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 (Δorf01830, Δorf01609, and Δorf03641). The physiological characteristics of these mutants were investigated. In all these mutant strains, protease activity decreased, and Δorf01609, and Δorf01830 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.

Keywords: Aeromonas hydrophila; extracellular proteomics; iTRAQ; iron starvation; vaccine candidate.

Figure 1

Characteristics of Aeromonas hydrophila LP-2 grown in iron-limited conditions. (A) Coomassie brilliant blue(CBB) stained SDS-PAGE of extracelluar protein fractions from A. hydrophila treated with 150 μM DIP. Lane M, molecular mass standards. (B) The frequency distribution of protein iTRAQ log₂ ratios between the two biological replicates. (C) Volcano plot comparing the extracellular proteomics of A. hydrophila grown in iron-limited conditions and A. hydrophila grown in normal conditions. Solid lines indicate statistically significant differences in protein expression (iTRAQ ratio >1.5 or <0.66 and p < 0.05 for both biological replicates). Upregulated proteins are indicated by red circles; downregulated proteins are indicated by green circles; and unaltered proteins are indicated by black circles. (D) Subcellular locations predicted for all identified proteins and for all differentially expressed proteins.

Figure 2

Gene Ontology (GO) of differentially expressed proteins under iron starvation. (A,C,E) Upregulated proteins associated with biological processes, molecular functions, and KEGG pathways, respectively. (B,D,F) Downregulated proteins associated with biological processes, molecular functions, and KEGG pathways, respectively. The p-value cutoff was set at < 0.05. Different colors represent different GO levels, from 3 to 9.

Figure 3

Protein-protein interaction (PPI) networks of the extracellular proteins differentially expressed in A. hydrophila under iron starvation condition. The gradient color indicates iTRAQ protein ratio (on a log₂ scale), and size represents protein interaction frequency. Predicted extracellular proteins are displayed in yellow circles.

Figure 4

The characteristics of overexpressed and purified recombinant proteins. (A) SDS-PAGE of overexpressed proteins in the BL21 strain. Lane 1: negative control; Lanes 2–7: ORF01609, ORF01830, ORF01839, ORF02943, ORF03355, and ORF03641, respectively. (B) SDS-PAGE of purified proteins. Lanes 1–6: purified ORF01609, ORF01830, ORF01839, ORF02943, ORF03355, and ORF03641, respectively. (C) Restriction enzyme identification of recombinant extracellular protein plasmids carried by the pET-32a vector. M: DL5000 marker; Lanes 1–6: ORF01609, ORF01830, ORF01839, ORF02943, ORF03355, and ORF03641, respectively.

Figure 5

Correlations between selected genes and proteins. (A) Western blot of differentially expressed extracellular proteins in A. hydrophila LP-2 with and without DIP treatment. Coomassie R-350 stained on PVDF membrane was used as the loading control. (B) The correlation between mRNA expression and proteomics in nine upregulated genes (orf00614, orf01609, orf01830, orf01839, orf02793, orf02943, orf03355, orf03641, and orf04443), and three downregulated genes (orf03513, orf03984, and orf04406).

Figure 6

Confirmation of the knockout mutant strains Δorf01609, Δorf01830, and Δorf03641. M: DL5000 marker; a: The fragment of genomic deletion mutant DNA amplified using cloning primers. b: The fragment of genomic wild-type (LP-2) DNA amplified using cloning primers. c: The fragment of genomic deletion mutant DNA amplified using the primer pair P7/P8. d: The fragment of genomic wild-type (LP-2) DNA amplified using the primer pair P7/P8.

Figure 7

Expression of immune-related genes in zebrafish immunized with recombinant proteins as compared to controls treated with BSA, as quantified by qPCR. Internal organs were collected before the A. hydrophila LP-2 challenge.

Figure 8

Cumulative survival rates of zebrafish after challenge with A. hydrophila. (A) Zebrafish vaccinated with six iron-related extracellular proteins (ORF01609, ORF01830, ORF01839, ORF02943, ORF03355, and ORF03641) and a control treated with BSA were challenged with A. hydrophila LP-2 for 14 days. (B,C) Zebrafish vaccinated with six recombinant proteins and a control treated with BSA were challenged with A. hydrophila LP-3 and YT-1 for 7 days, respectively. Mortalities were recorded for 14 days. Data represent the mean ± SEM of n ≥ two independent replicates.