Elucidating the Mechanistic Basis for Phagotrophy in the Protozoan Trypanosoma cruzi (equipment supplement)

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Being able to efficiently extract nutrients from one’s environment is an essential activity for any heterotroph ranging from predators to parasites. Among the free-living planktonic protozoans, the extremely diverse class of phagotrophic predators have devised a variety of methods to capture and consume the sources of organic carbon they need to grow and reproduce. One of the most widespread modes of mechanical predation employed by these protozoa involves cilia or flagella supported filter feeding. Captured prey are ultimately endocytosed via a cell spanning tubular invagination, originating at a pore in the plasma membrane, and ending in budding vesicles targeted for lysosomal digestion. This pore (cytostome), and its emanating tubule structure (cytopharynx), are collectively referred to here as the cytostome/cytopharynx complex (SPC). Despite its ubiquitous presence, almost nothing is known about how the SPC is generated or functions at the molecular level in any organism. It is worth noting that, collectively, the SPC containing protozoa play critical roles in diverse activities ranging from the global microbial food web to human parasitism. Intriguingly, the genetically tractable flagellate, Trypanosoma cruzi, has retained this ancestral mode of endocytosis and, much like it’s free-living bacterivorous relatives (e.g. Bodo saltans), actively endocytoses its host’s material sustenance via the SPC as well. How protozoans capture food at their surface, signal internally to initiate endocytosis and ultimately traffic this material down the SPC to the lysosome for digestion remains a mystery and is at the core of the questions we seek to answer in this proposal. We have found that SPC- mediated endocytosis is dispensable for T. cruzi when grown in vitro, and as a result, we are uniquely positioned to be able to conduct extensive knockout (KO) and complementation studies to functionally dissect multiple dimensions of SPC function without impacting cell viability for the first time. As a continuation of our prior published work, this proposal seeks to generate a holistic understanding of how SPC mediated endocytosis fundamentally functions. We will begin by dismantling the unified activity of endocytosis into its constituent processes; cargo capture through surface receptors (Aim1), receptor signal transduction and activation of endocytic machinery (Aim2) and finally active transport of phagocytosed cargo along the SPC for digestion (Aim3). Each of these aims will address important basic aspects of protozoan biology that continue to remain poorly understood. As studies currently underway have highlighted, the use of standard confocal live microscopy is too slow and toxic to study this highly dynamic and rapid process. As a result, this proposal requests funding to support the acquisition of a spinning disk confocal microscope that will be capable of imaging extremely fast protozoan endocytic activities across long periods of time without the concerns associated with phototoxicity hampering analysis. This equipment will no doubt enhance ongoing studies which seeks to elucidate the mechanistic underpinnings of the enigmatic process of protozoan phagotrophy.