Proteomic profile of epidermal mucus from Labeo rohita reveals differentially abundant proteins after Aeromonas hydrophila infection

Abstract

We report the proteomic profile of Epidermal Mucus (EM) from Labeo rohita and identified the differentially abundant proteins (DAPs) against Aeromonas hydrophila infection through label-free liquid chromatography-mass spectrometry (LC-MS/MS). Using discovery-based proteomics, a total of 2039 proteins were quantified in nontreated group and 1,328 proteins in the treated group, of which 114 were identified as DAPs in both the groups. Of the 114 DAPs, 68 proteins were upregulated and 46 proteins were downregulated in the treated group compared to nontreated group. Functional annotations of these DAPs shows their association with metabolism, cellular process, molecular process, cytoskeletal, stress, and particularly immune system. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and Fisher's exact test between the two groups shows that most of the proteins were immune-related, which were significantly associated with the proteasome, phagosome, and Salmonella infection pathways. Overall, this study shows a basic and primary way for further functional research of the involvement of vitellogenin 2, alpha-2-macroglobulin-like protein, toll-like receptors (TLR-13), calpain, keratin-like proteins, and heat shock proteins against bacterial infection. Nonetheless, this first-ever comprehensive report of a proteomic sketch of EM from L. rohita after A. hydrophila infection provides systematic protein information to broadly understand the biological role of fish EM against bacterial infection.

Keywords: Aeromonas hydrophila; Epidermal mucus; Immune-related proteins; Labeo rohita; Proteomic analysis.

Fig. 1

SDS-PAGE showed the separation of proteins extracted from all samples of EM of L. rohita of nontreated NT-LR 1-3 EM and treated T-LR 1-3 with A. hydrophila: Lane 1 (N-T1), Lane 2 (N-T2), Lane 3 (N-T3), Lane 4 (T1), Lane-5 (T2), Lane-6 (T6). Smobio Enhanced 3-colour High Range Protein Marker (5-245 kDa) was used as a standard. Approximately 10 μg protein was loaded/lane.

Fig. 2

Gene Ontology annotations of the DAPs of both NT-LR and T-LR groups based on the BLAST2GO (P ≤ 0.05). Bar graph is the identified proteins enriched with gene ontology (GO) after classification into 3-categories on the basis of enrichment score. GO terms: A): Biological Process (GO:0065007); developmental process (GO: 0032502); regulation of biological process (GO: 0048856); metabolic process (GO: 0008152); response to stimulus (GO: 0050896); B): Cellular Component (GO:0005575); Intracellular anatomical entity (GO:0110165); Protein containing complex (GO: 0032991); membranes (GO: 00432246): C): Molecular Function (GO: 0003674); Structural molecular activity (GO: 0005515): binding (GO: 0003824).

Fig. 3

Fisher Exact Test (Enrichment analysis): Fisher exact test was performed for the comparison of GO terms based on sequence percentage (Biological process, Cellular components, and Molecular function) of the DAPs (Upregulated v downregulated).

Fig. 4

KEGG pathways enrichment analysis for the identified DAPs. Each row denotes an enriched function, and the count denotes the enriched ratio which is reflected as the "input proteins number". The colour of the bar differentiates Up and downregulated pathways.