Constanza Cárdenas

A natural peptide and its variants derived from the processing of infectious pancreatic necrosis virus (IPNV) displaying enhanced antimicrobial activity: a novel alternative for the control of bacterial diseases.

The larger segment of the infectious pancreatic necrosis virus (IPNV) codifies most of the structural and non-structural proteins of the virus in two overlapping open reading frames (ORFs). The longer of the two ORF is expressed as a polyprotein which generates a number of variable length peptides of unknown function during processing. Since an appealing hypothesis would be that these peptides are generated by the virus to act as antimicrobial agents that favor viral infectivity in their fish host, we decided to test this possibility by selecting a master peptide and using it to generate substitution variants that may enhance their antimicrobial potential. A 20-residue master peptide (p20) was selected from the well-described maturation process of the structural viral protein VP2; several variants were then designed and chemically synthesized, ranging in size from 16 to 20 residues. The synthesized peptides were tested for in vitro activity against several prototype bacterial pathogens using standardized laboratory procedures. Chemically synthesized p20 and all its variants displayed broad activity against the tested bacteria and none of them were toxic to eukaryotic cells at least 10× the concentration used against the bacteria. Interestingly, when p20 was tested against the very aggressive bacterial pathogen Piscirickettsia salmonis, a common co-infectant of IPNV in salmonid fish, the specific activity of the novel peptide was significantly higher than that displayed for bactericidal fish farm antibiotics such as oxolinic acid, flumequine and florfenicol, which are commonly used to control Piscirickettsiosis in the field. It is potentially significant that the approach presented in this report provides a novel alternative for generating new and ideally more efficient and friendly safeguards for bacterial prophylaxis.

Novel strategy to evaluate infectious salmon anemia virus variants by high resolution melting.

Genetic variability is a key problem in the prevention and therapy of RNA-based virus infections. Infectious Salmon Anemia virus (ISAv) is an RNA virus which aggressively attacks salmon producing farms worldwide and in particular in Chile. Just as with most of the Orthomyxovirus, ISAv displays high variability in its genome which is reflected by a wider infection potential, thus hampering management and prevention of the disease. Although a number of widely validated detection procedures exist, in this case there is a need of a more complex approach to the characterization of virus variability. We have adapted a procedure of High Resolution Melting (HRM) as a fine-tuning technique to fully differentiate viral variants detected in Chile and projected to other infective variants reported elsewhere. Out of the eight viral coding segments, the technique was adapted using natural Chilean variants for two of them, namely segments 5 and 6, recognized as virulence-associated factors. Our work demonstrates the versatility of the technique as well as its superior resolution capacity compared with standard techniques currently in use as key diagnostic tools.

Denaturing Gradient Gel Electrophoresis (DGGE) as a Powerful Novel Alternative for Differentiation of Epizootic ISA Virus Variants

Infectious Salmon Anemia is a devastating disease critically affecting world-wide salmon production. Chile has been particularly stricken by this disease which in all cases has been directly related with its causative agent, a novel orthomyxovirus which presents specific and distinctive infective features. Among these, two molecular markers have been directly associated with pathogenicity in two of the eight RNA sub genomic coding units of the virus: an insertion hot spot region present in viral segment 5 and a Highly Polymorphic Region (HPR) located in viral segment 6. Here we report the successful adaptation of a PCR-dependent denaturing gel electrophoresis technique (DGGE), which enables differentiation of selected reported HPR epizootic variants detected in Chile. At the same time, the technique allows us to distinguish one nucleotide differences in sequences associated with the intriguing, and still not well-understood, insertion events which tend to occur on RNA Segment 5. Thus, the versatility of the technique opens new opportunities for improved understanding of the complex biology of all ISA variants as well as possible applications to other highly variable pathogens.

Molecular modeling of class I and II alleles of the major histocompatibility complex in Salmo salar

Knowledge of the 3D structure of the binding groove of major histocompatibility (MHC) molecules, which play a central role in the immune response, is crucial to shed light into the details of peptide recognition and polymorphism. This work reports molecular modeling studies aimed at providing 3D models for two class I and two class II MHC alleles from Salmo salar (Sasa), as the lack of experimental structures of fish MHC molecules represents a serious limitation to understand the specific preferences for peptide binding. The reliability of the structural models built up using bioinformatic tools was explored by means of molecular dynamics simulations of their complexes with representative peptides, and the energetics of the MHC-peptide interaction was determined by combining molecular mechanics interaction energies and implicit continuum solvation calculations. The structural models revealed the occurrence of notable differences in the nature of residues at specific positions in the binding groove not only between human and Sasa MHC proteins, but also between different Sasa alleles. Those differences lead to distinct trends in the structural features that mediate the binding of peptides to both class I and II MHC molecules, which are qualitatively reflected in the relative binding affinities. Overall, the structural models presented here are a valuable starting point to explore the interactions between MHC receptors and pathogen-specific interactions and to design vaccines against viral pathogens.

