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

Abstract

Chlamydia trachomatis is an obligate intracellular pathogen that is the etiological agent of a variety of human diseases, including blinding trachoma and sexually transmitted infections. Chlamydiae replicate within a membrane-bound compartment, termed an inclusion, which they extensively modify by the insertion of type III secreted proteins called Inc proteins. IncA is an inclusion membrane protein that encodes two coiled-coil domains that are homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) motifs. Recent biochemical evidence suggests that a functional core, composed of SNARE-like domain 1 (SLD-1) and part of SNARE-like domain 2 (SLD-2), is required for the characteristic homotypic fusion of C. trachomatis inclusions in multiply infected cells. To verify the importance of IncA in homotypic fusion in Chlamydia, we generated an incA::bla mutant. Insertional inactivation of incA resulted in the formation of nonfusogenic inclusions, a phenotype that was completely rescued by complementation with full-length IncA. Rescue of homotypic inclusion fusion was dependent on the presence of the functional core consisting of SLD-1 and part of SLD-2. Collectively, these results confirm in vitro membrane fusion assays identifying functional domains of IncA and expand the genetic tools available for identification of chlamydia with a method for complementation of site-specific mutants.

Absence of Specific Chlamydia trachomatis Inclusion Membrane Proteins Triggers Premature Inclusion Membrane Lysis and Host Cell Death

“…Despite the growth defect noted for CT288:: bla (Figure 1A), inclusions were similar in size to wild-type (Figure 1B). Furthermore, the CT229:: bla , incC::bla , and CT383:: bla formed non-fusogenic inclusions, comparable to the phenotype associated with incA::bla (Johnson and Fisher, 2013; Weber et al, 2016). This defect in fusion appears to be independent of IncA as IncA expression and localization on the inclusion was not impaired.…”

“…TargeTron mutagenesis (Johnson and Fisher, 2013; Weber et al, 2016) was used to generate eleven unique Inc mutants. DNA sequencing and PCR was performed to confirm appropriate targeting (Figure S1A).…”

“…Using the primers listed in Table S1, the intron was retargeted, amplified using the Qiagen core PCR kit (Qiagen), and the resulting PCR products were cloned into the BsrGI/HindIII site of pACT (Weber et al, 2016). The integrity of all constructs was verified by sequencing.…”

“…C. trachomatis serovar L2 was transformed with each TargeTron construct as previously described (Weber et al, 2016). Genes were amplified with gene specific primers and sequenced to verify intron orientation.…”

“…Genes were amplified with gene specific primers and sequenced to verify intron orientation. Southern blots were conducted as previously described (Weber et al, 2016). To complement specific Inc mutants, Renografin purified mutants were transformed with the complementation vector as previously described (Weber et al, 2016) and complements were selected for with 250 μg/ml spectinomycin.…”