Comparative genomic analysis between Corynebacterium pseudotuberculosis strains isolated from buffalo

Abstract

Corynebacterium pseudotuberculosis is a Gram-positive, pleomorphic, facultative intracellular pathogen that causes Oedematous Skin Disease (OSD) in buffalo. To better understand the pathogenic mechanisms of OSD, we performed a comparative genomic analysis of 11 strains of C. pseudotuberculosis isolated from different buffalo found to be infected in Egypt during an outbreak that occurred in 2008. Sixteen previously described pathogenicity islands (PiCp) were present in all of the new buffalo strains, but one of them, PiCp12, had an insertion that contained both a corynephage and a diphtheria toxin gene, both of which may play a role in the adaptation of C. pseudotuberculosis to this new host. Synteny analysis showed variations in the site of insertion of the corynephage during the same outbreak. A gene functional comparison showed the presence of a nitrate reductase operon that included genes involved in molybdenum cofactor biosynthesis, which is necessary for a positive nitrate reductase phenotype and is a possible adaptation for intracellular survival. Genomes from the buffalo strains also had fusions in minor pilin genes in the spaA and spaD gene cluster (spaCX and spaYEF), which could suggest either an adaptation to this particular host, or mutation events in the immediate ancestor before this particular epidemic. A phylogenomic analysis confirmed a clear separation between the Ovis and Equi biovars, but also showed what appears to be a clustering by host species within the Equi strains.

Fig 1. Workflow of the genome assembly and annotation of 11 Corynebacterium pseudotuberculosis strains isolated from buffalo, and comparative genomics analysis with other strains.

Fig 2. A circular genomic map that compares 11 Corynebacterium pseudotuberculosis strains isolated from Egyptian buffalo.

The rings, from the inner to outer circle, are: strain 31, GC skew, GC content, strains 32, 33, 34, 35, 36, 38, 39, 43, 46, and 48, and pathogenicity islands.

Fig 3. Gene content of the intact prophage predicted by PHAST.

The prophage is inserted in pathogenicity island PiCp12 of the strains isolated from buffalo.

Fig 4. Phylogeny of diphtheria toxin gene (tox) from Corynebacterium pseudotuberculosis, C. diphtheriae and C. ulcerans inferred by using the maximum likelihood method based on the Tamura 3-parameter model, on Mega v6.

Fig 5. Synteny graph of 11 Corynebacterium pseudotuberculosis strains isolated from Egyptian buffalo.

Fig 6. Circular genomic maps comparing Corynebacterium pseudotuberculosis 1002B (biovar Ovis) with other Equi strains.

The rings, from the inner to outer circle, are strain 1002B, CG Skew, CG Content, strains 31, 258, E19, MB11, MB14, MB30, MB66, 316, CIP52.97, 1/06-A, Cp162 and 262, and pathogenicity islands.

Fig 7. Circular genomic maps comparing Corynebacterium pseudotuberculosis 31 with other Equi strains.

The rings, from the inner to outer circle, are strain 31, CG Skew, CG Content, strains 258, E19, MB11, MB14, MB30, MB66, 316, CIP52.97, 1/06-A, Cp162 and 262, and pathogenicity islands.

Fig 8. Phylogenomic tree of Corynebacterium pseudotuberculosis genomes based on the proteome of 44 complete genomes, generated by PEPR.

The core proteins were used to produce a tree, and additional protein families were added to refine subtrees with low bootstrap values.

Fig 9. Phylogenomic tree of Corynebacterium pseudotuberculosis genomes based on the variable content of 44 complete genomes.

The percentages of similarity were plotted on a heatmap generated by Gegenees 2.2.1, and then used to produce a phylogenomic tree using Splitstree v4.14.2 with the UPGMA method.

Fig 10. Organization of nitrate reductase and molybdenum cofactor biosynthesis genes in Corynebacterium pseudotuberculosis 31.

Genes with the same color are transcribed in the same operon.

Fig 11. Comparison of pilus gene cluster spaA and spaD in Corynebacterium pseudotuberculosis isolated from buffalo, represented by strain 31, and biovar Ovis, represented by strain FRC41.