ABSTRACT
The potent phagocytic and microbicidal activities of neutrophils and macrophages are among the first lines of defense against bacterial infections. Yet Staphylococcus aureus is often resistant to innate immune defense mechanisms, especially when organized as a biofilm. To investigate how S. aureus biofilms respond to macrophages and neutrophils, gene expression patterns were profiled using Affymetrix microarrays. The addition of macrophages to S. aureus static biofilms led to a global suppression of the biofilm transcriptome with a wide variety of genes downregulated. Notably, genes involved in metabolism, cell wall synthesis/structure, and transcription/translation/replication were among the most highly downregulated, which was most dramatic at 1 h compared to 24 h following macrophage addition to biofilms. Unexpectedly, few genes were enhanced in biofilms after macrophage challenge. Unlike coculture with macrophages, coculture of S. aureus static biofilms with neutrophils did not greatly influence the biofilm transcriptome. Collectively, these experiments demonstrate that S. aureus biofilms differentially modify their gene expression patterns depending on the leukocyte subset encountered.
INTRODUCTION
Staphylococcus aureus produces numerous virulence factors that facilitate its ability to invade, colonize, disseminate to distant sites, and impede host defenses to cause disease (1, 2). These characteristics can be amplified during formation of biofilm, which represents a complex multicellular community of organisms encased in a matrix composed primarily of polysaccharides, extracellular DNA (eDNA), and proteins (3–5). S. aureus biofilm infections are often difficult to treat due to their heterogeneity and altered metabolic and transcriptional activity (6), which likely contribute to the chronic and recurrent nature of biofilm infections (7–10). Our recent studies have demonstrated that S. aureus biofilms interfere with traditional microbial recognition and killing mechanisms of the innate immune system (7, 9). The subversion of these responses is another example of the remarkable success of S. aureus as a pathogen, and it is now clear that biofilm growth represents yet another immune resistance determinant. However, our understanding of the cross talk between S. aureus biofilms and the immune response is limited.
Neutrophils are important antimicrobial effectors that possess an arsenal of bactericidal compounds, including defensins, cathelicidins, and lysozyme (11, 12). In terms of their microbicidal activity, neutrophils are most notable for their ability to produce large amounts of reactive oxygen intermediates catalyzed by NADPH oxidase. In addition, activated neutrophils degranulate and release neutrophil extracellular traps (NETs), meshworks of DNA and enzymes that facilitate the extracellular killing of S. aureus as well as other bacteria (13). However, the short life span of neutrophils requires their constant recruitment to sites of infection, and their transcriptional capacity for inflammatory mediator production is more limited than that of other professional phagocytes (i.e., macrophages and dendritic cells). Macrophages reside in virtually all tissues and also serve as a critical first line of defense against microbial invasion. In addition, macrophages are a major source of proinflammatory mediators that are critical for amplifying leukocyte recruitment and activation cascades upon bacterial exposure, as well as providing potent phagocytic and antimicrobial effects (14, 15). Like neutrophils, macrophages can form macrophage extracellular traps (METs),which are believed to exert similar antimicrobial activity (16). Both macrophages and neutrophils are also equipped with an arsenal of pattern recognition receptors that sense invariant motifs expressed across a broad range of microbial species to trigger inflammatory mediator release (17, 18). Consequently, neutrophils and macrophages represent key antimicrobial effector populations, and their interactions with S. aureus biofilms is likely critical for dictating the outcome of infection. Our previous studies have demonstrated that S. aureus biofilms impair macrophage phagocytosis and induce cell death (7, 9, 19); however, the response of the biofilm itself to these leukocyte populations remains to be defined.
