Disparity in the In Vitro versus In Vivo Regulation of Fibronectin-Binding Proteins by 2 Global Regulators, saeRS and sigB, in Staphylococcus aureus
Ambrose L. Cheung
2
Soo-Jin Yang
1
Arnold S. Bayer
0
1
Yan Q. Xiong
(yxiong@ucla.edu)
0
1
0
The David Geffen School of Medicine at UCLA
,
Los Angeles, California
1
Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center
,
Torrance
2
Department of Microbiology, Dartmouth Medical School
,
Hanover, New Hampshire
Fibronectin-binding protein A plays an important role in Staphylococcus aureus endovascular infections. We characterized the sigB-saeRS-fnbA expression network with fibronectin binding in vitro and in an experimental infective endocarditis (IE) model using parental strains RN6390 and SH1000 and their respective isogenic saeRS mutants. In contrast to the in vitro data, there was no influence of saeRS on fnbA expression in the IE model, yet ex vivo fibronectin binding was reduced in saeRS mutants. Moreover, as opposed to the in vitro findings, sigB appeared to have a positive rather than a negative effect on saeRS expression within cardiac vegetations.
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endovascular infections, since (1) knockout of fnbA leads to
reduced virulence in experimental infective endocarditis (IE)
[2]; (2) adoptive transfer of fnbA into nonvirulent Lactococcus
lactis renders such constructs virulent in experimental IE [3];
and (3) FnBPA facilitates S. aureus endothelial colonization,
penetration, and damage both in vitro and in experimental IE
[2].
The regulatory pathway for fnbA expression directly involves
the S. aureus global regulon saeRS [4, 5]. The saeRS operon, a
2-component regulatory system, is a key element in the
regulatory network that governs the expression of many virulence
factors (eg, cell-wallassociated proteins and exotoxins) at the
transcriptional level. In addition, the stress response regulon,
sigma factor B (sigB), may have indirect effects on fnbA
expression by its composite effects on saeRS and other global
regulators (eg, agr and sarA) [5, 6]. Knowledge of the effects
that saeRS and/or sigB have on fnbA expression in vivo in S.
aureus endovascular infections is limited. The current study
focused on the regulatory impacts of 2 global regulons (saeRS
and sigB) on fnbA expression in vitro versus in vivo in the
context of S. aureus endovascular infections and their
subsequent net effects on fibronectin binding.
Methods. The S. aureus strains RN6390 and SH1000 were
selected because (1) RN6390 has a natural 11-bp deletion in
rsbU, the first gene of the sigB operon, that renders it
functionally sigB deficient, whereas SH1000 is a sigB-repaired variant
(rsbU+) of the RN6390 lineage strain (8325-4) that has full sigB
activity [7]; (2) these strains have been extensively used in the
study of staphylococcal genetics and pathogenesis; (3) both
strains have intact saeRS and fnbA genes; and (4) each are
virulent in animal IE models [7]. Isogenic saeRS mutants with
an sae:Tn551 knockout in RN6390 and SH1000 backgrounds
were used in the current study [7].
For Northern blot analyses, S. aureus cells were harvested
during exponential (2 h), early stationary (6 h), and late
stationary (12 h) growth phases for RNA extraction. Total RNA
was isolated using the RNeasy kit (Qiagen) and FastPrep FP120
instrument (BIO 101), according to the manufacturers
recommended protocols. Northern blot analyses were performed
as described elsewhere but with modifications [8]. Digoxigenin
(DIG)labeled fnbA- and sae-specific probes were synthesized
using the polymerase chain reaction (PCR)based DIG Probe
Synthesis kit (Roche; fnbA-F, 5
-GCTGCAGCATCAGAACAAAA-3 ; fnbA-R, 5 -CCTTCAGTCAACGTCCCTGT-3 ; sae-F,
5 -TGGTCACGAAGTCCCTATGC-3 ; and sae-R, 5
-TGCTTGCGTAATTTCCGTTAG-3 ). To confirm fnbA expression at 2
h, reverse-transcription PCR (RT-PCR) was performed as
described below [8].
To quantitate correlations between fnbA expression and
fibronectin adherence, we evaluated the in vitro
fibronectinbinding capacities of the S. aureus study strains by direct
binding to immobilized human fibronectin [9]. Briefly, plates were
coated with fibronectin (50 mg/mL; Sigma Chemicals), washed,
and treated with 3% bovine serum albumin (Fisher
BioReagents) to prevent nonspecific adhesion. Aliquots of S. aureus
cells (5 103 colony-forming units [CFUs]) were applied to
individual wells and then incubated for 1 h at 37 C. After
removing unbound S. aureus cells, tryptic soy agar (TSA)
(Difco) was overlain in all wells. After 24 h of incubation at
37 C, adherent colonies were counted. Bacterial binding was
quantified as the percentage of the initial inoculum bound.
