Molecular basis of virulence in Staphylococcus aureus mastitis.

Abstract

S. aureus is one of the main pathogens involved in ruminant mastitis worldwide. The severity of staphylococcal infection is highly variable, ranging from subclinical to gangrenous mastitis. This work represents an in-depth characterization of S. aureus mastitis isolates to identify bacterial factors involved in severity of mastitis infection.
We employed genomic, transcriptomic and proteomic approaches to comprehensively compare two clonally related S. aureus strains that reproducibly induce severe (strain O11) and milder (strain O46) mastitis in ewes. Variation in the content of mobile genetic elements, iron acquisition and metabolism, transcriptional regulation and exoprotein production was observed. In particular, O11 produced relatively high levels of exoproteins, including toxins and proteases known to be important in virulence. A characteristic we observed in other S. aureus strains isolated from clinical mastitis cases.
Our data are consistent with a dose-dependant role of some staphylococcal factors in the hypervirulence of strains isolated from severe mastitis. Mobile genetic elements, transcriptional regulators, exoproteins and iron acquisition pathways constitute good targets for further research to define the underlying mechanisms of mastitis severity.

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Molecular Basis of Virulence in Staphylococcus aureus
Mastitis
Caroline Le Mare ´chal1,2,3, Nubia Seyffert1,2,4, Julien Jardin1,2, David Hernandez5, Gwenae ¨l Jan1,2,
Lucie Rault1,2, Vasco Azevedo4, Patrice Franc ¸ois5, Jacques Schrenzel5, Maarten van de Guchte6,
Sergine Even1,2, Nadia Berkova1,2, Richard Thie ´ry3, J. Ross Fitzgerald7, Eric Vautor3.¤, Yves Le Loir1,2*.
1INRA, UMR1253, Science et Technologie du Lait et de l’Œuf, Rennes, France, 2AGROCAMPUS OUEST, UMR1253, Science et Technologie du Lait et de l’Œuf, Rennes,
France, 3ANSES, Laboratoire de Sophia-Antipolis, Unite ´ pathologie des ruminants, Sophia-Antipolis, France, 4Universidade Federal de Minas Gerais (UFMG), Instituto de
Cie ˆncias Biolo ´gicas (ICB), Departamento de Biologia Geral, Belo Horizonte, Minas Gerais, Brazil, 5Genomic Research Laboratory, Service of Infectious Diseases, University of
Geneva Hospitals HUG, Geneva, Switzerland, 6INRA, UMR1319, MICALIS, Jouy en Josas, France, 7The Roslin Institute and Centre for Infectious Diseases, Royal Dick School
of Veterinary Studies, University of Edinburgh, Edinburgh, Scotland, United Kingdom
Abstract
Background: S. aureus is one of the main pathogens involved in ruminant mastitis worldwide. The severity of staphylococcal
infection is highly variable, ranging from subclinical to gangrenous mastitis. This work represents an in-depth
characterization of S. aureus mastitis isolates to identify bacterial factors involved in severity of mastitis infection.
Methodology/Principal Findings: We employed genomic, transcriptomic and proteomic approaches to comprehensively
compare two clonally related S. aureus strains that reproducibly induce severe (strain O11) and milder (strain O46) mastitis in
ewes. Variation in the content of mobile genetic elements, iron acquisition and metabolism, transcriptional regulation and
exoprotein production was observed. In particular, O11 produced relatively high levels of exoproteins, including toxins and
proteases known to be important in virulence. A characteristic we observed in other S. aureus strains isolated from clinical
mastitis cases.
Conclusions/Significance: Our data are consistent with a dose-dependant role of some staphylococcal factors in the
hypervirulence ofstrains isolated from severe mastitis. Mobile genetic elements, transcriptionalregulators, exoproteins and iron
acquisition pathways constitute good targets for further research to define the underlying mechanisms of mastitis severity.
Citation: Le Mare ´chal C, Seyffert N, Jardin J, Hernandez D, Jan G, et al. (2011) Molecular Basis of Virulence in Staphylococcus aureus Mastitis. PLoS ONE 6(11):
e27354. doi:10.1371/journal.pone.0027354
Editor: Malcolm James Horsburgh, University of Liverpool, United Kingdom
Received March 1, 2011; Accepted October 14, 2011; Published November 11, 2011
Copyright: ? 2011 Le Mare ´chal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Yves.LeLoir@rennes.inra.fr
. These authors contributed equally to this work.
¤ Current address: LVD06, Sophia-Antipolis, France
Introduction
Mastitis is an inflammation of the mammary gland with local
and or general symptoms that occasionally result in a systemic
infection. This disease has a profound impact on animal welfare
and milk quality [1] leading to great economical losses in milk
production [2]. Staphylococcus aureus is a major cause of mastitis in
ruminants worldwide which is often difficult to cure and is prone
to resurgence. Beside mastitis, S. aureus is involved in a wide range
of infections. In several infection types (e.g. pneumonia, osteomy-
elitis, skin infections), extremely severe cases associated with
hypervirulent strains have been reported [3–6]. The existence of
hypervirulent strains emphasizes the need to define the strain
characteristics involved in the increased severity so as to better
monitor their dissemination and find relevant therapeutic targets
to reduce severity. It has been reported that severity can be linked
to the production of a single virulence factor that enhances the
virulence of producing strains. For example, Panton-Valentine
leukocidin, a bi-component pore-forming toxin, is particularly
prevalent in severe infections [4] and has been proposed as a
hypervirulent determinant [7], due to its involvement in leukocyte
destruction and tissue necrosis [8,9]. Furthermore, staphylococ-
cal superantigens or alpha-toxin function in a dose-dependant
manner, resulting in more severe infections caused by highly-
expressing strains [10–13]. Severity of mastitis caused by Escherichia
coli was shown to be mainly determined by host factors and not by
the strains features [14]. In contrast, in S. aureus mastitis, inter-
strain variations exist in terms of virulence potential [15]. Alpha-
toxin and LukM-F’ have been reported to be highly produced
during gangrenous S. aureus mastitis [13,16–19]. However, global
studies which examine the expression of all proteins have not been
carried out, and to date no gene has been identified as being a
severity marker [20–22]. A better understanding of the pathoge-
nicity of S. aureus is critical to develop more efficient and
satisfactory therapy to overcome mastitis.
