PACAP sequence modifications modulate the peptide antimicrobial activity against bacterial pathogens affecting aquaculture.

The global aquaculture industry has significant losses each year due to disease outbreaks. Antibiotics are one of the common methods to treat fish infections, but prolonged use can lead to the emergence of resistant strains. Aeromonas spp. Infections are a common and problematic disease in fish, and members of this genera can produce antibiotic resistant strains. Antimicrobial peptides (AMPs) have emerged as an alternative method to treat and prevent infections and pituitary adenylate cyclase activating polypeptide (PACAP) is a prominent member of this family. The objective of this research was to study PACAP's direct antimicrobial activity and its toxicity in fish cells. Four synthetic variants of the natural PACAP from Clarias gariepinus were tested in addition to the natural variant. The experimental results show a different antimicrobial activity against A. salmonicida and A. hydrophila of each PACAP variant, and for the first time show dependence on the culture broth used. Furthermore, the results suggest that the underlying mechanism of PACAP antimicrobial activity includes a bacterial membrane permeabilizing effect, classifying PACAP as a membrane disruptive AMP. This study also demonstrated that the five PACAP variants evaluated showed low toxicity in vitro, at concentrations relevant for in vivo applications. Therefore, PACAP could be a promising alternative to antibiotics in the aquaculture sector.

New Evidence for the Role of Pituitary Adenylate Cyclase-Activating Polypeptide as an Antimicrobial Peptide in Teleost Fish.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is a multifunctional neuropeptide that is widely distributed and conserved across species. We have previously shown that in teleost fish, PACAP not only possesses direct antimicrobial properties but also immunomodulatory effects against the bacterial pathogens Flavobacterium psychrophilum and Pseudomonas aeruginosa using in vitro and in vivo experiments. These previous results suggest PACAP can be used as an alternative to antibiotics to prevent and/or treat bacterial infections in the aquaculture industry. To accomplish this goal, more studies are needed to better understand the effect of PACAP on pathogens affecting fish in live infections. In the present study, the transcripts PACAP, PRP/PACAP, and VPAC2 receptor were examined in rainbow trout (Oncorhynchus mykiss) naturally infected with Yersinia ruckeri, which exhibited an increase in their expression in the spleen when compared to healthy fish. Synthetic Clarias gariepinus PACAP-38 has direct antimicrobial activity on Y. ruckeri and inhibits up to 60% of the bacterial growth when the peptide is at concentrations between 50 and 100 µM in TSB. The growth inhibition increased up to 90% in the presence of 12.5 µM of PACAP-38 when salt-free LB broth was used instead of TSB. It was also found to inhibit Y. ruckeri growth in a dose-dependent manner when the rainbow trout monocyte/macrophage-like cell line (RTS11) was pre-treated with lower concentrations of the peptide (0.02 and 0.1 µM) before going through infection. Differential gene expression was analyzed in this in vitro model. Overall, the results revealed new evidence to support the role of PACAP as an antimicrobial and immunomodulatory peptide treatment in teleosts.

PACAP modulates the transcription of TLR-1/TLR-5/MyD88 pathway genes and boosts antimicrobial defenses in Clarias gariepinus.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide that belongs to the secretin/glucagon/GHRH/VIP superfamily. Some of these molecules have antimicrobial activity and they are capable of stimulating the immune system. The present work studied the antibacterial and immunostimulatory activity of PACAP-38 from African catfish Clarias gariepinus against the Gram-negative bacterium Pseudomonas aeruginosa in an in vivo test. PACAP-38 improved antimicrobial activity of skin mucus molecules against P. aeruginosa. The peptide modulates the gene expression profile of TLR-1, TLR-5, MyD88, IL-1ß, TNF-ɑ, IL-8, pardaxin, hepcidin and G/C-type lysozymes in skin, spleen and head kidney. The influenced exerted depended on the time after infection and tissue analyzed. This study provides the first evidence of a link between PACAP and antimicrobial peptides hepcidin and pardaxin. Our results suggest further use of PACAP as antimicrobial agent that could potentially be used to control disease in aquaculture.

Monoclonal antibody against Nile tilapia (Oreochromis niloticus) IgM heavy chain: A valuable tool for detection and quantification of IgM and IgM+ cells.