While considerable progress has been made in defining S. aureus virulence factors and their regulatory networks, less is known about the organism's ability to cope with the host immune response during biofilm growth (20–22). Genome-wide transcriptional profiling of planktonic S. aureus following neutrophil exposure has previously been reported (23, 24); however, the transcriptional changes occurring in S. aureus biofilms in response to neutrophils or macrophages has not yet been investigated. We predicted that S. aureus biofilms modify their transcriptome in response to these leukocyte subsets to subvert immune recognition and killing, thus favoring biofilm persistence. This possibility was assessed by defining alterations in S. aureus biofilm gene expression profiles after coculture with macrophages or neutrophils utilizing S. aureus Affymetrix GeneChip arrays. Here we report that S. aureus biofilms respond differently to these leukocyte populations, with kinetic distinctions also observed. For example, macrophage addition induced a generalized repression of the biofilm transcriptome within 1 h, whereas at a later interval this inhibition had dissipated, which correlated with the biofilm's ability to induce macrophage cell death. In contrast, the biofilm transcriptome remained relatively stable following neutrophil addition, regardless of the interval examined. These results indicate that S. aureus biofilms discriminate between leukocyte subsets and alter their transcriptional profile accordingly, presumably in favor of avoiding detection by the host, leading to biofilm persistence.
MATERIALS AND METHODS
Bacterial strains.S. aureus USA300 LAC, obtained from Frank DeLeo at NIAID Rocky Mountain Laboratories (NIH, Hamilton, MT), was used for all experiments in this study. For confocal imaging, the USA300 LAC-GFP strain was used as previously described (25). The isogenic USA300 LAC agr mutant (Δagr) was provided by Alex Horswill (University of Iowa Medical Center, Iowa City, IA).
Generation of S. aureus static biofilms and visualization by confocal microscopy.USA300 LAC or USA300 LAC-GFP static biofilms were established as previously described (7). Briefly, sterile 2-well glass chamber slides (Nunc, Rochester, NY) were treated with 20% human plasma overnight at 4°C to facilitate bacterial attachment. The following day, plasma coating buffer was removed and each chamber inoculated with USA300 LAC, whereupon bacteria were incubated at 37°C under static aerobic conditions for a period of 4 to 6 days to generate immature and mature biofilms, respectively (7). The medium was carefully replenished every 24 h, and biofilms were visualized using a Zeiss laser scanning confocal microscope (LSM 510 META; Carl Zeiss, Oberkochen, Germany). z-stacks were collected from beneath the glass slide extending to above the point where bacteria could no longer be detected. Three-dimensional imaging of biofilms and measurements to demonstrate biofilm thickness were performed using Zen 2009 software (Carl Zeiss).
Mouse strains and primary macrophage and neutrophil isolation.Primary macrophages and neutrophils were isolated from either the bone marrow or peritoneal cavities of C57BL/6 mice (Charles River Laboratories, Frederick, MD). Peritoneal macrophages were recovered 4 days following an intraperitoneal (i.p.) injection of 4% Brewer's thioglycolate broth as previously described (7). Neutrophil isolation from the peritoneal cavity was performed as previously reported (26, 27). Briefly, C57BL/6 mice received two i.p. injections of 9% casein at 21 and 3 h before cell harvest. Mice were euthanized 3 h after the second injection, whereupon the peritoneal cavity was lavaged twice with 5 ml of sterile phosphate-buffered saline (PBS). Cells were washed and layered on a 90% Percoll gradient (GE Healthcare, Piscataway, NJ), and after centrifugation (65,000 × g, 25 min at 4°C, no brake), neutrophils were recovered from the intermediate layer. Cells were then processed using a Miltenyi Anti-Ly6G MicroBead kit (Miltenyi Biotec, Cologne, Germany) for further purification per the manufacturer's instructions. For experiments to examine S. aureus biofilm effects on leukocyte phagocytosis and cell death, bone marrow-derived macrophages were prepared as previously described (7), and neutrophils were isolated from the bone marrow (28) and purified using an anti-Ly6G kit as described above. Our prior studies have demonstrated that thioglycolate-elicited peritoneal macrophages and bone marrow-derived macrophages behave similarly in response to S. aureus biofilms in terms of their phagocytic defects and susceptibility to cell death (7). The animal use protocol, approved by the University of Nebraska Medical Center Animal Care and Use Committee, is in accord with the NIH guidelines for the use of rodents.
