Publikationsserver
Molecular Investigations on the Type-specific Antigen 47 of Orientia tsutsugamushi
Orientia tsutsugamushi (OT), an obligate intracellular, Gram-negative bacterium from the family of Rickettsiaceae, replicates in the cytosol. However, among its surface and periplasmic proteins, the definitive role of the type-specific antigen of 47 kD (TSA47) has not been understood. This research...
Saved in:
| Main Author: | |
|---|---|
| Contributors: | |
| Format: | Doctoral Thesis |
| Language: | English |
| Published: |
Philipps-Universität Marburg
2024
|
| Subjects: | |
| Online Access: | PDF Full Text |
| Tags: | Add Tag |
| Summary: | Orientia tsutsugamushi (OT), an obligate intracellular, Gram-negative bacterium from the family of Rickettsiaceae, replicates in the cytosol. However, among its surface and periplasmic proteins, the definitive role of the type-specific antigen of 47 kD (TSA47) has not been understood. This research established a unique protein overexpression system in the host cells and studied the role of TSA47 in three different aspects of host-pathogen interaction.
Primarily, the TSA47 gene was codon-optimised for mammalian expression and was cloned into the pCAGGS high-expression vector backbone. To detect the proteins by microscopy and Western blot, it was tagged with FLAG/myc tags (F47M). This system was then adopted to carry out studies in cells of human hepatoma origin, the HuH7 cell line.
First, since TSA47 has a high homology to the human serine protease A1 (HtrA1) which is a known microtubule-associated protein (MAP), the study investigated how TSA47 expression impacts the integrity of host cytoskeletal components. Using kinetic studies employing TSA47 overexpression, it was found that TSA47 sequesters cellular α-tubulin and impairs the integrity of microtubules in HuH7 cells. Additionally, Nocodazole treatment which destabilises microtubules, was found to increase TSA47-α-tubulin association. TSA47 contains 4 domains viz. the signal peptide, serine-protease, PDZ1 and PDZ2. However, it was also shown that upon overexpressing a PDZ2-domain deletion mutant (M47ΔP), recruitment of α-tubulin is decreased. Also, when overexpressed TSA47 from HuH7 cells was pulled down, α-tubulin was not found to co-immunoprecipitated with it. In a live-OT infection model in HuH7 cells, at early (48h) as well as late (72h), OT did not interact or significantly alter the integrity of the microtubule network.
Second, since TSA47 is also similar to the bacterial HtrA ortholog DegP from E.coli, the present study also investigated the overexpressed form of TSA47. It was found that TSA47 forms homo-oligomers of high-molecular weight ≥250 kD. Since oligomeric forms of exogenous proteins trigger aggrephagy via involvement of the cellular adapter protein p62, TSA47 was also investigated and found to associate with p62 in HuH7 cells. Third, since TSA47 from OT has a high homology to the human HtrA1 in trypsin-like serine protease domain, OT’s role in supporting IAV [A/Hamburg/05/2009(H1N1)] replication was investigated. This was deemed essential since cleavage of HA from IAV is indispensable for its spread and replication to host cells. Hence, IAV spread and replication was measured in OT infected MDCK II cells with IAV super-infection. It was found that OT supplements growth of IAV in the absence of exogenous protease. Upon co-expression of HA from the same IAV and TSA47 in HEK293T cells however, TSA47 was deemed insufficient to cleave the HA and promote spread of IAV in these cells. Together, these findings showed that OT could provide a factor, so far unknown, that supports the IAV spread in host cells.
Overall, the newly established overexpression system provided new insights into the functions of TSA47 in host-pathogen interaction, e.g. as a secreted bacterial protein. |
|---|---|
| DOI: | 10.17192/z2024.0270 |