A well-characterized rabbit model of aortic valve IE was used
to study the impact of saeRS and/or sigB on fnbA expression
and ex vivo fibronectin binding [9]. IE was produced by the
intravenous injection of each study strain (107 CFUs per
animal) at 24 h after catheterization. This inoculum encompasses
the 95% infection dose for all study strains, as established
previously [7]. At 24 h after infection, all animals were killed for
analysis of in vivo fnbA and saeRS expression and ex vivo
fibronectin binding. We did not analyze relative virulence of
parental strains versus their respective saeRS mutants since we
have confirmed a reduced virulence phenotype in this IE
model for saeRS mutants [7].
For in vivo RNA isolation, cardiac vegetation samples were
removed 24 h after infection and quickly placed into 20 mL
of ice-cold acetone-ethanol (1:1). Next, the samples were
homogenized and processed to remove tissue debris, as described
elsewhere [7]. Total RNA was then isolated using the RNeasy
kit as described above. RT-PCR was performed as described
elsewhere with the following modifications [8]. Briefly, fnbA
and sae complementary DNA (cDNA) were generated using
Moloney murine leukemia virus reverse transcriptase (New
England Biolabs) and the reverse primers fnbA-R and sae-R. The
fnbA and sae cDNA products were then detected by PCR using
the primer pair fnbA-F-RT and fnbA-R-RT and the pair
saeF-RT and sae-R-RT, respectively. The RT-PCR primers used
for the detection of the gyrA transcripts have been described
elsewhere (gyrA-F, 5 -CGTGAAGGTGACGAAGTTGTAGG-3 ;
gyrA-R, 5 -TAACTGGCGTACGTTTACCATAACC-3 ) [8].
AmFigure 2. A, Adherence of Staphylococcus aureus RN6390, SH1000, and
their respective saeRS mutants to fibronectin in vitro. B, Ex vivo adherence
of study strains to fibronectin. *P ! .05 for the comparison with the
respective parent strains.
plification was performed with initial denaturation at 95 C for
3 min; followed by 1820 cycles of denaturation at 95 C for 30
s, annealing at 54 C for 30 s, and extension at 72 C for 30 s;
followed by a final extension at 72 C for 5 min. We were
unsuccessful in detecting fnbA and saeRS expression in kidney
and spleen in this model. This probably relates to the
significantly lower S. aureus densities and greater host tissue cell
numbers in these organs than in vegetations.
To determine the relative ability of the study strains directly
obtained from vegetations to adhere to fibronectin, a
modification of the above in vitro fibronectin adherence assay was
used. Briefly, all vegetations were removed, washed,
homogenized, and filtered to remove tissue debris 24 h after infection.
Filtrates then underwent pelleting by centrifugation (2000 g,
10 min) and were washed in phosphate-buffered saline. S.
aureus cells (5 103 CFUs based on anticipated vegetation
densities) from each vegetation sample were then directly assessed
ex vivo for fibronectin-binding capacity by the in vitro assay
detailed above. Initial inocula approximations were confirmed
by formal quantitative cultures. The extent of fibronectin
binding was quantified as above for the in vitro assay.
To compare fibronectin adherence between the various S.
aureus strains, the unpaired t test was used. Differences with
P ! 0.05 were considered significant.
Results. Northern blot analysis disclosed that the
transcription of fnbA was maximal at 2 h of incubation in both the
RN6390 and SH1000 parental strains; no fnbA transcription
was detected in either saeRS mutant strain, as anticipated
(Figure 1A, middle panel). RT-PCR confirmed fnbA expression in
the parental strains at 2 h (Figure 1B). Also as expected, saeRS
transcription was detected only in the RN6390 and SH1000
parental strains. The sae-specific transcripts T1 and T3 were
discerned using probes specific for saeRS (Figure 1A); in some
lanes (eg, lane 1 at 2 h), the T2 transcript likely arose from
endoribonucleolytic processing of the T1 transcript, as has been
reported previously [10]. Moreover, indirect evidence of a
sigBmediated inhibitory effect on saeRS expression was also
discerned, in concordance with the findings observed for the strain
Newman background [10]. More specifically, the SH1000
parental strain had a lower saeR transcription level than the
RN6390 parental strain, especially at 6 and 12 h of incubation
(Figure 1A).
