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

Abstract

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.

Keywords: Chlamydiaceae; chlamydia; competence; horizontal gene transfer; plasmid; transformation rate; zoonoses.

Fig 1

Transformation of C. pecorum strains with shuttle vector pUC-Cpecpl-GFP. (A) Map of the shuttle vector pUC-Cpecpl-GFP comprising the whole plasmid of C. pecorum strain W73 (labeled in light brown; coding sequences CDS 1–8 labeled in yellow) and pUC-GFP consisting of a beta-lactamase (bla, pale red), the pUC origin of replication (ori, pale blue), and GFP (neon-green). Image was created with Geneious version 2025.0 by Biomatters. Available from https://www.geneious.com. (B) Representative epifluorescence microscopy images of C. pecorum ovine P787 and IPA, chamois PV7855, and koala MC/MarsBar_2018 strains successfully transformed with pUC-Cpecpl-GFP at the time of collection after three to four passages. Images for DAPI (blue) and GFP (green, FITC channel) were taken individually using a 60× objective, and then merged. The white size bar represents 20 µm.

Fig 2

GFP-carrying shuttle vectors yield transformants with higher fluorescence intensities than shuttle vectors with mNeonGreen carrying the same promoter. (A) Representative epifluorescence microscopy images of C. pecorum strains transformed with a shuttle vector containing the C. pecorum W73 whole-plasmid sequence and mNeonGreen. Images for DAPI (blue) and mNeonGreen (green, FITC channel) were taken individually using a 60× objective and merged. The white size bar represents 20 µm. (B) The mean fluorescence intensity of 30 C. pecorum inclusions expressing NeonGreen was compared to that of GFP using strain P787. A Mann-Whitney test was used for the statistical analysis. Four asterisks (****) represent P-values < 0.0001. Representative images for each fluorophore were taken with a 40× objective (top right: GFP, bottom right: mNeonGreen). The white size bar represents 50 µm.

Fig 3

Transformation of C. caviae with shuttle vector pUC-Ccavpl-GFP. The left panel displays the vector map of the shuttle vector pUC-Ccavpl-GFP comprising the whole plasmid of C. caviae strain GPIC (labeled in light brown; coding sequences CDS 1-8 labeled in yellow). The shuttle vector possesses pUC-GFP, which contains a beta-lactamase (bla, pale red), the pUC origin of replication (ori, pale blue), and GFP (neon green). The image was created with Geneious version 2025.0 by Biomatters. Available from https://www.geneious.com. The right panel shows a representative epifluorescence microscopy image of successfully transformed C. caviae strain GPIC. Images for DAPI (blue) and GFP (green, FITC channel) were taken individually using a 60× objective and merged. The white size bar represents 50 µm.

Fig 4

Transformation of C. abortus with the shuttle vector pUC-Cabpl-GFP_aadA. The top panel shows the shuttle vector map of pUC-Cabpl-GFP_aadA comprising the whole plasmid of C. abortus strain 15-70d24 (labeled in light brown; coding sequences CDS 1-8 labeled in yellow) and the aadA-GFP backbone (dark green) comprising spectinomycin resistance (aadA, pale red), an ori (pale blue), and GFP (neon-green). The image was created with Geneious version 2025.0 by Biomatters. Available from https://www.geneious.com. The bottom panel displays representative epifluorescence microscopy images of successfully transformed C. abortus strains 15-70d24 (avian, top) and S26/3 (ruminant, bottom). Images for DAPI (blue) and GFP (green, FITC channel) were taken individually using a 100× oil objective and merged. The white size bar represents 10 µm.