Turnover of Variant Surface Glycoprotein in Trypanosoma brucei Is Not Altered in Response to Specific Silencing.

African trypanosomes evade the immune system of the mammalian host by the antigenic variation of the predominant glycosylphosphatidylinositol (GPI)-anchored surface protein, variant surface glycoprotein (VSG). VSG is a very stable protein that is turned over from the cell surface with a long half-life (~26 h), allowing newly synthesized VSG to populate the surface. We have recently demonstrated that VSG turnover under normal growth is mediated by a combination of GPI hydrolysis and direct shedding with intact GPI anchors. VSG synthesis is tightly regulated in dividing trypanosomes, and when subjected to RNA interference (RNAi) silencing, cells display rapid cell cycle arrest in order to conserve VSG density on the cell surface (K. Sheader, S. Vaughan, J. Minchin, K. Hughes, et al., Proc Natl Acad Sci U S A 102:8716-8721, 2005, https://doi.org/10.1073/pnas.0501886102). Arrested cells also display an altered morphology of secretory organelles-engorgement of the trans-Golgi cisternae-that may reflect a disruption of post-Golgi secretory transport. We now ask whether trypanosomes under VSG silencing also reduce the rate of VSG turnover to further conserve coat density. Our data indicate that trypanosomes do not regulate VSG turnover according to VSG protein abundance, nor was there any effect on the post-Golgi transport of soluble or GPI-anchored secretory cargo. However, the surface morphology of silenced cells was altered from a typically rugose topology to a smoother profile, consistent with reduced overall membrane trafficking to the cell surface. IMPORTANCE African trypanosomes evade the host immune system by altering the expression of variant surface glycoproteins (VSGs) in a process called antigenic variation. VSG is essential, and when its synthesis is ablated by RNAi silencing, cells enter precytokinesis growth arrest as a means to maintain constant cell surface VSG levels. We have investigated whether arrested cells also alter the rate of natural VSG turnover as a means to conserve the surface coat. This work provides insights into the natural biology of the glycocalyx of this important human and veterinary parasite.

The role of glycosylphosphatidylinositol phospholipase C in membrane trafficking in Trypanosoma brucei.

Glycosylphosphatidylinositol-phospholipase C (GPI-PLC) is an enzyme that has been implicated in GPI-dependent protein trafficking and phosphoinositide metabolism in the bloodstream stage of African trypanosomes. However, despite the fact that it is associated with the cytoplasmic face of internal organellar compartments, its role in general membrane trafficking has not been investigated. Using a GPI-PLC null cell line, we determine the effect of GPI-PLC deficiency on these processes. Biosynthetic trafficking of lysosomal cargo, soluble cathepsin L and membrane bound p67, are unaffected. Likewise, secretory transport, recycling and ultimate lysosomal turnover of the GPI-anchored and transmembrane glycoproteins, transferrin receptor and invariant surface glycoprotein 65, respectively, were unaffected. A significant decrease in the endocytic uptake of transferrin was observed, confirming a prior report, but ultimate delivery to the lysosome was unimpacted. These results contribute to our understanding of the roles of this enigmatic enzyme in trypanosome cell biology.

Turnover of Variant Surface Glycoprotein in Trypanosoma brucei Is a Bimodal Process.

African trypanosomes utilize glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) to evade the host immune system. VSG turnover is thought to be mediated via cleavage of the GPI anchor by endogenous GPI-specific phospholipase C (GPI-PLC). However, GPI-PLC is topologically sequestered from VSG substrates in intact cells. Recently, A. J. Szempruch, S. E. Sykes, R. Kieft, L. Dennison, et al. (Cell 164:246-257, 2016, https://doi.org/10.1016/j.cell.2015.11.051) demonstrated the release of nanotubes that septate to form free VSG+ extracellular vesicles (EVs). Here, we evaluated the relative contributions of GPI hydrolysis and EV formation to VSG turnover in wild-type (WT) and GPI-PLC null cells. The turnover rate of VSG was consistent with prior measurements (half-life [t1/2] of ∼26 h) but dropped significantly in the absence of GPI-PLC (t1/2 of ∼36 h). Ectopic complementation restored normal turnover rates, confirming the role of GPI-PLC in turnover. However, physical characterization of shed VSG in WT cells indicated that at least 50% is released directly from cell membranes with intact GPI anchors. Shedding of EVs plays an insignificant role in total VSG turnover in both WT and null cells. In additional studies, GPI-PLC was found to have no role in biosynthetic and endocytic trafficking to the lysosome but did influence the rate of receptor-mediated endocytosis. These results indicate that VSG turnover is a bimodal process involving both direct shedding and GPI hydrolysis. IMPORTANCE African trypanosomes, the protozoan agent of human African trypanosomaisis, avoid the host immune system by switching expression of the variant surface glycoprotein (VSG). VSG is a long-lived protein that has long been thought to be turned over by hydrolysis of its glycolipid membrane anchor. Recent work demonstrating the shedding of VSG-containing extracellular vesicles has led us to reinvestigate the mode of VSG turnover. We found that VSG is shed in part by glycolipid hydrolysis but also in approximately equal part by direct shedding of protein with intact lipid anchors. Shedding of exocytic vesicles made a very minor contribution to overall VSG turnover. These results indicate that VSG turnover is a bimodal process and significantly alter our understanding of the "life cycle" of this critical virulence factor.

p97 Inhibitor CB-5083 Blocks ERAD in Trypanosoma brucei.

Misfolded proteins trapped in the endoplasmic reticulum (ER) are specifically recognized and retrotranslocated to the cytosol by the ER-Associated Degradation (ERAD) system and delivered to the proteasome for destruction. This process was recently described in Trypanosoma brucei (T. brucei) using the misfolded epitope tagged Transferrin Receptor subunits ESAG7:Ty and HA:ESAG6 (HA:E6). Critical to this work was the proteasomal inhibitor MG132. However, MG132 has off-target inhibitory effects on lysosomal Cathepsin L that could cause misinterpretation of turnover results. Here, we evaluate an orally bioavailable p97 inhibitor, CB-5083, for use in T. brucei. p97 is a ubiquitous protein involved in many cellular events including the membrane extraction step of ERAD. CB-5083 strongly inhibits turnover of HA:E6, with comparable protein recovery to MG132 treatment. Interestingly, little deglycosylated cytoplasmic species accumulates, though it normally emerges with MG132 treatment. This suggests that CB-5083 blocks ERAD upstream of the proteasome, as expected for inhibition of the trypanosomal p97 orthologue TbVCP. Under CB-5083 treatment, HA:E6 is also strongly membrane-associated, suggesting ER localization. Finally, we provide an experimental example where CB-5083 treatment offers clarity to the off-target effects of MG132 treatment.

Concurrent hidradenitis suppurativa, inflammatory acne, dissecting cellulitis of the scalp, and pyoderma gangrenosum in a 16-year-old boy.

We report the case of a 16-year-old boy who presented to our clinic with concomitant hidradenitis suppurativa, inflammatory acne, dissecting cellulitis of the scalp, and pyoderma gangrenosum. Recent reports describe the co-occurrence of pyoderma gangrenosum, acne, and hidradenitis suppurativa. This case further expands the spectrum of concomitant pyoderma gangrenosum and dissecting cellulitis of the scalp.