Development of shuttle vector-based transformation systems for veterinary and zoonotic chlamydiae

Area of Science:

• Veterinary microbiology
• Bacterial genetics
• Molecular biology

Background:

• Obligate intracellular bacteria Chlamydia abortus, Chlamydia caviae, and Chlamydia pecorum cause significant diseases in animals, including ovine enzootic abortion and ocular/urogenital infections.
• A lack of genetic tools has hindered the study of these veterinary Chlamydia species.
• These Chlamydia species are also zoonotic, posing risks to human health.

Purpose of the Study:

• To establish stable transformation systems for Chlamydia abortus, Chlamydia pecorum, and Chlamydia caviae.
• To compare the fluorescence intensity of Green Fluorescent Protein (GFP) and mNeonGreen for tracking Chlamydiae in vitro.
• To refine existing transformation protocols for improved efficiency.

Main Methods:

• Development and comparison of two transformation protocols (Protocol A and Protocol B) using calcium chloride for competence induction.
• Introduction of shuttle vectors containing species-specific plasmids, Escherichia coli origin of replication, antibiotic resistance genes (bla or aadA), and fluorescent proteins (GFP or mNeonGreen).
• Optimization of protocols, including pre-incubation with trypsin-EDTA for Chlamydia abortus.

Main Results:

• Stable transformants were successfully obtained for Chlamydia pecorum and Chlamydia caviae using optimized protocols.
• Heterologous expression of GFP exhibited higher fluorescence intensity compared to mNeonGreen.
• Pre-treatment with trypsin-EDTA was crucial for achieving transformation in Chlamydia abortus.

Conclusions:

• Established stable calcium chloride-mediated transformation protocols for Chlamydia pecorum and Chlamydia abortus.
• Expanded the genetic toolbox for Chlamydia caviae.
• Demonstrated the necessity of species-specific protocol refinement for efficient genetic transformation of Chlamydiae.