Photorhabus asymbiotica Quorum Sensing Gene PauR may also Participate in Insect Cell Sensing Open Access
Rationale. Photorhabdus asymbiotica (Pa) is a Gram-negative bacterial pathogen that efficiently infects both insects and humans (skin ulcer). Apart from apoptosis induction and septicemia by variety of toxin effectors, quorum sensing/interbacterial communication mediated by PauR gene is yet another mechanism recently linked to its virulence. To thrive in either host, Pa regulates its gene expression leading to metabolic shift between 30°C (insect) and 37°C (human). This project attempts to explore the involvement of PauR in P. asymbiotica pathogenicity. Here, we focus on the effect of Pa growing temperatures on the PauR gene expression in the presence or absence of Drosophila melanogaster S2 cells. Methods. Estimation of S2 cell viability is aimed at confirming the kill efficiency of Pa grown at 30°C and 37°C. We firstly seeded 12-well plate with S2 cells and challenged them with 30°C-Pa or 37°C-Pa the following day. All incubation after infection was at 30°C. We measured S2 cell viability by Trypan Blue counting and determined the time points at which relative viability reaches 90%, 50%, and 10%. qRT-PCR was subsequently performed to estimate PauR gene expression. We also measured the level of pau00087, a Pa gene with putative role in temperature and environment sensing, to contrast the profile of the quorum sensing gene. Results. Pa grown at 30°C killed S2 cells faster than 37°C-Pa, with 50% relative viability reached at 4hr and 8hr, and 10% relative viability at 6hr and 10hr, respectively. Notably, PauR was elevated upon contact with S2 cells, compared to medium-only control, then plummeted to control level after 2hr. In contrast to immediate spike of pau00087 originating from 37°C-Pa, its 30°C-Pa counterpart was only elevated after 2hr incubation in S2 cells. Conclusion. P. asymbiotica grown at 30°C is more pathogenic to insect cells compared to those grown at 37°C. However, growing temperature is not involved in regulating PauR response in pathogenicity. In contrast, pau00087 of 37°C-Pa is more sensitive to the presence of S2 cells and temperature change than 30°C-Pa counterpart. Nevertheless, quorum sensing gene PauR is consistently and significantly upregulated upon contact with insect cells, which eludes us to suspect a second role of PauR in host cell detection.
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|Heryanto, Christa.pdf||2018-08-27||Open Access||