Activation and Transcriptional Interaction between agr RNAII and RNAIII in Staphylococcus aureus In Vitro and in an Experimental Endocarditis Model

Journal of Infectious Diseases, Sep 2002

This study compared the promoter activation profiles of the 2 major transcripts of the Staphylococcus aureus global regulon, agr (RNAII and RNAIII). In vitro, RNAIII activation temporally followed RNAII activation and was absent in agr mutants. In experimental endocarditis, maximal RNAII activation in vegetations occurred early, followed by progressive increases in RNAIII activation (P<.05; 2 vs. 48 h); this paralleled significant increases in vegetation bacterial densities over time (P<.05; 2 and 6 vs. 48 h). At 48 h of infection, S. aureus densities in vegetations were significantly higher than those in kidney or spleen tissue (P<.05), paralleling a significantly greater RNAIII activation profile in vegetations than in the latter tissues (P<.05). Of importance, RNAIII activation was observed in vegetations in 2 agr mutants. These data demonstrate that RNAIII activation in vivo is time and cell density dependent, may be tissue specific, and can occur through RNAII-dependent and -independent mechanisms

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Activation and Transcriptional Interaction between agr RNAII and RNAIII in Staphylococcus aureus In Vitro and in an Experimental Endocarditis Model

Yan-Qiong Xiong 0 2 William Van Wamel 3 Cynthia C. Nast 1 2 Michael R. Yeaman 0 2 Ambrose L. Cheung () 3 Arnold S. Bayer 0 2 0 School of Medicine, University of California , Los Angeles 1 Department of Pathology, Cedars-Sinai Medical Center 2 Division of Infectious Diseases, Harbor-University of California, Los Angeles, Research and Education Institute , Torrance 3 Department of Microbiology and Immunology, Dartmouth Medical School , Hanover, New Hampshire This study compared the promoter activation profiles of the 2 major transcripts of the Staphylococcus aureus global regulon, agr (RNAII and RNAIII). In vitro, RNAIII activation temporally followed RNAII activation and was absent in agr mutants. In experimental endocarditis, maximal RNAII activation in vegetations occurred early, followed by progressive increases in RNAIII activation (P ! .05; 2 vs. 48 h); this paralleled significant increases in vegetation bacterial densities over time (P ! .05; 2 and 6 vs. 48 h). At 48 h of infection, S. aureus densities in vegetations were significantly higher than those in kidney or spleen tissue (P ! .05), paralleling a significantly greater RNAIII activation profile in vegetations than in the latter tissues (P ! .05). Of importance, RNAIII activation was observed in vegetations in 2 agr mutants. These data demonstrate that RNAIII activation in vivo is time and cell density dependent, may be tissue specific, and can occur through RNAII-dependent and -independent mechanisms. - Staphylococcus aureus is a major human pathogen that causes a broad spectrum of severe infections, including systemic and life-threatening diseases, such as osteomyelitis and endocarditis [13]. The pathogenicity and virulence of S. aureus appear to depend on both expressions of cell wallassociated adhesins (e.g., microbial surface components recognizing adhesive matrix molecules [MSCRAMMs]) and secretion of extracellular proteins (e.g., hemolysins, enterotoxins, and proteases) [46]. Many of the purported virulence genes in S. aureus are coordinately controlled by a complex global regulatory locus known as the accessory gene regulator (agr). This locus suppresses expression of surface proteins and up-regulates expression of secreted proteins postexponentially in laboratory cultures [712]. The agr locus consists of 2 divergent transcripts, RNAII and RNAIII, initiated from 2 distinct promoters, P2 and P3, respectively. RNAII encodes 4 genes (agrB, agrD, agrC, and agrA), and all seem to be required for the activation of the P3 promoter. In turn, the P3 promoter drives the expression of RNAIII, the major effector molecule of the agr operon [1315]. In vitro studies have shown that in-frame deletions of any of the 4 genes within RNAII result in diminished transcription of RNAIII, thus indicating the dependence of RNAIII transcription on prior activation of RNAII in vitro [16]. However, the activation profiles of RNAII and RNAIII and the genetic requirements for RNAIII activation in vivo have not been defined. The aim of this study was to delineate the temporal profile and extent of activation and transcriptional interaction between RNAII and RNAIII of agr in vitro and in vivo within several distinct target tissues during the course of a multisystem S. aureus infection. To assess the RNAII and RNAIII activation in vivo, the well-characterized rabbit endocarditis model was used; this model was used previously to investigate the expression