A significantly reduced fibronectin binding profile was
observed in both saeRS mutants studied, compared with that of
their respective parental strains (P ! .05) (Figure 2A). In
addition, in agreement with the in vitro fnbA transcription data,
the SH1000 parental strain had substantially lower fibronectin
binding than did the RN6390 parental strain (Figure 2A).
In contrast to our in vitro findings, no influence of saeRS
on fnbA was observed within the cardiac vegetations in either
the RN6390 or the SH1000 strain set (Figure 1B). For example,
fnbA expression was comparable between the parental and
saeRS mutants in 2 different vegetation preparations from
distinct animals. Interestingly, the SH1000 parental strain had
higher saeRS expression than did the RN6390 parental strain
in vivo (Figure 1B).
For S. aureus cells directly isolated from vegetations 24 h
after infection, saeRS mutant strains adhered significantly less
to fibronectin than did their respective parental strains (P !
0.05) (Figure 2B), similar to the above in vitro findings.
Discussion. Our in vitro analyses confirmed existing
paradigms for saeRS-fnbA interactions noted for different
published strain backgrounds: (1) fnbA expression was maximal
during early exponential growth and was not noted during late
exponential or stationary phases [4, 9]; (2) saeRS significantly
affected fnbA expression [4]; and (3) by using strain pair
backgrounds that were either sigB intact (SH1000) or sigB deficient
(RN6390), indirect evidence of a sigB inhibitory effect on saeR
expression was demonstrated [5].
However, several novel observations emanated from our in
vivo investigation. We previously reported a number of
examples of in vitroin vivo disparities in S. aureus virulence gene
expression in the IE model using a green fluorescent protein
(GFP)based gene reporter system, including sarA, agr, and
cap5 [11, 12]. Importantly, these studies emphasized the
different patterns of target organspecific in vivo gene expression,
comparing cardiac vegetations versus kidneys versus spleens [9,
11, 12]. For the current study, we substituted an RT-PCRbased
assay for detecting fnbA and saeR expression for several reasons:
(1) saeR:GFP and fnbA:GFP tend to produce low fluorescent
signals in this model, an issue that is circumvented by the
RTPCR technique; (2) the GFP system requires a
double-fluorescent signal in which the organism is tagged with a fluorophore
of different wavelength to distinguish tissue particles from
bacterial cells (such modifications are not required in the RT-PCR
method); and (3) use of RT-PCR enables simultaneous
interrogation of both saeR and fnbA gene expression from the same
target tissue. To our knowledge, this is the first report to use
this method to detect expression of virulence genes in this
model.
Using the RT-PCRbased technique and in contrast with our
in vitro findings, we have shown the in vivo saeRS-fnbA
regulatory paradigm to be distinct. First, for both strains fnbA
expression was maintained in saeRS-positive parental strains,
as well as in their respective saeRS knockouts, within cardiac
vegetations (Figure 1C). This contrasts with the finding of fnbA
expression in vitro, where fnbA expression was down-regulated
in both the saeRS mutant and the parent strains at 2 h
incubation point (Figure 1A). We and others have reported that
fnbA expression was positively regulated by sarA and negatively
regulated by agr both in vitro and in cardiac vegetations in the
IE model [9, 13]. These observations suggest that
environmental cues or other regulatory loci (such as agr and sarA)
may well contribute to fnbA expression in the absence of saeRS.
Second, despite the ability of the saeRS knockouts to maintain
fnbA expression in vivo, ex vivo fibronectin binding by
intravegetation strains was significantly reduced. These data
underscore that the net S. aureus fibronectin-binding capacity in
vivo is multifactorial and likely includes saeRS regulation of
other FnBPs (eg, FnbB, Ebh [host extracellular matrix binding
protein homologue], or Emp [extracellular matrix protein])
[14, 15]. Third, although relatively repressed in vitro in the
sigB-intact parental strain SH1000 (compared with the
sigBdeficient strain RN6390), saeR expression appeared to be
relatively enhanced in SH1000 within cardiac vegetations. These
data suggest that sigB may play a positive (rather than a
negative) regulatory role in saeR expression in vivo and/or that
the sigB-saeRS interaction axis may be responsive to diverse
environmental signals or host parameters (eg, pH, host defense
peptides, inflammatory cells) [5].
We thank Yin Li and Wessam Abdel Hady for their excellent technical
assistance.