Journal: bioRxiv

Article Title: Temperature affects the host range of Rhabdochlamydia porcellionis and the infectivity of Waddlia chondrophila and Chlamydia trachomatis elementary bodies

doi: 10.1101/2022.09.02.506449

Figure Lengend Snippet: (A) Permissivity of arthropod and mammalian cell lines to R. porcellionis . The y-axis represents the log2 fold change of the number of genome copies per μL compared to the initial timepoint (mean +/− standard deviation, n=2) (B) Infectious progeny production. The supernatant from Sf9 cells infected with R. porcellionis was collected every other day (time points indicated on the x axis) and used to infect fresh Sf9 cells. The cells were then collected at zero (dots) and 6 days (triangles) post infection to quantify bacterial growth by qPCR (mean +/− standard deviation, n=2) (C) R. porcellionis in mammalian and arthropod cell lines at 6 days post-infection. Growth could only be seen in Sf9 cells. The reticulate bodies seem to replicate directly in the cytoplasm, without being enclosed in an inclusion. The enlarged bodies in the C6/36 cell lines are likely aberrant bodies. Bacteria appear to have been internalized in all the other cell lines, but failed to replicate. White arrows indicate enlarged bacteria in C6/36 cells and internalized EBs in the other cell lines. Cells were stained with concanavalin A (red), DAPI (blue) and anti- Simkania antibody (green), scale bar: 10 μm.

Article Snippet: After the incubation with the primary antibody, the coverslips were washed three times in PBS with 0.1% saponin and incubated for one hour at room temperature in blocking solution with 1.6 μg/ml DAPI dilactate (Molecular Probes, Thermo Fisher Scientific, Waltham, USA), 100 μg/ml Concanavalin A-Texas red conjugate (Invitrogen, Thermo Fisher Scientific, Waltham, USA) and Alexa-488 conjugated chicken anti-goat or goat anti-rabbit antibodies (1:1000 dilution) (Life Technologies, Thermo Fisher Scientific, Waltham, USA).

Techniques: Standard Deviation, Infection, Bacteria, Staining

Journal: bioRxiv

Article Title: Temperature affects the host range of Rhabdochlamydia porcellionis and the infectivity of Waddlia chondrophila and Chlamydia trachomatis elementary bodies

doi: 10.1101/2022.09.02.506449

Figure Lengend Snippet: Effect of different incubation temperatures on the infectivity of elementary bodies. (A) Evolution of the number of IFU after incubation at 20 °C or 37 °C, normalized to the initial IFU count (mean +/− standard deviation, n=3) (B) IFU counts at the initial timepoint (mean +/− standard deviation, n=3) (C) Confocal images of McCoy cells infected with serial dilutions of EBs incubated at 20 °C or 37 °C. This highlights the deleterious effect of an incubation at 37 °C for the EBs of all three species. Interestingly, the inclusions formed by EBs incubated at 37 °C also tended to be smaller. The difference between the well-formed inclusions for C. trachomatis and W. chondrophila and the dissemination of RBs in the host cytoplasm for R. porcellionis is also striking. Cells were fixed at 1 (for C. trachomatis and W. chondrophila) or 6 days (for R. porcellionis ) post-infection and stained with Concanavalin A (red), DAPI (blue) and antibodies against the different bacteria (green). Scale bar: 10μm. Inc.: incubation duration.

Article Snippet: After the incubation with the primary antibody, the coverslips were washed three times in PBS with 0.1% saponin and incubated for one hour at room temperature in blocking solution with 1.6 μg/ml DAPI dilactate (Molecular Probes, Thermo Fisher Scientific, Waltham, USA), 100 μg/ml Concanavalin A-Texas red conjugate (Invitrogen, Thermo Fisher Scientific, Waltham, USA) and Alexa-488 conjugated chicken anti-goat or goat anti-rabbit antibodies (1:1000 dilution) (Life Technologies, Thermo Fisher Scientific, Waltham, USA).

Techniques: Incubation, Infection, Standard Deviation, Staining, Bacteria

Journal: bioRxiv

Article Title: Temperature affects the host range of Rhabdochlamydia porcellionis and the infectivity of Waddlia chondrophila and Chlamydia trachomatis elementary bodies

doi: 10.1101/2022.09.02.506449

Figure Lengend Snippet: R. porcellionis grows in mammalian cells incubated at 28 °C (A) Growth kinetics of R. porcellionis in Sf9 incubated at 20, 28, 33 and 37 °C. (B) Growth kinetics of R. porcellionis in mammalian cells, incubated at 28 °C. In both (A) and (B) , the y-axis represent the log2 fold change compared to time 0 (mean +/− standard deviation, n=3) (C) IFU count in different cell lines infected with the same suspension of R. porcellionis (mean +/− standard deviation, n=3). All cells were all grown at 28 °C. A one-way ANOVA revealed that there was a statistically significant difference between at least two cell lines (p-value=0.0002). The plot also shows the results of Tukey HSD test for the pairwise comparison of the different cell lines (** < 0.01, *** < 0.001). (D) McCoy, A549 and Ishikawa cells infected with R. porcellionis and incubated at 28 °C. The two enlarged bodies in McCoy and A549 cells are likely aberrant bodies (white arrow). Cells were fixed 6 days post infection and stained with concanavalin A (red), DAPI (blue) and anti- Simkania antibodies (green). Scale bar: 10 μm.

Article Snippet: After the incubation with the primary antibody, the coverslips were washed three times in PBS with 0.1% saponin and incubated for one hour at room temperature in blocking solution with 1.6 μg/ml DAPI dilactate (Molecular Probes, Thermo Fisher Scientific, Waltham, USA), 100 μg/ml Concanavalin A-Texas red conjugate (Invitrogen, Thermo Fisher Scientific, Waltham, USA) and Alexa-488 conjugated chicken anti-goat or goat anti-rabbit antibodies (1:1000 dilution) (Life Technologies, Thermo Fisher Scientific, Waltham, USA).

Techniques: Incubation, Standard Deviation, Infection, Suspension, Comparison, Staining