profiles of other global regulatory elements of S. aureus in vivo [17]. In addition, we used a green fluorescent protein (GFP) reporter gene system and an S. aureus protein A immunodetection technique to discern S. aureusspecific expression in vivo. Materials and Methods Organisms. Table 1 lists the strains and plasmids used in this study. The plasmid pALC1484 was constructed from the Escherichia coliS. aureus shuttle vector (pSK236) that contains a promoterless red-shifted gfp (gfpuvr) reporter gene preceded by a polylinker region. The gfpuvr gene was constructed by introducing an S65T mutation into the gfpuv gene (Clontech) and shifting the optimal excitation and emission spectra from UV to longer wavelengths (488 and 507 nm, respectively), to facilitate argon laser detection by flow cytometry, as described elsewhere [18, 19]. The Staphylococcus aureus strains and plasmids used in a rabbit endocarditis model. Escherichia coliS. aureus shuttle plasmid pSK236 with a promoterless gfpuvr gene Derivative of pALC1484 in which agr RNAII promoter (nt 15281756) [19] was cloned upstream of gfpuvr reporter Derivative of pALC1484 in which agr RNAIII promoter (nt 17561528) was cloned upstream of gfpuvr reporter DNA constructs containing either RNAII or RNAIII promoter sequences of agr were cloned upstream of the promoterless gfpuvr gene in the shuttle plasmid. The recombinant plasmids were then electroporated into S. aureus RN4220 and subsequently transduced into strain RN6390 with phage F11. In addition, this plasmid construct was also introduced into an agrA mutant and into an agr deletion mutant in which the entire agr operon was deleted from strain RN6390 (15 plasmid copies/cell in all constructs, as revealed by semiquantitative plasmid analyses [data not shown]). Preliminary experiments indicated that the expression of GFP fluorescence in pALC1484 is dependent on the strength of the promoter inserted into the polylinker region [17]. Of more importance, the level of GFP expression in this vector, even with weak promoters, can be readily detected by flow cytometry. All S. aureus strains were kept at 70 C in trypticase soy broth (TSB; Difco) containing 10% glycerol (vol/vol). In vitro determination of RNAII and RNAIII promoter activation by flow cytometry. S. aureus strains were cultured overnight at 37 C in TSB (without shaking, to prevent GFP fluorescence, as described elsewhere [17, 20]) and then pelleted by centrifugation (2000 g for 10 min). Pellets were washed twice, resuspended in PBS (pH 7.2), and adjusted to an optical density at 600 nm of 1.0 (109 cfu/mL). Dilutions of the S. aureus suspension were made in fresh TSB to achieve a starting inoculum of 107 cfu/mL and then incubated at 37 C on a rotary shaker. At selected time points (i.e., 0, 1, 2, 3, 4, 5, 6, 7, and 24 h), 0.5mL aliquots were obtained for assessment of RNAII or RNAIII gene expression by flow cytometry, as described below (FACScalibur; Becton Dickinson). For quantitation of RNAII or RNAIII promoter activities, 10,000 S. aureus cells were acquired to provide 2 distinct data sets: the extent of GFP fluorescence and the percentage of the S. aureus cell population exhibiting GFP fluorescence. A promoterless gfpuvr construct in RN6390 (ALC1745) was used as the GFP-negative control. Since this latter construct exhibited low background GFP fluorescence, at !10 fluorescence units (FU; detected by the FL-1 channel), this value was set as the minimal threshold for positive fluorescence (figure 1A). To examine the effect of plasma and serum factors on RNAIII expression in vitro among the various constructs, we conducted parallel studies in which each construct was coincubated in media containing either 10% rabbit serum or plasma. Rabbit endocarditis model. We used a well-characterized experimental rabbit endocarditis model to evaluate the temporal profile of RNAII and RNAIII promoter activation and the requirements for RNAIII activation in vivo [17, 21]. In brief, New Zealand White female rabbits (2.02.5 kg; Irish Farms Products and Services) were anesthetized by intramuscular injection of ketamine hydrochloride (35 mg/kg) and xylazine hydrochloride (15 mg/kg). An indwelling polyethylene catheter was positioned in the left ventricle of each rabbit via the transcarotid artery, with the tip passing across the aortic valve, to induce sterile vegetations. At 24 h after catheterization, separate groups of 68 animals were challenged intravenously with each S. aureus construct (109 cfu/rabbit for the 2-, 6-, and 8-h studies and 107 cfu/rabbit for the 48-h studies). These inocula were chosen on the basis of extensive pilot studies to ensure adequate numbers of S. aureus cells within target tissues for detection of GFP expression by both flow cytometry and fluorescence microscopy. Moreover, although agr mutants are less virulent in this