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Title (eng)
Exploring host-pathogen interactions
Chlamydia caviae infection in A549 lung epithelial cell line
Author
Madeleine Haas
Degree supervisor
Aleksandra Inic-Kanada
Degree supervisor
Andrea Ladinig
Description (eng)
Master thesis - University of Veterinary Medicine Vienna - 2023
Abstract (eng)
Background: Chlamydia caviae is a species of the Chlamydia genus that mostly infects guinea pigs (Cavia porcellus). While C. caviae infections have primarily been studied in guinea pigs, there have also been limited reports of human infections. The clinical significance and prevalence of C. caviae infections in humans are not well-established, and further research is needed to better understand its impact on human health. Objective: We aimed to investigate the effects of C. caviae infection on lung epithelial cells utilizing the human alveolar basal epithelial cells (A549 cell line), examining alterations in the expression of E-cadherin, a guardian of epithelial phenotype, α-smooth muscle (α-SMA), a mesenchymal marker, as well as in TGF-β1 and TGF-β2, involved in epithelial-mesenchymal transition-inducing signaling pathways. We also measured cytokine secretion by the A549 cell line following C. caviae infection. Methods: The A549 cells were cultured in T75 cell culture flasks until reaching 70 % confluence. Cells were then infected with 1.0 x 107 IFU C. caviae; the MOI was calculated to be 3. Uninfected A549 cells were used as a control. After 48 hours, the cells were collected, and RNA, DNA, and proteins were isolated using an Allprep kit (QIAGEN). The expression of TGF-β1 and TGF-β2 was analyzed by qRT-PCR. E-cadherin, α-SMA gene, and protein expression were analyzed by qRT-PCR and SDS-PAGE, followed by western blot analysis. For ELISA analysis, A549 cells were grown in 6-well plates at 5x105 cells/ml density until reaching 70 % confluence. The cells were then infected with 1x103 and 1x105 IFU C. caviae. To get a comprehensive understanding of the immune response dynamics, we tested IL-8, TNF-α, IL-1β, IL-6, IL-13, and IFN-γ cytokine levels in supernatants at different time points: 6, 12, 24, and 48 hours post-infection by ELISA. Results: The qRT-PCR analysis revealed an increasing trend in gene expression levels for TGF-β1, TGF-β2, E-cadherin, and α-SMA. However, these results did not reach statistical significance. In contrast, the Western blot analysis displayed a significant reduction in E-cadherin expression in C. caviae-infected A549 cells compared to the control cells. No significant differences were observed in the expression of α-SMA between infected and control cells. Furthermore, the expression levels of the tested cytokines did not exhibit any significant differences between A549 cells infected with C. caviae and their respective non-infected controls. Conclusion: Based on the findings, the infection of A549 cells with C. caviae may not be productive/efficient. The downregulation of E-cadherin, a guardian of the epithelial phenotype, suggests a disruption in the epithelial cell integrity. However, the lack of increased expression in the mesenchymal marker α-SMA further indicates that the infection may not induce a transition toward a mesenchymal phenotype. The unaltered expression levels of the tested proinflammatory cytokines suggest that the C. caviae infection may not significantly activate the immune response in A549 cells.
Description (deu)
Masterarbeit - Veterinärmedizinische Universität Wien - 2023
Type (eng)
Master theses
Language
[eng]
Persistent identifier
https://phaidra.vetmeduni.ac.at/o:4115
AC number
AC17419887
Number of pages
V, 55
Date issued
2023
License
All rights reserved