Protozoan phagotrophy from predators to parasites: An overview of the enigmatic cytostome-cytopharynx complex of Trypanosoma cruzi

Eating is fundamental and from this basic principle, living organisms have evolved innumerable strategies to capture energy and nutrients from their environment. As part of the world’s aquatic ecosystems, the expansive family of heterotrophic protozoans uses self-generated currents to funnel prokaryotic prey into an ancient, yet highly enigmatic, oral apparatus known as the cytostome-cytopharynx complex prior to digestion. Despite its near ubiquitous presence in protozoans, little is known mechanistically about how this feeding organelle functions. Intriguingly, one class of these flagellated phagotrophic predators known as the kinetoplastids gave rise to a lineage of obligate parasitic protozoa, the trypanosomatids, that can infect a wide variety of organisms ranging from plants to humans. One parasitic species of humans, Trypanosoma cruzi, has retained this ancestral organelle much like its free-living relatives and continues to use it as its primary mode of endocytosis. In this review, we will highlight foundational observations made regarding the cytostome-cytopharynx complex and examine some of the most pressing questions regarding the mechanistic basis for its function. We propose that T. cruzi has the potential to serve as an excellent model system to dissect the enigmatic process of protozoal phagotrophy and thus enhance our overall understanding of fundamental eukaryotic biology.

Obtaining energy from your environment is a clear biological imperative, and for protozoan parasites, that environment, unfortunately for us, is our own bodies.The development of targeted approaches to impede these microscopic predators' ability to raid our cellular resources has long been the dream of those searching to find ways to combat innumerable parasitic diseases.Currently, however, we suffer from an acute lack of understanding of even the basic mechanistic underpinnings of endocytosis in countless species of protozoans, whether they are free-living or parasitic.One such medically important family of protozoan parasites transmitted by blood-sucking invertebrates, the trypanosomatids (class kinetoplastida), infect millions of people globally causing significant levels of morbidity and mortality in endemic areas.Human infections are dominated by three main species of trypanosomatids (Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp.) with T. cruzi, the etiological agent of Chagas disease (i.e.American trypanosomiasis), being the focus of this review.
Trypanosoma cruzi chronically infects an estimated 7 million people in the Americas, with an at-risk population of 70 million, making it the most severe parasitic disease of the Americas (Perez-Molina & Molina, 2018;World Health Organization, 2015).Despite its impact, Chagas disease remains underreported, understudied, and underfunded and continues to result in a yearly loss of more than 50,000 lives and 0.586 × 10 6 disabilityadjusted life years (Mathers et al., 2006).Even in the United States, the burden is significant, with estimates of those currently infected ranging from 238,000 to 347,000 (Manne-Goehler et al., 2016).Infection is lifelong and current therapeutics are not curative, often resulting in debilitating cardiomyopathy in one-third of those infected (Groom et al., 2017).Like other trypanosomatids that cause disease in humans, T. cruzi is characterized by having a dixenous (two-host) life cycle.Its definitive host (or vector), the triatomine insect (family Reduviidae), is considered to be the primary transmission vector for humans, although other known forms of parasite transmission are possible including oral infection, blood transfusion, organ transplantation, and congenital infection (Nickerson et al., 1989;Pelosse & Kribs-Zaleta, 2012;Zapparoli et al., 2022).The life cycle of this parasite can be divided into four major developmental stages each serving a particular role (summarized in Figure 1); in the insect vector, you find the actively replicating epimastigotes (green) and infectious metacyclic trypomastigotes (blue), while in the mammalian host, you find replicating amastigotes (red) and infectious trypomastigotes (blue; De Souza, 2002;Kariuki et al., 2019;Sousa, 1999).It should be noted, however, that intermediate stages distinct from these major developmental forms have also been observed (De Souza & Barrias, 2020).Natural transmission by the insect vector occurs during the course of a blood meal when the parasite, released in the vector's excrement as a metacyclic trypomastigote (blue), enters the host bloodstream either through the bite wound or nearby mucous membrane.The excreted parasite is capable of invading any nucleated cell it encounters.Upon reaching the host cell cytosol, T. cruzi converts into its amastigote (red) form and replicates for ~3-5 days until the host cell is completely filled with potentially over a hundred parasites.At this point, T. cruzi will elongate its flagellum and transform into infectious trypomastigotes (blue), lyse the host cell, and either initiate another round of host cell invasion or be taken up by a new insect vector where it will propagate as epimastigotes (green) and complete the transmission cycle (Barrett et al., 2003;Souza et al., 2010).
In order to complete their life cycle effectively, however, it is essential that parasites ensure unimpeded access to their host's nutrient stores.To achieve this, the trypanosomatids have been shown to employ both integral membrane transporters, to bring in simple nutrients, and bulk endocytosis, to bring in larger more complex extracellular material such as proteins and lipid complexes (Marchese et al., 2018;Michels et al., 2021;Souza et al., 2009).It is worth noting that for these protozoans, this process of endocytosis can only occur at specialized regions of the plasma membrane (PM) that are not obstructed by the rigid microtubule cytoskeleton that covers most of the organism (Figure 3 gray lines; Morgan et al., 2002Morgan et al., , 2002;;Souza et al., 2008).To date, most of what we know of the endocytic process in this parasitic family has come from studies of a single species, Trypanosoma brucei, the causative agent of African trypanosomiasis (Overath & Engstler, 2004).This focus is due, primarily, to the relative ease of in vitro culture and the wide array of high-quality genetic tools, including robust RNAi-mediated gene knockdown, that are unavailable in the study of related trypanosomatids (Meissner et al., 2007).Unfortunately, T. brucei and members of the salivarian trypanosomatids (i.e.those transmitted through the mouth of biting insects) are far from representative of the global family of kinetoplastids, or even parasitic trypanosomatids for that matter, as they endocytose extracellular material exclusively at the membranous invagination known as the flagellar pocket (FP;Field & Carrington, 2009).All remaining free-living and parasitic kinetoplastids, it turns out, employ a completely different and far more ancient mechanism of nutrient uptake using an oral apparatus known as the cytostomecytopharynx complex or SPC for short.This feeding structure is analogous to a mouth (cytostome) and gullet (cytopharynx) and begins as a stable opening adjacent to the flagellar pocket that extends internally as a tubular invagination that carries endocytosed material to the posterior end of the cell for digestion (summarized in Figure 3; Adl et al., 2012;De Souza, 2002;Losinno et al., 2020).Due to historical limitations in the molecular tools available to study T. cruzi, investigators seeking to understand the SPC in trypanosomatids have been forced to rely heavily on the use of observational techniques such as electron microscopy (EM)-based freeze fracture, fracture flip, transmission (TEM), scanning (SEM), tomography, and focused ion beam SEM (FIB-SEM; Chiurillo & Lander, 2021;Lander et al., 2019).These techniques and the resulting high-quality structural data have revealed, with ever-increasing levels of detail, the remarkable complexity of this fascinating organelle (Alcantara et al., 2014(Alcantara et al., , 2017(Alcantara et al., , 2021;;Chiari et al., 1978;De Souza et al., 1978;Girard-Dias et al., 2012;Martinez-Palomo et al., 1976;Milder & Deane, 1969;Okuda et al., 1999;Pimenta et al., 1989;Pitelka, 1961;Porto-Carreiro et al., 2000;Preston, 1969;Ramos et al., 2011;Souza, 1999;Souza et al., 1978Souza et al., , 2009;;Steinert & Novikoff, 1960;Vatarunakamura et al., 2005;Vidal et al., 2016).Where this work has been found wanting, however, is in its inability to provide a clear mechanistic explanation for how the SPC actually brings in food.Our laboratory's recently published work on SPC-targeted myosin motors has only just begun to elucidate the molecular machinery central to SPC function with an array of important questions still remaining to be answered (Chasen et al., 2020).Far from being comprehensive, our goals in this review are to briefly summarize our current conception of the T. cruzi SPC with regard to its evolutionary history and physical structure and use a variety of prior seminal observations in order to focus our attention on two broad questions which we think, when answered, will significantly enhance our understanding of how the SPC organelle functions as a whole: (1) How is food captured at the surface?and (2) How does the endocytic machinery pull food in?It is our hope that answers to these questions will not only bring us closer to a more complete view of the SPC in this important human pathogen, but also contribute more broadly to our understanding of basic protozoan biology and the uniquely eukaryotic process of endocytosis as well.

