Exploring differentially expressed genes of Staphylococcus aureus exposed to human tonsillar cells using RNA sequencing

Abstract

Background The nose and the throat are the most predominant colonizing sites of Staphylococcus aureus, and colonization is a risk factor for infection. Nasal colonization is well described; however, we have limited knowledge about S. aureus throat colonization. The main objective of this study was to explore differentially expressed genes (DEGs) in S. aureus throat isolate TR145 exposed to human tonsil epithelial cells (HTEpiC) by using RNA sequencing (RNA-seq) and pathway analysis. DEGs in S. aureus at 1 or 3 hours (h) interaction with its host were explored. Results S. aureus was co-cultured in absence and presence of tonsillar cells at 1 or 3 h. Over the 3 h time frame, the bacteria multiplied, but still caused only minor cytotoxicity. Upon exposure to tonsillar cell line, S. aureus changed its transcriptomic profile. A total of 508 DEGs were identified including unique (1 h, 160 DEGs and 3 h, 78 DEGs) and commonly shared genes (1 and 3 h, 270 DEGs). Among the DEGs, were genes encoding proteins involved in adhesion and immune evasion, as well as iron acquisition and transport. Reverse transcription qPCR was done on selected genes, and the results correlated with the RNA-seq data. Conclusion We have shown the suitability of using HTEpiC as an in vitro model for investigating key determinants in S. aureus during co-incubation with host cells. Several DEGs were unique after 1 or 3 h exposure to host cells, while others were commonly expressed at both time points. As their expression is induced upon meeting with the host, they might be explored further for future targets for intervention to prevent either colonization or infection in the throat.

Co-culturing with Streptococcus anginosus alters Staphylococcus aureus transcriptome when exposed to tonsillar cells

“…The Streptococcus anginosus (ATCC 33397) strain originated from human throat tissue, it is ß-hemolytic and typed as Lancefield’s group G ( Bauer et al., 2020 ; Kuryłek et al., 2022 ). S. anginosus was grown according to the handling information provided by ATCC and S. aureus was grown as described previously ( Bastakoti et al., 2023 ). Briefly, bacterial cultures were incubated separately in Brain Heart Infusion (BHI) media overnight at 37°C with shaking at 220 revolutions per minute (rpm).…”

“…Two different time points (1 h and 3 h) were selected for triplicate in-vitro experiments in 6-well plates. Following incubation at 37°C in the presence of 5% CO 2 with tonsillar cell medium (TEpiCM), bacteria in the presence or absence of host cells were collected and total RNA was subjected to RNA-seq according to a previously described protocol ( Bastakoti et al., 2023 ). Briefly, all unbound co-cultured bacteria were washed away, host cells were trypsinized and only those bacteria which had managed to attach to tonsillar cells were collected and processed for RNA-seq.…”

“…#C754532, ThomasScientifc, USA) to detect S. anginosus , and CHROMagar plate to detect S. aureus , before incubation for 24 h at 37°C The plating was done in triplicate, and the average CFU/ml was calculated. For better understanding of host stress levels during the exposure time points, cytotoxicity assays were performed on co-culturing plates, as described earlier ( Bastakoti et al., 2023 ).…”

“…The RNA extraction was performed according to a previously described protocol ( Bastakoti et al., 2023 ) following the recommendations from the manufacturer (RNeasy Mini Kit, Cat. No.…”

“…The success of S. aureus depends not only on adhesins and/or virulence genes and the ability to escape antibiotic treatment, but also on the coordinated and timely expression of genes upon infection of its host ( Xu et al., 2016 ), which may change in the presence of another microbe. We have previously identified differentially expressed key determinants in S. aureus in the presence of primary human tonsillar epithelial cell using RNA sequencing and pathway analysis ( Bastakoti et al., 2023 ). In the present study, we aimed to identify differentially expressed genes (DEGs) in S. aureus when co-cultured with S. anginosus in the presence or absence of a tonsillar cell line using the same experimental set up.…”