Macrophage and neutrophil cocultures with S. aureus biofilms.Macrophages or neutrophils (107 and 106, respectively) were cocultured with S. aureus biofilms for various periods to assess their impact on the biofilm transcriptome, which equated to a multiplicity of infection (MOI) of 10:1 (bacteria/macrophage ratio) or 100:1 (bacteria/neutrophil ratio). Macrophages were used at a higher MOI based on our recent studies demonstrating their inability to affect S. aureus biofilm growth (7), whereas neutrophils are considered more effective phagocytes in terms of staphylococcal immunity, which is supported by their ability to phagocytose S. aureus biofilm in these studies. Neutrophils or macrophages were incubated with biofilms in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) at 37°C under static aerobic conditions until they were harvested by mechanical dissociation at two different time points (1 and 24 h for macrophages; 1 and 4 h for neutrophils) to collect RNA for microarray analysis. These time points were selected based on pilot studies and our prior work (7).
For visualization of macrophage- or neutrophil-biofilm interactions, leukocytes were labeled with 5 μM CellTracker Orange or CellTracker Blue (both from Molecular Probes, San Diego, CA), depending on the experimental setup. Macrophage and neutrophil cell death was assessed using propidium iodide staining solution (eBioscience Inc., San Diego, CA). Leukocyte-biofilm interactions were visualized using a Zeiss laser scanning confocal microscope (LSM 510 META), where z-stacks were collected from beneath the glass slide extending to above the point where labeled macrophages or neutrophils could no longer be detected. Three-dimensional imaging and measurements to demonstrate leukocyte invasion into the biofilm were performed using Zen 2009 software and the Zeiss LSM Image Browser (both from Carl Zeiss).
RNA isolation.At the appropriate intervals after biofilm-leukocyte coculture, excess medium was removed from biofilm chambers and 2× the remaining volume of RNAprotect (Qiagen, Hilden, Germany) was added. Biofilms were collected from the bottom of the chamber slide using a cell scraper. RNA isolated from biofilms alone (i.e., with no leukocyte addition) was included as a control for comparisons. The resulting suspension of biofilm cells was transferred to a tube and sonicated for 5 min to facilitate dispersal. After sonication, cells were pelleted by centrifugation for 5 min and RNAprotect was decanted. The resulting pellet was resuspended in 700 μl of RLT buffer supplemented with β-mercaptoethanol and transferred to a 2 ml FastPrep lysing tube (MP Biomedicals, Santa Ana, CA). Biofilm cells were lysed in a FastPrep high-speed homogenizer (MP Biomedicals) for 20 s on a speed setting of 6. The resulting lysate was incubated for 5 min on ice and then centrifuged at 14,000 rpm at 4°C for 15 min. RNA was isolated from the clarified supernatant using a RNeasy minikit (Qiagen), and contaminating DNA was removed by on-column DNase digestion using the RNase-Free DNase set (Qiagen). RNA was isolated from eight samples for each time point and coculture condition, with three independent experimental replicates performed to assess the reproducibility of microarray results. RNA quality and quantity were determined using an Agilent RNA6000 Nano kit and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
RNA labeling and DNA microarray analysis.Following the manufacturer's recommendations, 75 ng of total RNA from each sample was amplified using ExpressArt bacterial mRNA amplification Nano kits (AmpTec GmbH, Germany) and labeled using BioArray high-yield RNA transcript labeling kits (Enzo Life Sciences, Inc., Farmingdale, NY). Three micrograms of the resulting labeled RNA was hybridized to a S. aureus GeneChip following the manufacturer's recommendations for antisense prokaryotic arrays (Affymetrix, Santa Clara, CA) and then washed, stained, and scanned as previously described (29, 30). Commercially available GeneChips were used in this study, representing >3,300 S. aureus open reading frames (ORFs) and >4,800 intergenic regions from strains N315, Mu50, NCTC 8325, and COL (Affymetrix). GeneChip signal intensity values for each biofilm sample at each replicate time point (n ≥ 3) were normalized to the median signal intensity value for each GeneChip and averaged using GENESPRING 7.2 software (Agilent Technologies, Redwood City, CA). Transcripts that (i) demonstrated at least a 2-fold change in expression, (ii) had a greater-than-background signal intensity value and were determined to be “Present” by Affymetrix algorithms, and (iii) had a value that was significant by Student's t test (P = 0.05) were considered differentially expressed.
