Nitrogen limitation induces hypervirulence in M. abscessus. (A) Simplified diagram of the experimental workflow used to induce lipid-loaded M. abscessus by nitrogen deprivation prior to infection of zebrafish embryos. Zebrafish embryos were infected via caudal vein injection at 30 hpf with approximately 200 CFU of M. abscessus S or R cultured for 48 h in either 7H9 or MSM NL Gly 1%. (B) Nitrogen limitation results in significantly earlier mortality in M. abscessus R-infected zebrafish. Zebrafish embryo survival was monitored daily over a 12-day period following infection. Each group consisted of approximately 20 embryos, with each curve reproduced in triplicate. Statistical analysis was completed using the Mantel-Cox log-rank test. (C) Representative images displaying significantly increased pathology phenotypes at 6 dpi in zebrafish embryos infected with the M. abscessus R morphotype following nitrogen limitation. Transgenic reporter line zebrafish embryos harbouring fluorescent macrophages (mpeg:mCherry) (red overlay) were infected with M. abscessus harbouring pTEC15::mWasabi (green overlay) and the merge was observed in yellow. The open arrow (top) displays an intact abscess, while the closed arrow (bottom) displays a ruptured abscess. Scale bars represent 500 µm. **p-value ≤ 0.01.
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