Structural and functional annotation of the porcine immunome

Abstract

Background: The domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems.

Results: The Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome.

Conclusions: This extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig's adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response.

Figure 1

Greater pig-human similarity revealed by orthology preservation analysis. As shown in the graph, pigs have 11-, 6- and 2- fold less unique genes than do the mouse, cow or human.

Figure 2

Biolayout Express^3D co-expression network of immune response expression patterns. On the left is shown the network. Nodes assigned to each MCL cluster are shown in a different color. MCL is a Markov Clustering (MCL) algorithm for graph clustering (micans.org/mcl); see also http://www.biolayout.org. At right, the immune-response gene enriched cluster 4 is highlighted by increasing the size of nodes for Cluster 4 and reduction of node size in all other clusters.

Figure 3

Expression pattern of MCL cluster 4 shows gene activation after immune stimulation/infection in multiple experimental datasets. In light blue is shown the average expression of the 619 probesets in cluster 4 from Figure 2. Details on each dataset are shown below the graph; some example patterns are highlighted in red; specific dataset patterns are boxed to show contrasts of interest. LN: lymph node; PRRSV: porcine respiratory and reproductive syndrome virus; PCV2: porcine circovirus type 2; Mac: alveolar macrophage; Ctrl: Control (uninfected) sample. Hpi: hours post infection or inoculation; Dpi: days post infection or inoculation. LPS: lipopolysaccharide.

Figure 4

Results of positive selection calculation are visualized onto 3D structure of PPP2R5C, HEXB, GBP1 and CASP8 pig proteins. The color scale from light to dark green represents purifying selection, while orange and red represent positive selection with posterior probabilities greater than 95% or 99%, respectively. The orange arrow points to the serine at position 427 of GBP1 protein that is under positive selection.