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.
All Chlamydia species are obligate intracellular bacteria that undergo a unique biphasic developmental cycle strictly in the lumen of a membrane bound compartment, the inclusion. Chlamydia specific Type III secreted effectors, known as inclusion membrane proteins (Inc), are embedded into the inclusion membrane. Progression through the developmental cycle, in particular early events of conversion from infectious (EB) to replicative (RB) bacteria, is important for intracellular replication, but poorly understood. Here, we identified the inclusion membrane protein IncS as a critical factor for Chlamydia development. We show that a C. trachomatis conditional mutant is impaired in transition from EB to RB in human cells, and C. muridarum mutant bacteria fail to develop in a mouse model of Chlamydia infection. Thus, IncS represents a promising target for therapeutic intervention of the leading cause of sexually transmitted infections of bacterial origin.
The obligate intracellular bacterium Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections. Once internalized in host cells, C. trachomatis undergoes a biphasic developmental cycle within a membrane-bound compartment, known as the inclusion. Successful establishment of the intracellular niche relies on bacterial Type III effector proteins, such as Inc proteins. In vitro and in vivo systems have contributed to elucidating the intracellular lifestyle of C. trachomatis, but additional models combining the archetypal environment of infection with the advantages of in vitro systems are needed. Organoids are three-dimensional structures that recapitulate the microanatomy of an organ's epithelial layer, bridging the gap between in vitro and in vivo systems. Organoids are emerging as relevant model systems to study interactions between bacterial pathogens and their hosts. Here, we took advantage of recently developed murine endometrial organoids (EMOs) and present a C. trachomatis-murine EMO infection model system. Confocal microscopy of EMOs infected with fluorescent protein-expressing bacteria revealed that inclusions are formed within the cytosol of epithelial cells. Moreover, infection with a C. trachomatis strain that allows for the tracking of RB to EB transition indicated that the bacteria undergo a full developmental cycle, which was confirmed by harvesting infectious bacteria from infected EMOs. Finally, the inducible gene expression and cellular localization of a Chlamydia Inc protein within infected EMOs further demonstrated that this model is compatible with the study of Type III secreted effectors. Altogether, we describe a novel and relevant system for the study of Chlamydia-host interactions.
The obligate intracellular bacterium
Chlamydia trachomatis
is the causative agent of the most frequently reported bacterial sexually transmitted disease. Upon internalization into host cells,
C. trachomatis
remains within a membrane-bound compartment known as an inclusion, where it undergoes its developmental cycle.
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