Reduced Glycolysis and Cytotoxicity in Staphylococcus aureus Isolates from Chronic Rhinosinusitis as Strategies for Host Adaptation

Abstract

Chronic rhinosinusitis (CRS) is a multifactorial infection of the nasal cavity and sinuses. In this study, nasal swabs from control donors (N = 128) and patients with CRS (N = 246) were analysed. Culture methods and metagenomics revealed no obvious differences in the composition of the bacterial communities between the two groups. However, at the functional level, several metabolic pathways were significantly enriched in the CRS group compared to the control group. Pathways such as carbohydrate transport metabolism, ATP synthesis, cofactors and vitamins, photosynthesis and transcription were highly enriched in CRS. In contrast, pathways related to lipid metabolism were more representative in the control microbiome. As S. aureus is one of the main species found in the nasal cavity, staphylococcal isolates from control and CRS samples were analysed by microarray and functional assays. Although no significant genetic differences were detected by microarray, S. aureus from CRS induced less cytotoxicity to lung cells and lower rates of glycolysis in host cells than control isolates. These results suggest the differential modulation of staphylococcal virulence by the environment created by other microorganisms and their interactions with host cells in control and CRS samples. These changes were reflected in the differential expression of cytokines and in the expression of Agr, the most important quorum-sensing regulator of virulence in S. aureus. In addition, the CRS isolates remained stable in their cytotoxicity, whereas the cytotoxic activity of S. aureus isolated from control subjects decreased over time during in vitro passage. These results suggest that host factors influence the virulence of S. aureus and promote its adaptation to the nasal environment during CRS.

Keywords: Staphylococcus aureus; chronic rhinosinusitis; microbiome.

Figure 1

Distribution of the bacterial community in control and CRS samples. (A) Bacterial community composition at the species level of the nasal cavities of control and CRS patients was analysed by conventional culture-dependent techniques. Differences in the proportions between both groups were compared using Fisher’s exact test (** p < 0.01). (B) Sinonasal microbial composition of control and CRS samples was analysed by metagenomics. The composition of the bacterial community at the genus level is shown.

Figure 2

Metabolic profiles from control and CRS samples. The heatmap depicts metagenomic bacterial community functional annotation based on PFAM, KEGG orthologs and enzyme commission (EC) abundance for both groups. The heatmap’s colour gradient is based on the pathway enrichment in each group (high enrichment in red to low enrichment in blue) and the unit is transcripts per million (TPM).

Figure 3

Cell death induction by S. aureus isolates from control (MIN) and CRS (CSS) donors. (A) Staphylococcal isolates from control and CRS donors from the main clonal complexes (CC7, CC30, CC45 and CC15) were selected. Human epithelial lung A549 cells (ATCC: CCL-185) were infected, and cell death induction was measured by staining the cells with propidium iodide after 24 h. (B) Proportions of dead cells obtained for staphylococcal isolates from the control and CRS groups. Differences between the proportions of dead cells infected with isolates from both groups were analysed by unpaired t tests (* p < 0.05, ** p < 0.01 and *** p < 0.001). Data are presented as the average of triplicate determinations, and error bars represent the standard deviation.

Figure 4

IL-6 induced by S. aureus-infected cells. Staphylococcal isolates from control and CRS donors were selected from the major clonal complexes (CC7, CC30, CC45 and CC15). Human lung epithelial A549 cells (ATCC: CCL-185) were infected and the supernatant was analysed by ELISA at 24 h post-infection. Differences in IL-6 release between the isolates from each pair infected were analysed by unpaired t tests (* p < 0.05, ** p < 0.01, *** p < 0.01). Data are presented as the mean of triplicate determinations, and error bars represent the standard deviation.

Figure 5

S. aureus isolates from the control and CRS groups induced glycolysis in different manners in A549 cells. Metabolic activity of A549 cells was monitored by measuring the extracellular acidification rate (ECAR) of uninfected and infected cells by Seahorse. A549 cells were infected with four different control and CRS S. aureus strains. The additions of glucose, oligomycin and 2-DG (deoxyglucosoe) are indicated. Statistical significance in the assay was compared to PBS alone and between the strains by two-way ANOVA (Table S4A,B).

Figure 6

Differential cytotoxicity induced by S. aureus isolates and their serially passaged strains. Cytotoxicity induction of S. aureus isolates and serially passaged strains from staphylococcal isolates from the control and CRS groups on human epithelial lung A549 cells. Data are presented as the average of triplicate determinations, and error bars represent the standard deviation. Differences in cell death between the original and serially passed isolates from each pair were analysed by unpaired t tests (* p < 0.05, ** p < 0.01, **** p < 0.01).