An Autobioluminescent Method for Evaluating and Growth of Rhodococcus Equi

Overview

Journal Microbiol Spectr

Specialty Microbiology

Date 2022 May 31

PMID 35638814

Authors

Yasunori Suzuki

Naho Sakaizawa

Shinji Takai

Hiroaki Kubota

Noeru Hasegawa

Yukako Sasaki

Tsutomu Kakuda

Affiliations

Soon will be listed here.

Abstract

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 using luciferase (). First, we connected to the functional promoter P 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 is functionally and quantitatively expressed in . 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 being selected as candidates. Luminescence was detected in each transformant expressing the using these upstream sequences. We examined the luminescence intensity by coexpressing the gene, an enhancer of the luciferase reaction, with . The luminescence intensity of the coexpressing transformant was significantly enhanced in J774A.1 compared with the non-coexpressing transformant. Finally, we examined the luminescence . 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 and quantitative analysis of proliferation. We established an autologous bioluminescent strain of and a method to evaluate its proliferation and 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 and growth evaluation method with excellent quantitative properties. In addition, it was suggested that the selection of a promoter that expresses 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.

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