Development of a Rhodococcus equi-Escherichia coli plasmid shuttle vector

Area of Science:

• Microbiology
• Molecular Biology
• Veterinary Science

Background:

• Rhodococcus equi isolates from pneumonic foals carry virulence plasmids (85- or 90-kb).
• VapA, a thermoregulated surface antigen encoded by these plasmids, is crucial for R. equi virulence.
• Understanding R. equi virulence mechanisms is vital for foal health.

Purpose of the Study:

• To develop a stable and functional shuttle plasmid for Rhodococcus equi and Escherichia coli.
• To facilitate the study of R. equi virulence-associated genes, specifically VapA.
• To investigate the regulation of virulence gene expression in R. equi.

Main Methods:

• Identification and sequencing of a 135-kb fragment containing the origin of replication (ori) from the R. equi virulence plasmid pOTS.
• Construction of a simple R. equi-E. coli shuttle plasmid (pRE-1) using pACYC177 and the pOTS ori.
• Development of an improved shuttle vector (pRE-7) by combining pRE-1 with pBluescript.
• Subcloning of the vapA gene into pRE-7 for expression analysis in both R. equi and E. coli.

Main Results:

• A stable R. equi-E. coli shuttle plasmid (pRE-1) was successfully constructed and demonstrated stability in both hosts.
• The shuttle plasmid expressed kanamycin resistance in R. equi but not beta-lactamase.
• An improved vector, pRE-7, was created, enabling subcloning of virulence genes.
• Subcloning of vapA resulted in weak gene expression in both E. coli and R. equi, suggesting potential regulatory mechanisms.

Conclusions:

• The developed shuttle vectors (pRE-1 and pRE-7) are valuable tools for genetic manipulation in R. equi.
• These vectors can be utilized to study the regulation of virulence gene expression in R. equi.
• Further research using these vectors may elucidate the role of VapA and other virulence factors in R. equi pathogenesis.