An Autobioluminescent Method for Evaluating In Vitro and In Vivo Growth of Rhodococcus equi.

A previously reported method for evaluating the intracellular growth of Rhodococcus equi using enhanced green fluorescent protein is unsuitable for the quantitative evaluation of the entire sample because the signal can be detected only in the excitation region. Therefore, we created an autobioluminescent R. equi using luciferase (luxABCDE). First, we connected luxABCDE to the functional promoter PaphII and introduced it into the chromosomes of ATCC33701 and ATCC33701_P-. Luminescence was detected in both transformants, and a correlation between the bacterial number and luminescence intensity in the logarithmic phase was observed, indicating that luxABCDE is functionally and quantitatively expressed in R. equi. The luminescence of ATCC33701 was significantly higher than that of ATCC33701_P- at 24 h after infection with J774A.1. Next, RNA-Seq analysis of ATCC33701 to search for endogenous high-expression promoters resulted in the upstream sequences of RS29370, RS41760, and vapA being selected as candidates. Luminescence was detected in each transformant expressing the luxABCDE using these upstream sequences. We examined the luminescence intensity by coexpressing the frp gene, an enhancer of the luciferase reaction, with luxABCDE. The luminescence intensity of the coexpressing transformant was significantly enhanced in J774A.1 compared with the non-coexpressing transformant. Finally, we examined the luminescence in vivo. The luminescence signals in the organs peaked on the third day following the administration of ATCC33701 derivatives in mice, but no luminescence signal was detected when the ATCC33701_P- derivative was administered. The autologous bioluminescent method described herein will enhance the in vitro and in vivo quantitative analysis of R. equi proliferation. IMPORTANCE We established an autologous bioluminescent strain of R. equi and a method to evaluate its proliferation in vitro and in vivo quantitatively. This method overcomes the weakness of the fluorescence detection system that only measures the site of excitation light irradiation. It is expected to be used as an in vitro and in vivo growth evaluation method with excellent quantitative properties. In addition, it was suggested that the selection of a promoter that expresses luxABCDE could produce a luminescence with high intensity. Although this method needs further improvement, such as creating transformants that can maintain high luminescence intensity regardless of environmental changes such as temperature fluctuations, it is possible to observe bacterial growth over time in mice without killing them. Therefore, this method can be used to not only evaluate the pathogenicity of various wild and gene-deficient strains but also to screen preventive and therapeutic methods such as vaccines.

Rhodococcus equiU19 strain harbors a nonmobilizable virulence plasmid.

Rhodococcus equiis the causative agent of pyogenic pneumonia in foals, and a virulence-associated protein A (VapA) encoded on the pVAPA virulence plasmid is important for its pathogenicity. In this study, we analyzed the virulence of R. equi strain U19, originally isolated in the Netherlands in 1997 and the genetic characteristics of the pVAPA_U19 plasmid. U19 expressed VapA that was regulated by temperature and pH and underwent significant intracellular proliferation in macrophages. The restriction fragment length polymorphism of pVAPA_U19 digested with EcoRI was similar to that of pREAT701 (85 kb Type I) harbored by R. equi ATCC33701, although the band pattern at 10-20 kb differed. Whole-genome sequencing showed that pVAPA_U19 was 51,684 bp in length and that the vapA pathogenicity island region and the replication/participation were almost identical to those in pREAT701. By contrast, the open reading frames (ORF26-ORF45) genes of pREAT701 (approximately 29,000 bp) were absent from pVAPA_U19. In this lacking region, mobility (MOB) genes, such as relaxase, which allow conjugative DNA processing, and the mating pair formation (MPF) genes, which are a form of the Type IV secretion system and provide the mating channel, were present. Coculture between U19 and five different recipient strains (two plasmid-cured strains and three cryptic plasmid-harboring strains) demonstrated that pVAPA_U19 could not support conjugation. Therefore, pVAPA_U19 does not differ significantly from the previously reported pVAPA in terms of virulence and plasmid replication and maintenance but is a nonmobilizable plasmid unable to cause conjugation because of the absence of genes related to MOB and MPF.