Coexistence in field samples of two variants of the infectious salmon anemia virus: a putative shift to pathogenicity.

Genetic reassortment plays an important role in the evolution of several segmented RNA viruses and in the epidemiology of their associated diseases. In particular, orthomyxoviruses show rapid fluctuation in the proportion of viral variants coexisting in an infected individual, especially under strong selective pressure. This is particularly relevant in salmon production carried out under confined and stressful conditions where one of the most feared pathogenic agents is the Infectious Salmon Anemia Virus, an orthomyxovirus family member whose biological behavior is only recently beginning to be understood. Pathogenicity of the virus has been mainly associated with deletions of the HPR region in coding segment 6 and the presence or absence of a specific insertion in a key region in coding segment 5. In this study we report, for the first time in Chile, the coexistence of two variants in fully asymptomatic fish. Of five samples analyzed, two were identified as the non-pathogenic variant, HPR0, and two as the highly pathogenic HPR7b variant, though with no clinical signs detectable in the fish. Interestingly, one of the samples unequivocally carried both variants, again without any clinical signs. Considering that in none of the samples the typical insertion in coding segment 5 was detected, it is our impression that this may represent a shift from the non-pathogenic HPR0 variant towards the highly infective HPR7b variant. If this were the case, the transition may be triggered first by deleting the corresponding sequence of the HPR region of segment 6, followed by the putative insertion in segment 5 to generate a virulent strain.

Design and activity of novel lactoferrampin analogues against O157:H7 enterohemorrhagic escherichia coli

Lactoferrampin 265-284 (LFampin 265-284) is a peptide consisting of residues 265-284 of N1-domain of bovine Lactoferrin (LF). This peptide has several cationic groups in the C-terminal lobe, exhibiting an antibacterial activity against a wide range of microorganisms. However, LFampin 265-284 exhibits low antimicrobial activity against the O157:H7 enterohaemorrhagic Escherichia coli (EHEC O157:H7) when compared with Lactoferrin chimera and Lactoferricin. Here, we have designed three analogues of LFampin 265-284 based on the distribution of cationic groups, hydrophobicity, size, and sequence. Analogues were synthesized by solid phase chemistry using Fmoc methodology obtaining peptides with 95% purity. All peptides maintain the ability to adopt helical conformations (checked by circular dichroism spectra and molecular simulations). Some of these analogues exhibited a significant increase in antimicrobial activity by counting colony forming units against EHEC O157:H7 compared to native LFampin 265-284, with MIC of 10 and 40 µM for 264G-D265K and 264G-D265K/S272R, respectively. The incorporation of a GKLI sequence in the N-terminal lobe increased dramatically its antibacterial activity, an effect which has been attributed to the addition of cationic groups in the N-terminal side that may stabilize the helical conformation of the new designed peptides.

Effect of secondary anchor amino acid substitutions on the immunogenic properties of an HLA-A*0201-restricted T cell epitope derived from the Trypanosoma cruzi KMP-11 protein

The TcTLE peptide (TLEEFSAKL) is a CD8(+) T cell HLA-A*0201-restricted epitope derived from the Trypanosoma cruzi KMP-11 protein that is efficiently processed, presented and recognized by CD8(+) T cells from chagasic patients. Since the immunogenic properties of wild-type epitopes may be enhanced by suitable substitutions in secondary anchor residues, we have studied the effect of introducing specific mutations at position 3, 6 and 7 of the TcTLE peptide. Mutations (E3L, S6V and A7F) were chosen on the basis of in silico predictions and in vitro assays were performed to determine the TcTLE-modified peptide binding capacity to the HLA-A*0201 molecule. In addition, the functional activity of peptide-specific CD8(+) T cells in HLA-A2(+) chagasic patients was also interrogated. In contrast to bioinformatics predictions, the TcTLE-modified peptide was found to have lower binding affinity and stability than the original peptide. Nevertheless, CD8(+) T cells from chronic chagasic patients recognized the TcTLE-modified peptide producing TNF-α and INF-γ and expressing CD107a/b, though in less extension than the response triggered by the original peptide. Overall, although the amino acids at positions 3, 6 and 7 of TcTLE are critical for the peptide affinity, they have a limited effect on the immunogenic properties of the TcTLE epitope.