Nile tilapia (Oreochromis niloticus) is a freshwater fish, which is extensively cultivated worldwide and constitutes one of the model species for the study of fish immunology. Monoclonal antibodies are very advantageous molecular tools for studying teleost immune system. Specifically, monoclonal antibodies that react with immunoglobulins are used successfully in the study of the humoral immune response of several fish species. In the present study, we produced and characterized a monoclonal antibody against tilapia IgM heavy chain using a peptide-based strategy. The peptide sequence was selected from the surface-exposed region between CH3-CH4 domains. The specificity of the polyclonal serum and the hybridoma culture supernatant obtained by immunization with the peptide conjugated to keyhole limpet hemocyanin were evaluated by western blotting, both showing reactivity against tilapia serum IgM. The purified mAb was able to recognize secreted IgM by western blotting and ELISA and membrane IgM by flow cytometry. We also demonstrated that the antibody doesn't cross-react with a recombinant IgT fragment. This tool allowed us to study for the first time the stimulation of mucosal immunity after Pituitary Adenylate Cyclase Activating Polypeptide administration. Overall, the results demonstrated the utility of this mAb to characterize humoral immune response in O. niloticus.

First in vivo evidence of pituitary adenylate cyclase-activating polypeptide antiviral activity in teleost.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide belonging to the glucagon/secretin superfamily. In teleost fish, PACAP has been demonstrated to have an immunomodulatory role. Although previous studies have shown that viral/bacterial infections can influence the transcription of PACAP splicing variants and associated receptors in salmonids, the antiviral activity of PACAP has never been studied in teleost. Thus, in the present work, we investigated in vitro the influence of synthetic Clarias gariepinus PACAP-38 on the transcription of genes related to viral immunity using the rainbow trout monocyte/macrophage-like cell line RTS11 as a model. Positive transcriptional modulation of interferon gamma (IFNγ), interferon alpha (FNα1,2), interleukin 8 (IL-8), Mx and Toll-like receptor 3 (TLR3) genes was found in a dose and time dependent manner. We also explored how a pre-treatment with PACAP could enhance antiviral immune response using poly (I:C) as viral mimic. Interferons and IL-8 transcription levels were enhanced when PACAP was added 24 h previous to poly (I:C) exposure. With these evidences, we tested in vivo how PACAP administration by immersion bath affected the survival of rainbow trout fry to a challenge with viral hemorrhagic septicemia virus (VHSV). After challenge, PACAP-treated fish had increased survival compared to non-treated/challenge fish. Furthermore, PACAP was able to decrease the viral load in spleen/kidney and stimulate the transcription of IFNs and Mx when compared to untreated infected fish. Altogether, the results of this work provide valuable insights regarding the role of teleost PACAP in antiviral immunity and point to a potential application of this peptide to reduce the impact of viral infections in aquaculture.

Promiscuous T cell epitopes boosts specific IgM immune response against a P0 peptide antigen from sea lice in different teleost species.

The development of vaccines employing conserved protein antigens, for instance ribosomal protein P0, has as disadvantage the high degree of identity between pathogen and host proteins due to possible induction of tolerance or auto antibodies in the host organism. To overcome this drawback, peptide-based vaccines have been designed with a proved high efficacy. The use of defined peptides as antigens has the problem that they are generally poor immunogenic unless coupled to a carrier protein. Several studies have established the potential for promiscuous T cell epitopes incorporated into chimeric peptides to enhance the immunogenicity in mammals. On the contrary, studies about the role of these epitopes on teleost immune system are scarce. Therefore, the main objective of our present study was to evaluate the potential of promiscuous T cell epitopes to boost specific IgM immune response in teleost fish against a peptide antigen. With this aim, we used a peptide of 35 amino acids from the ribosomal P0 protein of Lepeophtheirus salmonis, an important parasite in salmon aquaculture. We fused this peptide to the C-terminal of T cell epitopes from tetanus toxin and measles virus and produced the chimeric protein in Escherichia coli. Following vaccination, IgM antibody production was monitored in different immunization schemes in Tilapia, African catfish and Atlantic salmon. The results demonstrated for first time that the addition of T cell epitopes at the N-terminal of a target peptide increased IgM specific response in different teleost species, revealing the potential of this approach to develop peptide-based vaccines for aquaculture. The results are also of great importance in the context of vaccine development against sea lice using ribosomal protein P0 as antigen taking into account the key role of P0 in protein synthesis and other essential physiological processes.