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. Author manuscript; available in PMC: 2017 May 1.
Published in final edited form as: Cell Microbiol. 2016 Jan 12;18(5):720–732. doi: 10.1111/cmi.12543

Development of an in vitro colonization model to investigate Staphylococcus aureus interactions with airway epithelia

Megan R Kiedrowski 1, Alexandra E Paharik 1, Laynez W Ackermann 1, Annie U Shelton 2, Sachinkumar B Singh 2, Timothy D Starner 2, Alexander R Horswill 1,*
PMCID: PMC4840028  NIHMSID: NIHMS750565  PMID: 26566259

SUMMARY

Staphylococcus aureus is a bacterial pathogen responsible for a wide range of diseases and is also a human commensal colonizing the upper respiratory tract. Strains belonging to the clonal complex group CC30 are associated with colonization, although the colonization state itself is not clearly defined. In this work, we developed a co-culture model with S. aureus colonizing the apical surface of polarized human airway epithelial cells. The S. aureus are grown at the air-liquid interface to allow an in-depth evaluation of a simulated colonization state. Exposure to wild-type S. aureus bacteria or conditioned media killed airway epithelial cells within one day, while mutant S. aureus strains lacking alpha-toxin (hla) persisted on viable cells for at least two days. Recent S. aureus CC30 isolates are natural hla mutants, and we observed that these strains displayed reduced toxicity toward airway epithelial cells. Quantitative real-time PCR of known virulence factors showed the expression profile of S. aureus grown in co-culture correlates with results from previous human colonization studies. Microarray analysis indicated significant shifts in S. aureus physiology in the co-culture model toward lipid and amino acid metabolism. The development of the in vitro colonization model will enable further study of specific S. aureus interactions with the host epithelia.

INTRODUCTION

Staphylococcus aureus is a bacterial pathogen known for causing severe acute and chronic infections. Numerous surface and secreted virulence factors facilitate this infection diversity, and their regulation is under control of a complex network of signal transduction and global regulatory systems (Lowy, 1998). Despite this pathogenic prowess, S. aureus is also a human commensal that colonizes 30% of the healthy adult population (Gorwitz et al., 2008), predominantly in the anterior nares, although the throat and skin are also colonized at high rates (Nilsson et al., 2006, Andrews et al., 2009, Lee et al., 2011). While colonization is known to be a risk factor for disease and has been tracked extensively (Wertheim et al., 2005), the specific S. aureus – host interaction mechanisms that occur during colonization are only beginning to be unraveled.

Through nasal carriage sampling and multi-locus sequence typing (MLST), a series of studies identified the S. aureus clonal complexes most frequently associated with colonization. Analysis of 179 asymptomatic nasal isolates in the United Kingdom indicated the dominant lineage was clonal complex 30 (CC30) at 26% of strains (Feil et al., 2003). Similar observations were made in nasal isolate collections from Germany at 27% CC30 (Holtfreter et al., 2007) and the Netherlands at 26% CC30 (Melles et al., 2004). In the United States, the dominant lineage in nasal collections is pulsed-field gel electrophoresis (PFGE) type USA200 at 25–29% (Tenover et al., 2008), depending on the sampling year. PFGE type USA200 consists mainly of strains classified as MLST ST30 (McDougal et al., 2003, Tenover et al., 2008), which is part of the CC30 group, indicating the CC30 lineage is also the dominant nasal carriage isolate in the United States. Nasal carriage strains isolated from the 1980s to present have been categorized as “contemporary CC30” due to the presence of conserved nucleotide polymorphisms, including an alpha-toxin (hla) nonsense mutation and an agrC G55R mutation that limits AgrC function (Deleo et al., 2011). These adaptations attenuate acute virulence in animal models, an advantage that has been suggested to promote long-term colonization.

Animal and in vitro studies have demonstrated that a number of S. aureus surface adhesins are important for colonization. Clumping factor B (ClfB) is required to bind type I cytokeratin 10 on desquamated nasal epithelia (O’Brien et al., 2002, Walsh et al., 2004). The surface adhesins SasG (Roche et al., 2003), IsdA (Corrigan et al., 2009), SdrC and SdrD (Corrigan et al., 2009), as well as the fibronectin binding proteins (Mongodin et al., 2002), have also been demonstrated to have roles in adherence to host cells. ClfB and IsdA are essential in rodent models of nasal colonization (Clarke et al., 2006, Schaffer et al., 2006), and not surprisingly, the extracellular enzyme that localizes all of these surface adhesins to the cell wall, sortase (SrtA), is also required for colonization (Schaffer et al., 2006). In human studies, ClfB was again found to be necessary for colonization (Wertheim et al., 2008), supporting in vitro studies and prompting development of ClfB as a vaccine candidate (Schaffer et al., 2006). One drawback of most studies utilizing human cell lines is that S. aureus exposure to airway epithelial cells in vitro is brief, addressing initial attachment rather than long-term colonization (Mongodin et al., 2002, O’Brien et al., 2002, Roche et al., 2003, Corrigan et al., 2009). Aside from the role of specific adhesins or binding proteins, these studies provide little information on the physiological state of the bacterial cells in this host niche.

