Complete, closed and curated genome sequences of Photobacterium damselae subsp. piscicida isolates from Australia indicate mobilome-driven localized evolution and novel pathogenicity determinants

Abstract

Despite the recent advances in sequencing technologies, the complete assembly of multi-chromosome genomes of the Vibrionaceae , often containing several plasmids, remains challenging. Using a combination of Oxford Nanopore MinION long reads and short Illumina reads, we fully sequenced, closed and curated the genomes of two strains of a primary aquatic pathogen Photobacterium damselae subsp. piscicida isolated in Australia. These are also the first genome sequences of P. damselae subsp. piscicida isolated in Oceania and, to our knowledge, in the Southern hemisphere. We also investigated the phylogenetic relationships between Australian and overseas isolates, revealing that Australian P. damselae subsp. piscicida are more closely related to the Asian and American strains rather than to the European ones. We investigated the mobilome and present new evidence showing that a host specialization process and progressive adaptive evolution to fish are ongoing in P. damselae subsp. piscicida , and are largely mediated by transposable elements, predominantly in chromosome 2, and by plasmids. Finally, we identified two novel potential virulence determinants in P. damselae subsp. piscicida – a chorismate mutase gene, which is ubiquitously retained and co-localized with the AIP56 apoptogenic toxin-encoding gene on the pPHDP10 plasmid, and transfer-messenger RNA gene ssrA located on the main chromosome, homologous to a critical-to-virulence determinant in Yersinia pseudotuberculosis . Our study describes, to our knowledge, the only fully closed and manually curated genomes of P. damselae subsp. piscicida available to date, offering new insights into this important fish pathogen and its evolution.

Susceptibility of Sea Bream (Sparus aurata) to AIP56, an AB-Type Toxin Secreted by Photobacterium damselae subsp. piscicida

“…However, the screening of 49 Phdp isolates recovered between 2014 and 2020 from diseased sea bream and sea bass in different European countries showed a 100% prevalence of the AIP56-encoding gene. This result, which is in agreement with previous studies reporting a ubiquitous distribution of the aip56 gene in Phdp strains recovered worldwide from different fish species in the last 30 years [18,29,30], suggests the existence of selective pressure to maintain the toxin gene and supports the hypothesis that in the majority of hosts the toxin may be required for virulence. However, it cannot be excluded that, in some hosts, AIP56 may have a less important contribution for virulence and that other virulence determinants, so far unidentified, may assume a more prominent virulence role.…”

The Evolution of a Specialized, Highly Virulent Fish Pathogen through Gene Loss and Acquisition of Host-Specific Survival Mechanisms

“…Within the Pdp clade, geographical endemism clearly separates European from Asian and Australian isolates, as previously described ( 33 ). The Pdp clade is nested within one of the Pdd groups, indicating that some Pdd strains are more closely related to Pdp than to other Pdd.…”

“…However, it was not until 1990, when Pdp mortality events were first recorded in Europe ( 30 ), that these two plasmids were identified together ( 31 , 32 ). Other potential virulence factors have been proposed, but no functional studies have been conducted to date to assess their role ( 30 , 33 – 35 ). Similar to other host-adapted pathogens, the Pdp genome is reduced in size and functional gene content compared to Pdd ( 33 ).…”

“…Other potential virulence factors have been proposed, but no functional studies have been conducted to date to assess their role ( 30 , 33 – 35 ). Similar to other host-adapted pathogens, the Pdp genome is reduced in size and functional gene content compared to Pdd ( 33 ). An extensive proliferation of insertion sequence elements has led to a generalized pseudogenization, reduced metabolic plasticity and genomic streamlining ( 36 ).…”

“…However, it is still unclear how Pdp persists within the highly oxidative environment of macrophages. Furthermore, little is known to date about Pdp route of transmission and the existence of potential reservoirs or carriers ( 33 , 41 ).…”