Paul T. Manna

The autophagy receptor TAX1BP1 and the molecular motor myosin VI are required for clearance of Salmonella typhimurium by autophagy

Autophagy plays a key role during Salmonella infection, by eliminating these pathogens following escape into the cytosol. In this process, selective autophagy receptors, including the myosin VI adaptor proteins optineurin and NDP52, have been shown to recognize cytosolic pathogens. Here, we demonstrate that myosin VI and TAX1BP1 are recruited to ubiquitylated Salmonella and play a key role in xenophagy. The absence of TAX1BP1 causes an accumulation of ubiquitin-positive Salmonella, whereas loss of myosin VI leads to an increase in ubiquitylated and LC3-positive bacteria. Our structural studies demonstrate that the ubiquitin-binding site of TAX1BP1 overlaps with the myosin VI binding site and point mutations in the TAX1BP1 zinc finger domains that affect ubiquitin binding also ablate binding to myosin VI. This mutually exclusive binding and the association of TAX1BP1 with LC3 on the outer limiting membrane of autophagosomes may suggest a molecular mechanism for recruitment of this motor to autophagosomes. The predominant role of TAX1BP1, a paralogue of NDP52, in xenophagy is supported by our evolutionary analysis, which demonstrates that functionally intact NDP52 is missing in Xenopus and mice, whereas TAX1BP1 is expressed in all vertebrates analysed. In summary, this work highlights the importance of TAX1BP1 as a novel autophagy receptor in myosin VI-mediated xenophagy. Our study identifies essential new machinery for the autophagy-dependent clearance of Salmonella typhimurium and suggests modulation of myosin VI motor activity as a potential therapeutic target in cellular immunity.

Adaptin evolution in kinetoplastids and emergence of the variant surface glycoprotein coat in African trypanosomatids

Graphical abstract Highlights ► We identify the adaptin gene cohort from currently available kinetoplastid genomes. ► Further, we present analysis of newly sequenced Trypanosoma grayi adaptins. ► We describe the evolutionary history of kinetoplastid adaptins. ► Kinetoplastid adaptin cohorts are compared to major surface protein families. ► Adaptin evolution and immune evasion mechanisms are correlated in salivarian trypanosomes.

Life and times: synthesis, trafficking, and evolution of VSG

Highlights • Variant surface glycoprotein (VSG) is a paradigm for antigenic variation.• VSG provides a mechanism for immune evasion.• Rapid transport, turnover, and endocytosis contribute to VSG function.• VSG has provided, and continues to offer, important insights into trypanosome biology.

A draft genome for the African crocodilian trypanosome Trypanosoma grayi.

The availability of genome sequence data has greatly enhanced our understanding of the adaptations of trypanosomatid parasites to their respective host environments. However, these studies remain somewhat restricted by modest taxon sampling, generally due to focus on the most important pathogens of humans. To address this problem, at least in part, we are releasing a draft genome sequence for the African crocodilian trypanosome, Trypanosoma grayi ANR4. This dataset comprises genomic DNA sequences assembled de novo into contigs, encompassing over 10,000 annotated putative open reading frames and predicted protein products. Using phylogenomic approaches we demonstrate that T. grayi is more closely related to Trypanosoma cruzi than it is to the African trypanosomes T. brucei, T. congolense and T. vivax, despite the fact T. grayi and the African trypanosomes are each transmitted by tsetse flies. The data are deposited in publicly accessible repositories where we hope they will prove useful to the community in evolutionary studies of the trypanosomatids.

Tetherin is an exosomal tether

Exosomes are extracellular vesicles that are released when endosomes fuse with the plasma membrane. They have been implicated in various functions in both health and disease, including intercellular communication, antigen presentation, prion transmission, and tumour cell metastasis. Here we show that inactivating the vacuolar ATPase in HeLa cells causes a dramatic increase in the production of exosomes, which display endocytosed tracers, cholesterol, and CD63. The exosomes remain clustered on the cell surface, similar to retroviruses, which are attached to the plasma membrane by tetherin. To determine whether tetherin also attaches exosomes, we knocked it out and found a 4-fold reduction in plasma membrane-associated exosomes, with a concomitant increase in exosomes discharged into the medium. This phenotype could be rescued by wild-type tetherin but not tetherin lacking its GPI anchor. We propose that tetherin may play a key role in exosome fate, determining whether they participate in long-range or short-range interactions. DOI: http://dx.doi.org/10.7554/eLife.17180.001

ENTH and ANTH domain proteins participate in AP2-independent clathrin-mediated endocytosis

ABSTRACT Clathrin-mediated endocytosis (CME) is a major route of entry into eukaryotic cells. A core of evolutionarily ancient genes encodes many components of this system but much of our mechanistic understanding of CME is derived from a phylogenetically narrow sampling of a few model organisms. In the parasite Trypanosoma brucei, which is distantly related to the better characterised animals and fungi, exceptionally fast endocytic turnover aids its evasion of the host immune system. Although clathrin is absolutely essential for this process, the adaptor protein complex 2 (AP2) has been secondarily lost, suggesting mechanistic divergence. Here, we characterise two phosphoinositide-binding monomeric clathrin adaptors, T. brucei (Tb)EpsinR and TbCALM, which in trypanosomes are represented by single genes, unlike the expansions present in animals and fungi. Depletion of these gene products reveals essential, but partially redundant, activities in CME. Ultrastructural analysis of TbCALM and TbEpsinR double-knockdown cells demonstrated severe defects to clathrin-coated pit formation and morphology associated with a dramatic inhibition of endocytosis. Depletion of TbCALM alone, however, produced a distinct lysosomal segregation phenotype, indicating an additional non-redundant role for this protein. Therefore, TbEpsinR and TbCALM represent ancient phosphoinositide-binding proteins with distinct and vital roles in AP2-independent endocytosis.

