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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  in vitro -grown microbial pathogens have been performed,  in vivo  proteomic approaches are still rare. In addition, increasing evidence supports that  in vitro  studies inadequately reflect  in vivo  conditions. Choosing the proper host is essential to detect the expression of proteins from the pathogen  in vivo . Numerous studies have demonstrated the suitability of zebrafish ( Danio rerio ) embryos as a model to  in vivo  studies of  Pseudomonas aeruginosa  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  P. aeruginosa  PAO1 by immersion and injection and tracked the  in vivo  immune response by the zebrafish. Additionally, by using non-isotopic (Q-exactive) metaproteomics we simultaneously evaluated the proteomic response of the pathogen ( P. aeruginosa  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  P. aeruginosa  that were enriched only after exposure by injection, such as the Type-III secretion system and flagella-associated proteins. On the other hand,  P. aeruginosa  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  in vivo  host-pathogen based proteomic studies in  P. aeruginosa . Our global proteomic profiling identifies novel molecular signatures that give systematic insight into zebrafish- Pseudomonas  interaction.

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