C., Beaudeau, F., Production effects

related to mastitis and mastitis economics in dairy cattle

herds. Vet. Res. 2003, 34, 475–491.

[4] Bradley, A., Bovine mastitis: an evolving disease. Vet.

J. 2002, 164, 116–128.

[5] Sutra, L., Poutrel, B., Virulence factors involved in the

pathogenesis of bovine intramammary infections due to

Staphylococcus aureus. J. Med. Microbiol. 1994, 40, 79–89.

[6] Peacock, S. J., Moore, C. E., Justice, A., Kantzanou, M. et al.,

Virulent combinations of adhesin and toxin genes in natural

populations of Staphylococcus aureus. Infect. Immun. 2002,

70, 4987–4996.

[7] Melles, D. C., Gorkink, R. F., Boelens, H. A., Snijders, S. V.

et al., Natural population dynamics and expansion of

pathogenic clones of Staphylococcus aureus. J. Clin. Invest.

2004, 114, 1732–1740.

[8] Feil, E. J., Cooper, J. E., Grundmann, H., Robinson, D. A.

et al., How clonal is Staphylococcus aureus? J. Bacteriol.

2003, 185, 3307–3316.

[9] Maiden, M. C., Bygraves, J. A., Feil, E., Morelli, G. et al.,

Multilocus sequence typing: a portable approach to the iden-

tification of clones within populations of pathogenic micro-

organisms. Proc. Natl. Acad. Sci. USA 1998, 95, 3140–3145.

[10] Monecke, S., Jatzwauk, L., Weber, S., Slickers, P., Ehricht,

R., DNA microarray-based genotyping of methicillin-resis-

tant Staphylococcus aureus strains from Eastern Saxony.

Clin. Microbiol. Infect. 2008, 14, 534–545.

[11] Fitzgerald, J. R., Meaney, W. J., Hartigan, P. J., Smyth, C. J.,

Kapur, V., Fine-structure molecular epidemiological analy-

sis of Staphylococcus aureus recovered from cows. Epide-

miol. Infect. 1997, 119, 261–269.

[12] Kapur, V., Sischo, W. M., Greer, R. S., Whittam, T. S.,

Musser, J. M., Molecular population genetic analysis of

Staphylococcus aureus recovered from cows. J. Clin.

Microbiol. 1995, 33, 376–380.

[13] Sung, J. M., Lloyd, D. H., Lindsay, J. A., Staphylococcus

aureus host specificity: comparative genomics of human

versus animal isolates by multi-strain microarray. Micro-

biology 2008, 154, 1949–1959.

[14] Lindsay, J. A., Holden, M. T., Staphylococcus aureus:

superbug, super genome? Trends Microbiol. 2004, 12, 378–385.

[15] Lindsay, J. A., Moore, C. E., Day, N. P., Peacock, S. J. et al.,

Microarrays reveal that each of the ten dominant lineages of

Staphylococcus aureus has a unique combination of

surface-associated and regulatory genes. J. Bacteriol. 2006,

188, 669–676.

[16] Hacker, J., Carniel, E., Ecological fitness, genomic islands

and bacterial pathogenicity. A Darwinian view of the

evolution of microbes. EMBO Rep. 2001, 2, 376–381.

[17] Herron-Olson, L., Fitzgerald, J. R., Musser, J. M., Kapur, V.,

Molecular correlates of host specialization in Staphylo-

coccus aureus. PLoS One 2007, 2, e1120.

[18] Kozytska, S., StauX, D., Pawlik, M. C., Hensen, S. et al.,

Identification of specific genes in Staphylococcus aureus

strains associated with bovine mastitis. Vet. Microbiol.

2010, 145, 360–365.

[19] Shafer, W. M., Iandolo, J. J., Genetics of staphylococcal

enterotoxin B in methicillin-resistant isolates of Staphylo-

coccus aureus. Infect. Immun. 1979, 25, 902–911.

[20] Novick, R., Properties of a cryptic high-frequency transdu-

cing phage in Staphylococcus aureus. Virology 1967, 33,

155–166.

[21] Kuroda, M., Ohta, T., Uchiyama, I., Baba, T. et al., Whole

genome sequencing of meticillin-resistant Staphylococcus

aureus. Lancet 2001, 357, 1225–1240.