Despite epidemiological advances and initial successes with animal models, the definition of what morphologically and physiologically constitutes the S. aureus colonized state is not completely clear. It is widely acknowledged that S. aureus has a distinct lifestyle during growth in biofilms compared to growth in a planktonic state, with over 500 genes differentially regulated (Beenken et al., 2004), and there have been recent suggestions that S. aureus also colonizes in a biofilm-like state (Iwase et al., 2010). However, studies using animal models indicated that during colonization, S. aureus persists as single cells or in small clumps on the mucosal surface (Burian et al., 2010a), prompting proposals that colonization is a unique state of growth (Krismer et al., 2011). As S. aureus is also known to bind mucosal secretions (Shuter et al., 1996), a mixed population of cell-attached and mucosal-bound bacteria could be present in the colonization environment, adding another level of complexity.

In this study, we provide an in-depth examination of S. aureus interactions with airway epithelia using a novel in vitro co-culture model that allows us to observe S. aureus growth for a couple days following initial inoculation. We identified alpha-toxin production as a crucial factor in determining whether a strain will colonize airway cells or induce epithelial cell death. Quantitative real-time PCR and microarray analysis of S. aureus grown in the co-culture model show differential expression patterns of virulence factors and genes involved in metabolism, shedding light on what it means to exist in a colonizing state. Overall, this work takes an important step toward better understanding the nature of S. aureus colonization of the human respiratory tract and has resulted in the development of a colonization model that will allow us to further study specific S. aureus interactions with the host epithelia.

RESULTS

S. aureus damages airway epithelia

To model S. aureus colonization of mucosal surfaces, we selected Calu-3 cells as a representative airway epithelial cell line. Calu-3 cells polarizes and forms tight junctions in vitro at the air liquid interface (ALI), and the morphology and electrical resistance of these cells is similar to the in vivo airway (Grainger et al., 2006). Furthermore, air-liquid interface Calu-3 cell cultures and submerged primary human airway epithelial cultures displayed similar degrees of relatedness to in vivo airway epithelia based on transcriptional profiling and unsupervised hierarchical clustering analysis (Pezzulo et al., 2011). Calu-3 cells have been successfully used to investigate Haemophilus influenzae interactions with polarized airway epithelia (Starner et al., 2006), and we used a similar approach in a co-culture model with S. aureus (Fig. 1A). In our initial studies, we tested a USA300 clinical isolate of the community-associated methicillin resistant S. aureus (CA-MRSA) called LAC as well as the laboratory strain SH1000 (see Table 1). We observed that both strains markedly reduced the viability of the Calu-3 airway epithelial cell monolayer after one day of co-incubation through transepithelial resistance (TR) measurements (Fig. 1B) and gentian violet staining (Supplemental Fig. 1). We hypothesized that S. aureus was secreting a damaging agent that was toxic to the epithelial cells, and to test this question, we compared filtered, conditioned media from LAC bacterial cultures to whole bacteria in viability assays. As shown in Figure 1C, LAC whole bacteria induced killing of Calu-3 airway epithelial cells twenty-four hours following inoculation. Conditioned media retained the killing activity, demonstrating that the unknown toxic factor was secreted rather than localized to the bacterium (Fig. 1C).

Figure 1. Modeling S. aureus interactions with airway epithelia.

Figure 1

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A co-culture model utilizing S. aureus and polarized Calu-3 airway epithelial cells grown at the air-liquid interface. Bacteria or conditioned media may be added to the apical chamber of the transwell to observe effects on Calu-3 cells (A). Transepithelial resistance (TR) measurements of polarized Calu-3 monolayers following addition of a PBS control or whole bacteria (B). TR measurements of polarized Calu-3 monolayers following addition of either PBS control, bacterial media control, 1 × 108 CFU whole bacteria, 2 × 107 CFU whole bacteria, bacterial conditioned media, or diluted bacterial conditioned media to the apical surface (C). Experiments performed at least three times, with statistical significance relative to 0 hour time point by unpaired, two-tailed Student’s T test (** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001).

Table 1.

Strains and plasmids

Strain or Plasmid Description Source or reference
Strains
S. aureus
  SH1000 rsbU+ version of 8325-4 (Horsburgh et al., 2002)
  AH1263 USA300 CA-MRSA Erms (Boles et al., 2010)
  AH1292 AH1263/Δagr∷TetM (Mootz et al., 2013)
  AH1589 AH1263/hla∷Erm (Olson et al., 2013)
  AH1754 AH1263/hla∷Erm/pCM30 This work
  AH1726 AH1263/pCM29 (Chiu et al., 2013)
  AH1792 AH1589/pCM29 This work
  AH2216 AH1263/ΔsaePQRS (Flack et al., 2014)
S. aureus CC30
  UAMS-1 Osteomyelitis isolate (Gillaspy et al., 1995)
  3636 1567493 (Deleo et al., 2011)
  11512 MRSA252 (Holden et al., 2004)
  21203 MN8 (Lin et al., 2011)
  21247 HT 2000 0328 (Deleo et al., 2011)
  21295 No. 426; 5442 (Deleo et al., 2011)
  21345 HT 20020470 (Deleo et al., 2011)
  22030 M-1015 (Deleo et al., 2011)
  22033 WBG 10049 (Deleo et al., 2011)
  22251 65–20 (Deleo et al., 2011)
Plasmids
 pCM29 pCE with PsarA − sGFP, CamR (Pang et al., 2010)
 pCM30 hla complementing clone, CamR (Nygaard et al., 2012)
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Alpha-toxin is the primary secreted damaging agent

