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- Chapman, Joanne R., et al.
(författare)
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The Evolution of Innate Immune Genes : Purifying and Balancing Selection on beta-Defensins in Waterfowl
- 2016
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Ingår i: Molecular biology and evolution. - : Oxford University Press (OUP). - 0737-4038 .- 1537-1719. ; 33:12, s. 3075-3087
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Tidskriftsartikel (refereegranskat)abstract
- In disease dynamics, high immune gene diversity can confer a selective advantage to hosts in the face of a rapidly evolving and diverse pathogen fauna. This is supported empirically for genes involved in pathogen recognition and signalling. In contrast, effector genes involved in pathogen clearance may be more constrained. beta-Defensins are innate immune effector genes; their main mode of action is via disruption of microbial membranes. Here, five beta-defensin genes were characterized in mallards (Anas platyrhynchos) and other waterfowl; key reservoir species for many zoonotic diseases. All five genes showed remarkably low diversity at the individual-, population-, and species-level. Furthermore, there was widespread sharing of identical alleles across species divides. Thus, specific beta-defensin alleles were maintained not only spatially but also over long temporal scales, with many amino acid residues being fixed across all species investigated. Purifying selection to maintain individual, highly efficacious alleles was the primary evolutionary driver of these genes in waterfowl. However, we also found evidence for balancing selection acting on the most recently duplicated beta-defensin gene (AvBD3b). For this gene, we found that amino acid replacements were more likely to be radical changes, suggesting that duplication of beta-defensin genes allows exploration of wider functional space. Structural conservation to maintain function appears to be crucial for avian beta-defensin effector molecules, resulting in low tolerance for new allelic variants. This contrasts with other types of innate immune genes, such as receptor and signalling molecules, where balancing selection to maintain allelic diversity has been shown to be a strong evolutionary force.
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| 5. |
- Helin, Anu S., et al.
(författare)
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From genes to function : variation in antimicrobial activity of avian β-defensin peptides from mallards
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Avian β-defensins are an important class of antimicrobial peptides in birds. These short cationic peptides are directly involved in the clearance of infections by membrane disruption, but can also act as immunomodulators and chemotactic agents recruiting immune cells. Recent genomic studies have shown the presence of several different avian β-defensin (AvBD) genes across the avian phylogeny, but also significant copy number variation and occurrence of pseudogenes. In mallard (Anas platyrhynchos) and other waterfowl AvBD genes are conserved and seem to be maintained by purifying selection. Due to their relatively simple peptide structure and direct mode of action, AvBDs is a potential tractable system to investigate how small differences in the gene sequence translates into differences in immune function. Here, we used genomic information from three different mallard defensin loci (AvBD4, AvBD10 and AvBD13) and synthesized the linear peptides from the most common allele of each locus, plus two rare alleles from AvBD13 locus and measured their antimicrobial activity against Gram-negative (E. coli and S. Typhimurium) and Gram-positive (S. aureus and M. luteus) bacteria. In these assays, AvBD4 showed the most potent antibacterial activity against both Gram-negative and Gram-positive bacteria, with an IC50 value of 0.48 mM against S. Typhimurium. Among AvBD13 peptides, the less frequently observed AvBD13:2 variant, was most potent, with IC50 value against S. aureus approximately 15 times lower than that of the most common AvBD13:1. Interestingly, AvBD10 had no antibacterial effect on the tested bacteria. Thus, antimicrobial function varied substantially among loci, but also within the AvBD13 locus, suggesting a direct link between genetic variation and immune function variation. Interestingly, results from assays with AvBD4 and AvBD13 seem to indicate that a higher positive net charge in peptides is associated with a more potent antibacterial effect.
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| 6. |
- Helin, Anu S., et al.
