Both the N- and C- terminal regions of the Chlamydial inclusion protein D (IncD) are required for interaction with the pleckstrin homology domain of the ceramide transport protein CERT

Abstract

An obligate intracellular bacterium Chlamydia trachomatis requires host lipid ceramide for their replication within an intracellular membranous compartment, the inclusion. Chlamydial inclusion membrane protein D (IncD) composed of two closely linked long hydrophobic domains with their N- and C-termini exposed to the host cytosol, binds directly to the pleckstrin homology (PH) domain of the ceramide transport protein (CERT), likely redirecting ceramide to the inclusion. The precise regions of IncD required for this interaction have not been delineated. Using co-transfection studies together with phylogenetic studies, we demonstrate that both the IncD N- and C-terminal regions are required for binding to the CERT PH domain and define key interaction residues. Native gel electrophoresis analysis demonstrates that the transmembrane region of IncD forms SDS-resistant but dithiothreitol-sensitive homodimers, which in turn can assemble to form higher order oligomers through additional N- and C-terminal domain contacts. We propose that IncD oligomerization may facilitate high affinity binding to CERT, allowing C. trachomatis to efficiently redirect host ceramide to the inclusion.

Functions of Sphingolipids in Pathogenesis During Host–Pathogen Interactions

“…Moreover, the C. trachomatis inclusion membrane protein IncD specifically interacts with the PH (pleckstrin homology) domain of CERT, suggesting that IncD is involved in the recruitment of CERT to C. trachomatis inclusions. Co-transfection studies together with phylogenetic analysis revealed that both the N- and C-terminal regions of IncD are required for binding to the CERT PH domain, allowing C. trachomatis to redirect host cell-derived ceramides to inclusions more efficiently ( Kumagai et al, 2018 ). In cells depleted of CERT ( via short interfering RNAs and siRNAs), CERT was no longer detected on the surfaces of C. trachomatis inclusions, which resulted in smaller inclusions than cells treated with control siRNAs ( Derre et al, 2011 ; Elwell et al, 2011 ).…”

Structure, functions and regulation of CERT, a lipid‐transfer protein for the delivery of ceramide at the ER–Golgi membrane contact sites

“…When cells are infected with Chlamydia trachomatis , an obligate intracellular bacterium, CERT is hijacked by the bacterial protein IncD to deliver the host‐derived ceramide to the pathogen . PtdIns 4‐kinase IIα is also hijacked by C. trachomatis to modulate host trafficking by generating a PtdIns(4) pool in the inclusion membrane.…”

Chlamydia trachomatis‐infected human cells convert ceramide to sphingomyelin without sphingomyelin synthases 1 and 2

“…In addition, CERT is required for the proliferation of several Chlamydia species ( C. trachomatis , C. muridarum , and C. psittaci ). CERT is recruited to the inclusion membrane by binding to the inclusion membrane protein IncD , which is encoded by the C. trachomatis genome. In Chlamydia ‐infected cells, the Golgi apparatus is fragmented, and the resultant Golgi mini‐stacks are scattered around the inclusions .…”