Based on the evidence that S. aureus secretes a factor that is toxic to airway epithelial cells, we tested the cytotoxicity of mutants in global regulators of secreted virulence factor gene expression. Strains with mutations in the agr and sae regulators were selected for initial testing, as these are major toxin regulators in the USA300 LAC strain background (Nygaard et al., 2010, Pang et al., 2010, Thoendel et al., 2011). Experiments were also performed in parallel with a nasal epithelial cell line, RPMI2650 (Quinn et al., 2007), to better simulate the nasal environment. After one day of exposure to different amounts of conditioned media from wild-type (WT) LAC and the mutant strains, we observed using an LDH release assay that both agr and sae mutants showed a significant reduction in toxicity toward the Calu-3 (Fig. 2A) and RPMI2650 (Fig. 2B) cells compared to WT, and results were similar across both cell lines.

Figure 2. Cytotoxicity of conditioned media from S. aureus mutants deficient in Hla production.

Figure 2

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Percent lysis of Calu-3 (A) and RPMI2650 (B) human airway cells following addition of filtered conditioned media from cultures of the following S. aureus strains: wild-type (WT); hla knockout (Δhla); hla complemented strain (Δhla/Comp); agr two-component system deletion mutant (Δagr); and sae two-component system deletion mutant (Δsae). Percent lysis determined by LDH release assay. White bars indicate 5% conditioned media and black bars indicate 20%. Experiments performed at least three times at each concentration, with statistical significance relative to WT at the 20% conditioned media level, two-tailed Student’s T test (** = p ≤ 0.01; *** = p ≤ 0.001).

In light of a recent report that S. aureus secreted alpha-toxin (Hla) damages A549 airway epithelial cells (Wilke et al., 2010), and knowing that the hla gene is positively regulated by both the agr and sae systems (Nygaard et al., 2010, Pang et al., 2010), we predicted that Hla was the primary damaging agent in our experiments. In support of this proposal, conditioned media from a hla knockout mutant displayed minimal toxicity toward both the Calu-3 and RPMI2650 cell lines, and this phenotype could be complemented (Fig. 2). Real time PCR confirmed that transcript for a disintegrin and metalloprotease 10 (ADAM-10), the receptor for Hla (Wilke et al., 2010), is produced in both the Calu-3 and RPMI2650 cell lines (Supplemental Table 2), with levels over four times higher in Calu-3 cells. Transepithelial resistance (TR) is a measure of polarized airway epithelial integrity, where cytotoxicity or disruption of the apical tight junctions causes a lower TR. Using the ALI co-culture model with Calu-3 cells, the same phenotype of reduced toxicity was observed with the hla knockout (Fig. 3A). Calu-3 cell monolayers co-cultured with WT S. aureus appeared significantly disrupted, while cell monolayers exposed to hla knockout remained intact over two days of co-culture. Thus, from these data we conclude that alpha-toxin is the primary airway epithelial damaging agent produced by S. aureus, although after long-term co-culture additional toxic effects are observed after two days whose cause remains unknown.

Figure 3. Cytotoxicity of S. aureus Hla non-producing strains in co-culture with epithelial cells.

Figure 3

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TR measurements of polarized Calu-3 cell monolayers co-cultured with a S. aureus hla knockout (hla∷Erm, black squares) or wild-type S. aureus (WT, white diamonds) (A). Values for the knockout were statistically significant relative to WT by two-tailed Student’s T test (* = p ≤ 0.1; ** = p ≤ 0.01). TR measurements of Calu-3 cells co-cultured with CC30 strains 21345 (white squares), 21247 (black circles) and 3636 (white triangles) with naturally occurring hla nonsense mutations (B). Compared to WT, values for 21247 (p=0.018) and 3636 (p=0.02) were statistically significant at day 2.

Interactions of S. aureus CC30 strains with airway epithelia

To extend our studies on S. aureus – airway interactions, we tested ten strains belonging to clonal complex 30 (CC30), the dominant nasal carriage isolates identified in recent sampling studies (Holtfreter et al., 2007, Melles et al., 2008). We asked whether selected isolates representative of recent and historical CC30 strains (Table 2) would show similar levels of toxicity toward airway epithelial cells as the USA300 LAC strain. Included in this group is the strain UAMS-1, which is a clinical isolate used in many studies of S. aureus pathogensis (Cassat et al., 2005). Of the group, three isolates with naturally occurring hla nonsense mutations (21345, 21247, and 3636) were selected to test in the ALI co-culture model (Fig. 3B). All three strains showed a similar phenotype of decreased toxicity toward cells as the LAC hla knockout. Calu-3 monolayer integrity was maintained after one day of co-culture with the CC30 isolates, and then viability decreased at day two (Fig. 3B). Conditioned media from all ten isolates was tested. Media from three strains (21295, 22030, and 22033) demonstrated potent toxicity toward the Calu-3 (Fig. 4A) and RPMI2650 (Fig. 4B) cells to a level similar as the USA300 LAC strain, while conditioned media from the other seven isolates displayed minimal toxicity to both airway cell lines, similar to the hla knockout. Toxicity toward airway epithelia correlated with whether strains encoded an intact hla gene to produce alpha-toxin (Table 2).

