Lopez A et al. (2021), Developing Tadpole Xenopus laevis as a Comparat...

XB-ART-57738

Int J Mol Sci 2021 Jan 15;222:. doi: 10.3390/ijms22020806.

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Developing Tadpole Xenopus laevis as a Comparative Animal Model to Study Mycobacterium abscessus Pathogenicity.

Lopez A , Shoen C , Cynamon M , Dimitrakopoulou D , Paiola M , Pavelka MS , Robert J .

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Mycobacterium abscessus (Mab) is an emerging, nontuberculosis mycobacterium (NTM) that infects humans. Mab has two morphotypes, smooth (S) and rough (R), related to the production of glycopeptidolipid (GPL), that differ in pathogenesis. To further understand the pathogenicity of these morphotypes in vivo, the amphibian Xenopus laevis was used as an alternative animal model. Mab infections have been previously modeled in zebrafish embryos and mice, but Mab are cleared early from immunocompetent mice, preventing the study of chronic infection, and the zebrafish model cannot be used to model a pulmonary infection and T cell involvement. Here, we show that X. laevis tadpoles, which have lungs and T cells, can be used as a complementary model for persistent Mab infection and pathogenesis. Intraperitoneal (IP) inoculation of S and R Mab morphotypes disseminated to tadpole tissues including liver and lungs, persisting for up to 40 days without significant mortality. Furthermore, the R morphotype was more persistent, maintaining a higher bacterial load at 40 days postinoculation. In contrast, the intracardiac (IC) inoculation with S Mab induced significantly greater mortality than inoculation with the R Mab form. These data suggest that X. laevis tadpoles can serve as a useful comparative experimental organism to investigate pathogenesis and host resistance to M. abscessus.

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Species referenced: Xenopus laevis

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	Figure 1. Colony morphology and bacterial abundance in C57BL/6 mice 1, 7, and 14 dpi. (A) Picture of a smooth (S) Mab colony cultured 5 days at 37 °C on agar plates showing a circular shape and a moist appearance; (C) Rough (R) Mab colony with rugged edges and dry appearance. Mice were infected with high (107 CFUs) and low (104 CFUs) of (B) S (3044) and (D) R (3492) Mab by intranasal infection. The right lung was collected from four mice per timepoint, and lysates were plated on agarose media. Average bacterial load decreased from 1 to 14 days postinfection (dpi) for each morphotype and dose.
	Figure 2. Bacterial recovery from tadpoles 3, 14, and 40 days after intraperitoneal (IP) inoculation of either R or S Mab. (A) Timeline and (B) two-by-two comparison. Tadpoles were IP-injected with 5 × 105 CFU in a 10 μL volume. Spleen, liver, peritoneal leukocytes (PLs), and lungs were collected, suspended in 500 µL of aPBS, lysed by bead beating, and plated on agar media. The bacterial abundance was determined by the number of CFUs/tissue. p < 0.005, p < 0.0005, and ***p < 0.0001 (two-way ANOVA followed by post hoc analysis using Tukey’s multiple comparisons test).
	Figure 3. Tadpole survival following IP (A) or IC (B) inoculation with either R or S Mab. (A) Twenty tadpoles, ten in each group, were inoculated with 5 × 105 CFU of R or S Mab by IP infection and monitored for 40 days. (B) Seventy-two tadpoles were inoculated with 5 × 105 CFU of R or S Mab by IC infection and monitored for 50 days for survival. There was a significant difference in the survival of S and R IC inoculated tadpoles (p < 0.005) based on the log-rank (Mantel–Cox) test.
	Figure 4. Bacterial recovery from tadpoles 3 and 14 days after intracardiac (IC) inoculation of either R or S Mab. (A) Comparison of R and S Mab CFUs for different tissues at 3 and 14 dpi. (B) CPU comparison between 3 and 14 dpi for R and S Mab. Tadpoles were IC-injected with 5 × 105 CFU in a 10 μL volume. Spleen, liver, PLs, and lungs were collected, suspended in 500 µL of aPBS, lysed by bead beating, and plated on agar media. The bacterial abundance was determined by the number of CFUs/tissue. * p < 0.05, p < 0.005, and ** p < 0.0001 (two-way ANOVA followed by post hoc analysis using Tukey’s multiple comparisons test).
	Figure 5. Comparison of bacterial recovery between IP and IC inoculation at 3 and 14 dpi for S and R Mab. * p < 0.05, p < 0.005, * p < 0.0005, and **** p < 0.0001 (two-way ANOVA followed by post hoc analysis using Tukey’s multiple comparisons test).

References [+] :

Alderwick, The Mycobacterial Cell Wall--Peptidoglycan and Arabinogalactan. 2015, Pubmed

	Figure 1. Colony morphology and bacterial abundance in C57BL/6 mice 1, 7, and 14 dpi. (A) Picture of a smooth (S) Mab colony cultured 5 days at 37 °C on agar plates showing a circular shape and a moist appearance; (C) Rough (R) Mab colony with rugged edges and dry appearance. Mice were infected with high (107 CFUs) and low (104 CFUs) of (B) S (3044) and (D) R (3492) Mab by intranasal infection. The right lung was collected from four mice per timepoint, and lysates were plated on agarose media. Average bacterial load decreased from 1 to 14 days postinfection (dpi) for each morphotype and dose.
	Figure 2. Bacterial recovery from tadpoles 3, 14, and 40 days after intraperitoneal (IP) inoculation of either R or S Mab. (A) Timeline and (B) two-by-two comparison. Tadpoles were IP-injected with 5 × 105 CFU in a 10 μL volume. Spleen, liver, peritoneal leukocytes (PLs), and lungs were collected, suspended in 500 µL of aPBS, lysed by bead beating, and plated on agar media. The bacterial abundance was determined by the number of CFUs/tissue. p < 0.005, p < 0.0005, and ***p < 0.0001 (two-way ANOVA followed by post hoc analysis using Tukey’s multiple comparisons test).
	Figure 3. Tadpole survival following IP (A) or IC (B) inoculation with either R or S Mab. (A) Twenty tadpoles, ten in each group, were inoculated with 5 × 105 CFU of R or S Mab by IP infection and monitored for 40 days. (B) Seventy-two tadpoles were inoculated with 5 × 105 CFU of R or S Mab by IC infection and monitored for 50 days for survival. There was a significant difference in the survival of S and R IC inoculated tadpoles (p < 0.005) based on the log-rank (Mantel–Cox) test.
	Figure 4. Bacterial recovery from tadpoles 3 and 14 days after intracardiac (IC) inoculation of either R or S Mab. (A) Comparison of R and S Mab CFUs for different tissues at 3 and 14 dpi. (B) CPU comparison between 3 and 14 dpi for R and S Mab. Tadpoles were IC-injected with 5 × 105 CFU in a 10 μL volume. Spleen, liver, PLs, and lungs were collected, suspended in 500 µL of aPBS, lysed by bead beating, and plated on agar media. The bacterial abundance was determined by the number of CFUs/tissue. * p < 0.05, p < 0.005, and ** p < 0.0001 (two-way ANOVA followed by post hoc analysis using Tukey’s multiple comparisons test).
	Figure 5. Comparison of bacterial recovery between IP and IC inoculation at 3 and 14 dpi for S and R Mab. * p < 0.05, p < 0.005, * p < 0.0005, and **** p < 0.0001 (two-way ANOVA followed by post hoc analysis using Tukey’s multiple comparisons test).