S. aureus strains O11 and 046 were isolated from gangrenous
mastitis and subclinical mastitis of ewes, respectively. These strains
were shown to reproducibly induce severe (O11) or mild (O46)
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mastitis in experimental infections [15]. In the current study, they
were comprehensively analyzed by a comparative genomic,
transcriptomic and proteomic approach to identify staphylococcal
factors that can be linked to mastitis severity in order to define
strain characteristics associated with hypervirulence in mastitis.
Results
Genome analysis reveals minor differences between O11
and O46
In order to investigate the genetic bases for the high virulence of
strain O11 in ewe mastitis, we determined and compared the
genome sequences of strains O11 and O46 [23]. The great
majority of the genes were found in both strains except for an
additional serogroup B prophage (42 CDS) in O46 genome
(Figure 1). O11 and O46 share high similarity with the recently
sequenced ED133 genome [24] (Figure 1), a S. aureus strain
isolated from ovine mastitis. Yet, ED133 belongs to the clonal
complex CC133 (MLST) whereas O11 and O46 clustered in the
same lineage as bovine strains found in CC130 [25]. In a study by
Guinane et al, comparative genome analysis of ED133 in addition
to other ruminant and human strains revealed molecular evidence
for host-adaptation and several novel mobile genetic elements
(MGE) encoding virulence proteins with attenuated or enhanced
activity in ruminants [19]. In the current study, we found that
most of the genes present in ED133 genome are present in O11 or
O46 genomes (Figure 1). For example, both O11 and O46 carry
the newly described phages related to the wSaov1 and wSaov3
phages from ED133 but do not contain wSaov2, reportedly unique
to ED133, or SaPIov1, carrying an ovine allelic variant of sec
(encoding staphylococcal enterotoxin type C). Nevertheless scn
(staphylococcal complement inhibitor), vwb (von Willebrand
factor-binding protein) and SAOV_2050 (hypothetical protein)
carried by SaPIov2 pathogenicity island are identified in O11 and
O46 sequences. In contrast to ED133, putative virulence factors
edin-B and a homolog of etd carried by a putative pathogenicity
island are present in both O11 and O46 [26].
Although O11 and O46 are clonally related as demonstrated by
spa typing, PFGE analysis [27] and genome content, they contain
Single Nucleotide Polymorphims (SNP) (around 1600 synonymous
SNP and 1250 non synonymous SNP detected). SNP mediated
diversification of genes encoding cell-wall associated proteins was
previously observed [24,28]. Here, comparison of O11 and O46
showed that the SNPs were evenly distributed around the genome
and did not correlate with protein location or function.
O11 and O46 comparison also revealed 103 truncated genes
(listed in supplemental data, Table S1) present in one strain or the
other, corresponding to point mutations or indels causing a
frameshift or leading to a premature stop codon. Among these
103 truncated genes, 37% are involved in cellular machinery,
notably in gene regulation (8.7%), iron metabolism (3%), virulence
(11%), and proteins of unknown function (36%). Truncated genes
that may play a role in phenotype differences observed between
O11 and O46 have been identified. For instance, 2 genes encoding
enzymes involved in restriction/modification systems are found
intact in O46 (046_2610 similar to type III restriction protein [29]
and 046_0485 similar to HsdR type I restriction endonuclease [30]
whereas they are truncated in O11 (Table S1). Transformation tests
(electroporation with pMAD plasmid DNA directly extracted from
E.coli DH5) on O11 and O46 revealed that only O11 is
transformable with transformation efficiency comparable to that
of S. aureus RN4220, bearing the same mutations [30]. Plasmidic
DNA extracted from O11 transformants was successfully intro-
duced into S. aureus MW2 and, to a lesser extend, into O46,
suggesting that additional feature(s) impairs O46 transformability
(see supplemental data, Table S2). Similarly, icaC is truncated in
O11, and this correlates with a lower capacity for biofilm formation
in O11 when compared to O46 (biofilm formation tested as
described in [31]; see supplemental data, figure S1). Some of these
differences have direct consequences on transcription as revealed by
transcriptomic differences (18% of the truncated genes appeared
underexpressed in O11 or O46 (Table S1)).
Comparison of O11 and O46 transcriptome during
growth in mastitis-like conditions reveals major
differences
Total RNA samples were prepared from O11 and O46 strains
grown in deferoxamine-RPMI under anaerobic conditions to
simulate the in vivo context [32]. Cells were harvested in
exponential and stationary phase, and gene expression profiles
determined. Fold changes in Table S3, S4, and S5 indicate the
gene expression ratio between O11 and O46. Only ratios higher
than 2 (overexpression in O11) and lower than 0.5 (overexpression
in O46) were considered. Microarray analyses showed that 269
genes and 308 genes (Table S4 and S5) were differentially
expressed between O11 and O46 during log and stationary phases
Figure 1. Graphical mapping of the genomes of S. aureus O11 and O46 and the recently released S. aureus ED133 genome. Left panel:
S. aureus O11 (left side) and S. aureus ED133 (right side), middle panel: S. aureus O46 (left side) and ED133 (right side) and right panel: O11 (left side)
and O46 (right side) genomes. Homologuous sequences between strains are linked by colored ribbons. Sequences are ordered in a way to minimize
ribbons crossing. Arrows indicate the contig containing the additional phage in O46 (see text for details).
