Trichomonas vaginalis adherence phenotypes and extracellular vesicles impact parasite survival in a novel in vivo model of pathogenesis.

Trichomonas vaginalis is a human infective parasite responsible for trichomoniasis-the most common, non-viral, sexually transmitted infection worldwide. T. vaginalis resides exclusively in the urogenital tract of both men and women. In women, T. vaginalis has been found colonizing the cervix and vaginal tract while in men it has been identified in the upper and lower urogenital tract and in secreted fluids such as semen, urethral discharge, urine, and prostatic fluid. Despite the over 270 million cases of trichomoniasis annually worldwide, T. vaginalis continues to be a highly neglected organism and thus poorly studied. Here we have developed a male mouse model for studying T. vaginalis pathogenesis in vivo by delivering parasites into the murine urogenital tract (MUT) via transurethral catheterization. Parasite burden was assessed ex-vivo using a nanoluciferase-based gene expression assay which allowed quantification of parasites pre- and post-inoculation. Using this model and read-out approach, we show that T. vaginalis can be found within MUT tissue up to 72 hrs post-inoculation. Furthermore, we also demonstrate that parasites that exhibit increased parasite adherence in vitro also have higher parasite burden in mice in vivo. These data provide evidence that parasite adherence to host cells aids in parasite persistence in vivo and molecular determinants found to correlate with host cell adherence in vitro are applicable to infection in vivo. Finally, we show that co-inoculation of T. vaginalis extracellular vesicles (TvEVs) and parasites results in higher parasite burden in vivo. These findings confirm our previous in vitro-based predictions that TvEVs assist the parasite in colonizing the host. The establishment of this pathogenesis model for T. vaginalis sets the stage for identifying and examining parasite factors that contribute to and influence infection outcomes.

Sizing lipid droplets from adult and geriatric mouse liver tissue via nanoparticle tracking analysis.

The significance of lipid droplets in lipid metabolism, cell signaling, and regulating longevity is increasingly recognized, yet the lipid droplet's unique properties and architecture make it difficult to size and study using conventional methods. To begin to address this issue, we demonstrate the capabilities of nanoparticle tracking analysis (NTA) for sizing of lipid droplets. NTA was found to be adequate to assess lipid droplet stability over time, indicating that lipid droplet preparations are stable for up to 24 h. NTA had the ability to compare the size distributions of lipid droplets from adult and geriatric mouse liver tissue, suggesting an age-related decrease in lipid droplet size. This is the first report on the use of NTA to size intracellular organelles. Graphical Abstract Light scattering reveals the temporal positions of individual lipid droplets, which are recorded with a camera. The two-dimensional diffusion constant of each lipid droplet is extracted from the data set, which is then used to calculate a hydrodynamic radius using the Stokes-Einstein equation.

Capillary Electrophoresis with Laser-Induced Fluorescent Detection of Immunolabeled Individual Autophagy Organelles Isolated from Liver Tissue.

Macroautophagy is a cellular degradation process responsible for the clearance of excess intracellular cargo. Existing methods for bulk quantification of autophagy rely on organelle markers that bind to multiple autophagy organelle types, making it difficult to tease apart the subcellular mechanisms implicated in autophagy dysfunction in liver and other pathologies. To address this issue, methods based on individual organelle measurements are needed. Capillary electrophoresis with laser-induced fluorescent detection (CE-LIF) was previously used to count and determine properties of individual autophagy organelles isolated from an LC3-GFP expressing cell line, but has never been used on autophagy organelles originating from a tissue sample. Here, we used DyLight488-labeled anti-LC3 antibodies to label endogenous LC3 present on organelles isolated from murine liver tissue prior to CE-LIF analysis. We evaluated the ability of this method to detect changes in a known model system of altered autophagy, as well as confirmed the specificity and reproducibility of the antibody in the labeling of autophagy organelles from liver tissue. This is both the first demonstration of CE-LIF to analyze individual organelles labeled with fluorophore-conjugated antibodies, and the first application of individual organelle CE-LIF to measure the properties of autophagy organelles isolated from tissue. The observations described here demonstrate that CE-LIF of immunolabeled autophagy organelles is a powerful technique useful to investigate the complexity of autophagy in any tissue sample of interest.

