Floxed-Cassette Allelic Exchange Mutagenesis Enables Markerless Gene Deletion in Chlamydia trachomatis and Can Reverse Cassette-Induced Polar Effects

Abstract

As obligate intracellular bacteria, spp. have evolved numerous, likely intricate, mechanisms to create and maintain a privileged intracellular niche. Recent progress in elucidating and characterizing these processes has been bolstered by the development of techniques enabling basic genetic tractability. Florescence-reported allelic exchange mutagenesis (FRAEM) couples chromosomal gene deletion with the insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Similar to other bacteria, many chlamydial genes exist within polycistronic operons, raising the possibility of polar effects mediated by insertion cassettes. Indeed, FRAEM-mediated deletion of negatively impacts the expression of We have adapted FRAEM technology by employing a cassette flanked by sites. Conditional expression of Cre recombinase in containing a floxed cassette resulted in deletion of the marker and restoration of expression. infections represent a significant burden to human health. The ability to genetically manipulate spp. is overcoming historic confounding barriers that have impeded rapid progress in understanding overall chlamydial pathogenesis. The current state of genetic manipulation in spp. requires further development, including mechanisms to generate markerless gene disruption. We leveraged a stepwise Cre-lox approach to excise selection marker genes from a deleted gene locus. We found this process to be efficient, and the removal of extraneous elements resulted in the reversal of a negative polar effect on a downstream gene. This technique facilitates a more direct assessment of gene function and adds to the molecular toolbox by facilitating the deletion of genes within operons.

The Chlamydia trachomatis secreted effector TmeA hijacks the N-WASP-ARP2/3 actin remodeling axis to facilitate cellular invasion

“…Proteomic comparison of EBs and RBs has identified several effector proteins that are enriched in EBs and are presumed to be prepackaged at the end of the developmental cycle to initiate new rounds of infection [7]. While insertional inactivation of the early-stage effectors TepP [6,14] and TmeB [15] does not impair invasion, TmeA [15,16] and TarP [13] null mutants are significantly impaired in host cell invasion, highlighting these two effectors as key regulators of chlamydial entry. In this study, we sought to determine how TmeA promotes chlamydial invasion.…”

“…has been shown to be necessary for bacterial invasion and pathogenesis [15,16]; however the mechanisms utilized by TmeA to promote invasion are unknown. TmeA was previously shown to bind a large scaffolding protein, AHNAK, yet depletion of AHNAK does not impair bacterial invasion and AHNAK is still recruited to the entry site in the absence of TmeA [15,17].…”

Forward and Reverse Genetic Analysis of Chlamydia

“…A detailed protocol describing gene exchange by FRAEM in Chlamydia has previously been published [25] and will not be mentioned here. Null alleles within operons can be generated without incurring polar effects on downstream genes by utilizing a modified Cre-loxP FRAEMs approach [19]. Here, we describe a protocol for generating loss of function alleles in non-essential ORFs using a C. trachomatis adapted Group II intron (TargeTron system) [17].…”

“…Culture infected cell monolayers at 37 °C for 40-48 hours in a 5% CO 2 humidified incubator (see Note 21). 19. Chlamydia preparations from crude cell lysates of infected host cells exhibit higher transformation efficiencies relative to gradient purified EB preparations.…”

“…These include the use of a C. trachomatis adapted type II intron (TargeTron ® , Sigma Inc) for targeted insertional mutagenesis [16,17] and targeted gene replacement by Fluorescence Reported Allelic Exchange Mutagenesis (FRAEM) [18]. An upgrade to the FRAEM approach with a Cre-loxP system now allows for recycling of selectable markers during mutagenesis and disruption of individual genes within operons without incurring polar effects on transcription of downstream genes [19].…”