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. 2020 Sep 21;10:15396. doi: 10.1038/s41598-020-72340-9

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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AnkG_NM mediates host tolerance to C. burnetii infection. (a) Survival of infected Galleria mellonella larvae was analyzed over a 7 day period. 10 larvae each were infected with 10⁶ bacteria (C. burnetii wildtype (WT), C. burnetii ΔdotA (ΔdotA), C. burnetii pFlag-AnkG (AnkG) or C. burnetii pFlag-AnkF (AnkF)) per larvae in 20 µl PBS or with a PBS control. Survival was controlled every 24 h. Shown is the mean of three experiments ± SD. **p < 0.01; ***p < 0.001. (b) AnkG level was measured by RT-PCR 1, 3 and 7 days post-induction with 2 mM IPTG and compared to the AnkG level of respective the untreated control. RNA was isolated and reverse transcribed in cDNA. Fold replication was calculated using the ΔΔCT method. Shown is the mean of three independent experiments. *p < 0.05. (c) C. burnetii replication between day 1 and day 5 post-infection was measured via RT-PCR. At days 1 and 5 post-infection, bacterial DNA was isolated from two larvae and genomic equivalents (GE) were measured by RT-PCR. The fold replication was determined by dividing the GE from day 5 post-infection by the GE from day 1 post-infection. Shown is the mean of three independent experiments. *p < 0.05. (d, e) The infection rate of Galleria mellonella hemocytes on days 1 and 5 post-infection was determined. Hemocytes from three pooled larvae per infected strain were isolated on days 1 and 5 post-infection and stained for immunofluorescence. (d) Representative immunofluorescence micrographs of infected hemocytes are shown. The cytoskeleton was stained with phalloidin (red), C. burnetii was stained with an anti-Coxiella antibody (green) and the nuclei were stained with DAPI (blue). Scale bars, 10 µm. (e) The infection rate of at least 100 hemocytes were scored in three independent experiments. Shown is the mean of three experiments. *p < 0.05; **p < 0.01; ***p < 0.001, n.s. not significant. (f) Survival of infected Galleria mellonella larvae was analyzed over a period of 8 days. Ten larvae each were infected with 10⁶ bacteria (C. burnetii wildtype (WT) or C. burnetii ΔankG (ΔAnkG)) per larvae in 20 µl PBS or with a PBS control. Survival was controlled every 24 h. Shown is the mean of three experiments ± SD. ***p < 0.001.