Table 2.

CC30 strain properties

Strain
MSSA/MRSA
AgrC statusa
Alpha-toxin production
Airway damagec
UAMS-1 MSSA Mutant − −
3636 MSSA Mutant − −
11512 MRSA Mutant − −
21203 MSSA Mutant − −
21247 MSSA Mutant − −
21295 MSSA Wild-type + +
21345 MSSA Mutant − −
22030 MRSA Wild-type + +
22033 MSSA Wild-type + +
22251 MSSA Wild-typeb − −
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a

Strains with AgrC mutations have a G55R substitution.

b

Strain 22251 has an AgrA C123F mutation that blocks alpha-toxin (Hla) production.

c

Damage to Calu-3 and RPMI2650 cell lines based on LDH assay (Fig. 4).

Figure 4. Effects of conditioned media from colonizing CC30 strains on airway epithelia.

Figure 4

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Percent lysis of Calu-3 (A) and RPMI2650 (B) cells following incubation with with 5% (white bars) or 20% (black bars) conditioned media from cultures of S. aureus CC30 strains as measured by LDH release. Strains tested indicated on X axis, along with ability to produce alpha-toxin (hla+ indicates alpha-toxin produced; hla- indicates non-producer). WT indicates conditioned media from wild-type S. aureus. Experiments performed at least three times, with statistical significance relative to WT at the 20% conditioned media level, two-tailed Student’s T test (* = p ≤ 0.1; ** = p ≤ 0.01; *** = p ≤ 0.001).

Growth of S. aureus occurs at the ALI

In the ALI co-culture model, no nutrients are supplemented to the apical surface, and S. aureus must obtain all necessary requirements for survival and growth directly from the Calu-3 cells. We assessed the growth of the LAC hla knockout strain on Calu-3 cells at the ALI to determine whether adherent S. aureus cells grow or remain static in this environment. By performing cell counts of adherent cells at three time points, we observed the number of viable S. aureus increased substantially over two days (Fig. 5A), demonstrating growth at the ALI. Growth of three Hla-deficient CC30 isolates (21345, 21247, and 3636) was also examined, and colony-forming units per mL increased over the two-day co-culture period for all three strains (Fig. 5B).

Figure 5. Growth of S. aureus on airway epithelia at the air-liquid interface.

Figure 5

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Determination of colony forming units per mL (CFU/mL) of a S. aureus hla knockout (LAC hla∷Erm) (A) and three CC30 strains (B), 21345 (diamond), 21247 (square) and 3636 (circle), by plating samples taken 4 hours, 1 day, and 2 days post-inoculation in the co-culture model. Growth difference of the hla knockout in CFU/mL (A) was statistically significant at day 2 versus 4 hr (p = 0.018 by Student’s T test). The CC30 strains (B) showed a positive trend in growth (r2 values 0.87–0.99).

Microscopic characterization of S. aureus at the ALI

We next examined the morphological state of growth of S. aureus at the ALI using scanning electron microscopy (SEM). Focusing on the colonized regions of the Calu-3 cells, clumps of S. aureus hla knockout were observed binding to themselves and the surface of the epithelia (Fig. 6A–D). Although reports of cilia-like structures on the apical surface of Calu-3 cells have been mixed (Grainger et al., 2006), microvilli are clearly visible, and the S. aureus clusters appear to be attaching at or close to these structures (Fig. 6C, D). At higher magnification, some extracellular material can be seen bound to the surface of S. aureus cells (Fig. 6C, D, black arrows), but whether the source of this material is the bacteria or the epithelial cells is unclear. Actively dividing bacteria can also be observed (Fig. 6C, D, white arrows).

Figure 6. Scanning electron microscopy to evaluate interactions between S. aureus and airway epithelial cells.

Figure 6

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S. aureus- Calu-3 co-culture samples were argon-coated and imaged on a Hitachi S-4000 scanning electron microscope at scales of 30 μm (A), 20.0 μm (B), 3.0 μm (C) and 2.0 μm (D). Images depict S. aureus cells bound to the apical surface of Calu-3 cells grown on transwell filters. White arrowheads indicate cells displaying division septa. Black arrowheads indicate extracellular matrix material.

Since SEM captures only the apical surface of the cell monolayer, confocal microscopy was performed using GFP-labeled LAC to achieve a more comprehensive view of interactions between the bacteria and airway cells over time (Fig. 7). Following co-culture with WT LAC, viable Calu-3 cells could only be visualized at the earliest four-hour time point. At four hours, singly attached GFP-expressing S. aureus cells and some small cell clusters on select regions of the apical surface could be observed (Fig. 7A). After one or two days of exposure to LAC WT, the Calu-3 cells were no longer viable, and cell monolayers were completely disrupted, exposing the Transwell filter (Fig. 7B, C). A two-day time course was also performed with GFP-labeled hla knockout cells. At four hours, the phenotype of the hla knockout was similar to LAC WT, with some single GFP-labeled cells seen attached to the surface of the Calu-3 monolayer (Fig. 7D). After one day of co-culture, the density of the attached hla knockout was significantly greater than it had been at four hours (Fig. 7E). The increased number of hla knockout bacteria visualized on day one correlates with the increase in adherent biomass measured at this time point (Fig. 5A). Both viable Calu-3s and attached hla knockout bacteria could be visualized after two days of co-culture (Fig. 7F). 3D renderings and cross-section views of images taken at one day of co-culture with the hla knockout appear to show small clusters of S. aureus adhering to the Calu-3 cells (Fig. 7G, H). In some instances, bacteria in cross-sections appear to be attached not to the surface of the Calu-3 cells but are instead seen in-between cells (Fig. 7G). Coupled with the SEM, these microscopy studies provide visual confirmation of the growth of S. aureus on the Calu-3 cells.