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respectively (Figure 2). The two strains had significantly different
gene expression profiles suggesting that O11 and O46 respond to
these growth conditions in distinct ways (Table S3, S4, S5).
Whatever the growth phase, O46 overexpressed genes encoding
surface components, e.g. cap operon or adhesin genes (fnbB and
clfA), in addition to other genes such as clpP and phage genes,
whereas O11 overexpressed genes encoding secreted virulence
factors (hla, hlgA, scpA, splE) and genes carried by pathogenicity
islands as well as genes involved in iron metabolism (sir operon,
sbnc, isdH). Overexpression of these latter genes in O11 may
account for the higher sensitivity of O11 (compared to O46) to
streptonigrin, an antibiotic which is toxic to cells in the presence of
intracellular free iron (minimum inhibitory concentration was at
least 4-fold higher for O46 than for O11).
O11 and O46 comparison revealed differences in two s-factors:
the sigS gene is indeed truncated in O46 (Table S1) and is found
transcribed in O11, only, whatever the growth phases (confirmed
by RT-qPCR, Table S3); the rsbU gene, part of sigB operon, is
overexpressed in O11 during stationary phase (Table S3). Genome
analysis of the two strains revealed that spoVG gene is truncated in
O11 (Table S1). Both sBand sSappear to dramatically differ
between O11 and O46. This may have huge consequences
considering their central role in gene regulation and, subsequently,
virulence expression.
Inasmuch as the accessory gene regulator (agr) system is central
to the control of virulence gene expression, we specifically tested
the agr functionality using RT-qPCR targeting hld (RNAIII) and
agrA (RNAII). Both genes are expressed at similar levels in the two
strains suggesting that agr does not contribute the differences
observed between O11 and O46 gene expression profiles.
Differences in extracellular proteomes between O11 and
O46: overproduction of exoproteins by O11
Protein samples representing total (whole-cell lysate), cell wall,
and extracellular fractions were prepared from O11 and O46
strains grown in conditions identical to those of transcriptomic
experiments [32]. At least 3 gels from 3 independent cultures for
each strain and each compartment were compared. Image analysis
identified 41 spots as being differentially expressed. The majority
of differences were observed in extracellular samples, as illustrated
in figure 3 (21 spots varied between O11 and O46 in supernatant
gels whereas only 20 spots differed in both total and cell wall gels).
It is important to note that each spot may contain more than
one unique protein as well as a given protein can be found in
several spots. Hereafter, we present data resulting from protein
identification (i.e. numbers refer to proteins and not to spots).
Protein identification was carried out using Nano-LC analysis and
results are listed in Table S6 for extracellular samples, table S7 and
S8 (additional files) for total and cell wall samples respectively.
Most proteins were common to O11 and O46 as indicated by
protein patterns on the 2-D gels from total and cell wall extracts
(figure S2 and S3, additional files). Some differences were however
observed in both compartments but were mostly due to volume
differences and few were due to the absence of the protein in one
of the 2 strains. Some proteins were present in both strains but at
clearly different positions on the 2-D gels, or they were found in
Figure 2. Transcriptomic comparison of S. aureus O11 and S. aureus O46 after growth in deferoxamine-RPMI during log and
stationary phase. Genes differentially expressed were categorized by functional annotation. Genes overexpressed by each strain are indicated on
the right side of the figure. Right side, numbers of genes differentially expressed belonging to the core genome (italic black) and mobile genetic
elements (grey) are indicated.
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several spots (see table S7), like the alkyl hydroperoxide reductase
subunit C (spots T9, T10 on figure S2) or the phosphoglycerate
kinase (spot T3 in O46 samples and T4 in O11 samples; figure S2).
It should be noted that some spots corresponding to different Mr
and/or pI contained the same protein (e.g. spots P7 and P8, or T5,
T6 and T12, T13 containing the fructose 1,6-biphosphate
aldolase; tables S5 and S7, and figures S1 and S2). In summary,
17 proteins were overproduced by O11 and 8 by O46 in total and
cell wall fractions representing proteins from various functional
categories including metabolism (14), cellular processes and
signalling (4), information storage and processing (5) and unknown
functions (2).
In contrast, the extracellular proteomes revealed more pro-
nounced differences (figure 4A and 4B, and additional files table
S6, S8, figure S4). A majority of proteins (28 out of the 35 proteins
that differed between O11 and O46) were overproduced in O11
extracellular 2-D gels compared to O46. They are directly
implicated in virulence (32%; e.g. LukE, LukM, Hla, or Hlg, or
Sbi), or predicted to play a role in metabolism or other cellular
processes (e.g. IsdA,B,C, and H, involved in iron metabolism).
Surprisingly, when considering the predicted location of the
proteins (according to the SurfG+ analysis of O11 and O46
genome sequences), many (43%) are predicted to be cytoplasmic
(e.g. GAPDH, CspA, or PurH).
