Use of aminoglycoside 3′ adenyltransferase as a selection marker for Chlamydia trachomatis intron-mutagenesis and in vivo intron stability

Abstract

BackgroundChlamydia spp. are obligate, intracellular bacteria that infect humans and animals. Research on these important pathogens has been hindered due to a paucity of genetic tools. We recently adapted a group II intron (GII) mutagenesis platform for creation of ampicillin-selectable gene insertions in C. trachomatis L2. The aims of this study were: (1) to assess the stability of the intron-insertion in an in vivo infection model to gauge the efficacy of this genetic tool for long term animal studies and (2) to expand upon the utility of the method by validating a second selection marker (aadA, conferring spectinomycin resistance) for mutant construction.ResultsIntron stability was assessed using a mouse vaginal tract infection model with a C. trachomatis L2 434/Bu incA::GII(bla) mutant. Infections were performed in the absence of selection and isolates shed into the vaginal tract were isolated and expanded in cell culture (also without selection). PCR and inclusion phenotype analysis indicated that the intron was stable for at least 27 days post-infection (at which point bacteria were no longer recovered from the mouse). The aminoglycoside 3′ adenyltransferase (aadA) gene was used to create a spectinomycin-selectable GII intron, facilitating the construction of an incA::GII[aadA] C. trachomatis L2 insertion mutant. Both the GII(aadA) intron and our previously reported GII(bla) intron were then used to create an incA::GII(aadA), rsbV1::GII(bla) double mutant. Mutants were confirmed via PCR, sequencing, inclusion morphology (incA only), and western blot.ConclusionsThe stability of the intron-insertion during in vivo growth indicates that the GII-insertion mutants can be used to study pathogenesis using the well-established mouse infection model. In addition, the validation of an additional marker for mutagenesis in Chlamydia allows for gene complementation approaches and construction of targeted, double mutants in Chlamydia. The aadA marker also could be useful for other genetic methods. Collectively, our results expand upon the rapidly growing chlamydial genetic toolkit and will aid in the implementation of studies dissecting the contribution of individual genes to infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1542-9) contains supplementary material, which is available to authorized users.

A Functional Core of IncA Is Required for Chlamydia trachomatis Inclusion Fusion

“…In 2011, Wang et al (25) described a system for plasmid transformation of C. trachomatis EBs using calcium chloride and penicillin selection. Since that seminal study, the genetic toolbox available for use in C. trachomatis studies has been expanded to include expression of a variety of fluorescent tags (14,31), epitope tags (14), inducible promoters (14), and alternative selectable markers (30,32). Additionally, dendrimers have been used to silence gene expression (33,34) and a library of random mutants has been generated using chemical mutagenesis (26,27).…”

“…Additionally, dendrimers have been used to silence gene expression (33,34) and a library of random mutants has been generated using chemical mutagenesis (26,27). Recently, a mobile group II intron was adapted for use in C. trachomatis that allows selectable insertional inactivation of target genes (28) and this technology has been extended to insertionally inactivate multiple genes (32). While complementation has been demonstrated for random chemical mutants, the ability to complement specific mutants harboring a selectable marker has been lacking.…”

Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

“…Furthermore, the higher MICs required for the delivery of these antibiotics into the inclusion can lead to toxicity in the host cell, which imposes further limits on the use of antibiotics for selection. Several antibiotics have been used successfully in genetic selections, including chloramphenicol, kasugamycin, nalidixic acid, rifampin, spectinomycin, trimethoprim, tetracycline (only for naturally resistant veterinary strains), ␤-lactams (only for LGV serovars), and blasticidin S (12,14,18,19,27,31,(50)(51)(52)(53). However, the use of mutant versions of chlamydial factors, such as 16S rRNA, RpoB, and GyrA, that render them resistant to antibiotics as selectable markers is limited because the gene mutations that confer resistance to these antibiotics are often recessive.…”

“…A second TargeTron vector (pDFTT3-aadA) (Fig. 3), carrying a spectinomycin resistance cassette (aadA), was also generated (53) and successfully adapted to generate a C. trachomatis strain bearing an inactivated rsbV1 gene (rsbV1::GII[aadA]), encoding the anti-anti-sigma factor RsbV1 (52). A similar approach also enabled the generation of a double mutant strain bearing loss-of-function alleles of incA and rsbV1 (incA::GII[aadA] and rsbV1::GII[bla]) (53).…”

“…3), carrying a spectinomycin resistance cassette (aadA), was also generated (53) and successfully adapted to generate a C. trachomatis strain bearing an inactivated rsbV1 gene (rsbV1::GII[aadA]), encoding the anti-anti-sigma factor RsbV1 (52). A similar approach also enabled the generation of a double mutant strain bearing loss-of-function alleles of incA and rsbV1 (incA::GII[aadA] and rsbV1::GII[bla]) (53). The pDFTT3-aadA TargeTron vector provides an additional selectable marker for use in inactivation of multiple genes in Chlamydia strains, especially for non-LGV serovars, and allows for the use of the aadA cassette for complementation studies.…”