model at low-challenge inocula (103106 cfu) [22], at the higher inocula used (107 or 109 cfu), agr mutants exhibit virulence equivalent to the parent strain. This approach enabled us to sharply focus our analyses on differences in agr regulation in distinct genetic contexts. As in the in vitro studies, each S. aureus construct was grown overnight in TSB without shaking to minimize GFP fluorescence before animal inoculation. Mitigation of promoter activation in the challenge inoculum was confirmed by flow cytometry before animal challenge (i.e., !10 FU). Catheters remained in place until animals were killed by lethal injection of sodium pentobarbital (100 mg/ kg) at 2, 6, 8, or 48 h after infection. At death, vegetations were removed at all time points. However, since kidney and spleen are seeded relatively late in this model, these tissues were obtained for analyses only at the 48 h postinfection time of death. At the time of death, one part of the vegetations was placed in a tissue fixative solution (Zeus Scientific) for analysis by routine and fluorescence microscopy. The remaining target tissues were weighed, homogenized, and processed in parallel for quantitative culture and promoter activation detection by flow cytometry. For quantitative culture and promoter activation within the bloodstream, we collected a large volume of blood (50 mL) at the time of death, to assure an adequate S. aureus quantity for flow cytometry analysis, because the level of bacteremia is relatively low in this model (102103 cfu/ mL). Animals were included in the analysis only if the catheters were correctly positioned across the aortic valve, and macroscopic vegetations were detected at the time of death. Microbiologic evaluation. Blood and homogenized tissue samples from each rabbit were serially diluted and quantitatively cultured onto TSB agar plates. Plates were then incubated at 37 C for 24 h. S. aureus densities in the tissue samples were calculated and expressed as mean ( SD) log10 colony-forming units per gram of tissue or mean ( SD) log10 colony-forming units per milliliter of blood. In vivo detection of RNAII or RNAIII promoter activation by flow cytometry. Blood and tissue samples were prepared for flow cytometry by a series of processing steps to remove tissue debris: lowspeed centrifugation (120 g for 10 min), exposure to deionized water (5 min) followed by sonication (Branson Sonifer) with a microtip (35 s, 25 W) to lyse host cells, and filtration of the lysate through 5-mm pore-sized filters (Millipore) to trap residual host cell debris. Filtrates (containing S. aureus cells) then underwent pelleting by high-speed centrifugation (2000 g for 10 min) and a wash in PBS. In addition, we analyzed forward scatter (FSC), representing relative cell sizes in a population of acquired cells, and side scatter (SSC), representing cell granularity or internal complexity in a population of acquired cells, in all target tissue samples. FSC and SSC ranges in all target tissue samples were similar to those of in vitro grown S. aureus constructs, which suggests that most host tissue cells were removed after the above series of processing steps (data not shown). To enhance the specificity of our flow cytometric analyses, an immunodetection system was developed in our laboratory to precisely distinguish S. aureus cells from any residual host tissue debris. This system was based on the expression of protein A, a cell wall associated MSCRAMM produced by virtually all S. aureus strains [2, 23, 24]. A highly specific affinity-purified chicken antistaphylococcal protein A antibody (Accurate Chemical & Scientific; purity, 195%) and Alexa Fluor 594 goat antichicken IgG conjugate (Molecular Probes) were used as primary and secondary antibodies, respectively. The secondary antibody has an excitation and emission maximum of 590 and 617 nm, respectively. These are distinct from GFP fluorescence profiles and caused no quenching of the GFP signal. In brief, after the initial processing steps described, blood and tissues samples were preincubated with 3% PBSbovine serum albumin (BSA; Sigma) at 37 C for 2 h to block nonspecific binding. Samples were then exposed to the chicken antistaphylococcal protein A antibody (7.5 mg/mL in 1% PBS-BSA) at 37 C for 1 h, washed once in PBS, and exposed to the secondary antibody (7.5 mg/mL in 1% PBS-BSA) at 37 C for 1 h. In pilot studies, these antibody concentrations were delineated to represent conditions that labeled S. aureus cells most efficiently (80% of 10,000 acquired cells exhibiting red fluorescence of 110 FU). After labeling, all samples were washed once in PBS and resuspended in PBS for GFP expression detection by flow cytometry. To minimize S. aureus clumping and to ensure that only single cells were acquired, each S. aureus suspension was sonicated briefly before flow