Forward and Reverse Genetic Analysis of Chlamydia

Abstract

Chlamydia is a major etiological agent of human disease that affects millions of individuals worldwide. Historically, our understanding of the mechanisms that contribute to its pathogenesis has been limited. However, the recent development of powerful genetic tools for manipulating Chlamydia has resulted in significant gains in our ability to dissect its virulence mechanisms. These tools have overcome several barriers for manipulating intracellular pathogens and are amenable for the routine genetic engineering of Chlamydia. Here, we provide several detailed protocols for performing genetic analysis in Chlamydia trachomatis allowing investigators to elucidate how this obligate intracellular pathogen causes disease.

The acetylase activity of Cdu1 regulates bacterial exit from infected cells by protectingChlamydiaeffectors from degradation

“…Ligations were transformed into E. coli DH5α, clones isolated, and cdu1 redirected pDFTT3-aadA plasmids identified by restriction digest and verified by Sanger sequencing (Eton Bioscience) using a T7-promoter specific primer. The resulting plasmid was transformed into a C. trachomatis LGV L2 434 Bu strain and transformants selected with 150 μg/mL spectinomycin and plaque purified as previously described (Kędzior and Bastidas., 2019). Insertion of the GII aadA intron at the cdu1 locus was verified by PCR analysis (S. Figure1) using primers that amplify amplicons spanning the cdu1 ::GII 5’ (RBP409 and RBP436) and 3’ (RBP468 and RBP118) junctions, the cdu1 CDS (RBP409 and RBP118), and the aadA CDS (RBP512 and RBP513).…”

“…pBOMB4-MCI based plasmids and p2TK2_SW2-InaC-3X Flag plasmids were transformed into corresponding Chlamydia strains, and transformants were selected with 10 U/mL Penicillin G and plaque purified as previously described (Kędzior and Bastidas., 2019). All primer sequences and plasmids generated in this study are listed in S. Table 10 and Key Resources Table.…”

The acetylase activity of Cdu1 regulates bacterial exit from infected cells by protecting Chlamydia effectors from degradation

“…Ligations were transformed into E. coli DH5α, clones isolated, and cdu1 redirected pDFTT3-aadA plasmids identified by restriction digest and verified by Sanger sequencing (Eton Bioscience) using a T7-promoter specific primer. The resulting plasmid was transformed into a C. trachomatis LGV L2 434 Bu strain and transformants selected with 150 μg/mL spectinomycin and plaque purified as previously described (Kędzior and Bastidas., 2019). Insertion of the GII aadA intron at the cdu1 locus was verified by PCR analysis (S. Figure 1) using primers that amplify amplicons spanning the cdu1::GII 5' (RBP409 and RBP436) and 3' (RBP468 and RBP118) junctions, the cdu1 CDS (RBP409 and RBP118), and the aadA CDS (RBP512 and RBP513).…”

Advances in genetic manipulation of Chlamydia trachomatis

“…Due to its advantages of simple, rapid, cheap and repeatable operation, the CaCl 2 transformation method is widely used as a general transformation method for Chlamydia. For the CaCl 2 transformation, crude preparations of Chlamydia from host cell lysates exhibit more efficient than gradient purified EB preparations (54)(55)(56). Based on this method, group II intronbased targeted gene knockout (21-30), fluorescence-reported allelic exchange mutagenesis (FRAEM) (31)(32)(33)(34)(35)(36)(37)(38), CRISPR interference (CRISPRi) (39-43) and transposon insertion mutagenesis (44)(45)(46) have been realized in C. trachomatis.…”