Verification of differentially expressed genes by qRT-PCR.A subset of biofilm genes that were differentially regulated after leukocyte coculture (i.e., sodA, saeS, saeR, agrB, rsbU, atl, recA, and nuc) was verified by quantitative reverse transcription-PCR (qRT-PCR) using Sybr green. Primers were designed using Primer 3.0 software (Table 1), and melt curve analysis was performed at the end of each amplification run to verify signal specificity. Results are presented as the relative expression compared to that in biofilms that were not cocultured with leukocytes as a reference standard.
Primers used for qRT-PCR validation
Statistical analysis.Significant differences between experimental groups were determined using an unpaired two-tailed Student t test in Prism 4 (GraphPad, La Jolla, CA). For all analyses, a P value of <0.05 was considered statistically significant.
Microarray data accession number.A complete listing of microarray results has been placed in the GEO repository (accession number GSE50675).
RESULTS
Differences in neutrophil and macrophage interactions with S. aureus biofilms in vitro.To evaluate the impact of leukocyte subsets on S. aureus biofilm transcriptional profiles, biofilms that were propagated for either 4 or 6 days were selected for analysis based on their differences in structural maturity (7). Specifically, 4-day-old biofilms were considered more immature in terms of average thickness (32 μm) and irregular density (Fig. 1A and C), whereas 6-day-old biofilms were classified as more mature based on a relatively uniform average thickness (47 μm) and a more consistent density (Fig. 1B and C).
S. aureus biofilm growth states. (A and B) USA300 LAC-GFP was inoculated into sterile 2-well glass chamber slides and incubated at 37°C under static aerobic conditions for a period of 4 (A) or 6 (B) days in RPMI 1640 supplemented with 10% FBS with daily medium replacement. Biofilms were visualized using confocal microscopy (magnification, ×63; 1-μm slices), and representative three-dimensional images were constructed. (C) Quantification of 4- and 6-day-old biofilm thickness. Significant differences are denoted with asterisks (***, P < 0.001 using an unpaired two-tailed Student t test; n = 30 biofilms/time point).
Previous studies from our group have demonstrated that S. aureus biofilms are capable of circumventing macrophage phagocytosis and inducing macrophage death (7, 9, 19). Since the goal of this study was to compare the impact of macrophages versus neutrophils on the biofilm transcriptome, side-by-side comparisons of how both populations interact with the biofilm were required. S. aureus USA300 LAC-GFP static biofilms were grown for 4 or 6 days, whereupon macrophages or neutrophils were cocultured with biofilms for an additional 1, 4, or 24 h. Three-dimensional confocal microscopy images were constructed to demonstrate the proximity of macrophages or neutrophils to the biofilm surface and the extent of phagocytosis (Fig. 2A and B, respectively). Macrophages incubated with S. aureus biofilms for either a 1- or 24-h period contained few internalized bacteria, a phenomenon that was independent of biofilm age, as we have previously described (Fig. 2A and 3) (7). In contrast, intracellular bacteria were readily discernible in neutrophil biofilm cocultures at 1, 4, and 24 h (Fig. 2B and 3; see Fig. S1 in the supplemental material). In addition, the majority of neutrophils were found in close association with the biofilm surface, whereas most macrophages remained distant from the biofilm (Fig. 2).
S. aureus biofilm-leukocyte coculture paradigm. USA300 LAC-GFP static biofilms (green) were grown for 6 days, whereupon bone marrow-derived macrophages (A) (orange) or bone marrow-isolated neutrophils (B) (blue) were incubated with biofilms for 24 h and 4 h, respectively. Biofilm cocultures were visualized using confocal microscopy (magnification, ×63; 1-μm slices), and representative three-dimensional images were constructed. Insets show a higher magnification to highlight the absence of macrophage phagocytosis (A) and the presence of neutrophil phagocytosis (B) of staphylococcal biofilms. Results are representative of two independent experiments examining three individual biofilms each.
