Comparative Extracellular Proteomics of Aeromonas hydrophila Reveals Iron-Regulated Secreted Proteins as Potential Vaccine Candidates

Abstract

In our previous study, several iron-related outer membrane proteins in Aeromonas hydrophila , a serious pathogen of farmed fish, conferred high immunoprotectivity to fish, and were proposed as potential vaccine candidates. However, the protective efficacy of these extracellular proteins against A. hydrophila remains largely unknown. Here, we identified secreted proteins that were differentially expressed in A. hydrophila LP-2 in response to iron starvation using an iTRAQ-based quantitative proteomics method. We identified 341 proteins, of which 9 were upregulated in response to iron starvation and 24 were downregulated. Many of the differently expressed proteins were associated with protease activity. We confirmed our proteomics results with Western blotting and qPCR. We constructed three mutants by knocking out three genes encoding differentially expressed proteins (Δ orf01830 , Δ orf01609 , and Δ orf03641 ). The physiological characteristics of these mutants were investigated. In all these mutant strains, protease activity decreased, and Δ orf01609 , and Δ orf01830 were less virulent in zebrafish. This indicated that the proteins encoded by these genes may play important roles in bacterial infection. We next evaluated the immune response provoked by the six iron-related recombinant proteins (ORF01609, ORF01830, ORF01839, ORF02943, ORF03355, and ORF03641) in zebrafish as well as the immunization efficacy of these proteins. Immunization with these proteins significantly increased the zebrafish immune response. In addition, the relative percent survival (RPS) of the immunized zebrafish was 50–80% when challenged with three virulent A. hydrophila strains, respectively. Thus, these extracellular secreted proteins might be effective vaccine candidates against A. hydrophila infection in fish.

Glycine, serine and threonine metabolism confounds efficacy of complement-mediated killing

“…Western blot, far-Western blot, and Co-IP were performed as follows 54–56 . For Western blot, samples were dissolved in 15% SDS-PAGE.…”

Analysis of virulence and immunogenic factors in Aeromonas hydrophila: Towards the development of live vaccines

“…A high‐throughput proteomics technology has been advanced recently, which has been frequently used to identify novel antigens for the development of new vaccines (Wang et al., 2019). Much recent researches have been focused on the immunoprotective properties of a few extracellular or secreted proteins, as these typically affect bacterial virulence (Zhang, Pridgeon, & Klesius, 2014).…”

Malate enhances survival of zebrafish against Vibrio alginolyticus infection in the same manner as taurine

“…For the pathway enrichment analysis, the metabolic pathways involving the metabolites that showed differences between the test groups were identified using the MetaboAnalyst online website (www. metaboanalyst.ca) [61,62]. The -log(p) value ** [63] and a value reflecting the impact of each metabolic pathway were calculated using a hypergeometric test, and the metabolic pathways with p < 0.05 were retained.…”

“…qRT-PCR was performed as previously described [62]. Zebrafish were randomly divided into seven groups, 25 each.…”