Figure 3. Venn diagram of S. aureus O11 and S. aureus O46 spots, constructed after analysis of total, cell wall and extracellular
fraction 2D-gels with Image Master 2D. Numbers in black-shaded regions represent proteins identified in both O11 and O46 samples. Numbers
in dark grey- or light grey-shaded regions indicate proteins specifically identified in S. aureus O11 or S. aureus O46 respectively. Results are derived
from three independent experiments.
doi:10.1371/journal.pone.0027354.g003
Figure 4. Comparison of exoproteins produced by S. aureus strains isolated from clinical or subclinical mastitis. Culture supernatants
were harvested after growth in deferoxamine-RPMI during 24 h. 2-DE comparison was carried out by image analysis with Image Master 2D. Spots
corresponding to differentially produced protein(s) are indicated by arrows and numbers (S1 to S22). Protein identification was carried out using
NanoLC MS/MS (see Table S5 for details). A: a representative 2-DE gel of S. aureus O11 secreted proteins, B: a representative 2-DE gel of S. aureus O46
secreted proteins, C: production of 6 protein spots in 6 different strain supernatants are depicted with their spot. Strains O11, 1628, and 1624 were
isolated from clinical mastitis cases; Strains O46, O82, and O55 were isolated from subclinical mastitis cases. Mr: Molecular weight marker.
doi:10.1371/journal.pone.0027354.g004
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Some differences in protein expression can be explained by the
presence of indels in one of the 2 strains, which most likely result in
a difference in transcriptome and proteome. This is the case for
IsdH, whose gene contains a deletion of 1215 bp in O46, and
IsdA, whose gene contains an insertion of 45 bp in O46. For some
proteins, transcriptomic results (overexpression of the correspond-
ing genes) corroborated proteomic results as shown for Asp23, 2,3-
bisphosphoglycerate-dependent phosphoglycerate mutase, IsaA,
DapA that are overexpressed by O46 or general stress protein
20 U, ClpL, Hla, mercury(II) reductase that are overexpressed by
O11. Some proteins are overexpressed by one strain in log phase
and by the other one during stationary phase. For instance, lukE
and nuc are overexpressed in log phase at transcriptomic and
proteomic levels (data not shown) by O46 but appeared to be
overproduced by O11 during late stationary phase at proteomic
level (S8 and S15 figure 4 and figure S4). Finally the
overproduction of many proteins by O11 is not always explained
by a difference in genome sequence or by higher gene expression.
Post-transcriptional regulation, modifications or stability of
proteins may contribute to these differences.
Some proteins differentially produced by O11 and O46
are distributed among a panel of strains isolated from
clinical vs subclinical ewe mastitis
In order to identify protein candidates to characterize strains
isolated from clinical versus subclinical mastitis, we screened an
additional 4 strains isolated from subclinical (n=2) and clinical
(n=2) cases of ewe mastitis for the presence of the previously
identified proteins by proteome analysis of extracellular samples
(2-D gels and Coomassie blue staining). Twenty two proteins that
were identified as differentially produced by O11 and O46 were
checked in other strains. At least 7 spots of these proteins appeared
to be also overproduced either by clinical strains or by subclinical
strains (figure 4C). SspB, AdhA, LdH, Gap, AhpC, SspA, CspA,
Hla, LukM, LukF-PV were overproduced by O11, 1624 and 1628
(severe mastitis isolates) whereas O46_2740 gene product (with
similarity to exfoliative toxin family) was overproduced by O46,
O82 and O55 (subclinical mastitis isolates).
Discussion
S. aureus mastitis outcomes are highly variable and depend, in
part, on strain-dependent features. Here we have achieved the first
in-depth characterization of 2 S. aureus strains that were shown to
reproducibly induce different symptoms in experimental mastitis
despite close genotypic relatedness [15]. Complementary ap-
proaches were used to gain insight in the molecular basis of S.
aureus virulence variability in mastitis. Taken together, the results
show limited divergence in gene content and clear differences in
gene expression. The combined results suggest that differences in
iron metabolism, transcriptional regulators
production capacity may contribute to the differences observed
in mastitis severity induced by these two strains.
and exoprotein
Ability to acquire DNA of exogenous origin and mobile
genetic elements
Around 15% of the S. aureus genome is composed of MGE, i.e.
bacteriophages, transposons, plasmids or pathogenicity islands that
can be horizontally transferred from one isolate to another. The
restriction-modification systems control in part, the uptake of
foreign DNA by bacteria, by identifying and modifying specific
DNA sequences so as to prevent the uptake of deleterious DNA for
the bacteria (lysogenic bacteriophages or superfluous genes). Four
restriction-modification systems have been described in S. aureus
[30]. The enhanced ability of S. aureus strain RN4220 to accept
foreign DNA is due to frameshift mutations in hsdR gene, a gene
belonging to a type 1 restriction-modification system [30] and in a
gene encoding a type III-like restriction endonuclease [29]. These
two genes were found truncated in O11. This likely explains how
O11 was amenable to transformation by plasmid DNA directly
extracted from E. coli, whereas O46, which contains intact
restriction modification genes was not. O11 is thus a naturally
transformable strain, which can be useful to further study gene
function in the pathogenesis of mastitis. S. aureus strains that are
deficient in these restriction systems are hypersusceptible to the
horizontal transfer of DNA [29]. In addition to being highly
virulent, O11 strain may become a reservoir of horizontally
acquired antibiotic resistance genes. Occurrence and spread of
such strains in lifestock thus deserve special attention.
Surprisingly, O46, and not the transformable O11, contains an
additional prophage. Furthermore, phage genes are expressed at
higher levels in O46 compared to O11. Phages have been shown
to play a crucial role in virulence [33–35]. The potential role of the
additional prophage found in O46 in mastitis pathogenesis has to
be further determined. In contrast, O11 overexpressed genes
carried by pathogenicity islands although these latters are found in
both O11 and O46 strains. Differences in the expression of MGE-
related genes between strains O46 and O11 may contribute to the
relative pathogenic potential of the 2 strains.