(författare)
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Relation between structure and function of three AvBD3b variants from mallard (Anas platyrhynchos)
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Defensins are multifunctional antimicrobial peptides expressed in several tissue types and leucocytes as part of the innate immune response against microbes. Based on the three-dimensional structure and disulfide connectivity, vertebrate defensins are subdivided into α-, β-, and θ-defensins. While all three types have been found in mammals, only β-defensins have been identified in birds. Genetic studies have revealed dozens of different avian β-defensin (AvBD) genes in different bird species, as well as allelic variation for different genes. Knowledge of the relation between avian peptide structure features and antimicrobial activity is however limited. In this study, the structure-functional relations of three variants of AvBD3b, a mallard (Anas platyrhynchos) defensin of evolutionary interest, was investigated. Gene alleles encoding two of these peptides, AvBD3b:1 and AvBD3b:2 are common in mallards, whereas AvBD3b:3 occurs rare. These β-defensin peptides were synthesized as linear peptides and subjected to oxidative folding. The three-dimensional structure of AvBD3b:1, including disulfide bond connectivity, was determined using NMR, and those of AvBD3b:2 and AvBD3b:3 respectively, were modelled using AvBD3b:1 as the template. The antimicrobial activities of folded peptides were compared to those of linear peptides. The NMR analysis showed that folded AvBD3b adopts a three-dimensional structure typical for β-defensins, including C-terminal antiparallel β-sheets and disulfide bond organization between six cysteine (C) residues: C6-C34, C13-C28, and C18-C35. Analyses of antimicrobial activity showed that both folded and linear variants of the three peptides inhibited bacterial growth. However, differences in activity were observed, suggesting that folded AvBD3b:3 was the most efficient against both Gram-negative and Gram-positive bacteria. Taken together, these findings provide additional insight into the influence of amino acid sequence variation and three-dimensional structure on the antimicrobial activity of mallard AvBD3b.
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| 7. |
- Chapman, Joanne R., et al.
(författare)
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A Panel of Stably Expressed Reference Genes for Real-Time qPCR Gene Expression Studies of Mallards (Anas platyrhynchos)
- 2016
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:2
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Tidskriftsartikel (refereegranskat)abstract
- Determining which reference genes have the highest stability, and are therefore appropriate for normalising data, is a crucial step in the design of real-time quantitative PCR (qPCR) gene expression studies. This is particularly warranted in non-model and ecologically important species for which appropriate reference genes are lacking, such as the mallard-a key reservoir of many diseases with relevance for human and livestock health. Previous studies assessing gene expression changes as a consequence of infection in mallards have nearly universally used beta-actin and/or GAPDH as reference genes without confirming their suitability as normalisers. The use of reference genes at random, without regard for stability of expression across treatment groups, can result in erroneous interpretation of data. Here, eleven putative reference genes for use in gene expression studies of the mallard were evaluated, across six different tissues, using a low pathogenic avian influenza A virus infection model. Tissue type influenced the selection of reference genes, whereby different genes were stable in blood, spleen, lung, gastrointestinal tract and colon. beta-actin and GAPDH generally displayed low stability and are therefore inappropriate reference genes in many cases. The use of different algorithms (GeNorm and NormFinder) affected stability rankings, but for both algorithms it was possible to find a combination of two stable reference genes with which to normalise qPCR data in mallards. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies in ducks. The fact that nearly all previous studies of the influence of pathogen infection on mallard gene expression have used a single, non-validated reference gene is problematic. The toolkit of putative reference genes provided here offers a solid foundation for future studies of gene expression in mallards and other waterfowl.
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| 8. |
- Helin, Anu S., et al.
