Staphylococcus aureus Biofilm Growth on Cystic Fibrosis Airway Epithelial Cells Is Enhanced during Respiratory Syncytial Virus Coinfection

Abstract

Staphylococcus aureus is a major cause of chronic respiratory infection in patients with cystic fibrosis (CF). We recently showed that Pseudomonas aeruginosa exhibits enhanced biofilm formation during respiratory syncytial virus (RSV) coinfection on human CF airway epithelial cells (AECs). The impact of respiratory viruses on other bacterial pathogens during polymicrobial infections in CF remains largely unknown. To investigate if S. aureus biofilm growth in the CF airways is impacted by virus coinfection, we evaluated S. aureus growth on CF AECs. Initial studies showed an increase in S. aureus growth over 24 h, and microscopy revealed biofilm-like clusters of bacteria on CF AECs. Biofilm growth was enhanced when CF AECs were coinfected with RSV, and this observation was confirmed with S. aureus CF clinical isolates. Apical conditioned medium from RSV-infected cells promoted S. aureus biofilms in the absence of the host epithelium, suggesting that a secreted factor produced during virus infection benefits S. aureus biofilms. Exogenous iron addition did not significantly alter biofilm formation, suggesting that it is not likely the secreted factor. We further characterized S. aureus-RSV coinfection in our model using dual host-pathogen RNA sequencing, allowing us to observe specific contributions of S. aureus and RSV to the host response during coinfection. Using the dual host-pathogen RNA sequencing approach, we observed increased availability of nutrients from the host and upregulation of S. aureus genes involved in growth, protein translation and export, and amino acid metabolism during RSV coinfection.IMPORTANCE The airways of individuals with cystic fibrosis (CF) are commonly chronically infected, and Staphylococcus aureus is the dominant bacterial respiratory pathogen in CF children. CF patients also experience frequent respiratory virus infections, and it has been hypothesized that virus coinfection increases the severity of S. aureus lung infections in CF. We investigated the relationship between S. aureus and the CF airway epithelium and observed that coinfection with respiratory syncytial virus (RSV) enhances S. aureus biofilm growth. However, iron, which was previously found to be a significant factor influencing Pseudomonas aeruginosa biofilms during virus coinfection, plays a minor role in S. aureus coinfections. Transcriptomic analyses provided new insight into how bacterial and viral pathogens alter host defense and suggest potential pathways by which dampening of host responses to one pathogen may favor persistence of another in the CF airways, highlighting complex interactions occurring between bacteria, viruses, and the host during polymicrobial infections.

Keywords: biofilms; coinfection; host-pathogen interaction; polymicrobial.

FIG 1

Characterizing S. aureus growth on cystic fibrosis airway epithelial cells. Coculture of S. aureus strains USA100, USA300, and 502A on polarized CF AECs over a 24-h time course (A). CFU per milliliter were measured at 1 h, 7 h, and 24 h postinfection. Polarized monolayers infected with GFP-expressing S. aureus USA100 (green) and then fixed and stained with Hoechst stain (blue) for confocal imaging at specified time points (B). Images show three-dimensional volume renderings at ×20 magnification generated in Nikon Elements. S. aureus gene expression on CF AECs alone and during RSV coinfection (C). Relative amount of mRNA transcript from S. aureus USA100 grown in coculture with control CF AECs or RSV-infected CF AECs, compared to S. aureus stationary-phase culture and measured by qRT-PCR. ***, P ≤ 0.001.

FIG 2

Effects of RSV coinfection on S. aureus attachment and biofilm growth on CF AECs. Live-cell imaging of S. aureus USA100 coculture with CF AECs in the absence and presence of 24-h RSV infection was observed using a closed flow-cell chamber and visualized on a Nikon Ti inverted wide-field microscope (A). Images were obtained at 2 h, 4 h, and 7 h after inoculation of S. aureus USA100 expressing GFP (green), with CF AEC nuclei stained with Hoechst stain (blue). Imaging experiments were repeated in triplicate, with 5 fields per chamber sampled. Static coculture biofilm assays with S. aureus USA100 were performed on polarized, air-liquid interface control CF AECs or CF AECs infected with RSV for 24 h, 48 h, or 72 h. Cocultures were allowed to proceed for 1 h to measure initial S. aureus attachment (B) and 7 h to measure biofilm growth (C). Bacteria were enumerated by plating after the desired incubation time and calculating CFU per milliliter. S. aureus clinical isolates cultured from sinonasal swabs collected from CF patients with chronic rhinosinusitis were evaluated in static biofilm coculture assays on CF AECs in the presence and absence of RSV infection (D). Black bars, control virus-free CF AECs; red bars, RSV-infected CF AECs. Experiments repeated in triplicate. Significance determined by unpaired Student’s t test; *, P ≤ 0.05; ***, P ≤ 0.001.