Dramatic differences between O11 and O46 gene
expression profiles with regard to iron acquisition and
metabolism
Iron is an absolute requirement for the growth of most
microorganisms and serves as a cofactor in many enzymatic
reactions and as a catalyst in electron transport processes [36]. It is
however present at a very low concentration in many environ-
ments (e.g. in milk, the concentration of available iron is around
10212mM) [37]. Bacteria have developed various mechanisms to
overcome iron restriction [38] and S. aureus is able to grow in the
presence of extremely low (0.04 mM) iron concentrations [39].
Growth of strains O11 and O46 in deferoxamine-RPMI was
carried out to mimic nutritional deficiencies relevant to the
mammary gland. Differences regarding genes involved in iron
acquisition were revealed through genome, transcriptome and
proteome analyses. Indeed, O46 contains truncated genes related
to different systems involved in iron metabolism (isdH, hrtB and
feoA). Moreover, several genes involved in iron uptake (sir operon,
some genes of isd operon, fer, sstA, sbnC) were overexpressed in
O11. Such overexpression was confirmed at the proteomic level
for some gene products, like IsdA, B, C, H, that were found
overproduced by O11.
The S. aureus requirements for iron during infection can be
satisfied through several different systems. Heme acquisition by the
Isd system is required for full virulence in several models of
pathogenesis [40]. It involves 9 proteins, 4 of which were found
truncated (isdH) and or differentially expressed (isdA, isdB, isdC, and
isdH) in strain O46. High intracellular concentrations of heme are
toxic and S. aureus possesses de-toxification systems such as the
HrtAB system, a hemin-regulated ABC transporter that protects S.
aureus against hemin toxicity [41]. In O46, hrtB gene is truncated
and it appears that the hemin uptake (isd system) and
detoxification (hrtAB) pathways are attenuated in O46. In addition
to its role in heme uptake, IsdH was shown to inhibit complement
binding at the cell surface and so to contribute to the host immune
evasion [42]. Interruption of isdH induced a reduced virulence in a
mouse sepsis model [42] and a truncated isdH in O46 may take
part in the reduced severity observed in ewe mastitis as well. Other
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iron uptake systems differ between O11 and O46. Iron can be
acquired in its ferrous form via the widespread bacterial FeoAB
system or via siderophore through the SstABCD, SirABC,
FhuBCD systems. It is worth noting that feoA is truncated in
O46 whereas several siderophore genes are overexpressed in O11.
Notably sbnC, a gene encoding staphylobactin, an S. aureus
siderophore and sirABC, the genes encoding its cell receptor are
overexpressed in O11. It has been shown that siderophore
production enhances the virulence of S. aureus [38]. S. aureus
strains isolated from bovine mastitis have been shown to be able to
overcome iron starvation [43]. However, the underlying mecha-
nisms may vary between human and ruminant isolates. Genes
involved in iron uptake or metabolism may reflect host tropism, as
suggested by allelic variations reported in these genes in the
recently released genome sequences of bovine and ovine isolates
[24,28]. O11 was more sensitive to streptonigrin when grown on
deferoxamine-RPMI which suggests, together with higher expres-
sion of siderophore genes, that it has a better iron acquisition
compared to O46. Strain-dependant expression of genes involved
in iron acquisition or metabolism may impact on the severity of
infection in vivo, in the mammary gland.
Overexpression of exoproteins by O11
Transcriptomic and proteomic comparison revealed that O11
overexpressed genes encoding exoproteins whereas O46 overex-
pressed genes encoding surface components. Notably, proteomic
analysis of the culture supernatants revealed differences in toxin
and protease expression. This was confirmed by the analysis of
extracellular fraction in 4 additional strains. This global trend was
also observed at the transcriptomic level but the difference in
exoprotein levels did not always correlate with transcription
implying differences in mRNA half-life or post-translational
regulation. It is worth noting that some genes involved in the sec
pathway (spsB and 4.5S RNA) and genes of the accessory sec system
(secY2 and asp21) are overexpressed in O11 but a link between
expression of these genes and the overproduction of exoproteins
has still to be demonstrated.
Of note, differences were observed between the two strains with
regard to transcriptional regulators. The sigS gene (sSplays a role
in virulence in vivo [44]) is truncated in O46 whereas it is intact and
transcribed in O11. The rsbU gene (part of sigB operon) is
overexpressed in O11 compared to O46 (Table S3). Consequently
in O11, one would expect an up-regulation of sB regulon
including surface components like cap operon and surface proteins
and a down-regulation of many toxins and secreted proteases [45].
Yet we observed the opposite situation in O11, and further found
that the spoVG gene, part of yabJ-spoVG locus, a sBeffector that
modulates sB control over its dependent genes lacking an
apparent sB promoter, is truncated in O11 (Table S1). This
correlates well with the lower nuclease activity observed in O11, as
expected according to [46] (Figure S5, supplemental data).
Altogether, these differences might account for the differences in
expression levels of genes encoding exoproteins (and putatively in
mastitis severity) observed for the two strains [15]. Among the
oversecreted proteins in O11, some candidates are indeed of
special interest and make sense when considering the mastitis
context. LukM/F’ has been reported to be produced at higher
levels during severe mastitis than in moderate mastitis [18,19].
Production of a hemolysin has also been reported to play a role in
mastitis severity [47] and to be involved in gangrenous mastitis
[16,17].