cytometry analysis. In all, 10,000 cells from blood and tissue samples were acquired by flow cytometry and analyzed. To confirm that this immunolabeling method was specific for protein A, we used a protein A knockout S. aureus mutant strain (ALC1645) as the negative control. Minimal red fluorescence ( 10 FU) was detected in this strain after immunolabeling by flow cytometry (FL-4 channel): only 5% of cells exhibited positive red fluorescence 110 FU. Therefore, only cells with FL-4 10 FU were considered as protein Apositive S. aureus cells (figure 1B). Sterile vegetations in this model did not yield either FL-1 (GFP) or FL-4 (protein A) signals. Therefore, GFP quantitation was limited to protein Apositive S. aureus cells (figure 1C, left and right upper quadrants), and flow cytometry was designed to optimally detect and quantify dually labeled GFP/protein Aexpressing S. aureus cells. Detection of GFP expression by in situ fluorescence microscopy. To further characterize the temporal profile of RNAII and RNAIII promoter activations in vivo, selected target tissues were evaluated by fluorescence microscopy. Since the microscopic detection of bacterial cells in target tissues is dependent on tissue bacterial densities, we focused on infected vegetations, where the bacterial densities were consistently 16.5 log10 cfu/g at all death time points [17]. In contrast, bacterial densities in other target tissues (i.e., kidney and spleen) were, in general, 12 log10 cfu/g lower than those in vegetations, representing a level below the microscopy detection limits. Infected vegetations were placed into fixative solution, as described above, washed in citrate buffer, embedded (OCT compound; Sakura Finetek USA), and frozen. Frozen sections (4 mm) were placed on slides, air dried, and covered with coverslips with aqueous mounting media. Slides were examined with an Olympus DH2RFCA fluorescence microscope by using a filter system preset at excitation and emission of 485 and 515 nm, respectively, to detect the GFP signal. The slides were also stained with Giemsa to localize S. aureus cells within the vegetation lesion. The slides were read blindly by one of us (C.C.N.) as to the infecting S. aureus construct and scored by use of a semiquantitative system for in situ GFP tissue fluorescence [17]. Successive (serial adjacent) microscopy fields of individual lesions were used for both Giemsa and fluorescence microscopy. Microscopy fields were selected for analyses if they showed both individual bacterial cells and large bacterial colonies, to delineate gene expression profiles in relation to in vivo cell densities. Stability of RNAIII promoter:gfpuvr plasmid and agr knockout phenotype after in vivo passage of the agr knockout constructs. To confirm the in vivo stability of the plasmid RNAIII promoter: gfpuvr construct in the 2 agr mutant backgrounds, phenotypic promoter expression and plasmid profiling were carried out by using colonies directly isolated from vegetation culture plates of animals infected with these constructs. Postanimal passage S. aureus colonies were inoculated directly onto antibiotic-containing TSB plates (chloramphenicol [10 mg/mL] and tetracycline [5 mg/mL] for the agr deletion construct or chloramphenicol [10 mg/mL] and erythromycin [5 mg/mL] for the agrA mutant), to test the retention of the antibiotic resistance phenotypes of the constructs. The stability of these S. aureus constructs after in vivo passage was also verified by flow cytometry, as described above, in terms of the absence of in vitro RNAIII activation in the agrA or agr deletion mutants. Finally, the retention of the original recombinant RNAIII promoter:gfpuvr plasmid construct was confirmed by plasmid restriction analysis. Statistical analysis. To statistically compare S. aureus tissue densities (log10 colony-forming units per gram) and quantitative GFP fluorescence among the various groups, we used the KruskalWallis test with the Tukey posthoc correction for multiple comparisons, as needed. Significance was determined at P ! .05. We used regression analyses to analyze the relationship between target tissue S. aureus densities and RNAII or RNAIII promoter activation profiles (r 2 0.5 was considered to be significant). Activation of RNAII or RNAIII promoters in vitro. The agr promoters, P2 and P3, are divergent and initiate the transcription of RNAII and RNAIII, respectively. The P2 and P3 transcript starts are 191 nt apart; this intergenic region also represents the binding region of SarA, an important regulatory factor of agr [25, 26]. To assay activation of the RNAII and RNAIII in vitro, we cloned the P2 and P3 promoters upstream of the gfpuvr reporter gene. Figure 2 shows the temporal activation profiles of the RNAII and RNAIII promoters in vitro. RNAII promoter activation clearly precedes that of RNAIII. For