The Chlamydia trachomatis IncA protein is required for homotypic vesicle fusion

Abstract

Chlamydiae replicate within an intracellular vacuole, termed an inclusion, that is non‐fusogenic with vesicles of the endosomal or lysosomal compartments. Instead, the inclusion appears to intersect an exocytic pathway from which chlamydiae intercept sphingomyelin en route from the Golgi apparatus to the plasma membrane. Chlamydial protein synthesis is required to establish this interaction. In an effort to identify those chlamydial proteins controlling vesicle fusion, we have prepared polyclonal antibodies against several Chlamydia trachomatis inclusion membrane proteins. Microinjection of polyclonal antibodies against three C. trachomatis inclusion membrane proteins, IncA, F and G, into the cytosol of cells infected with C. trachomatis demonstrates reactivity with antigens on the cytoplasmic face of the inclusion membrane, without apparent inhibition of chlamydial multiplication. Microinjection of antibodies against the C. trachomatis IncA protein, however, results in the development of an aberrant multilobed inclusion structure remarkably similar to that of C. psittaci GPIC. These results suggest that the C. trachomatis IncA protein is involved in homotypic vesicle fusion and/or septation of the inclusion membrane that is believed to accompany bacterial cell division in C. psittaci. This proposal is corroborated by the expression of C. trachomatis and C. psittaci IncA in a yeast two‐hybrid system to demonstrate C. trachomatis, but not C. psittaci, IncA interactions. Despite the inhibition of homotypic fusion of C. trachomatis inclusions, fusion of sphingomyelin‐containing vesicles with the inclusion was not suppressed.

Biochemical and Localization Analyses of Putative Type III Secretion Translocator Proteins CopB and CopB2 of Chlamydia trachomatis Reveal Significant Distinctions

“…We regard this as unlikely, given that midcycle T3S effectors such as IncA and CT847 are secreted during this time. Both IncA-mediated homotypic fusion of chlamydial inclusions (20) and degradation of the CT847 target protein GCIP (13) are detectible by 12 h postinfection. Alternatively, a low-abundance pool of CopB could be present in the inclusion membrane during this time.…”

Chlamydial Intracellular Survival Strategies

“…However, in vitro liposome fusion assays indicate that instead of promoting fusion with endocytic compartments, IncA acts as an inhibitory SNARE to limit fusion with these compartments (Paumet et al 2009). IncA has also been shown to participate in homotypic fusion of individual inclusions, as naturally occurring IncA-deficient variants or infected cells microinjected with anti-IncA antibodies display an aberrant multilobed inclusion structure (Hackstadt et al 1999;Delevoye et al 2004;Xia et al 2005).…”

A small-molecule inhibitor of type III secretion inhibits different stages of the infectious cycle of Chlamydia trachomatis

“…IncA transcript can be detected 10-12 h p.i. and continue to be produced during the mid and late phase of the infectious cycle (13). We therefore infected McCoy cells with C. trachomatis at a MOI of 5 and treated the cells with INP0400 at 50 M after 8 h of infection.…”

“…Little is known about the function of these early-phase proteins that are displayed at the interface of intravacuolar Chlamydia and host cell. IncA, a protein induced and secreted during chlamydial mid-cycle, has been demonstrated to be involved in homotypic fusion between Chlamydia-containing vesicles at high multiplicities of infection (13). In addition to the family of Inc-proteins, other chlamydial proteins secreted into the inclusion membrane have been identified (14,15).…”