Figure 7. Evaluation of S. aureus growth in co-culture via confocal microscopy.

Figure 7

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Images from a time course growth experiment of a S. aureus WT and hla knockout strains expressing GFP (green/yellow) and Calu-3 cells (red) after 4 hours (A, D), 1 day (B, E) and 2 days (C, F) of co-culture. Depiction of the XY, YZ, and XZ planes of one slice in a Z-series image of S. aureus hla∷Erm Calu-3 co-culture after 1 day (G). A 3D rendering of S. aureus hla∷Erm co-cultured with Calu-3 cells after 1 day of growth (H).

Regulation of S. aureus gene expression at the ALI

A study on S. aureus human nasal carriage isolates was recently performed (Burian et al., 2010b) in which the expression levels of a select group of genes were examined in bacteria collected from nasal swabs of persistently colonized carriers. From the data gathered, a profile of genes showing increased and repressed transcription during colonization was created. We reasoned that this nasal carriage regulatory profile could serve as a biomarker for the S. aureus colonization state. If S. aureus grown in the ALI co-culture model mimics colonization, a similar regulatory profile should be observed. We selected twelve targets that overlapped with the genes reported in the carriage expression study (Burian et al., 2010b) and compared their expression level to S. aureus grown in vitro (Fig. 8). In the ALI model, surface adhesins (clfB, spa) and cell wall biosynthesis components (atlA, sceD, and tarK) were up-regulated. Expression of global regulators varied, with agr (RNAIII) down-regulated, sae similar to the in vitro level, and walKR up-regulated. Of the secreted virulence factors, chp, sak, and nuc were all up-regulated in the ALI model, while PSMβ was down-regulated.

Figure 8. S. aureus gene expression when grown in co-culture with airway epithelial cells.

Figure 8

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Relative amount of mRNA transcript from S. aureus hla∷Erm grown in co-culture with airway epithelial cells compared to bacteria grown in broth culture as measured by qRT-PCR. S. aureus genes are grouped according to the functional roles of the encoded proteins in S. aureus (X axis labels).

Microarray analysis of S. aureus hla∷Erm co-cultured with Calu-3 cells

To expand the genetic profile of S. aureus in the co-culture model, we performed a microarray comparing the S. aureus LAC hla mutant grown in liquid culture to growth in co-culture with Calu-3 cells. In accordance with the RT-PCR results, up-regulated genes included the surface adhesin clfB, and down-regulated genes included the secreted toxins PSMβ1 and PSMβ2, which were 10.1-fold and 9.4-fold, respectively (Fig. 9 and Supplemental Table 3). We also observed differential expression of several metabolic pathway genes. The fatty acid biosynthesis genes fadA, fadB, fadD, and fadE were the most strongly up-regulated genes, with 38.7- to 67.9-fold increases relative to liquid culture growth. Genes in the histidine biosynthesis operon hisIEFAHBCDG were also found to be up-regulated between 4.5- and 8.8-fold (Fig. 9). These results support findings from our RT-PCR experiments and reveal additional differentially regulated genes for further study.

Figure 9. Microarray analysis to evaluate global changes in S. aureus gene expression during growth in co-culture.

Figure 9

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Altered gene expression of a S. aureus hla mutant following two days of ALI co-culture compared to growth in broth culture. The array was performed using an Affymetrix chip. The heat map shows the top 30 S. aureus genes found to be differentially expressed by at least two-fold, with a p-value cutoff of 0.1 based on one-way ANOVA. The lightest shade corresponds to the smallest fold change and darkest shade to the greatest fold change.

DISCUSSION

In this work, we have developed a co-culture model to examine interactions of S. aureus with airway epithelia at the ALI. This work has demonstrated that alpha-toxin (Hla) is a key damaging agent to the epithelia, and S. aureus strains unable to make the toxin, such as representative strains from the CC30 lineage, maintain a long-term interaction in vitro. Using microscopy approaches, S. aureus was found to interact with the airways in single cells or clumps, and the regulatory profile observed in the co-culture model mimicked human nasal carriage.