example, at 5 h of incubation, the percentage of RNAII-expressing S. aureus cells was 40%, compared with 10% of RNAIII-expressing S. aureus cells. In contrast, at 56 h of incubation, RNAIII promoter activation increased substantially, with 70% of cells expressing from this promoter (figure 2A). This temporal profile paralleled the data obtained for intensity of GFP fluorescence at 7 h of growth (figure 2B). For example, by 6 h of incubation, the Table 2. Staphylococcus aureus densities in blood and target tissues of animals with endocarditis caused by the RNAII: gfpuvr and RNAII:gfpuvr promoter constructs. RN6390 RNAII:gfpuvr RN6390 RNAIII:gfpuvr ND ND a Data are log10 colony-forming units per milliliter ( SD). b Data are log10 colony-forming units per gram ( SD). c P ! .05, 48-h vegetations vs. 2- and 6-h vegetations and vs. 48-h kidney and spleen tissues. extent of RNAII promoter activation was near maximal, whereas RNAIII promoter activity was minimal. However, at 67 h of incubation, the extent of RNAIII promoter activation increased rapidly. Moreover, between 7 and 24 h of incubation, the extent of RNAII promoter activation did not increase, whereas that of the RNAIII promoter increased 3-fold (150450 FU). This in vitro augmentation in the extent of RNAIII-induced GFP fluorescence (figure 2B) between 7 and 24 h of growth far exceeds the moderate increase in the percentage of GFP-positive cells expressing RNAIII versus RNAII (figure 2A). As anticipated from previous studies of agrA or agr deletion mutants in vitro [16], RNAIII activation was not detectable in either strain (agrA mutant data not shown). Coincubation of the latter mutants in 10% rabbit serum or plasma did not stimulate the activation of RNAIII in vitro (data not shown). Microbiologic evaluation and activation of RNAII and RNAIII promoters in experimental rabbit endocarditis. S. aureus densities of the parental and mutant strains achieved in blood and target tissues in the experimental endocarditis model are shown in table 2 and table 3, respectively. For vegetations, significant increases in S. aureus densities were observed when we compared early (2 and 6 h) and late (48 h) time points for both RNAII:gfpuvr and RNAIII:gfpuvr constructs (P ! .05; table 2). In addition, at 48 h of infection, S. aureus vegetation densities were significantly higher than kidney or splenic densities for both parental constructs (P ! .05; table 2). In contrast, a relatively constant, low-level bacteremia was demonstrated over the 48-h infection period for both parental constructs (table 2). Of importance, there were no significant differences in achievable S. aureus densities in animals infected with either the RNAII:gfpuvr or RNAIII:gfpuvr constructs at any death time point (table 2). For the RNAIII:gfpuvr constructs in either the agrA or agr deletion mutant backgrounds, tissue densities did not differ significantly from those in animals infected with the RNAII:gfpuvr and RNAIII:gfpuvr constructs in the wild-type background at 48 h of infection (table 3). RNAII and RNAIII promoter activation in target tissues, as assessed by flow cytometry. Figure 3 depicts the temporal activation profile of the RNAII (figure 3A and 3B) and RNAIII (figure 3C and 3D) promoters in blood and in the target tissues. A relatively constant and low-level activation of the RNAII and RNAIII promoters was observed in blood over the 48-h postinfection period (figure 3A and 3C), paralleling the constant low-level bacteremia noted above. In contrast, RNAII promoter activation increased in vegetations over time and reached a maximal level by 8 h after infection (figure 3A). For RNAIII, paralleling the increases in S. aureus densities, the extent of activation in vegetations significantly increased over time (figure 3 and table 2). The mean GFP fluorescence reflecting RNAIII promoter activation increased 3-fold between 2 and 68 h of infection and continued to increase progressively between 8 and 48 h after infection (figure 3C). At 48 h, the extent of RNAII promoter activation was slightly higher in vegetations than in kidney and spleen (figure 3B). However, the tissue-specific profile of RNAIII promoter activation was more prominent than that observed with the RNAII promoter, and S. aureus cells in vegetations exhibited a significantly more activation than in the other target tissues where the tissue bacterial densities were lower (figure 3D; P ! .05). Given that bacterial densities in vegetations were comparable between RNAII and RNAIII constructs (table 2), these data emphasize the significant augmentation in bacterial fluorescence due to RNAIII (but not RNAII) promoter activation as a consequence of increasing bacterial densities within vegetations over time. Correlation of S. aureus densities with RNAII or RNAIII promoter activation. To further characterize the