SspB and SspA belong to a proteolytic cascade where a
metalloprotease aureolysin (Aur) activates a serine protease
zymogen proSspA, which in turn activates the SspB cysteine
protease [48]. SspA and SspB play an important role in virulence
in a mouse abscess model [49] and they are both involved in the
degradation of conjonctive tissue [50]. SspB plays a role in local
inflammation of the tissue [51] and in blocking phagocytosis by
neutrophils and inhibiting their chemotactic activity [52]. SspA
and SspB have also been reported to be produced in vivo during
gangrenous mastitis [15]. Other exoproteins (e.g. Gap, CspA,
AhpC) were found overproduced in the extracellular fraction of
O11 and 2 additional strains isolated from severe mastitis although
they were predicted to be cytoplasmic. Gap has been reported to
be surface located and able to bind to bovine transferrin, which is
another high-affinity iron-scavenging mechanism [53]. CspA, a
general stress protein is also a strong antigen in human sepsis
caused by S. aureus [54]. AhpC is not required for virulence in vivo
but plays a role in nasal colonization and has an important role in
host-pathogen interaction [55]. Except Gap, these proteins have
not previously been reported to be produced by mastitis S. aureus
isolates. These three proteins have been reported to be present in
S. aureus supernatant in other studies [56,57] but are predicted to
be localized in the cytoplasm. A new secretion system involved in
protein export via vesicule secretion has recently been described
[58]. The secretion mechanism of these proteins is still unknown
but this may play a role in the oversecretion of some proteins by
O11.
Only one protein was shown to be specifically associated to S.
aureus strains isolated from moderate mastitis. This analog of
exfoliative toxin D has also been shown to be produced in vivo [15].
Interestingly, a highly similar exfoliative toxin (76% homology) is
also produced by coagulase negative staphylococci, which are the
predominant pathogens responsible for subclinical mastitis.
Whether this newly identified toxin is active and plays a role in
mastitis remains to be determined.
These results clearly show that some exoproteins are specifically
produced by isolates associated with severe mastitis. Proteins, like
LukM/F’ or a hemolysin, have been previously reported to be
associated with severe mastitis. To our knowledge, this is the first
time a link between proteins other than LukM/F’ and Hla and
mastitis severity is suggested. These proteins are thus good
candidates for further investigation of their exact role in mastitis
onset and severity.
Conclusion
The current study provides the first high resolution comparison
of gene content and expression for S. aureus mastitis isolates from
ovine origin. The results indicate several systems that may
contribute to mastitis severity, including MGE, iron metabolism,
sigma regulators and exoprotein production. These pathways
represent excellent candidates for targeted studies of the molecular
basis of S. aureus pathogenesis in ruminant mastitis.
Materials and Methods
Bacterial strains, growth conditions
Staphylococcus aureus O46 was isolated from a case of ovine
subclinical mastitis and O11 from a lethal gangrenous mastitis
[25]. S. aureus O46 and O11 are representative of the major
lineage found associated to ewe mastitis in southeast of France
[27,59]. Four other S. aureus strains isolated from gangrenous and
clinical ewe mastitis (1628 and 1624, respectively) and subclinical
ewe mastitis (O55 and O82) were used in this study and were
previously described [27]. Growth conditions and preparation of
protein extracts were as described in Le Mare ´chal et al. 2009 [32].
Briefly, all cultures were performed as follows: Overnight cultures
in BHI were diluted 1:1000 in fresh RPMI 1640 medium (Sigma,
In-Depth Comparison of S. aureus Mastitis Strains
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Page 7

Saint Quentin fallavier, France). Deferoxamine (0.15 mM; Sigma),
an iron chelator, was added to RPMI (hereafter referred to as
deferoxamine-RPMI). S. aureus strains were grown anaerobically in
falcon tubes (50 ml) or in flasks (250 ml) filled up with medium and
incubated at 37uC without agitation in anaerobic conditions. The
same anaerobic conditions were used to compare O11 and O46
transcriptome and proteome. Minimum inhibitory concentration
for streptonigrin (Sigma) was determined as follows. Overnight
cultures of O11 and O46, on BHI, were 1/100 diluted and used to
inoculate fresh deferoxamine-RPMI (2.5 mL, in 15-mL tubes)
containing increasing concentrations of streptonigrin (0, 1.25, 2.5,
5, 10, and 20 ng/mL). Cultures were incubated at 37uC under
agitation.
RNA, DNA and protein extraction
Protein samples for extracellular, cell wall or total fraction,
RNA extraction and purification and genomic DNA extraction
were exactly done as previously described [32,59,60].
Genome sequencing, assembly, annotation and
comparison of O11 and O46
Whole genome sequencing and assembling strategy are
described in [23]. Comparison and graphical mapping were
performed using the MUMmer software package [61], the Circos
visualization software [62] as well as an application developed in
house. Coding sequences (CDSs) detection was performed with the
Glimmer software application [63]. Annotations were imported
from already annotated S. aureus strains and mapped to the
corresponding CDSs by using an application developed in house
as well as the Exonerate sequence alignment program [64]. These
genome sequences have been deposited at DDBJ/EMBL/
GenBank under the accession AEUQ00000000 (O11) and
AEUR00000000 (O46) [23].
Microarray design and manufacturing
The microarray was manufactured by in situ synthesis of 60-base
oligonucleotide probes (Agilent, Palo Alto, CA), selected as
previously described [65]. The array covers 98% of all open
reading frames (ORFs) annotated in strains N315, Mu50, COL,
MRSA252, MSSA476, MW2, USA300_FPR3757, NCTC8325,
RF122 including their respective plasmids.