The Trypanosome Exocyst: A Conserved Structure Revealing a New Role in Endocytosis.

Membrane transport is an essential component of pathogenesis for most infectious organisms. In African trypanosomes, transport to and from the plasma membrane is closely coupled to immune evasion and antigenic variation. In mammals and fungi an octameric exocyst complex mediates late steps in exocytosis, but comparative genomics suggested that trypanosomes retain only six canonical subunits, implying mechanistic divergence. We directly determined the composition of the Trypanosoma brucei exocyst by affinity isolation and demonstrate that the parasite complex is nonameric, retaining all eight canonical subunits (albeit highly divergent at the sequence level) plus a novel essential subunit, Exo99. Exo99 and Sec15 knockdowns have remarkably similar phenotypes in terms of viability and impact on morphology and trafficking pathways. Significantly, both Sec15 and Exo99 have a clear function in endocytosis, and global proteomic analysis indicates an important role in maintaining the surface proteome. Taken together these data indicate additional exocyst functions in trypanosomes, which likely include endocytosis, recycling and control of surface composition. Knockdowns in HeLa cells suggest that the role in endocytosis is shared with metazoan cells. We conclude that, whilst the trypanosome exocyst has novel components, overall functionality appears conserved, and suggest that the unique subunit may provide therapeutic opportunities.

Lineage-specific proteins essential for endocytosis in trypanosomes

ABSTRACT Clathrin-mediated endocytosis (CME) is the most evolutionarily ancient endocytic mechanism known, and in many lineages the sole mechanism for internalisation. Significantly, in mammalian cells CME is responsible for the vast bulk of endocytic flux and has likely undergone multiple adaptations to accommodate specific requirements by individual species. In African trypanosomes, we previously demonstrated that CME is independent of the AP-2 adaptor protein complex, that orthologues to many of the animal and fungal CME protein cohort are absent, and that a novel, trypanosome-restricted protein cohort interacts with clathrin and drives CME. Here, we used a novel cryomilling affinity isolation strategy to preserve transient low-affinity interactions, giving the most comprehensive trypanosome clathrin interactome to date. We identified the trypanosome AP-1 complex, Trypanosoma brucei (Tb)EpsinR, several endosomal SNAREs plus orthologues of SMAP and the AP-2 associated kinase AAK1 as interacting with clathrin. Novel lineage-specific proteins were identified, which we designate TbCAP80 and TbCAP141. Their depletion produced extensive defects in endocytosis and endomembrane system organisation, revealing a novel molecular pathway subtending an early-branching and highly divergent form of CME, which is conserved and likely functionally important across the kinetoplastid parasites. Summary: Endocytosis is a vital process in most cells, and here we identify important proteins required for this process in trypanosomes. Significantly, these are unique and not present in animals, fungi or plants.

Phosphoinositides, kinases and adaptors coordinating endocytosis in Trypanosoma brucei.

In the kinetoplastid parasite Trypanosoma brucei clathrin-mediated endocytosis is essential for survival and aids immune evasion in the mammalian host. The formation of endocytic clathrin coated vesicles in T. brucei is via a unique mechanism owing to an evolutionarily recent loss of the adaptor protein (AP)2 complex, a central hub in endocytic vesicle assembly. Despite this loss, recent studies examining endocytic clathrin coat assembly have highlighted a high degree of conservation between trypanosomes and their mammalian hosts. In particular phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and its putative effectors, TbCALM and TbEpsinR, are central to clathrin-mediated endocytosis in the trypanosome, just as they are in animal cells. In addition to providing insights into the cell biology of T. brucei, these studies also suggest an ancient, possibly pan-eukaryotic connection between PtdIns(4,5)P2 and endocytosis.

The WDR11 complex facilitates the tethering of AP-1-derived vesicles

Vesicluar transport of proteins from endosomes to the trans-Golgi network (TGN) is an essential cellular pathway, but much of its machinery is still unknown. A screen for genes involved in endosome-to-TGN trafficking produced two hits, the adaptor protein-1 (AP-1 complex), which facilitates vesicle budding, and WDR11. Here we demonstrate that WDR11 forms a stable complex with two other proteins, which localises to the TGN region and does not appear to be associated with AP-1, suggesting it may act downstream from budding. In a vesicle tethering assay, capture of vesicles by golgin-245 was substantially reduced in WDR11-knockout cells. Moreover, structured illumination microscopy and relocation assays indicate that the WDR11 complex is initially recruited onto vesicles rather than the TGN, where it may in turn recruit the golgin binding partner TBC1D23. We propose that the complex acts together with TBC1D23 to facilitate the golgin-mediated capture of vesicles that were generated using AP-1.Trafficking from endosomes to the trans-Golgi network requires recognition of vesicle tethers during membrane docking. Here, the authors identify a complex localised to AP-1 generated vesicles containing WDR11, C17orf75 and FAM91A, which together with TBC1D23 facilitates vesicle capture on Golgi membranes