[22] Holtfreter, S., Grumann, D., Schmudde, M., Nguyen, H. T.

et al., Clonal distribution of superantigen genes in clinical

Staphylococcus aureus isolates. J. Clin. Microbiol. 2007, 45,

2669–2680.

[23] Jarraud, S., Peyrat, M. A., Lim, A., Tristan, A. et al., egc, a

highly prevalent operon of enterotoxin gene, forms a

putative nursery of superantigens in Staphylococcus

aureus. J. Immunol. 2001, 166, 669–677.

[24] Enright, M. C., Day, N. P., Davies, C. E., Peacock, S. J., Spratt, B.

G., Multilocus sequence typing for characterization of methi-

2500 C. Wolf et al. Proteomics 2011, 11, 2491–2502

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

cillin-resistant and methicillin-susceptible clones of Staphylo-

coccus aureus. J. Clin. Microbiol. 2000, 38, 1008–1015.

[25] Feil, E. J., Li, B. C., Aanensen, D. M., Hanage, W. P., Spratt, B.

G., eBURST: inferring patterns of evolutionary descent among

clusters of related bacterial genotypes from multilocus

sequence typing data. J. Bacteriol. 2004, 186, 1518–1530.

[26] Monecke, S., Slickers, P., Ehricht, R., Assignment of

Staphylococcus aureus isolates to clonal complexes based

on microarray analysis and pattern recognition. FEMS

Immunol. Med. Microbiol. 2008, 53, 237–251.

[27] B .uttner, K., Bernhardt, J., Scharf, C., Schmid, R. et al.,

A comprehensive two-dimensional map of cytosolic proteins

of Bacillus subtilis. Electrophoresis 2001, 22, 2908–2935.

[28] Bernhardt, J., B .uttner, K., Scharf, C., Hecker, M., Dual

channel imaging of two-dimensional electropherograms in

Bacillus subtilis. Electrophoresis 1999, 20, 2225–2240.

[29] Eymann, C., Dreisbach, A., Albrecht, D., Bernhardt, J. et al.,

A comprehensive proteome map of growing Bacillus

subtilis cells. Proteomics 2004, 4, 2849–2876.

[30] Candiano, G., Bruschi, M., Musante, L., Santucci, L. et al.,

Blue silver: a very sensitive colloidal Coomassie G-250

staining for proteome analysis. Electrophoresis 2004, 25,

1327–1333.

[31] Wolf, C., Hochgr .afe, F., Kusch,H., Albrecht, D. et al.,

Proteomic analysis of antioxidant strategies of Staphylo-

coccus aureus: diverse responses to different oxidants.

Proteomics 2008, 8, 3139–3153.

[32] Ziebandt, A. K., Kusch, H., Degner, M., Jaglitz, S. et al.,

Proteomics uncovers extreme heterogeneity in the Staphylo-

coccus aureus exoproteome due to genomic plasticity and

variant gene regulation. Proteomics 2010, 10, 1634–1644.

[33] Gertz, S., Engelmann, S., Schmid, R., Ohlsen, K. et al.,

Regulation of sigmaB-dependent transcription of sigB and

asp23 in two different Staphylococcus aureus strains. Mol.

Gen. Genet. 1999, 261, 558–566.

[34] Fuchs, S., Pane-Farre, J., Kohler, C., Hecker, M., Engelmann,

S., Anaerobic gene expression in Staphylococcus aureus. J.

Bacteriol. 2007, 189, 4275–4289.

[35] Wetzstein, M., Volker, U., Dedio, J., Lobau,S. et al., Cloning,

sequencing, and molecular analysis of the dnaK locus from

Bacillus subtilis. J. Bacteriol. 1992, 174, 3300–3310.

[36] Holtfreter, S., Bauer, K., Thomas, D., Feig, C. et al., egc-

encoded superantigens from Staphylococcus aureus are

neutralized by human sera much less efficiently than are

classical staphylococcal enterotoxins or toxic shock

syndrome toxin. Infect. Immun. 2004, 72, 4061–4071.