Preparation of labeled nucleic acids for expression
microarrays
Total RNA was purified from bacteria grown in deferoxamine-
RPMI during log phase (OD600=0.5) and stationary phase
(OD600=1). For each strain total RNA of three independent
cultures was extracted as previously described [60]. After
additional DNase treatment, noncontamination of the RNA
sample by genomic DNA (gDNA) was confirmed by quantitative
PCR on gyrB. Batches of 8 mg of total S. aureus RNA were labeled
with Cy3-dCTP using SuperScript II (Invitrogen, Basel, Switzer-
land) following the manufacturer’s instructions. Labeled products
were then purified onto QiaQuick columns (Qiagen). The
following steps were performed as described in [66].
Microarray analysis
Fluorescence intensities were extracted using Feature Extraction
software (version 8; Agilent). Local background-subtracted signals
were corrected for unequal dye incorporation or unequal load of
the labelled product. Per chip normalizations were performed
using the 50th percentile of all measurements for different
hybridisations to make comparisons between different experiments
valid. Data consisting of three independent biological experiments
were analyzed using GeneSpring, version 8.0 (Silicon Genetics,
Redwood City, CA) after per gene and per chip normalization.
Statistical significance of differential gene expression was calculat-
ed by analysis of variance using GeneSpring, including the
Benjamini and Hochberg false discovery rate correction of 5% (P
value cutoff, 0.05) and higher than 2-fold induction or reduction of
expression was accepted as differential expression.
Microarray data accession number
The microarray design and the complete dataset were deposited
in the public repository database Gene Expresion Omnibus under
the accession numbers GPL11137 and GSE25084, respectively.
qRT-PCR
To confirm microarray data, expression profiles of clfA, sigS, sirA,
urea, hld, ahpF, phoP, agrA, capA were determined by quantitative
reverse transcription-PCR (qRT-PCR) analyses. Primer sequences
are given in Table S9. All primer efficiencies were tested for each
strain and ranged between 85% and 110%. cDNA was synthesized
using the high-capacity cDNA archive kit as recommended by the
manufacturer (Applied Biosystems, Warrington, United Kingdom).
Quantitative real-time PCR was performed using an Opticon 2
real-time PCR detector (Bio-Rad, Hercules, CA). The reaction
mixture contained power Sybr green PCR master mix (1X; Applied
Biosystems, Warrington, United Kingdom), each primer (0.5 mM),
and 1 mg cDNA template. Thermal cycling consisted of 10 min at
95uC, followed by 40 cycles of 15 s at 95uC and 60 s at 60uC. qRT-
PCR analyses for all experimental time points were performed in
triplicate (using three independent biological replicates). Calibration
curvesweregeneratedtocalculatethecopynumberforeachgenein
eachsample.gyrA,ftsZ,hu and sodA [67] weretestedtodeterminethe
best internal standards for normalization using the geNorm VBA
applet for Microsoft Excel. gyrA, ftsZ and sodA were used as internal
standards for exponential phase and ftsZ and sodA for stationary
phase. The Ct values of genes of interest were transformed to
quantities (number of copies) by using standard curves. Gene
expression levels were calculated by dividing gene of interest
quantities by the previously calculated normalization factor
(according to the geNorm user manual). Statistical analyses were
performed as in [68].
2-Dimensional Gel Electrophoresis
Protein samples (200 mg) were precipitated with 2D clean up kit
(GE Healthcare, Orsay, France) according to the manufacturer’s
instructions. Pellets were solubilised in sample solution containing
7 M urea, 2 M thio-urea, 25 mM dithiothreitol (DTT), 4% (w/v)
3-[(3-Cholamidopropyl)dimethylammonio]-1-propane-sulfonate
(CHAPS) and 2% (w/v) ampholyte containing buffer (IPG-Buffer
4–7 or 3–10 NL, GE Healthcare). Isoelectric focusing was carried
out using pH 4 to 7 (Cell wall and total proteins) or 3 to 10 NL
(extracellular fraction) 13 cm Immobiline Dry Strips on a Multi-
phor II electrophoresissystem (AmershamBiosciences) as described
previously [15]. The second dimension separation was performed
on an Ettan dalt electrophoresis system (GE Healthcare) using 14%
acrylamide separating gels without a stacking gel at a voltage of
50 V for 1 h and 180 V for 7 h. Low molecular weight markers
(GE Healthcare) were used as the standards. Gels were stained
with R250 Coomassie blue (Serva, Heildelberg, Germany). Three
extractions from three different cultures were carried out to
perform 2-D gels. Stained 2-D gels were scanned with Image
Scanner II (Amersham biosciences) and image analysis was per-
formed with ImageMaster 2D platinum software as previously
described [69,70].
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Nano-LC analysis
Proteins were identified by tandem mass spectrometry (MS/MS)
after an in-gel trypsin digestion adapted from Shevchenko [71] and
described in details in [15]. Briefly, gel pieces were excised from the
gel. In-gel trypsin digestion was performed overnight at 37uC. After
concentration, the supernatants containing peptides were analyzed
using an on-line liquid chromatography tandem mass spectrometry
(MS/MS) setup. A full continuous MS scan was carried out
followed by three data-dependent MS/MS scans. Spectra were
collected in the selected mass range 400 to 2,000 m/z for MS and
60 to 2,000 m/z for MS/MS spectra. The three most intense ions
from the MS scan were selected individually for collision-induced
dissociation(1+ to 4+ chargedionswere considered for the MS/MS
analysis). The mass spectrometer was operated in data-dependent
mode automatically switching between MS and MS/MS acquisi-
tion. The proteins present in the samples were identified from MS
and MS/MS data by using MASCOT v.2.2 software for search
into two concatenated databases: (i) a homemade database
containing all the predicted proteins of the O11 and O46 strains
used in this study as deduced from their genome [23] and (ii) a
portion of the UniProtKB database corresponding to the
Staphylococcus aureus taxonomic group (http://www.uniprot.org/).
