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.

Chlamydia trachomatis, an obligate intracellular pathogen, grows inside of a vacuole, termed the inclusion. Within the inclusion, the organisms differentiate from the infectious elementary body (EB) into the reticulate body (RB). The RB communicates with the host cell through the inclusion membrane to obtain the nutrients necessary to divide, thus expanding the chlamydial population. At late time points within the developmental cycle, the RBs respond to unknown molecular signals to redifferentiate into infectious EBs to perpetuate the infection cycle. One strategy for Chlamydia to obtain necessary nutrients and metabolites from the host is to intercept host vesicular trafficking pathways. In this study we demonstrate that a trans-Golgi soluble N-ethylmaleimide–sensitive factor attachment protein (SNARE), syntaxin 10, and/or syntaxin 10-associated Golgi elements colocalize with the chlamydial inclusion. We hypothesized that Chlamydia utilizes the molecular machinery of syntaxin 10 at the inclusion membrane to intercept specific vesicular trafficking pathways in order to create and maintain an optimal intra-inclusion environment. To test this hypothesis, we used siRNA knockdown of syntaxin 10 to examine the impact of the loss of syntaxin 10 on chlamydial growth and development. Our results demonstrate that loss of syntaxin 10 leads to defects in normal chlamydial maturation including: variable inclusion size with fewer chlamydial organisms per inclusion, fewer infectious progeny, and delayed or halted RB-EB differentiation. These defects in chlamydial development correlate with an overabundance of NBD-lipid retained by inclusions cultured in syntaxin 10 knockdown cells. Overall, loss of syntaxin 10 at the inclusion membrane negatively affects Chlamydia. Understanding host machinery involved in maintaining an optimal inclusion environment to support chlamydial growth and development is critical toward understanding the molecular signals involved in successful progression through the chlamydial developmental cycle.

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