[37] Smith, E. M., Green, L. E., Medley, G. F., Bird, H. E. et al.,

Multilocus sequence typing of intercontinental bovine

Staphylococcus aureus isolates. J. Clin. Microbiol. 2005, 43,

4737–4743.

[38] Monecke, S., Kuhnert, P., Hotzel, H., Slickers, P., Ehricht, R.,

Microarray based study on virulence-associated genes and

resistance determinants of Staphylococcus aureus isolates

from cattle. Vet. Microbiol. 2007, 125, 128–140.

[39] Rabello, R. F., Moreira, B. M., Lopes, R. M., Teixeira, L. M.

et al., Multilocus sequence typing of Staphylococcus aureus

isolates recovered from cows with mastitis in Brazilian dairy

herds. J. Med. Microbiol. 2007, 56, 1505–1511.

[40] Jorgensen, H. J., Mork, T., Caugant, D. A., Kearns, A.,

Rorvik, L. M., Genetic variation among Staphylococcus

aureus strains from Norwegian bulk milk. Appl. Environ.

Microbiol. 2005, 71, 8352–8361.

[41] Smyth, D. S., Feil, E. J., Meaney, W. J., Hartigan, P. J. et al.,

Molecular genetic typing reveals further insights into the

diversity of animal-associated Staphylococcus aureus.

J. Med. Microbiol. 2009, 58, 1343–1353.

[42] Enright, M. C., Robinson, D. A., Randle, G., Feil, E. J. et al.,

The evolutionary history of methicillin-resistant Staphylo-

coccus aureus (MRSA). Proc. Natl. Acad. Sci. USA 2002, 99,

7687–7692.

[43] Fitzgerald, J. R., Monday, S. R., Foster, T. J., Bohach, G. A.

et al., Characterization of a putative pathogenicity island

from bovine Staphylococcus aureus encoding multiple

superantigens. J. Bacteriol. 2001, 183, 63–70.

[44] Coleman, D. C., Sullivan, D. J., Russell, R. J., Arbuthnott,

J. P. et al., Staphylococcus aureus bacteriophages

mediating the simultaneous lysogenic conversion of beta-

lysin, staphylokinase and enterotoxin A: molecular

mechanism of triple conversion. J. Gen. Microbiol. 1989,

135, 1679–1697.

[45] Ziebandt, A. K., Becher, D., Ohlsen, K., Hacker, J. et al., The

influence of agr and sigmaB in growth phase dependent

regulation of virulence factors in Staphylococcus aureus.

Proteomics 2004, 4, 3034–3047.

[46] Abdelnour, A., Arvidson, S., Bremell, T., Ryden, C.,

Tarkowski, A., The accessory gene regulator (agr) controls

Staphylococcus aureus virulence in a murine arthritis

model. Infect. Immun. 1993, 61, 3879–3885.

[47] Cheung, A. L., Eberhardt, K. J., Chung, E., Yeaman, M. R.

et al., Diminished virulence of a sar-/agr- mutant of

Staphylococcus aureus in the rabbit model of endocarditis.

J. Clin. Invest. 1994, 94, 1815–1822.

[48] Gillaspy, A. F., Hickmon, S. G., Skinner, R. A., Thomas, J. R.

et al., Role of the accessory gene regulator (agr) in patho-

genesis of staphylococcal osteomyelitis. Infect. Immun.

1995, 63, 3373–3380.

[49] Wright, J. S., 3rd, Jin, R., Novick, R. P., Transient interference

with staphylococcal quorum sensing blocks abscess forma-

tion. Proc. Natl. Acad. Sci. USA 2005, 102, 1691–1696.

[50] Tjalsma, H., Bolhuis, A., Jongbloed, J. D., Bron, S., van Dijl,

J. M., Signal peptide-dependent protein transport in Bacil-

lus subtilis: a genome-based survey of the secretome.

Microbiol. Mol. Biol. Rev. 2000, 64, 515–547.

[51] Antelmann, H., Tjalsma, H., Voigt, B., Ohlmeier, S. et al., A

proteomic view on genome-based signal peptide predic-

tions. Genome Res. 2001, 11, 1484–1502.

[52] Gr .undling, A., Schneewind, O., Synthesis of glycerol

phosphate lipoteichoic acid in Staphylococcus aureus. Proc.

