In vivo Host-Pathogen Interaction as Revealed by Global Proteomic Profiling of Zebrafish Larvae

Abstract

The outcome of a host-pathogen interaction is determined by the conditions of the host, the pathogen, and the environment. Although numerous proteomic studies of <i>in vitro</i>-grown microbial pathogens have been performed, <i>in vivo</i> proteomic approaches are still rare. In addition, increasing evidence supports that <i>in vitro</i> studies inadequately reflect <i>in vivo</i> conditions. Choosing the proper host is essential to detect the expression of proteins from the pathogen <i>in vivo</i>. Numerous studies have demonstrated the suitability of zebrafish (<i>Danio rerio</i>) embryos as a model to <i>in vivo</i> studies of <i>Pseudomonas aeruginosa</i> infection. In most zebrafish-pathogen studies, infection is achieved by microinjection of bacteria into the larvae. However, few reports using static immersion of bacterial pathogens have been published. In this study we infected 3 days post-fertilization (DPF) zebrafish larvae with <i>P. aeruginosa</i> PAO1 by immersion and injection and tracked the <i>in vivo</i> immune response by the zebrafish. Additionally, by using non-isotopic (Q-exactive) metaproteomics we simultaneously evaluated the proteomic response of the pathogen (<i>P. aeruginosa</i> PAO1) and the host (zebrafish). We found some zebrafish metabolic pathways, such as hypoxia response via HIF activation pathway, were exclusively enriched in the larvae exposed by static immersion. In contrast, we found that inflammation mediated by chemokine and cytokine signaling pathways was exclusively enriched in the larvae exposed by injection, while the integrin signaling pathway and angiogenesis were solely enriched in the larvae exposed by immersion. We also found important virulence factors from <i>P. aeruginosa</i> that were enriched only after exposure by injection, such as the Type-III secretion system and flagella-associated proteins. On the other hand, <i>P. aeruginosa</i> proteins involved in processes like biofilm formation, and cellular responses to antibiotic and starvation were enriched exclusively after exposure by immersion. We demonstrated the suitability of zebrafish embryos as a model for <i>in vivo</i> host-pathogen based proteomic studies in <i>P. aeruginosa</i>. Our global proteomic profiling identifies novel molecular signatures that give systematic insight into zebrafish-<i>Pseudomonas</i> interaction.