The complete destruction of airway epithelia observed during co-culture with WT S. aureus was somewhat unexpected (Fig. 1). This pathogen is a human commensal that is predominantly carried on upper respiratory mucosal surfaces, suggesting it has evolved to co-exist at this site in a non-inflammatory state (van Belkum, 2011). Other commensals, such as H. influenzae, can colonize airway epithelia at the ALI for days in vitro with no evidence of detrimental effects (Starner et al., 2006). Most reports of S. aureus in vitro interactions with epithelia cell lines use brief incubation times (less than four hours) to examine adherence (Mongodin et al., 2000, Mongodin et al., 2002, Escotte et al., 2006), although interactions at time points of up to 20 hours have been reported using low inocula with no damage to the airway cells (Quinn et al., 2007). The reason for these short-term experiments could be due to the in vitro toxicity of S. aureus that results from alpha-toxin. Studies with lung epithelial (A549) and cystic fibrosis epithelial (CFT-1) cells have both demonstrated that alpha-toxin is a potent cytotoxic agent (Jarry et al., 2008, Wilke et al., 2010). In time course studies using a human tracheal gland cell line (MM-39), S. aureus was found to induce damage over time, while alpha-toxin levels increased (da Silva et al., 2004). Using mutant and complemented strains in this report, we have made a direct link to alpha-toxin as the primary source of airway damage (Fig. 2, 3). S. aureus alpha-toxin is known to alter tight junction integrity by interfering with processing of E-cadherin (Inoshima et al., 2011, Kwak et al., 2012), which could be the reason for the rapid loss of transepithelial resistance.

Our observations with CC30 strains coincide with findings on S. aureus production of alpha-toxin (Deleo et al., 2011). The CC30 isolates that make significant quantities of alpha-toxin in vitro, such as the 22033 South Pacific clone, destroy the airway epithelia to the same degree as USA300 isolates (Fig. 4). Isolates carrying the hla nonsense mutation cause damage at a similar level to hla knockout mutants of other lineages (Fig. 3B, 4). Given that CC30 strains are the dominant human nasal carriage isolates (Holtfreter et al., 2007, Melles et al., 2008), the fact that many strains belonging to this group do not make alpha-toxin is striking. These isolates also possess an agrC mutation (G55R) that attenuates agr function (Deleo et al., 2011), which could additionally reduce the levels of other potentially damaging virulence factors. Whether these features of many CC30 isolates give them a competitive advantage in the upper respiratory carriage environment remains to be determined.

SEM and confocal microscopy of S. aureus grown on Calu-3 cells in the co-culture model shed light on the bacterial lifestyle during colonization (Fig. 5, 6). There is some debate as to whether S. aureus exists in a biofilm-like state when colonizing nasal mucosal tissue (Krismer et al., 2011), although some groups have drawn this parallel (Iwase et al., 2010). While many cocci are seen clumping to one another and adhering to host tissue projections, there is relatively little extracellular matrix material observed (Fig. 6), unlike traditional S. aureus biofilms that accumulate large amounts of extracellular DNA, proteins, and sometimes polysaccharide (Kiedrowski et al., 2011). Whether a decreased amount of extracellular material is being produced by S. aureus in this environment, or if this decrease can be attributed to a lack of other host cells known to contribute material to the matrix in vivo, is unclear. Confocal images show S. aureus cells migrating down through the Calu-3 monolayer and attaching between cells (Fig. 7G). These images suggest that interacting with specific tight junction proteins is a mechanism that could be utilized by S. aureus for either persistence or invasion of the airway epithelia. Taken together, our microscopy suggests a unique mode of growth for S. aureus during colonization that is distinct from growth of a typical biofilm on an abiotic surface.

The results of the regulation experiments mirrored those of human and rat nasal carriage studies (O’Brien et al., 2002, Burian et al., 2010a, Burian et al., 2010b). Rodent models of nasal carriage, human colonization tests, and immunization experiments have demonstrated that clumping factor B (ClfB) is one of the most important factors in S. aureus nasal colonization (Schaffer et al., 2006, Wertheim et al., 2008, Jenkins et al., 2015). Multiple studies have demonstrated that this surface adhesin is expressed during human colonization (O’Brien et al., 2002, Wertheim et al., 2008), and it was also found to be up-regulated in the ALI co-culture model (Fig. 8). Similar to the findings of Burian et al. (Burian et al., 2010b), the agr system was repressed in the co-culture model, which was supported by the increased levels of spa and decrease in PSMs observed. The WalKR system was up-regulated, along with its outputs sceD, atlA, and sak. The sceD and atlA genes are also important for cell wall maintenance, along with tarK, all of which were up-regulated, again supporting previous observations. Burian et al. reported that the saeRS system was not activated during colonization (Burian et al., 2010b), although the reduction from in vitro expression was quite small. We also noted only a slight reduction in saeRS expression in the co-culture model, but some sae-regulated outputs, such as nuc and chp (Olson et al., 2013), are up-regulated. The chp gene was previously noted to be up-regulated (Burian et al., 2010b). The microarray analysis expanded on these findings and supported our observation that S. aureus in co-culture exhibits a pattern of increased expression of surface adhesins and decreased expression of secreted toxins relative to liquid culture growth (Fig. 9). Similar to the RT-PCR observations, genes within the agrBCDA operon, and those directly within the AgrA regulon (PSMβs) or have known links through Rot repressor (Mootz et al., 2015), are down-regulated in the microarray. Notably, the microarray studies also demonstrated a shift in metabolism to lipid and fatty acid substrates and amino acids. These changes may reflect the metabolic needs of S. aureus during growth on the Calu-3 cells and provide insight into metabolism that occurs in vivo. Interestingly, a recent study by Krismer et al. also described specific metabolic pathways that are required to persist in a synthetic medium that mimics the host nasal mileu (Krismer et al., 2014). Similar to our microarray, the transcriptome analysis of bacteria grown in the synthetic medium compared to basic medium had an overall increase in the expression of the amino acid biosynthesis genes. The Krismer et al. study reports a 5–10 fold increase in expression of the his operon for amino acid metabolism (Krismer et al., 2014), which mirrors our findings. Both studies show similar trends in iron-related genes, with an increase in sbnC expression (11.2 increase versus ours at 3.6) and isdB expression (2.6-fold increase versus ours at 3.3). Although we found a striking upregulation in fatty acid biosynthesis pathway genes, this was not observed in previous studies. These findings suggest that the limited nutrient environment of the host airway requires a specific metabolic profile of colonizing S. aureus. Further, the corroboration of our findings with other results supports our model as a tool for investigating the details of S. aureus colonization.