Supporting Information
Table S1
sequences.
(DOCX)
Truncated genes in S. aureus O11 or S. aureus O46
Table S2
(DOC)
Amenability of O11 and O46 strains to transformation.
Table S3
expressed during log and stationary phases between O11 and
O46.
(DOCX)
Genes discussed in this work that were differentially
Table S4
variations between O11 and O46 during log phase in deferox-
amine-RPMI medium.
(DOC)
Expression profiles of genes exhibiting significant
Table S5
variations between O11 and O46 during stationary phase in
deferoxamine-RPMI medium.
(DOC)
Expression profiles of genes exhibiting significant
Table S6
identified in this study.
(DOCX)
S. aureus O11 and S. aureus O46 extracellular proteins
Table S7
differentially produced by O11 or O46 after analysis of 2D gels of
total cell lysate samples (figure S2).
(DOC)
Proteins identified by nanoLC MS/MS as being
Table S8
differentially produced by O11 and O46 after analysis of 2D gels
of cell wall extract (figure S2).
(DOC)
Proteins identified by nanoLC MS/MS as being
Table S9
real-time PCR.
(DOC)
Oligonucleotides used in this study for quantitative
Figure S1
determined by Cristal violet staining assay. Biofilm staining assays
were performed as described previously [29]. Briefly, after
bacterial growth in iron-depleted RPMI, microtiter plates (Multi-
Biofilm production in S. aureus O11 and O46 as
wellTM 6 well, Becton Dickinson) were washed twice with
phosphate-buffered saline (PBS), fixed for 20 min at 80uC and
stained for 10 min with 1% (w/v) crystal violet solution freshly
diluted twofold in 1% (v/v) ethanol/distilled water. Plates were
then washed with water and photographed. The crystal violet was
dissolved in dimethyl sulfoxide (DMSO) for 1 h before OD600 nm
measurements. Biofilm formation was estimated for each strain, on
6 replicates, and the data were analysed by the student’s paired t
test. A P value of 0.05 or less (here, P=0.044).was considered
statistically significant.
(TIF)
Figure S2
O46 cell lysates. A: representative 2-DE gel of S. aureus O11 (upper
gel) and S. aureus O46 (lower gel) total lysates. Proteins were
prepared after growth in iron-depleted RMPI. 200 mg of protein
preparation was separated on 13 cm gels (pI 4–7, 14% SDS-
PAGE) and Coomassie Blue-stained. Image analysis with Image
Master 2D revealed differences in the protein spots indicated with
arrows and numbers. Identification was carried out by NanoLC
MS/MS (see Table S7). B: The expression of numbered spots are
depicted in three different gels prepared from three biological
replicates (R1, R2, R3) from O11 or O46 total lysates.
(TIF)
Proteomic comparison of S. aureus O11 and S. aureus
Figure S3
O46 cell wall proteins. A: representative 2D gel of O11 (upper gel)
and O46 (lower gel) cell wall extracts. Proteins were prepared after
growth in iron-depleted RMPI. 200 mg of protein preparation was
separated on 13 cm gels (pI 4–7, 14% SDS-PAGE) and Coomassie
Blue-stained. Image analysis with Image Master 2D revealed
differences in the protein spots indicated with arrows and
numbers. Identification was carried out by NanoLC MS/MS
(see Table S8). B: The expression of numbered spots are depicted
in three different gels prepared from three biological replicates
(R1, R2, R3) from O11 or O46 cell wall samples.
(TIF)
Proteomic comparison of S. aureus O11 and S. aureus
Figure S4
aureus O46 extracellular proteins highlighted by image analysis
with Image master 2D. Identification was carried out by NanoLC
MS/MS (see Table S6). The expression of numbered spots are
depicted in three different gels prepared from three biological
replicates (R1, R2, R3) from O11 (left panel) or O46 (right panel)
extracellular protein samples.
(TIF)
Proteomic differences between S. aureus O11 and S.
Figure S5
O46. A nuclease plate assay was carried out on supernatant of
O11 and O46 strains after overnight culture on deferoxamine-
RPMI. 10 mL of 0.2 mm filtered supernatant were spotted on
Toluidine Blue-DNA agar as described previously [72]. Plates
were incubated o.n. at 37uC and nuclease activity was revealed by
the development of a pink halo, which diameter is proportional to
the amount of Nuclease secreted. Presence of truncated spoVG in
O11 correlates with a lower nuclease production as previously
reported [46].
(TIF)
Nuclease activity assay on supernatant of O11 and
Author Contributions
Conceived and designed the experiments: EV YLL. Performed the
experiments: CLM NS DH JJ GJ LR. Analyzed the data: PF JRF NB
VA SE MvdG JS YLL. Contributed reagents/materials/analysis tools: RT
EV DH. Wrote the paper: YLL EV. Obtained permission from the farmers
to get access to the dairy sheep and collected the strains: EV.
In-Depth Comparison of S. aureus Mastitis Strains
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Page 9

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In-Depth Comparison of S. aureus Mastitis Strains
PLoS ONE | www.plosone.org 10November 2011 | Volume 6 | Issue 11 | e27354