Differential responses of innate immune cells to S. aureus biofilms. A total of 107 bone marrow-derived macrophages (Mϕ) or 5 × 106 bone marrow-isolated neutrophils (PMN) were incubated with 6 day-old USA300 LAC-GFP biofilms for 18 to 24 h, whereupon the percentage of leukocytes exhibiting phagocytosis or death was enumerated using confocal microscopy. Significant differences are denoted with asterisks (**, P < 0.01 using an unpaired two-tailed Student t test; n = 3).
Macrophages and neutrophils induce differential gene expression profiles in S. aureus biofilms.The disparity between the abilities of macrophages and neutrophils to phagocytose and invade S. aureus biofilms suggested that these cell types may differentially influence biofilm transcriptional activity. To investigate this possibility, Affymetrix S. aureus GeneChip profiles for biofilms incubated in the presence or absence of either macrophages or neutrophils were compared. Analysis of the number of differentially expressed genes revealed that macrophages caused the most substantive changes in the biofilm, primarily at an early time point (i.e., 1 h) (Fig. 4A and 5A and B; Tables 2 and 3), affecting genes involved in metabolism and transcription/translation/replication for both immature and mature biofilms (Fig. 5). Unexpectedly, most biofilm genes were downregulated in response to acute macrophage exposure, with the exception of several hypothetical genes and a lone gene involved in staphyloxanthin biosynthesis (see Table S1 in the supplemental material). However, the ability of macrophages to induce global gene repression was transient, as the number of genes altered was dramatically reduced after a 24-h coculture period (Fig. 4A and Tables 2 and 3; see Table S1 in the supplemental material). This is likely attributable to the fact that a large percentage (≥55%) of macrophages are dead at approximately 6 h after coculture with S. aureus biofilms and presumably are no longer able to produce factors that influence the biofilm transcriptome (7). A subset of genes identified as differentially expressed by biofilms upon macrophage addition in the microarray analysis were verified by qRT-PCR (Fig. 6).
Acute macrophage addition to S. aureus biofilms leads to the transcriptional repression of numerous genes. The total number of genes significantly up- or downregulated in response to macrophage (A) or neutrophil (B) coculture in immature (4-day-old) or mature (6-day-old) S. aureus biofilms is shown, including those encoding hypothetical proteins.
Classification of genes significantly altered by leukocyte addition in S. aureus biofilms. S. aureus USA300 LAC-GFP static biofilms were grown for 4 or 6 days, whereupon macrophages (A and B) or neutrophils (C and D) were incubated with biofilms for 1 h. The numbers of genes with defined functions (grouped into cell wall/membrane, virulence/defense, regulation, metabolism, transcription/translation/replication, and miscellaneous categories) that were significantly altered after macrophage or neutrophil challenge are shown. Genes encoding hypothetical proteins were not included.
Genes significantly downregulated in 6-day-old S. aureus biofilm versus 1-h biofilm-macrophage cocultures
Genes differentially expressed in 6-day-old S. aureus biofilm versus 24-h biofilm-macrophage cocultures
qRT-PCR validation of downregulated genes in S. aureus biofilms identified by microarray analysis. A subset of genes identified by microarray analysis was confirmed by qRT-PCR following either a 1- or 24-h coculture period of macrophages with 4-day-old USA300 LAC static biofilms. Results are presented as the relative gene expression after macrophage-biofilm coculture compared to that in biofilms that were not incubated with macrophages as a reference standard.
In comparison to macrophages, neutrophils were more limited in their ability to affect S. aureus biofilm gene transcription (Fig. 4B and 5C and D; Tables 4 and 5; see Table S2 in the supplemental material). Of note, many genes remained unaltered following macrophage or neutrophil addition to biofilms, suggesting that these genes are important for biofilm maintenance in the face of an immune challenge. Nonetheless, neutrophils and macrophages differentially impact the S. aureus biofilm transcriptome as revealed by the dichotomy in the observed gene expression patterns.
