Culture‐independent genomics of a novel chlamydial pathogen of fish provides new insight into host‐specific adaptations utilized by these intracellular bacteria

Abstract

Several Chlamydiales families are associated with epitheliocystis, a common condition of the fish gill epithelium. These families share common ancestors with the Chlamydiaceae and environmental Chlamydiae. Due to the lack of culture systems, little is known about the biology of these chlamydial fish pathogens. We investigated epitheliocystis in cultured Orange-spotted grouper (Epinephelus coioides) from North Queensland, Australia. Basophilic inclusions were present in the gills of 22/31 fish and the presence of the chlamydial pathogen in the cysts was confirmed by in situ hybridization. Giant grouper (Epinephelus lanceolatus) cultured in the same systems were epitheliocystis free. 16S rRNA gene sequencing revealed a novel member of the Candidatus Parilichlamydiaceae: Ca. Similichlamydia epinephelii. Using metagenomic approaches, we obtained an estimated 68% of the chlamydial genome, revealing that this novel chlamydial pathogen shares a number of key pathogenic hallmarks with the Chlamydiaceae, including an intact Type III Secretion system and several chlamydial virulence factors. This provides additional evidence that these pathogenic mechanisms were acquired early in the evolution of this unique bacterial phylum. The identification and genomic characterization of Ca. S. epinephelii provides new opportunities to study the biology of distantly-related chlamydial pathogens while shining a new light on the evolution of pathogenicity of the Chlamydiaceae.

Epitheliocystis in fish: An emerging aquaculture disease with a global impact

“…Molecular methods (e.g. sequencing and culture‐independent genomics) eventually supported this thesis, confirming the involvement of novel species belonging to a diversity of chlamydial families (Draghi et al., ; Taylor‐Brown et al., ). Subsequent studies have exposed a more complex aetiology, however, including the presence of γ‐ (Mendoza et al., ) and β‐proteobacteria (Mitchell et al., ; Toenshoff et al., ), and mixed infections (Guevara Soto, Vidondo et al., ; Schmidt‐Posthaus et al., ; Seth‐Smith et al., ; Wiik‐Nielsen et al., ).…”

“…In‐situ hybridization (ISH), where a labelled probe binds to the DNA or RNA in the cyst, thereby associating the PCR result with the cysts themselves, has also emerged as an important tool for identification and confirmation of epitheliocystis agents. Several authors have demonstrated the presence of chlamydial (Draghi et al., ; Karlsen et al., ; Nylund et al., ; Steigen et al., ; Stride, Polkinghorne, Powell et al., ; Taylor‐Brown et al., ), β‐proteobacterial (Mitchell et al., ; Toenshoff et al., ) and γ‐proteobacterial (Mendoza et al., ) DNA or RNA in the cysts in fish affected by epitheliocystis. ISH is particularly useful to confirm the infectious agent in cases of mixed infection (Katharios et al., ; Seth‐Smith et al., ; Toenshoff et al., ), with cysts belonging to different epitheliocystis agents occurring on the same gill lamellae.…”

“…The authors used a culture‐independent method whereby genomic DNA from microdissected cysts was subject to DNA treatment to enrich the microbial DNA prior to genome sequencing. The method has since been applied to novel β‐proteobacterial and chlamydial epitheliocystis agents (Qi et al., ; Seth‐Smith et al., ; Taylor‐Brown et al., ). These studies have revealed a breadth of biological diversity among these species which have converged to cause the same disease, albeit in separate host species.…”

“…An example of this is the recent detection and description of a chlamydial agent of epitheliocystis in farmed orange‐spotted grouper ( Epinephelus coioides ) and giant grouper ( Epinephelus lanceolatus ), farmed within the same systems. The giant grouper was free of epitheliocystis, while the orange‐spotted grouper had visible cysts in the gills that were positive by PCR and ISH for a novel chlamydial epitheliocystis agent (Taylor‐Brown et al., ).…”

“…Subsequent studies have exposed a more complex aetiology, however, including the presence of γ‐ (Mendoza et al., ) and β‐proteobacteria (Mitchell et al., ; Toenshoff et al., ), and mixed infections (Guevara Soto, Vidondo et al., ; Schmidt‐Posthaus et al., ; Seth‐Smith et al., ; Wiik‐Nielsen et al., ). Genomic characterization of these disease agents is helping researchers to unravel the biology of these bacteria, implying significant reliance on the host for basic metabolic activities (Katharios et al., ; Qi, Vaughan, Katharios, Schlapbach, & Seth‐Smith, ; Seth‐Smith et al., ; Taylor‐Brown et al., ). While several reviews have described the pathology associated with epitheliocystis (Nowak & LaPatra, ), in reference to chlamydial agents of epitheliocystis (Stride et al., ), to the best of our knowledge, no review exists has taking a broader perspective that includes the aetiological agents and multiple facets (e.g.…”