Chemical Synthesis and In Vitro Evaluation of a Phage Display-Derived Peptide Active against Infectious Salmon Anemia Virus

ABSTRACT Infectious salmon anemia virus (ISAV) is the etiological agent of the disease by the same name and causes major losses in the salmon industry worldwide. Epizootic ISAV outbreaks have occurred in Norway and, to a lesser degree, in Canada. In 2007, an ISAV outbreak in Chile destroyed most of the seasonal production and endangered the entire Chilean salmon industry. None of the existing prophylactic approaches have demonstrated efficacy in providing absolute protection from or even a palliative effect on ISAV proliferation. Sanitary control measures for ISAV, based on molecular epidemiology data, have proven insufficient, mainly due to high salmon culture densities and a constant presence of a nonpathogenic strain of the virus. This report describes an alternative treatment approach based on interfering peptides selected from a phage display library. The screening of a phage display heptapeptide library resulted in the selection of a novel peptide with significant in vitro antiviral activity against ISAV. This peptide specifically interacted with the viral hemagglutinin-esterase protein, thereby impairing virus binding, with plaque reduction assays showing a significant reduction in viral yields. The identified peptide acts at micromolar concentrations against at least two different pathogenic strains of the virus, without detectable cytotoxic effects on the tested fish cells. Therefore, antiviral peptides represent a novel alternative for controlling ISAV and, potentially, other fish pathogens. IMPORTANCE Identifying novel methods for the efficient control of infectious diseases is imperative for the future of global aquaculture. The present study used a phage display heptapeptide library to identify a peptide with interfering activity against a key protein of the infectious salmon anemia virus (ISAV). A piscine orthomyxovirus, ISAV is a continuous threat to the commercial sustainability of cultured salmon production worldwide. The complex epidemiological strategy of this pathogen has made prophylactic control extremely difficult. The identified antiviral peptide efficiently impairs ISAV infection in vitro by specifically blocking hemagglutinin-esterase, a pivotal surface protein of this virus. Peptide synthesis could further modify the primary structure of the identified peptide to improve specific activity and stability. The present results form the foundation for developing a new pharmacological treatment against ISAV.

Immobilization of lipases in hydrophobic chitosan for selective hydrolysis of fish oil: The impact of support functionalization on lipase activity, selectivity and stability.

The objective of this paper was to carry out an integral study of the use of hydrophobic chitosan as a low-cost support for immobilizing lipases and their further application in the selective hydrolysis of fish oil. Chitosan functionalized with different alkyl chains (C4, C8, C12) were characterized by FTIR, TGA, SEM, and Rose Bengal adsorption. Lipase B from Candida antarctica (CalB) and lipase from Rhizomucor miehei (RML) were immobilized obtaining a higher expressed activity at a longer alkyl chain length of support. Biocatalyst thermal stability showed that the impact of the alkyl chain length on enzyme stabilization varied according to the lipase source. The biocatalysts were applied in menhaden oil hydrolysis. Total polyunsaturated fatty acids released after 30 h of reaction with lipases immobilized in butyl, octyl and dodecyl-chitosan was 60, 107, and 90 mM for CalB biocatalysts, and 560, 392, and 50 mM for RML biocatalysts, respectively. Selectivity of CalB was not affected by the alkyl chain, while in the case of RML, a higher selectivity to cis-4,7,10,13,16,19-docohexaenoic acid release was obtained with dodecyl-chitosan. In conclusion, the adequate functionalization of chitosan varied according to lipase source, affecting their activity, stability and performance in the hydrolysis of fish oil.

Structure-activity relationships on the study of β-galactosidase folding/unfolding due to interactions with immobilization additives: Triton X-100 and ethanol

Improving the enzyme stability is a challenge for allowing their practical application. The surfactants are stabilizing agents, however, there are still questions about their influence on enzyme properties. The structure-activity/stability relationship for β-galactosidase from Bacillus circulans is studied here by Circular Dichroism and activity measurements, as a function of temperature and pH. The tendency of preserving the β-sheet and α-helix structures at temperatures below 65°C and different pH is the result of the balance between the large- and short-range effects, respecting to the active site. This information is fundamental for explaining the structural changes of this enzyme in the presence of Triton X-100 surfactant and ethanol. The enzyme thermal stabilization in the presence of this surfactant responds to the rearrangement of the secondary structure for having optimal activity/stability. The effect of ethanol is more related with changes in the dielectric properties of the aqueous solution than with protein structural transformations. These results contribute to understand the effects of surfactant-enzyme interactions on the enzyme behavior, from the structural point of view and to rationalize the surfactant-based stabilizing strategies for β-galactosidades.