Chlamydia trachomatis and Chlamydia muridarum spectinomycin resistant vectors and a transcriptional fluorescent reporter to monitor conversion from replicative to infectious bacteria

Abstract

Chlamydia trachomatis infections are the leading cause of sexually transmitted infections of bacterial origin. Lower genital tract infections are often asymptomatic, and therefore left untreated, leading to ascending infections that have long-term consequences on female reproductive health. Human pathology can be recapitulated in mice with the mouse adapted strain C . muridarum . Eight years into the post-genetic era, significant advances to expand the Chlamydia genetic toolbox have been made to facilitate the study of this important human pathogen. However, the need for additional tools remains, especially for C . muridarum . Here, we describe a new set of spectinomycin resistant E . coli-Chlamydia shuttle vectors, for C . trachomatis and C . muridarum . These versatile vectors allow for expression and localization studies of Chlamydia effectors, such as Inc proteins, and will be instrumental for mutant complementation studies. In addition, we have exploited the differential expression of specific Chlamydia genes during the developmental cycle to engineer an omcA :: gfp fluorescent transcriptional reporter. This novel tool allows for monitoring RB to EB conversion at the bacterial level. Spatiotemporal tracking of GFP expression within individual inclusions revealed that RB to EB conversion initiates in bacteria located at the edge of the inclusion and correlates with the time post initiation of bacterial replication and inclusion size. Comparison between primary and secondary inclusions potentially suggests that the environment in which the inclusions develop influences the timing of conversion. Altogether, the Chlamydia genetic tools described here will benefit the field, as we continue to investigate the molecular mechanisms underlying Chlamydia -host interaction and pathogenesis.

Reprogramming of host glutamine metabolism during Chlamydia trachomatis infection and its key role in peptidoglycan synthesis

“…5e). To investigate if Gln plays a role in EB to RB conversion in host cells, we used the EB-RB reporter strain Ct mCh(GroL2) GFP(OmcAL2) 23 . In the absence of Gln, Chlamydia, did not convert into the actively replicating RB form, with low GFP expression and constant mCherry fluorescence (Fig.…”

Detection of Chlamydia Developmental Forms and Secreted Effectors by Expansion Microscopy

“…EBs are more commonly found at later stages of infection (30 h) in wildtype Chlamydia (Figure 1; Movie S4). To confirm the distinguishability of EBs and RBs by size we made use of a recently developed Chlamydia strain Ct mCh(GroL2) GFP(OmcAL2) expressing mCherry under the control of the constitutive groESL operon promoter and GFP fused to the omc promotor (expressed in EBs; Cortina et al, 2019). Confocal images revealed the different chlamydial forms by fluorescence protein expression but differences in sizes are barely detectable.…”

“…They were grown in a humidified atmosphere containing 5% (v/v) CO 2 at 37°C. In this study C. trachomatis serovar L2/434/Bu (ATCC VR-902B tm ) and C. trachomatis mutant strains [cdu1:: Tn bla , CPAF (RSTE4 from Raphael Valdivia)] and the EB-RB reporter strain Ct mCh(GroL2) GFP(OmcAL2) (Cortina et al, 2019) were used. They were cultured and purified as previously described: Chlamydia were propagated in HeLa 229 cells at a multiplicity of infection (MOI) of 1 for 48 h. Afterwards the cells were detached and lysed using glass beads (3 mm, Roth).…”

Murine Endometrial Organoids to Model Chlamydia Infection

“…To determine if C. trachomatis underwent a complete developmental cycle within EMOs, we used our fluorescent reporter strain of C. trachomatis that allows for tracking of the RB to EB transition (Cortina et al, 2019 ). This strain expresses the mCherry fluorescent protein under the control of the groESL promoter, resulting in constitutive expression throughout the developmental cycle.…”

“…C. trachomatis Lymphogranuloma venereum, Type II were obtained from ATCC (L2/434/Bu VR-902B). C. trachomatis strain expressing mCherry alone and the RB-to-EB transition fluorescent reporter strain were described previously (Cortina et al, 2019 ). The C. trachomatis strain expressing mCherry constitutively and IncV-3xFLAG under the control of the anhydrotetracycline inducible promoter was described previously (Stanhope et al, 2017 ).…”