Determining the effect of treatment with an exogenous host defence peptide on Mycobacterium marinum-zebrafish infection

Abstract

We urgently require novel antimicrobial therapies for TB infections as a third of the world’s population is infected with latent TB and cases of multidrug-resistant TB are increasing. Naturally occurring host defence peptides (HDPs), components of the innate immune system, provide protection against infections in vivo through their direct antimicrobial and immunomodulatory functions. The effect of one HDP, bovine bactenecin-5 (Bac5), on mycobacterial infections was investigated in this study using the M. marinum-zebrafish TB infection model. Bacterial two-component systems (TCSs) regulate gene expression in response to external environmental changes, controlling physiological processes including virulence. Generation of TCSs deletion mutants in Mycobacterium marinum was attempted, unsuccessfully, by mycobacteria-adapted recombineering and sucrose counter-selection, with a view to studying the effects of targeting these systems during mycobacterial infections using zebrafish as a model organism.

The M. marinum-zebrafish embryo model permits the monitoring of pathogenesis in real time through live-imaging. Bacterial Distance Distribution and Fluorescent Pixel Count protocols were developed or optimised in Icy to quantify bacterial dissemination and bacterial burden from images of zebrafish embryo infections using fluorescently labelled bacterial strains. These techniques, coupled with zebrafish leukocyte recruitment quantification using transgenic zebrafish lines and our image processing protocol, permit simultaneous analysis of multiple infection progression parameters during longitudinal studies.

Injection with exogenous Bac5 alone and co-injection with M. marinum led to a significant initial increase in macrophage recruitment to the zebrafish injection site. We also observed trends following injections of zebrafish embryos with Bac5 for increased initial neutrophil recruitment, increased expression of the chemokine cxcl-c1c and pro-inflammatory cytokines IL-1β and MMP-9, and reduced M. marinum burdens. These results provide a basis for further investigations into the efficacy of Bac5 treatment of mycobacterial infections, in particular the optimal timings and dosage, with a view to using this exogenous HDP to treat human TB infections.