FIG 3

S. aureus attachment and biofilm growth on primary CF human bronchial epithelial cells during RSV coinfection. CFU counts of static biofilm coculture assays performed with S. aureus USA100 on primary CF HBE cells in the presence and absence of 72 h RSV coinfection at 1 h (A) and 7 h (B). Coculture assays were performed using at least two different CF HBE cell codes obtained from different patients and repeated for a minimum of n = 3 experiments. Significance determined by unpaired Student’s t test (*, P ≤ 0.05). GFP-expressing S. aureus USA100 (green) and CF HBE cell nuclei stained with Hoechst stain (blue) (C). Images were captured at ×60 magnification, with three-dimensional volume renderings and measurements obtained using Nikon Elements software. Scale, 20 µm.

FIG 4

Coinfection with human rhinovirus enhances S. aureus growth on CF AECs. Static coculture biofilm assays with S. aureus USA100 were performed on polarized, air-liquid interface control CF AECs or CF AECs infected with human rhinovirus 14 (hRV) for 24 h at an MOI of 1. Cocultures were allowed to proceed for 7 h to measure biofilm growth. Bacteria were enumerated by plating and calculating CFU per milliliter. Significance was determined by unpaired Student’s t test (*, P ≤ 0.05).

FIG 5

Conditioned medium from RSV-infected CF AECs enhances S. aureus biofilm growth. Conditioned medium containing apical secretions from polarized CF AECs was collected from control or RSV-infected cultures. GFP-expressing S. aureus USA100 was inoculated into CM and cultured in glass-bottom MatTek dishes for fluorescence microscopy. Microscopy was performed on a Nikon-Ti wide-field fluorescence microscope. Representative three-dimensional reconstructions of z-stacks obtained for S. aureus biofilm growth in control (A) and RSV (B) CM. Biomass quantification for control and RSV CM (C) was performed in Nikon Elements. Images and biomass quantification are representative of n = 3 individual experiments, with 5 random fields imaged per sample. Total protein content in CM and RSV CM (D). Biofilm growth in untreated control CM and RSV CM and CM pretreated with proteinase K (E) or subjected to 3-kDa centrifugal filtration (F). Biofilm assays were performed in microtiter plates, with a minimum of n = 3 biological replicates and 5 technical replicates per condition, per assay, with crystal violet absorbance quantified at 550 nm. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 6

Addition of iron does not enhance S. aureus biofilms on CF AECs in the absence of virus infection. Total concentration of metals measured in CM and RSV CM, as measured by individual assay kits for iron (Fe²⁺), zinc (Zn²⁺), and copper (Cu²⁺) (A). CFU counts from static biofilm coculture assays performed with S. aureus USA100 on CF AECs in MEM plus 2 mM l-glutamine alone or with addition of exogenous 250 µM FeCl₃, 12.5 µM apotransferrin (non-iron bound), or 12.5 µM holotransferrin (iron bound) diluted in MEM plus 2 mM l-glutamine (B).

FIG 7

S. aureus transcriptomic changes during coculture on RSV-infected CF AECs. Proteomaps depicting functional categories of differentially expressed S. aureus genes during S. aureus-RSV coinfection compared to S. aureus single infection of CF AECs (SaRSV v Sa) (A). Heat maps showing fold change gene expression in specific S. aureus genes during S. aureus-RSV coinfection compared to S. aureus single infection of CF AECs (B). In each heat map, column 1 shows gene name, column 2 shows S. aureus gene identifier (from S. aureus strain N315 genome annotation), column 3 shows fold change relative to single infection, and column 4 indicates false discovery rate P value significance, with ** indicating a false discovery rate P value of < 0.05.

FIG 8

Transcriptomic changes in CF AECs during coinfection with S. aureus and RSV. Venn diagram depicts differentially expressed transcripts under each of RSV (RSV v Control, pink), S. aureus (Sa v Control, blue), or S. aureus-RSV coinfection (SaRSV v Control, yellow) conditions (A). Proteomaps depict functional categories of differentially expressed host genes during S. aureus-RSV coinfection compared to either RSV (SaRSV v RSV) (B) or S. aureus (SaRSV v Sa) (C) single infections. Heat maps indicate fold change gene expression under SaRSV v RSV or SaRSV v Sa conditions (D).