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

In veterinary medicine, the obligate intracellular bacteria Chlamydia (C.) abortus, Chlamydia caviae, and Chlamydia pecorum are known to cause ovine enzootic abortion, conjunctivitis in guinea pigs, and ocular/urogenital disease in koalas, respectively. Studying the biology of these bacteria has been challenging due to a dearth of genetic tools. This study aimed to establish stable transformation systems for C. abortus, C. pecorum, and C. caviae by introducing shuttle vectors carrying green fluorescent proteins. With the aim to select the most suitable green fluorescent protein for the tracking of chlamydiae in vitro, we further compared the fluorescence intensity of GFP to that of mNeonGreen. Transformed shuttle vectors comprised the native plasmid of the chlamydial species of interest, an Escherichia coli origin of replication (ori), a beta-lactamase (bla) or spectinomycin (aadA) resistance gene, and GFP or mNeonGreen for heterologous fluorescence expression. We compared the success of a C. suis-tailored transformation protocol (Protocol A) to that of an alternative protocol for C. psittaci and C. trachomatis (Protocol B), both of which employ calcium chloride for competence induction. Stable transformants were obtained for C. pecorum and C. caviae using protocols A and B, respectively, and we found that the fluorescence intensity of heterologously expressed GFP is higher than that of mNeonGreen. In contrast, pre-incubation with trypsin-EDTA prior to the application of calcium chloride was needed to obtain transformants of C. abortus. In summary, we established protocols for stable calcium chloride-mediated transformation for C. pecorum and C. abortus and expanded upon the genetic toolbox of C. caviae.IMPORTANCEChlamydiae are a diverse group of bacteria impacting human and animal health. Many of the veterinary species, such as Chlamydia abortus, Chlamydia caviae, and Chlamydia pecorum, which cause reproductive disorders and/or conjunctivitis, are zoonotic pathogens leading to a potentially life-threatening disease in humans. Our understanding of these species has been hampered due to a lack of genetic tools. In this study, we developed calcium chloride-mediated transformation protocols for each of these species: chlamydiae are mixed with shuttle vectors containing the complete species-specific plasmid sequence, an Escherichia coli origin of replication, and an antibiotic resistance gene for selection. We could further show that certain chlamydial species become more susceptible to genetic modification if they are pre-treated with trypsin-EDTA prior to the addition of calcium chloride and the vector of interest. Overall, we demonstrate that species-specific protocol refinement is indispensable to render chlamydiae competent for genetic transformation.