Effect of MakA on zebrafish embryos and adult zebrafish and immunoblot analysis of MakA expression and secretion from different secretion system mutants of V. cholerae A) Survival of zebrafish embryos treated with 106 CFU/ml E. coli Top10 harboring either vector control or pmakC+B+A+ or pmakD+C+B+A+ operon at different time points; B) Enumeration of bacteria internalized by zebrafish embryos at different time points; C) Fluorescence micrographs showing bacterial colonization of zebrafish embryo intestine after 9 h treatment: phase contrast (i and iv); fluorescence (ii and v), overlay (iii and vi); D) Survival of adult zebrafish treated with 107 CFU/ml E. coli Top10 or V. cholerae strain A1552 harbouring vector control or pmakD+C+B+A+ plasmid at different time points; E) Immunoblot analysis of MakA expression and secretion in the wild type V. cholerae O1 strain TRH700 and its T2SS mutant derivatives, ΔepsC and ΔepsG (upper panels). Secretion of PrtV as a T2SS substrate control (lower panel); F) Immunoblot analysis of MakA expression and secretion in the wild type V. cholerae O1 strain A1552 and its T6SS mutant derivatives ΔvipA and ΔvgrG1 (upper panels). Secretion of Hcp as a T6SS substrate control (lower panel); G) Immunoblot analysis using anti-Crp antiserum in the culture supernatants of the wild type A1552 and its flagella cascade mutants as a marker for protein release due to cell lysis. H) Growth of bacteria before, during, and after treatment with indicated concentrations of CCCP.