Deterministic Absolute Negative Mobility for Micro- and Submicrometer Particles Induced in a Microfluidic Device.

Efficient separations of particles with micron and submicron dimensions are extremely useful in preparation and analysis of materials for nanotechnological and biological applications. Here, we demonstrate a nonintuitive, yet efficient, separation mechanism for µm and subµm colloidal particles and organelles, taking advantage of particle transport in a nonlinear post array in a microfluidic device under the periodic action of electrokinetic and dielectrophoretic forces. We reveal regimes in which deterministic particle migration opposite to the average applied force occurs for a larger particle, a typical signature of deterministic absolute negative mobility (dANM), whereas normal response is obtained for smaller particles. The coexistence of dANM and normal migration was characterized and optimized in numerical modeling and subsequently implemented in a microfluidic device demonstrating at least 2 orders of magnitude higher migration speeds as compared to previous ANM systems. We also induce dANM for mouse liver mitochondria and envision that the separation mechanisms described here provide size selectivity required in future separations of organelles, nanoparticles, and protein nanocrystals.

Surface proteins, ERAD and antigenic variation in Trypanosoma brucei.

Variant surface glycoprotein (VSG) is central to antigenic variation in African trypanosomes. Although much prior work documents that VSG is efficiently synthesized and exported to the cell surface, it was recently claimed that 2-3 fold more is synthesized than required, the excess being eliminated by ER-Associated Degradation (ERAD) (Field et al., ). We now reinvestigate VSG turnover and find no evidence for rapid degradation, consistent with a model whereby VSG synthesis is precisely regulated to match requirements for a functional surface coat on each daughter cell. However, using a mutated version of the ESAG7 subunit of the transferrin receptor (E7:Ty) we confirm functional ERAD in trypanosomes. E7:Ty fails to assemble into transferrin receptors and accumulates in the ER, consistent with retention of misfolded protein, and its turnover is selectively rescued by the proteasomal inhibitor MG132. We also show that ER accumulation of E7:Ty does not induce an unfolded protein response. These data, along with the presence of ERAD orthologues in the Trypanosoma brucei genome, confirm ERAD in trypanosomes. We discuss scenarios in which ERAD could be critical to bloodstream parasites, and how these may have contributed to the evolution of antigenic variation in trypanosomes.

Characterization of the late endosomal ESCRT machinery in Trypanosoma brucei.

The multivesicular body (MVB) is a specialized Rab7+ late endosome (LE) containing multiple intralumenal vesicles that function in targeting ubiquitinylated cell surface proteins to the lysosome for degradation. African trypanosomes lack a morphologically well-defined MVB, but contain orthologs of the ESCRT (Endosomal Sorting Complex Required for Transport) machinery that mediates MVB formation. We investigate the role of TbVps23, an early ESCRT component, and TbVps4, the terminal ESCRT ATPase, in lysosomal trafficking in bloodstream form trypanosomes. Both localize to the TbRab7+ LE and RNAi silencing of each rapidly blocks growth. TbVps4 silencing results in approximately threefold accumulation of TbVps23 at the LE, consistent with blocking terminal ESCRT disassembly. Trafficking of endocytic and biosynthetic cargo, but not default lysosomal reporters, is also negatively affected. Others reported that TbVps23 mediates ubiquitin-dependent lysosomal degradation of invariant surface glycoproteins (ISG65) (Leung et al., Traffic 2008;9:1698-1716). In contrast, we find that TbVps23 ablation does not affect ISG65 turnover, while TbVps4 silencing markedly enhances lysosomal degradation. We propose several models to accommodate these results, including that the ESCRT machinery actually retrieves ISG65 from the LE to earlier endocytic compartments, and in its absence ISG65 traffics more efficiently to the lysosome. Overall, these results confirm that the ESCRT machinery is essential in Trypanosoma brucei and plays important and novel role(s) in LE function in trypanosomes.