Our co-culture results lead us to question whether S. aureus toxicity toward airway epithelia is an in vitro phenomenon, or if the same is true in vivo. While we have observed potent toxicity for alpha-toxin in vitro, there is little evidence that this phenotype occurs in the nasal carriage state. In a xenograft model of S. aureus – airway interactions using human airway epithelia cells (HAEC), an Hla-positive strain (8325-4) was grown with HAEC and implanted in a mouse flank for extended periods without toxicity complications (Mongodin et al., 2002). Additionally, many nasal carriage isolates are known to have the capacity to produce high levels of alpha-toxin in vitro (Deleo et al., 2011). It is plausible that the presence of additional factors in vivo could alter the inflammatory state of S. aureus and impact its ability to colonize. For example, hemoglobin is present in nasal secretions and quenches agr activation (Pynnonen et al., 2011), which would in turn reduce alpha-toxin levels, but hemoglobin is not present in the co-culture model. Additionally, numerous native commensal microorganisms are also present in the natural carriage environment and could be impacting alpha-toxin regulation or activity through interactions with S. aureus. By modeling S. aureus – airway interactions, hopefully we can begin to address the remaining unexplained complexities of the carriage state.

EXPERIMENTAL PROCEDURES

Bacterial strains, growth conditions, and genetic techniques

Bacterial strains and plasmids used are described in Table 1. E. coli cultures were grown in Luria-Bertani (LB) broth or on LB agar, and S. aureus strains were grown in tryptic soy broth (TSB) or on tryptic soy agar (TSA) at 37°C unless otherwise indicated. Plasmids in S. aureus were maintained using chloramphenicol (Cam) at a concentration of 10 μg/ml. Plasmid DNA was prepared from E. coli and electroporated into S. aureus RN4220 as previously described (Schenk et al., 1992). DNA was moved from RN4220 into other S. aureus strains through transduction with bacteriophage 11 (Novick, 1991).

Quantitative real-time PCR

Cells were harvested after 24 hr incubation in co-culture and pelleted by centrifugation. The supernatant was removed and the pellet resuspended in RNAprotect (Qiagen). Bacteria were then pelleted by centrifugation and incubated in 100 uL of lysostaphin 1U/mL in 50mM TRIS, 0.15M NaCl pH 8.0 at 37° for 30–60 minutes. RNA was then extracted and isolated using the RNeasyR kit (Qiagen). 10 ng of RNA was amplified with Express SYBR Green One Step qRT-PCR Mix (Invitrogen) using the following conditions: 5 min at 50°C, 2 min at 95°C, 40 cycles of 15 sec at 95°C and 1min at 53°C followed by a disassociation curve. Oligonucleotides (Supplemental Table 1) were generated from published S. aureus genome sequences and synthesized by Integrated DNA Technologies (Coralville, IA). To test for contamination of genomic DNA, samples were run without reverse transcriptase. Transcript levels were calculated using the ΔΔCT method with DNA gyrase as a reference gene.

For assessing ADAM-10 levels of cell lines, RNA was purified from Calu-3 and RPMI2650 cells using TRIzol reagent (Thermo-Fisher) according to manufacturer’s instructions. The qRT-PCR was performed as described above using oligonucleotides specific to ADAM-10 and reference genes 18S and GAPDH (Supplemental Table 1).

Cell culture techniques

Cell culture medium was comprised of 500 mL MEM (Gibco), 50 mL HI fetal calf serum, 5 mL nonessential amino acid solution (100×) and 5 mL Penicillin/Streptomycin (100×). Calu-3 cells were cultured in 75 cm2 flasks using 10 mL medium and maintained in a humidified 5%CO2–95% atmospheric air incubator at 37°C. Medium was changed twice per week, and cells were passed weekly at 1:3 split ratio using TrypLE™ Express reagent (Gibco). RPMI2650 cells purchased from ATCC (Rockville Md.) were cultured in the same manner in medium without nonessential amino acids.

Cytotoxicity assays

The toxicity of S. aureus spent culture media (conditioned media) was determined by measuring LDH release from submerged Calu-3 and RPMI2650 cells following 24 hr of exposure. LDH levels were measured with the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega). The percent cell death was determined by assessing the maximal cell death (using the provided lysis buffer) compared to the amount of LDH release after addition of S. aureus bacteria or media. Each experiment was repeated at two different conditioned media concentrations, each with at least two biological replicates, and within these multiple technical replicates for each test.