(författare)
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A rapid and transient innate immune response to avian influenza infection in mallards
- 2018
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Ingår i: Molecular Immunology. - : Elsevier. - 0161-5890 .- 1872-9142. ; 95, s. 64-72
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Tidskriftsartikel (refereegranskat)abstract
- The vertebrate innate immune system provides hosts with a rapid, non-specific response to a wide range of invading pathogens. However, the speed and duration of innate responses will be influenced by the co-evolutionary dynamics of specific host-pathogen combinations. Here, we show that low pathogenic avian influenza virus (LPAI) subtype H1N1 elicits a strong but extremely transient innate immune response in its main wildlife reservoir, the mallard (Anas platyrhynchos). Using a series of experimental and methodological improvements over previous studies, we followed the expression of retinoic acid inducible gene 1 (RIG-I) and myxovirus resistance gene (Mx) in mallards semi-naturally infected with low pathogenic H1N1. One day post infection, both RIG-I and Mx were significantly upregulated in all investigated tissues. By two days post infection, the expression of both genes had generally returned to basal levels, and remained so for the remainder of the experiment. This is despite the fact that birds continued to actively shed viral particles throughout the study period. We additionally show that the spleen plays a particularly active role in the innate immune response to LPAI. Waterfowl and avian influenza viruses have a long co-evolutionary history, suggesting that the mallard innate immune response has evolved to provide a minimum effective response to LPAIs such that the viral infection is brought under control while minimising the damaging effects of a sustained immune response.
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| 9. |
- Helin, Anu S.
(författare)
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Eco-immunological studies of innate immunity in Mallards (Anas platyrhynchos)
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis comprises two sections, both of which explore eco-immunology of the innate immune system of mallards (Anas platyrhynchos). The innate immune system serves a pivotal role as the first line of defense against invading pathogens, and is comprised of physical and chemical barriers. Its main function is to inhibit and/or eliminate the pathogenic microorganisms while minimizing collateral damage to host cells.The first section investigates the allelic variation and selective forces acting on five avian β-defensins (AvBD) genes. Showing that purifying selection is the predominant selective force, although one gene AvBD3b, appeared to be subject to balancing selection. Moreover, the solution structure of the AvBD3b peptide was solved in this work, revealing that it contains a typical β-defensin fold with three β-sheets. Linear and folded AvBD3b peptides were shown to exhibit similar antibacterial properties, indicating that the tertiary structure was not the primary determinant of antimicrobial activity. Moreover, testing the antimicrobial activity of synthetic AvBD peptides showed that they mostly had higher activity against Gram-negative than Gram-positive bacteria.The second section investigates expression of two innate immune genes during avian influenza virus infection. Data quality in gene expression studies depends, in part, on the stability of the reference genes (RGs) used to normalize expression levels, meaning putative RGs must be validated prior to use. Eleven potential mallard RGs were tested and it was found that the stability varied across different tissue types, highlighting the importance of correct RG selection for the specific experimental conditions. Optimal RGs were then used in a gene expression study of retinoic acid inducible gene 1 (RIG-I) and myxovirus resistant gene (Mx) in mallards during a low pathogenic avian influenza (LPAI) infection. Upregulation of both genes was rapid and transient, returning back to basal levels two days post infection across most of the five tissue types analyzed.This thesis provides new insights into the tertiary structure and antimicrobial activity of AvBDs, and how this relates to selective pressures exerted in natural populations. It also highlights the importance of RGs validation, and confirms that RIG-I and Mx are involved in the early stages of the mallard immune response to LPAI infection.
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| 10. |
- Helin, Anu S., et al.
(författare)
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Expression of immune genes RIG-I and Mx in mallard ducks infected with low pathogenic avian influenza (LPAI) : A dataset
- 2018
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Ingår i: Data in Brief. - : Elsevier. - 2352-3409. ; 18, s. 1562-1566
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Tidskriftsartikel (refereegranskat)abstract
- This article provides data on primer sequences used to amplify the innate immune genes RIG-I and Mx and a set of normalizing reference genes in mallards (Anal platyrhynchos), and shows which reference genes are stable, per tissue, for our experimental settings. Data on the expressional changes of these two genes over a time-course of infection with low pathogenic avian influenza virus (LPAI) are provided. Individual-level data are also presented, including LPAI infection load, and per tissue gene expression of RIG-I and Mx. Gene expression in two outlier individuals is explored in more depth. (C) 2018 The Authors. Published by Elsevier Inc.
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