Genes differentially expressed in 6-day-old S. aureus biofilm versus 1-h biofilm-neutrophil cocultures
Genes differentially expressed in 6-day-old S. aureus biofilm versus 4-h biofilm-neutrophil cocultures
Increased agr transcription by S. aureus biofilms promotes resistance to neutrophil challenge.agrA and agrB were both significantly enhanced in 4-day-old S. aureus biofilms following a 1-h exposure to neutrophils (see Table S2 in the supplemental material). To evaluate the importance of increased agr transcriptional activity in the face of neutrophil challenge, we compared the extents of biofilm phagocytosis and death of neutrophils after coculture with USA300 LAC or its isogenic agr mutant (Δagr). While the Δagr mutant displayed significantly thicker biofilms as previously reported by others (Fig. 7A) (31, 32), neutrophils coincubated with the Δagr mutant demonstrated significantly less cell death and slightly enhanced phagocytosis of biofilm-associated bacteria, although the latter did not reach statistical significance (Fig. 7B). These results suggest that while the physical nature of the biofilm can limit the extent of phagocytosis, secreted factors also appear to play a role in evasion of neutrophil effector functions, at least in vitro, a hypothesis further corroborated by the types of genes known to be regulated by the agr operon (33, 34) as well as data previously reported by others (35). This functional study further substantiates our microarray data by illustrating how the enhanced expression of specific genes may facilitate biofilm evasion of innate immune mechanisms.
agr promotes S. aureus biofilm resistance to neutrophil challenge. S. aureus USA300 LAC-GFP wild-type (WT) and isogenic Δagr static biofilms were grown for 6 days and visualized using confocal microscopy. (A) Quantification of 4- and 6-day-old biofilm thickness. Significant differences are denoted with asterisks (***, P < 0.001 using an unpaired two-tailed Student t test; n = 60). (B) Neutrophils (5 × 106) were incubated with biofilms for 20 h, whereupon the percentages of phagocytic and dead neutrophils were enumerated. Significant differences are denoted with asterisks (*, P < 0.05 using an unpaired two-tailed Student t test; n = 3).
Differential responses of macrophages and neutrophils to S. aureus biofilms are cell autonomous.To determine whether the differences between macrophages and neutrophils in the ability to phagocytose biofilm-associated bacteria could be influenced by one another, both leukocyte populations were cocultured with USA300 LAC biofilms. The extent of macrophage and neutrophil invasion into the biofilm was similar whether cells were added together (Fig. 8A) or separately (Fig. 8C). Similarly, macrophages were incapable of phagocytosing biofilm-associated S. aureus whether cocultured with neutrophils (Fig. 8B) or alone (Fig. 8D), whereas neutrophils were equally phagocytic under both conditions (Fig. 8B and D). These data demonstrate that the phagocytic ability of neutrophils over macrophages in regard to S. aureus biofilm is cell autonomous and is not influenced by the other population in vitro.
Differential responses of macrophages and neutrophils to S. aureus biofilms are cell autonomous. Confocal microscopy quantification of invasion or phagocytosis of macrophages and neutrophils (5 × 106 each) cultured either together (A and B) (**, P < 0.01 using an unpaired two-tailed Student t test [n = 4]; ***, P < 0.001 using an unpaired two-tailed Student t test [n = 4]) or separately (C and D) (*, P < 0.05 using an unpaired two-tailed Student t test [n = 2]; **, P < 0.01 using an unpaired two-tailed Student t test [n = 2]) with 6-day-old S. aureus USA300 LAC-GFP static biofilms at 4 and 24 h.