Airway epithelia – S. aureus co-cultures

Polarized, antibiotic-free Calu-3 cell cultures were prepared by methods as previously described (Karp et al., 2002, Starner et al., 2006), with the exception that 1:1 DMEM:F12 with 0.5% HI FCS and 1% Pen/Strep was used as basolateral media instead of USG media. To establish bacterial colonization of the epithelial surface, the basolateral media was replaced with antibiotic-free DMEM:F12 + 0.5% HI FCS, and 1 × 108 or 2 × 107 colony forming-units (CFUs) of either wild-type or mutant S. aureus suspended in 50 μL of phosphate-buffered saline were pipetted onto the apical surface of polarized cells. When necessary to maintain plasmids, both the S. aureus suspension and the basolateral media were supplemented 1:1000 with 10 μg/μL Cam. Transepithelial resistance (TR) was measured using an EVOM volt-ohmmeter (World Precision Instruments) at 4 hours, 24 hours, and 48 hours following inoculation with bacteria using methods previously described (Starner et al., 2006). Following TR measurement, the Calu-3 cells were rinsed with 200 µL PBS, followed by application of 200 μL of 0.1% gentian violet stain (diluted in PBS) to the apical surface for 15 minutes. The stain was then aspirated, and cells were rinsed with PBS before imaging.

Scanning electron microscopy

Scanning electron microscopy (SEM) of Calu-3 – S. aureus co-cultures were performed as previously described (Starner et al., 2006). Briefly, samples were fixed in 1% osmium tetroxide (EMS) dissolved in perfluorocarbon (Fluorinert FC-72; 3M, St. Paul, MN) for 2 hours at room temperature, then dehydrated by three washes with 100% ethanol for 15 minutes each. Samples were next washed in hexamethyldislazane (Ted Pella, Inc.; Redding, CA) twice for 15 minutes each, dried overnight and mounted on stubs before coating with argon, and imaged with a Hitachi S-4000 scanning electron microscope. Calu-3 cells are known to produce a thick mucus layer, which was visible in the SEM and obstructed viewing of the bacteria. Fields of view were selected on the edges where S. aureus cells were visible.

Confocal microscopy

Imaging was performed on a Nikon Eclipse E600 microscope using the Radiance 2100 image capturing system (Biorad, Hercules, CA). All confocal microscopy experiments used S. aureus strain LAC or LAC hla∷Erm (AH1792) expressing GFP. Bacteria expressing GFP were imaged using the Argon 488 laser and are colored green in image renderings. Separate co-cultures of Calu-3 cells and S. aureus were prepared for each time point for confocal evaluation. Calu-3 cells were stained with 10 mM CellTracker Orange CMRA (Invitrogen, Eugene, OR) diluted 1:400 in the basolateral culture medium and incubated for 45 minutes in 5% CO2 at 37°C. The basolateral culture medium was then replaced with fresh culture medium and the cells incubated for another 45 minutes in 5% CO2 at 37°C. After the second incubation, cells were rinsed five times with PBS+Ca/Mg. The cell culture membrane was then cut out and prepared on a glass slide for confocal microscopy. Calu-3 cells are colored red in image renderings. For each time point, five Z-series at 200× magnification (382 × 382 μm) and one Z-series at 600× magnification (120 × 120 μm) were obtained. Four 200× Z-series were selected at pre-determined clock-wise positions around the edge of the transwell filter (12, 3, 6 and 9 o’clock), and one 200× Z-series was selected from the center of the filter. The 600× Z-series was taken from the center of the filter. Image rendering was performed using the Volocity program (Improvision). Confocal images presented here are representative of 4 trials.

Microarray analysis

S. aureus hla∷Erm mutant was grown for 48 hrs in the co-culture model. Adherent S. aureus were removed from the Calu-3 monolayer by washing the apical side of each transwell twice with 100 uL of 0.05% Triton X-100 in PBS. The wash was spun at 1000 RPM to pellet the Calu-3 cells membranes, then at 8000 RPM to pellet the bacteria. The bacterial pellet was resuspended in 100 uL lysostaphin (1U/uL in 50 mM Tris, 0.15 M NaCl, pH 8.0) and incubated at 37 °C for one hour to lyse the bacteria. RNA extraction was carried out using the RNeasyR kit (Qiagen) with DNAse digestion included. For the control condition, bacteria were grown overnight in TSB at 37 °C with shaking. RNA was isolated from these bacteria in the same manner. Both co-culture and liquid culture conditions were done in triplicate. The Affymetrix S. aureus microarray and statistical analysis were carried out by the DNA Core at the University of Iowa. A one-way ANOVA with multiple test correction was performed (Supplemental Table 3).

Supplementary Material

SuppTable1
SuppTable2
SuppTable3

Acknowledgments

We thank Dr. Barry Kreiswirth for providing strains. AEP was funded by an American Heart Association Pre-doctoral Fellowship. Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIH) under award numbers AI078921 to ARH. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

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Supplementary Materials

SuppTable1
NIHMS750565-supplement-SuppTable1.pdf (17.1KB, pdf)
SuppTable2
NIHMS750565-supplement-SuppTable2.pdf (8.4KB, pdf)
SuppTable3
NIHMS750565-supplement-SuppTable3.pdf (210.5KB, pdf)

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