Natl. Acad. Sci. USA 2007, 104, 8478–8483.

[53] Ben Zakour, N. L., Sturdevant, D. E., Even, S., Guinane,

C. M. et al., Genome-wide analysis of ruminant Staphylo-

coccus aureus reveals diversification of the core genome.

J. Bacteriol. 2008, 190, 6302–6317.

Proteomics 2011, 11, 2491–2502 2501

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

[54] Herron, L. L., Chakravarty, R., Dwan, C., Fitzgerald, J. R.

et al., Genome sequence survey identifies unique sequen-

ces and key virulence genes with unusual rates of amino

acid substitution in bovine Staphylococcus aureus. Infect.

Immun. 2002, 70, 3978–3981.

[55] Rogasch, K., R .uhmling, V., Pane-Farre, J., Hoper, D. et al.,

Influence of the two-component system SaeRS on global

gene expression in two different Staphylococcus aureus

strains. J. Bacteriol. 2006, 188, 7742–7758.

[56] Burlak, C., Hammer, C. H., Robinson, M. A., Whitney, A. R.

et al., Global analysis of community-associated methicillin-

resistant Staphylococcus aureus exoproteins reveals

molecules produced in vitro and during infection. Cell

Microbiol. 2007, 9, 1172–1190.

[57] Pocsfalvi, G., Cacace, G., Cuccurullo, M., Serluca, G. et al.,

Proteomic analysis of exoproteins expressed by enter-

otoxigenic Staphylococcus aureus strains. Proteomics 2008,

8, 2462–2476.

[58] Hempel, K., Pane-Farre, J., Otto, A., Sievers, S. et al.,

Quantitative cell surface proteome profiling for SigB-

dependent protein expression in the human pathogen

Staphylococcus aureus via biotinylation approach. J.

Proteome Res. 2010, 9, 1579–1590.

[59] Schuberth, H. J., Krueger, C., Zerbe, H., Bleckmann, E.,

Leibold, W., Characterization of leukocytotoxic and super-

antigen-like factors produced by Staphylococcus aureus

isolates from milk of cows with mastitis. Vet. Microbiol.

2001, 82, 187–199.

[60] Fueyo, J. M., Mendoza, M. C., Rodicio, M. R., Muniz, J. et al.,

Cytotoxin and pyrogenic toxin superantigen gene profiles

of Staphylococcus aureus associated with subclinical

mastitis in dairy cows and relationships with macrorestric-

tion genomic profiles. J. Clin. Microbiol. 2005, 43,

1278–1284.

[61] Dinges, M. M., Orwin, P. M., Schlievert, P. M., Exotoxins of

Staphylococcus aureus. Clin. Microbiol. Rev. 2000, 13,

16–34.

[62] Guinane, C. M., Ben Zakour, N. L., Tormo-Mas, M. A.,

Weinert, L. A. et al., Evolutionary genomics of Staphylo-

coccus aureus reveals insights into the origin and molecular

basis of ruminant host adaptation. Genome Biol. Evol. 2010,

2, 454–466.

[63] Viana, D., Blanco, J., Tormo-Mas, M. A., Selva, L. et al.,

Adaptation of Staphylococcus aureus to ruminant and

equine hosts involves SaPI-carried variants of von Will-

ebrand factor-binding protein. Mol. Microbiol. 2010, 77,

1583–1594.

[64] Petzl, W., Zerbe, H., G .unther, J., Yang, W. et al., Escherichia

coli, but not Staphylococcus aureus triggers an early

increased expression of factors contributing to the innate

immune defense in the udder of the cow. Vet. Res. 2008,

39, 18.

[65] Poutrel, B., Lerondelle, C., Induced staphylococcal infec-

tions in the bovine mammary gland. Influence of the month

of lactation and other factors related to the cow. Ann. Rech.

Vet. 1978, 9, 119–128.

[66] Prasad, L. B., Newbould, F. H., Inoculation of the bovine teat

duct with Staphylococcus aureus: the relationship of teat

duct length, milk yield and milking rate to development of

intramammary infection. Can. Vet. J. 1968, 9, 107–115.

2502 C. Wolf et al. Proteomics 2011, 11, 2491–2502

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