relationship between RNAII or RNAIII promoter activation and achievable Strain (no. of rabbits) RN6390 RNAIII:gfpuvr agrA mutant (8) RN6390 RNAIII:gfpuvr agr mutant (8) a Data are log10 colony-forming units per milliliter ( SD). b Data are log10 colony-forming units per gram ( SD). c P ! .05, vegetations vs. kidney and spleen. S. aureus densities, regression analyses were performed for these 2 parameters in individual vegetations 48 h after infection. At this infection time point, the range of achievable bacterial densities was 78.5 log10 cfu/g of vegetation. As shown in figure 4, there was a significant positive correlation between promoter activation and vegetation densities in individual infected vegetations for the RNAIII promoter construct (r 2 p 0.5371). In contrast, there was no positive correlation between these parameters for the RNAII promoter construct (r 2 p 0.0003). RNAIII promoter activation in vivo in agrA or agr deletion mutants. To delineate the relationship between RNAII and RNAIII promoter activations in vivo, we evaluated RNAIII promoter:gfpuvr S. aureus constructs in either agrA or agr deletion mutant backgrounds. Contrary to our in vitro findings, RNAIII promoter activation was observed in vegetations infected with both agr mutants 48 h after infection (figure 5). However, the extent of RNAIII promoter activation was significantly lower in these knockout constructs than in the parental background strain (117 34, 41 12, and 32 7 FU for the parental, agrA knockout, and agr knockout mutants, respectively; P ! .05). RNAII and RNAIII promoter activation in situ within vegetations detected by fluorescence microscopy. Figure 6 illustrates RNAII and RNAIII promoter activations within vegetations over the 48-h postinfection period. For the RNAII promoter, at 6 h after infection, minimal bacterial fluorescence was observed, despite a moderate number of organisms visible by Giemsa stain (RNAII:gfpuvr ; figure 6A and 6B). At 8 h of infection, there was focal 1 to 12 fluorescence (RNAII:gfpuvr ; figure 6C) involving more organisms within similarly sized colonies (RNAII:gfpuvr ; figure 6D). At 48 h of infection, most individual S. aureus cells were only weakly fluorescent (1 to 2); however, S. aureus cells within high density and large colonies were brightly fluorescent (2 to 4, RNAII:gfpuvr ; figure 6E). In contrast, for the RNAIII: gfpuvr construct over the same postinfection period, in situ GFP fluorescence progressively increased over time. At 6 h of infection, there was activation of RNAIII within bacterial clusters (1 to 2), with only weak fluorescence ( 1) of individual bacteria (RNAIII:gfpuvr ; figure 6A). At 8 h of infection, there was bright fluorescence of S. aureus bacterial clusters ( 3), although the isolated bacteria were still less fluorescent (1 to 2) than those within bacterial clusters (RNAIII:gfpuvr ; figure 6C). At 48 h of infection, there was intense ( 4) fluorescence of bacterial cells, both in clusters and in individual cells (RNAIII:gfpuvr ; figure 6E). At this same time point, S. aureus cells from vegetations infected with the agrA or agr mutants exhibited weak fluorescence (data not shown). Stability of RNAIII promoter:gfpuvr plasmid and agr knockout phenotype after in vivo passage of agr knockout constructs. The anticipated antibiotic resistance profiles of these constructs (i.e., chloramphenicol and either tetracycline or erythromycin) were retained in all in vivo colonies tested after passage. Moreover, postpassage flow cytometric analysis of these constructs demonstrated no RNAIII promoter activation, identical to the prepassage constructs (data not shown). Finally, plasmid restriction analyses confirmed that the bacteria harvested from the vegetations retained the identical RNAIII:gfpuvr plasmid constructs (data not shown). S. aureus pathogenicity is a complex process that involves the coordinate expression and activation of a number of regulatory and structural genes. The global regulator agr is thought to be a principal virulence factor in S. aureus because it up-regulates the in vitro expression of exotoxins while down-regulating adhesin genes postexponentially [811, 27] and because agr knockout mutants are less virulent in a number of experimental models than the isogenic parental strains (e.g., arthritis, osteomyelitis, endophthalmitis, and endocarditis [22, 2831]). Despite the impact of the agr regulon on virulence factor expression, there are few data on regulation of this locus in vivo. The in vitro expression of the major effector molecule of the agr operon, RNAIII, is highly dependent on preceding activation of a 2-component sensor-activator regulatory system within RNAII (i.e., agrC and agrA, respectively) [15, 16, 32]. However, it is unclear whether this agr activation cascade is operative in vivo. Recognizing that host factors (e.g., blood proteins and cells or vascular tissue) likely modulate the expression of many virulence determinants in S. aureus and that such host factors vary considerably in distinct target tissues, we investigated the temporal and tissue-specific expression profiles of RNAII and RNAIII in a multisystem S. aureus infection model, experimental endocarditis. This model was chosen because bacterial densities are high enough in selected target tissues (i.e., vegetations) to allow examination of the temporal gene promoter expression profiles by both flow cytometry and in situ tissue fluorescence [17]. This study produced several important findings. First, we confirmed previous data [15, 16, 32] that in vitro activation of RNAII temporally precedes RNAIII activation during mid- to late-logarithmic phases of growth. The steady increase in RNAII promoter expression during the logarithmic growth phase reached a plateau at 56 h of incubation, followed by a rapid increase in RNAIII expression. This activation pattern is consistent with the density-dependent (quorum-sensing) activation of RNAIII [15, 32]. As expected, deletion of agrA or of the agr operon abrogated activation of RNAIII in vitro. Second, the in vitro density-dependent quorum-sensing paradigm observation was paralleled in vivo in selected target tissues. For example, we observed a constant and low level of bacteremia in the endocarditis model (102103 cfu/mL) over a 48-h postinfection period. Neither the RNAII nor the RNAIII promoter exhibited a high level of activation in this compartment. However, in infected vegetations, where RNAII promoter activation reached a plateau 8 h after infection, as analyzed by flow cytometry, RNAIII promoter activation increased significantly over the entire 48-h postinfection period in this tissue, paralleling the progressive increases in S. aureus densities in this tissue. Therefore, there was a direct correlation between RNAIII expression and achievable bacterial densities in vegetations above a threshold of 7 log10 cfu/g, whereas RNAII expression plateaued over this same density range (figure 4). These flow cytometry observations (i.e., early peak in RNAII expression followed by progressive increases in RNAIII expression) essentially paralleled our fluorescence microscopy analyses. Collectively, the above in vivo data suggest that, after achieving a threshold bacterial density within vegetations, RNAII expression is triggered and followed by RNAIII promoter expression. Presumably, the expression of the RNAIII promoter continues to increase in parallel with increasing bacterial densities at this site. As noted elsewhere [9, 13], RNAIII, the major effector molecule of the agr operon, positively regulates toxin gene expression (e.g., hla, which is responsible for a-toxin secretion), while down-regulating adhesin gene expression. The continued high-level expression of RNAIII in S. aureus cells in areas of high bacterial density may reflect both decreased requirements for adhesion and an increased reliance on secreted toxins (e.g., a-toxin) within maturing vegetations over time. Such toxin production would theoretically destroy host tissues and phagocytic cells (e.g., platelets and leukocytes [33]), thereby facilitating the spread of bacteria from the vegetation into surrounding tissue sites. Another noteworthy finding in this study was the differential RNAII and RNAIII promoter activation profiles in S. aureus among distinct target tissues. Specifically, the extent of RNAII and RNAIII promoter activation was consistently greater in vegetations than in kidneys or spleen. These observations may further implicate distinct host factors within such target tissues as being pivotal to the overall gene expression profile in S. aureus, likely including the following 4 factors: first, influences of ionicity and osmotic strength differences, as found in the kidney [34]; second, differences in phagocytic cell accumulation (e.g., as found in vegetations vs. renal abscesses [1]); third, matrix molecule content differences; and fourth, differences in antibody presence. Alternatively, these differences in the overall extents of RNAIII expression in vegetations, compared with other target tissues, may simply reflect the larger bacterial populations available in vegetations to respond to the RNAII activation signal. Also of note, the RNAII:gfp and RNAIII:gfp reporter gene fusions used in our study were each encoded on multicopy plasmids (15 copies/cell). This raised the possibility that the differential gene expression profiles we observed in vivo between different isogenic constructs might be influenced by disproportionate titration of regulatory proteins due to multiple copies of the promoter. However, we consider this to be unlikely for the RNAIII promoter, since the plasmid copy number, as estimated by plasmid