A Proteomic Characterization of Innate Immune Peptides and Proteins in Fish Plasma

通過蛋白質組學來研究魚類血液中先天免疫蛋白

Student thesis: Doctoral Thesis

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Award date13 Jul 2017

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Abstract

Bacteria infection is a serious pathogenic condition for both freshwater and marine fish, causing great economic loss and threatening human health. The innate immunity of fish, which forms the primary safeguard against bacteria, consists of a large number of antibacterial factors mainly present in blood, but the types and functions of antimicrobial factors, especially proteins and peptides, are still not fully understood. The overarching aim of this PhD study is to systematically characterize the antimicrobial proteins or peptides in fish blood by using a proteomic approach, with the intention of discovering potentially novel antimicrobial factors and mechanisms.
In the first part of my PhD project, I characterized the proteins in the serum of Turbot fish (Scophthalmus maximus) that were stably immobilized on E.tarda bacteria, a common waterborne pathogen. I demonstrated that the serum of turbot, but not the heat-inactivated control, significantly reduced the number of viable E.tarda bacteria, confirming the bactericidal activity of fish serum. By conjugating fish serum proteins with fluorescent dyes, I showed that E.tarda was coated with multiple fish proteins. In order to systematically identify these bacteria-binding proteins, I used live E.tarda bacterial cells to capture turbot serum proteins and subjected the samples to shotgun proteomic analysis. A total of 76 fish proteins were identified, including many known antimicrobial proteins such as immunoglobulins and complement components. 34 proteins with no previously known immunological function were also identified to bind to E.tarda. This approach also allowed the study of the corresponding proteomic changes in E.tarda exposed to turbot serum. Our data indicated Turbot serum induced a general decrease of protein synthesis in E. Tarda, except for the upregulation of membrane components. Interestingly, fish serum also caused a rapid suppression of the expression of bacterial antioxidant enzymes, such as catalase, and led to an accumulation of reactive oxygen species. The suppression of the bacterial antioxidant defense by fish serum factors has not been documented previously and suggests a novel mechanism of antibacterial actions in fish.
Besides antimicrobial proteins, fish continually secrete different types of antimicrobial peptides (AMPs), which act as host defense peptides and play key roles in the innate immunity. In the second part of this project, I aimed to identify naturally occurring AMPs in fish blood. Antimicrobial peptides (AMPs) have been studied in many organisms but efforts on the systematic identification of AMPs in fish have been sporadic. In this study, plasma from medaka of different gender, age and infection status were combined, and thereby providing a resource of plasma macromolecules under various possible physiological conditions. Peptides under the molecular weight of 3 kDa were fractionated and purified, followed by mass spectrometry analysis. In total, 6399 unique and non-redundant peptide sequences were identified. A large number of the detected peptides were derived from a subset of non-abundant, intracellular proteins, suggesting that these peptides were not a result of non-specific degradation of serum proteins. These circulating peptides covered a wide range of isoelectric points, with a slight bias towards an anionic charge. They also exhibited a preference in nonpolar amino acids such as methionine, glycine and alanine, while aromatic amino acids are underrepresented.
After evaluation with a combination of web-based prediction tools and conserved physicochemical AMP properties, 430 potential antimicrobial peptides were predicted. Experimental validation of a selection of 53 predicted peptides resulted in the discovery of eight novel AMPs. One of them, an evolutionarily conserved 13-residue peptide, showed a broad-spectrum toxicity on fish and human pathogenic bacteria (gram+ or gram-) without significant toxicity against mammalian cell lines, and on medaka embryos and adults. BING (Blocker of INtermembrane protein folding in Gram-negative bacteria), showed a broad-spectrum toxicity on fish and human pathogenic bacteria including drug resistant strains, at concentrations that did not exhibit toxicity against mammalian cell lines and medaka fish. In vivo studies showed that this peptide could moderately protect medaka fish from lethality caused by E.tarda infection. Proteomic analysis indicated that it specifically suppressed the envelope stress response (ESR) in Gram-negative bacteria, suggesting that this peptide exerts its antibacterial activity by inducing periplasmic protein misfolding. I therefore name this novel AMP “Blocker of INtermembrane stress response in Gram negative bacteria” (BING). The RNA level of CPXR, a crucial part of the two-component system of ESR, was significantly reduced by BING, but not by another fish AMP (piscidin) nor by ampicillin. To my knowledge, BING is the first molecule demonstrated to target the Cpx two-component pathway, which is instrumental to the bacterial resistance against both AMPs and antibiotics. The identification of BING illustrates the feasibility of the proteomic approach in the discovery of potentially novel AMPs, and opens up potential applications in controlling antimicrobial resistance.
Taken together, this work offers a comprehensive view of the interactions between fish serum proteins and pathogenic bacteria, and reveals previously unknown factors and mechanisms in fish innate immunity. The small molecular proteins in fish innate immune system discovered in the project may have a wide application in aquaculture.

    Research areas

  • Antibacterial proteins, Antimicrobial peptides, immunology, proteomics