The M. marinum ppe10::tn mutant is attenuated in zebrafish embryo infections.

A) M. marinum E11 wild type strain and the isogenic mutants espG₅::tn, eccCb₁::tn and ppe10::tn, as well as the complemented ppe10::tn strain (PPE10-C), were pre-cultured in liquid medium containing Tween-80. 50-100 CFUs of bacteria were injected in the bloodstream of zebrafish embryos at 28 hours post fertilization. Embryos were homogenized five days post infection and plated to establish the number of CFU per embryo. Three independent experiments of six embryos per group were performed and the data were pooled. B) Visualization of M. marinum infection of zebrafish embryos. Wild-type M. marinum or ppe10::tn containing the plasmid pSMT3::mCherry was injected in the bloodstream of zebrafish embryos as described above. After 5 days of infection the embryos were examined by brightfield (top) or fluorescence (bottom) microscopy. C) Quantification of fluorescence in infected zebrafish embryos. M. marinum wild type (closed symbols) and the ppe10::tn (open symbols) mutants expressing mCherry were injected in the bloodstream of zebrafish embryos 28 hours post fertilization. Images were acquired by fluorescence microscopy at day 5 (blue) and 7 (red) post infection and were analyzed for fluorescent intensity by dedicated software [43]. Differences on day 5 and day 7 were analyzed by GraphPad Prism software using Mann-Whitney two-tailed test. * = p<0.05, ** = p<0.01, n.s. = not significant.