DISCUSSION
One facet of S. aureus pathogenesis is the organism's ability to maintain cellular homeostasis while enduring immune-mediated stresses (36). S. aureus has been shown to interfere with virtually every level of the host immune response, including increased resistance to antimicrobial peptides, impairment of phagocyte recruitment, escape from NETs, resistance to intracellular killing, and interference with complement function as well as antibody-mediated opsonization (37). The objective of this study was to examine how relevant innate immune cell populations, such as neutrophils and macrophages, alter the S. aureus biofilm transcriptome. To our knowledge, there have been no studies to date examining biofilm gene expression profiles after incubation with innate immune cell populations with any species of bacterial biofilm. Based on our preliminary data and the fact that neutrophils are generally shorter lived than macrophages, we chose early (i.e., 1- and 4-h) and late (i.e., 24-h) time points for biofilm cocultures, respectively. Surprisingly, in subsequent studies, we found that a significant number of neutrophils remained viable at 24 h after biofilm addition; therefore, neutrophil coincubation periods were extended in later confocal microscopy experiments.
In the current study, the greatest transcriptional impact on S. aureus biofilms was achieved during an early (i.e., 1-h) coculture period with macrophages. This effect was independent of phagocytosis, since we observed few internalized bacteria within macrophages incubated with S. aureus biofilms (7, 19). Although numerous genes were repressed following biofilm exposure to macrophages after 1 h of coculture, surprisingly, many genes remained unaltered. These unaffected genes are likely important for biofilm maintenance and may represent an essential core transcriptome needed to maintain biofilm survival and/or evade host antimicrobial effector mechanisms in the face of an immune challenge. In contrast, few genes were altered in S. aureus biofilms following a 24-h exposure to macrophages, which may be explained by changes in macrophage viability following extended biofilm coculture periods. Specifically, our previous work demonstrated that most macrophages remain viable during a 1-h coculture period with biofilms; however, by 6 h macrophage viability is significantly reduced as measured by the live/dead stain 7-aminoactinomycin D (7-ADD) (7). Together, these findings suggest that S. aureus biofilms rapidly transition into a transcriptionally dormant mode after macrophage challenge, which is reversed once the pressure of viable macrophages has dissipated. We cannot exclude the possibility that minor changes in biofilm gene expression could have occurred following leukocyte addition that were not detected due to limitations in microarray sensitivity. In addition, although extreme care was taken to ensure rapid processing of biofilms prior to RNA isolation, it remains possible that some transcriptional changes may have occurred during this interval due to the short half-lives of many bacterial mRNAs (38). However, since we compared the transcriptional profiles of biofilms alone concurrent with leukocyte coculture conditions, any changes in mRNA turnover would be expected to occur at the same rate.
Despite the extensive microbicidal mechanisms employed by neutrophils, these cells did not significantly alter the S. aureus biofilm transcriptome. This was unexpected, since we predicted that neutrophil challenge would enhance bacterial virulence factor expression to interfere with recognition/killing mechanisms. While recent studies have demonstrated the ability of S. aureus to survive intracellularly within neutrophils and, to a lesser extent, macrophages, one would expect this adaptation to necessitate large-scale transcriptional changes (39). One possibility to explain the discrepancy between macrophages and neutrophils in regulating biofilm transcriptional responses is that neutrophils were added to biofilms at a 10-fold-lower density than macrophages. However, our confocal analysis indicated that neutrophils invade the biofilm in greater numbers, remain viable for a longer period than macrophages, and exhibit phagocytosis. Nonetheless, phagocytosis appears to be futile, since neutrophil coculture with S. aureus biofilms does not significantly decrease bacterial numbers in vitro (19). Additionally, neutrophils have not proven to play a key microbicidal role in an S. aureus catheter-associated biofilm model in vivo, showing minimal impact on bacterial burdens (10, 19).
While macrophages and neutrophils perform many overlapping functions in terms of bactericidal activity, subtle specialization of labor could potentially account for the S. aureus biofilm transcriptional differences reported in this study. For example, while neutrophils are considered potent phagocytic effectors against numerous extracellular bacteria, activated macrophages are recognized as a signaling hub during bacterial infections through their secretion of numerous immune-stimulatory and bactericidal factors (14, 15, 40–42). Therefore, it is possible that S. aureus biofilms are more responsive to this secreted milieu than physical disruption via phagocytosis, although this remains speculative.
