Development of shuttle vector-based transformation systems forChlamydia pecorumandChlamydia caviae

Abstract

AbstractChlamydia (C.) abortus,C. caviaeandC. pecorumare obligate intracellular, zoonotic pathogens, which have all been associated with community-acquired pneumonia in humans.C. abortusis the causative agent of enzootic ovine abortion in small ruminants and can lead to miscarriage in women.C. caviaecauses conjunctivitis in guinea pigs, whileC. pecorumis found in livestock, resulting in economic losses and contributing to the decline of the koala population in Australia. Studying the biology of these bacteria has been challenging due to a dearth of genetic tools. This study aimed to establish transformation systems forC. abortusandC. pecorumusing shuttle vectors and to expand upon already existing protocols forC. caviae. Shuttle vectors comprised the cryptic plasmid of the chlamydial species of interest, the pUC19 origin of replication (ori), a beta-lactamase (bla), and genes that mediate heterologous expression of fluorescent proteins (GFP, mNeonGreen, mScarlet). AC. suis-tailored transformation protocol and a previously established protocol forC. psittaci, C. trachomatisandC. pneumoniaewere applied. WhileC. pecorumandC. caviaetransformation experiments were successful, transformation ofC. abortusremained ineffective. Shuttle vectors yielded stable transformants over several passages in the presence and absence of selective antibiotics while the fluorescence intensity of GFP was superior compared to mNeonGreen. Finally, we co-cultured GFP- and mScarlet-expressingC. pecorumstrains demonstrating that both fluorophores can be detected in the same cell or even inclusion, possibly promoting homologous recombination. These findings open new avenues into our understanding of interstrain and interspecies co-infection dynamics bothin vitroandin vivo.