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. - 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.