In addition to producing molecules to circumvent the host immune system, S. aureus biofilms must adapt to nutrient-limiting conditions encountered during growth. Interestingly, macrophage addition to S. aureus biofilms repressed numerous metabolism-associated genes, further suggesting that biofilms transition into a dormant mode to evade immune killing pathways. Correspondingly, macrophage exposure also leads to the repression of genes implicated in transcription, translation, and replication as well as cell wall synthesis, which would be expected to conserve energy during a decreased metabolic state. The repression of cell wall synthesis may also be a mechanism to evade innate immune recognition.
The agr and sarA regulatory systems are among the many controlling the production of staphylococcal virulence and defense factors. The agr locus encodes a quorum-sensing system involved in RNAIII production, which switches the synthesis of surface and adhesive molecules to toxin and exoprotein expression (2). SarA is a DNA-binding regulator protein that influences the expression of multiple genes, including those contributing to virulence and biofilm formation (29, 43). In this study, sarA transcription was reduced in S. aureus biofilms following macrophage exposure, which agrees with the generalized decrease in numerous genes regulated by SarA. In contrast, agr transcription was significantly increased in S. aureus biofilms after neutrophil addition. The enhanced expression of agr in this setting allowed us to examine its contribution to neutrophil reactivity using an isogenic agr mutant. The percentage of neutrophils exhibiting phagocytic activity was increased in Δagr biofilms, despite the significant increase in biofilm thickness. This phenotype may be explained, in part, by alterations in phenol-soluble modulin (PSM) expression. PSMs are surfactant peptides that have recently been implicated in bacterial biofilm formation across a number of species, including S. aureus (44). PSM expression is intricately linked to bacterial density and is agr regulated (44). Based on this evidence, it is reasonable to predict that although Δagr biofilms are thicker than wild-type biofilms, they are likely less structurally complex and unable to effectively circumvent neutrophil invasion. In addition, Δagr biofilms elicited less neutrophil death, likely due to the reduced expression of lytic toxins, such as alpha-toxin and PSMs, which have been shown to play key roles in S. aureus pathogenesis (45, 46). There were no significant effects on macrophage phagocytosis or viability in response to Δagr biofilms (data not shown), implying that enhanced agr transcription is selective for thwarting aspects of neutrophil function. This finding further substantiates a potential selective role for impaired PSM action on neutrophils, since its cytotoxic effects primarily target this cell type (47, 48). Finally, as PSM expression is induced by the stringent response resulting from the harsh conditions within the phagolysosome (49), impaired PSM activity provides a potential explanation for the increased intracellular burden of Δagr biofilms by neutrophils. The impressive ability of S. aureus biofilms to adapt to neutrophil challenge correlates with the modest transcriptome changes reported in this study.
In conclusion, the changes in S. aureus biofilm transcriptional profiles after leukocyte exposure reported here provide a comprehensive view of the molecules that may impact S. aureus immune evasion and survival during biofilm growth. Further investigations into the role of differentially regulated genes will provide a better understanding of the ability of S. aureus to adapt to environmental challenges and may provide novel strategies and therapeutic targets for staphylococcal biofilm infections.
ACKNOWLEDGMENTS
We thank Vinai C. Thomas for primer design and helpful suggestions regarding gene classification based on biofilm transcriptome analysis, Amy Aldrich for performing qRT-PCR analysis, Paul Fey and Kenneth Bayles for helpful discussions, and Paul Fey for critical review of the manuscript.
This work was supported by National Institute of Allergy and Infectious Disease (NIAID) P01 AI083211 project 4 to T.K.
FOOTNOTES
- Received 2 July 2013.
- Returned for modification 14 August 2013.
- Accepted 5 September 2013.
- Accepted manuscript posted online 16 September 2013.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00819-13.
- Copyright © 2013, American Society for Microbiology. All Rights Reserved.