yield of comparable cell numbers, did not differ substantially among the various isogenic constructs. Of interest, despite inactivation of the agrA or the entire agr locus (both eliminating RNAII expression), RNAIII promoter activity remained readily observable within infected vegetations, albeit to a significantly lower extent than that observed with the parental construct. These findings emphasize the amplification impact of RNAII activation on ensuing RNAIII activation in vivo. These data also implicate the existence of both RNAII-dependent and -independent pathways for the activation of RNAIII in vivo. These observations are consistent with a growing body of evidence that underscores the presence of distinct environmental cues in vivo that can affect virulence gene expression and the capacity of such environmental signals to bypass or act in concert with traditional virulence gene regulatory pathways, as established in vitro. For example, Vandenesch et al. [12] showed that, although the level of hla transcription (an agr-regulated gene) was dependent on a functional agr locus, an additional postexponential-phase signal independent of agr function, controlled the timing of RNAIII expression and subsequent hla transcription. It was postulated that this latter temporal signal could be generated by changes in environmental pH, nutrient concentrations, oxygen tension, or accumulation of metabolic products in an agr-independent signal-transduction pathway. Similarly, Goerke et al. [35] recently demonstrated that the expression of the hla gene was inducible in vivo in the absence of a functional agr locus. Thus, 2 agr-deficient isolates were shown to express hla in vivo to near parental levels by using a guinea pig model of subcutaneous device infection [35]. This same group [36] also studied the comparative profiles of RNAIII and hla expression in vitro and in vivo in human S. aureus sputum isolates from patients with cystic fibrosis. Of interest, among isolates that displayed an intact functional agr locus in vitro, none was able to express RNAIII transcripts in vivo by direct transcriptional analyses. However, a number of these strains still expressed hla in vivo, emphasizing the existence of an additional agr-independent regulatory mechanism in vivo. Although the above studies stress the likelihood of distinct physiologic cues for agr activation or repression in vivo, the specific host factor(s) within the vegetation microenvironment responsible for triggering RNAIII expression in the absence of the RNAII activation signal in the current study are not known. Thus, we were unable to induce RNAIII expression in the agr mutant constructs in vitro by incubation in either dilute rabbit serum or plasma, suggesting that other or additional environmental cues are required in vivo for such expression. There are several hypothetical pathways by which RNAIIindependent activation in vivo might occur. For example, in an agrA mutant, the P2-P3 intergenic region of 191 bp is still present. Thus, it is conceivable, but not proven, that SarA, in conjunction with additional in vivo microenvironmental signals, might cooperate to activate RNAIII expression [26]. Moreover, a number of new regulatory factors, homologous to SarA (e.g., SarU [also called Rlp] [37]) were recently discovered. These factors may well impact RNAIII expression via RNAII-independent pathways. Studies are in progress in our laboratories to further investigate the role of selected host factors and RNAII-independent regulons in mediating selective virulence gene expression in vivo. Figure 7 provides a theoretical in vivo agr activation model in this context, incorporating both existing regulatory paradigms, with several proposed in vivo regulatory pathways. Finally, to circumvent the potential problems of nonspecific host tissue fluorescence, as well as unintended acquisition of tissue debris by flow cytometry, in our studies, we developed an S. aureus protein A immunofluorescent detection system to assure that only S. aureus cells, not host cells, were analyzed for promoter activation profiles in vivo. The development of this immunodetection technique and the availability of gene promoter:reporter fusion constructs may facilitate the study of other putative S. aureus virulence genes in vivo in various model systems. We thank Yin Li for excellent technical assistance and Richard Proctor and Peter McNamara (University of Wisconsin) for invaluable discussions.


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Yan-Qiong Xiong, William Van Wamel, Cynthia C. Nast, Michael R. Yeaman, Ambrose L. Cheung, Arnold S. Bayer. Activation and Transcriptional Interaction between agr RNAII and RNAIII in Staphylococcus aureus In Vitro and in an Experimental Endocarditis Model, Journal of Infectious Diseases, 2002, 668-677, DOI: 10.1086/342046