The inclusion membrane protein IncS is critical for initiation of the Chlamydia intracellular developmental cycle

Abstract

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.

Homologues of the Chlamydia trachomatis and Chlamydia muridarum Inclusion Membrane Protein IncS Are Interchangeable for Early Development but Not for Inclusion Stability in the Late Developmental Cycle

“…Analysis of the samples by mass spectrometry revealed the presence of the C. trachomatis inclusion membrane protein CTL0402 in the coimmunoprecipitate of the infected samples. We have recently renamed CTL0402 IncS Ct ( 29 ); this nomenclature will be used here. To confirm the inclusion localization of endogenous IncS Ct , HeLa cells infected with wild-type C. trachomatis for 24 h were fixed and stained with anti-IncS antibodies.…”

“…We next investigated whether IncS Ct was necessary for STIM1 association with C. trachomatis inclusion. However, we recently showed that a C. trachomatis incS Ct mutant does not progress through the early stages of the C. trachomatis developmental cycle ( 29 ). To bypass the early developmental defect of the incS Ct mutant, we capitalized on the fact that the severe growth defect of the C. trachomatis incS Ct mutant can be complemented by inducing incS Ct transcription during the first 6 to 8 h p.i.…”

“…To bypass the early developmental defect of the incS Ct mutant, we capitalized on the fact that the severe growth defect of the C. trachomatis incS Ct mutant can be complemented by inducing incS Ct transcription during the first 6 to 8 h p.i. of the developmental cycle ( 29 ). Under this condition, we found that IncS Ct is present at the inclusion 8 h p.i.…”

“…In the past years, the Fluorescence-Reported Allelic Exchange Mutagenesis (FRAEM)/FLAEM methodology has been instrumental in achieving targeted gene inactivation and cis complementation ( 33 – 35 ). Additionally, we recently reported a blueprint for the FRAEM-based generation of conditional mutants in essential genes, such as genes critical for progression through the early stages of the developmental cycle, like incS ( 29 ). We showed that induction of a complementation allele exclusively during the first 8 h of the developmental cycle was sufficient to complement the early developmental defect of the C. trachomatis incS Ct mutant ( 29 ).…”

“…Additionally, we recently reported a blueprint for the FRAEM-based generation of conditional mutants in essential genes, such as genes critical for progression through the early stages of the developmental cycle, like incS ( 29 ). We showed that induction of a complementation allele exclusively during the first 8 h of the developmental cycle was sufficient to complement the early developmental defect of the C. trachomatis incS Ct mutant ( 29 ). Here, we show that temporal complementation, exclusively in the early stages of the developmental cycle, rescued the early developmental defect of the C. trachomatis incS Ct mutant, while revealing and allowing us to investigate an additional role of IncS Ct during the late stages of the developmental cycle.…”