Sequencing and characterization of Helcococcus ovis: a comprehensive comparative genomic analysis of virulence

Abstract

Background Helcococcus ovis (H. ovis) is an emerging bacterial pathogen that commonly causes opportunistic respiratory, mammary, and uterine infections across mammalian hosts. This study applied long- and short-read whole genome sequencing technologies to identify virulence factors in five H. ovis isolates with low, medium, and high virulence phenotypes. Results The resulting assemblies contained one circular chromosome ranging from 1,744,566 to 1,850,083 bp in length and had a mean GC content of 27.6%. Phylogenetic and nucleotide identity analyses found low virulence strain KG38 to be part of a clade that forms an outgroup apart from the rest of the H. ovis taxon. Assembling the first complete genomes of the species revealed major genomic rearrangements in KG38. One to six prophage regions were identified in each genome. A novel pathogenicity island was found exclusively in the two high virulence strains (KG37 and KG104), along with two hypothetical transmembrane proteins designated as putative VFs. Finally, three zinc ABC transporters and three Type-II/IV secretion systems were identified as possible virulence determinants in this species. The low virulence strain KG38 has fewer intact paralogs of these operons in its genome compared to the higher virulence isolates, which strongly suggests a role in virulence. This strain is also missing four putative virulence factors (VFs) found in other isolates associated with adherence (collagen adhesin precursor), immune evasion (choline-binding protein A and a PspA-like hypothetical protein) and cell wall synthesis (glycerol-3-phosphate cytidylyltransferase). Conclusions In this study, we assembled reference-quality complete genomes for five H. ovis strains to identify putative virulence factors. Phylogenetic analyses of H. ovis isolates revealed the presence of a clade representing a potentially novel species within the genus Helcococcus. A novel pathogenicity island and two hypothetical transmembrane proteins were found exclusively in high-virulence strains. The identification of Zinc ABC transporters and Type-II/IV secretion systems as possible virulence determinants, along with the differences in operon content between the low and high virulence isolates, strongly suggests they also play a role in the bacterium’s pathogenicity. Taken together, these findings are a valuable first step toward deciphering the pathogenesis of H. ovis infections.

Pangenomic analysis ofHelcococcus ovisreveals widespread tetracycline resistance and a novel bacterial species,Helcococcus bovis

“…This strain displayed an atypical biochemical profile for H. ovis and a 98.9% 16S rRNA sequence identity with the H. ovis type strain, which led researchers to question its place within the species taxon. Phylogenomic analyses have shown that, based on 16S rRNA sequence identity, the Tongji strain belongs to a subclade of the species also populated by H. ovis strain KG38 (15). Whole genome-based multi-locus phylogenomic analyses in this study confirmed these findings and identified three further strains (KG95, KG105, and KG197) belonging to the cryptic clade.…”

“…As shown in an invertebrate infection model, H. ovis strains originating from the uterus of dairy cows can display varying degrees of virulence (14). Based on the virulence phenotypes from that study, whole genome comparative analyses identified potential high virulence determinants in this organism, including Zinc ABC transporters, two hypothetical proteins, and a pathogenicity island (15). These comparative analyses created a blueprint for investigating H. ovis pathogenic capabilities, but the role these virulence factors may play in disease pathogenesis remains unexplored.…”

“…However, they were retained for other analyses since they can provide valuable taxonomic and gene presence information. Finally, we also included five complete H. ovis genomes (KG36, KG37, KG38, KG104, and KG106) from a previous study (15). Genome assembly statistics for all thirty-five genomes included in this study and their accession numbers are listed in Supplemental File 3.…”

“…In a recent study we identified a single putative H. ovis strain (KG38) whose average nucleotide identity (ANI) with other H. ovis strains is lower than the suggested 96% threshold for same species determination (15). Although the initial identification of H. ovis isolates for this experiment was conducted based on 16S rRNA sequence identity comparisons, 16S rRNA sequence variations are often not specific enough to discriminate between closely related species (19).…”

“…After using Abricate for mass screening of virulence factors in the 26 H. ovis genomes against the Virulence Factor Database, no positive hits were returned. To further investigate the virulome of 26 H. ovis strains in this study, we curated a set of 22 putative virulence factor genes based on previous comparative genome analyses (15). The resulting virulome is presented in Figure 8 as a heat map.…”