Vesicle-Associated Membrane Protein 4 and Syntaxin 6 Interactions at the Chlamydial Inclusion

Abstract

The predominant players in membrane fusion events are the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins. We hypothesize that SNARE proteins mediate fusion events at the chlamydial inclusion and are important for chlamydial lipid acquisition. We have previously demonstrated that trans-Golgi SNARE syntaxin 6 localizes to the chlamydial inclusion. To investigate the role of syntaxin 6 at the chlamydial inclusion, we examined the localization and function of another trans-Golgi SNARE and syntaxin 6-binding partner, vesicle-associated membrane protein 4 (VAMP4), at the chlamydial inclusion. In this study, we demonstrate that syntaxin 6 and VAMP4 colocalize to the chlamydial inclusion and interact at the chlamydial inclusion. Furthermore, in the absence of VAMP4, syntaxin 6 is not retained at the chlamydial inclusion. Small interfering RNA (siRNA) knockdown of VAMP4 inhibited chlamydial sphingomyelin acquisition, correlating with a log decrease in infectious progeny. VAMP4 retention at the inclusion was shown to be dependent on de novo chlamydial protein synthesis, but unlike syntaxin 6, VAMP4 recruitment is observed in a species-dependent manner. Notably, VAMP4 knockdown inhibits sphingomyelin trafficking only to inclusions in which it localizes. These data support the hypothesis that VAMP proteins play a central role in mediating eukaryotic vesicular interactions at the chlamydial inclusion and, thus, support chlamydial lipid acquisition and chlamydial development.

The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components

“…Interestingly, the localization of Stx6 to the inclusion has also been documented using microscopy and lack of Stx6 slightly but significantly reduced C . trachomatis infectious progeny [ 53 , 54 ]. Whether the strong inhibitory effect of Retro-2 treatment on C .…”

Chlamydia cell biology and pathogenesis

“…These include the trans -Golgi SNARE proteins syntaxin 6 (STX6) 58,59 and STX10 (REF. 60), vesicle-associated membrane protein 4 (VAMP4), a STX6 binding partner 61 , and GS15 (also known as BET1L) 62 , which regulates the acquisition of nutrients from the Golgi exocytic pathway. The recruitment of STX6 requires a Golgi-targeting signal (YGRL) and VAMP4 (REFS 59,61).…”

“…60), vesicle-associated membrane protein 4 (VAMP4), a STX6 binding partner 61 , and GS15 (also known as BET1L) 62 , which regulates the acquisition of nutrients from the Golgi exocytic pathway. The recruitment of STX6 requires a Golgi-targeting signal (YGRL) and VAMP4 (REFS 59,61). In an example of molecular mimicry, at least three Incs contain SNARE-like motifs (IncA (also known as CT119), InaC (also known as CT813) and inclusion protein acting on microtubules (IPAM; also known as CT223)), which act as inhibitory SNARE proteins to limit fusion with compartments that contain VAMP3, VAMP7 or VAMP8 (REFS 49,63,64).…”

“…Host proteins that are implicated in this vesicle-dependent acquisition include ARF GTPases 57,72,73 , the ARF guanine nucleotide exchange factor GBF1 (REFS 72,73), RAB GTPases (specifically, RAB6, RAB11, RAB14 and RAB39) 53,54 , RAB11FIP2 (REF. 55), VAMP4 (REF. 61), dynamin 71 and FYN kinase 21 .…”

Safe haven under constant attack-TheChlamydia-containing vacuole

“…Moreover, Chlamydia promote fusion of the inclusion with nutrient‐rich vesicles of the exocytic pathway to obtain lipids and other metabolites from the host cell (Carabeo, Mead, & Hackstadt, ; Hackstadt, Rockey, Heinzen, & Scidmore, ). This selective vesicle fusion is achieved by recruitment of specific members of Rab GTPases, phosphoinositide lipid kinases, and SNARE proteins with the help bacterial effector proteins present on the inclusion membrane—the so‐called Inc proteins (Damiani, Gambarte Tudela, & Capmany, ; Kabeiseman, Cichos, Hackstadt, Lucas, & Moore, ; Lucas, Ouellette, Kabeiseman, Cichos, & Rucks, ; Moorhead, Jung, Smirnov, Kaufer, & Scidmore, ). Inc proteins are secreted by a type 3 secretion system and are not well conserved between the individual Chlamydia species; however, all members of the Inc protein family share a bilobed hydrophobic domain composed of at least two transmembrane domains connected with a short hairpin loop (Bannantine, Griffiths, Viratyosin, Brown, & Rockey, ; Dehoux, Flores, Dauga, Zhong, & Subtil, ; Lutter, Martens, & Hackstadt, ).…”