Comparative Genome Analysis of Two Isolates of the Fish Pathogen Piscirickettsia salmonis from Different Hosts Reveals Major Differences in Virulence-Associated Secretion Systems

Abstract

Outbreaks caused by Piscirickettsia salmonis are one of the major threats to the sustainability of the Chilean salmon industry. We report here the annotated draft genomes of two P. salmonis isolates recovered from different salmonid species. A comparative analysis showed that the number of virulence-associated secretion systems constitutes a main genomic difference.

GENOME ANNOUNCEMENT

Piscirickettsia salmonis is a Gram-negative bacterium and the causative agent of a systemic infection known as piscirickettsiosis (1). The disease is a main concern due to its high prevalence in marine rearing areas; it is thus responsible for huge economic losses (2). P. salmonis was isolated for the first time in coho salmon (Oncorhynchus kisutch) in 1989, being described as an obligate intracellular parasite (3), a fact that delayed the study of its pathogenesis and the development of vaccines against the disease. However, the introduction of solid medium (4) allowed for a demonstration of the facultative intracellular nature of the pathogen and sequencing of the P. salmonis strains LF-89 (5) and AUSTRAL-005 (6).

An early phylogenetic study based on the sequence of the internal transcribed spacer (ITS) placed the P. salmonis EM-90 strain, which was isolated from Atlantic salmon (Salmo salar), apart from the LF-89-like isolates collected from three salmonid species (7). Using a similar approach, we identified EM-90-like isolates in Atlantic salmon only (our unpublished data), suggesting a common genogroup. These isolates were found to be susceptible to quinolones presenting a particular gyrA genotype (8). Moreover, their mucoid texture is quite different from the “sticky” phenotype shown by LF-89-like colonies. In order to decipher the genetic basis of such phenotypic traits, we sequenced two P. salmonis isolates: A1-15972 (EM-90-like) and B1-32597 (LF-89-like), recovered in 2010 and 2012 from Atlantic and coho salmon, respectively. Sequencing was performed at Macrogen, Inc. (Seoul, South Korea) on the Illumina HiSeq 2000 platform. A total of 103,741,344 and 116,881,806 reads (100 bp) were de novo assembled using Platanus version 1.2.1 (9), RepARK version 1.2.2 (10), and Novoalign version 3.02 into the draft genomes of 3,138,697 bp (360 contigs; N₅₀, 26,215) and 3,461,332 bp (308 contigs; N₅₀, 28,873) for A1-15972 and B1-32597, respectively. The genomes had a similar G+C content of ~38.0%. The assembled data were annotated with Prodigal (11), Blast2GO (12), tRNAscan (13), and RNAmmer (14), which predicted 3,478 coding sequences (CDSs), 12 rRNAs, and 56 tRNAs for A1-15972, and 3,840 CDSs, 12 rRNAs, and 56 tRNAs for B1-32597. A comparison using MUMmer version 3.23 (15) and CLC Genomic Workbench Version 6.5.1. revealed 123,822 single nucleotide polymorphisms (SNPs) and 998 insertions/deletions (indels), as well as 247 specific genes for A1-15972 and 351 for B1-32597.

Type IV secretion systems (T4SSs) are well-known virulence-associated multiprotein complexes (16). Recently, the expression of T4SS-related genes was reported in LF-89 (17). Consistent with this previous work, three T4SSs were found in the B1-32597 genome. Remarkably, a comparative analysis performed with Mauve (18) disclosed that the A1-15972 genome contains two T4SSs, lacking ~30 kb that bear a complete T4SS in B1-32597. A ~20-kb indel encoding tra genes was detected in B1-32597 only. Such indels may be related to the narrow host range exhibited by A1-like isolates. Regarding the colony phenotype, gross differences can be linked to polymorphisms on lipopolysaccharide (LPS) genes, specifically those encoding the O antigen (19). Interestingly, four glycosyltransferase genes were deleted in A1-15972. Additionally, the A1-15972 genome harbors more LPS-related kinase genes than those found in B1-32597. Further functional characterization is required to prove these hypotheses.

The new sequences will allow a more comprehensive phylogenetic analysis of P. salmonis.

Nucleotide sequence accession numbers.

The sequences of A1-15972 and B1-32597 are part of a sequencing project, which has been deposited at DDBJ/EMBL/GenBank under the accession numbers JRAV00000000 and JRAD00000000, respectively. The versions described in this paper are the second versions, JRAV02000000 and JRAD02000000, respectively.