PREDATORS TO PARASITES
Protozoal phagotrophy is an extremely ancient mode of nutrient acquisition and, with respect to the evolutionary history of eukaryotes, is considered to have been a necessary prerequisite for the acquisition of the endosymbiotic mitochondrial and photosynthetic organelles we see today (Cavalier-Smith, 2002).As a result, phagotrophy can be found in all of the major phylogenetic branches of the protistan family.It is estimated that phagotrophic predators, such as flagellates and ciliates, consume up to 75% of the daily phytoplankton production from oceanic ecosystems and, as such, play pivotal roles in carbon cycling within global microbial food webs (Sherr & Sherr, 2002).In addition to free-living predators, many of these protozoan phagotrophs also evolved into obligate human parasites and the kinetoplastids, an environmentally widespread and ecologically important group of singlecelled flagellates are a clear example (Flegontov et al., 2013;Lukes et al., 2018;Skalicky et al., 2017).Due to the severe impact that kinetoplastid parasites continue to have on human health, it should be no surprise that the majority of what we know of this protozoan class has been gleaned from the study of its disease-causing members (Burza et al., 2018;Buscher et al., 2017;Perez-Molina & Molina, 2018).In order to better understand the basic biology of trypanosomatids, it is worth discussing their likely evolution from free-living aquatic excavates who used their cytostomes to hunt, capture, and ultimately digest bacterial prey (analogous process for T. cruzi shown in Figure 3; Lukes et al., 2014;Simpson et al., 2002).In an attempt to trace the evolutionary origins of this parasitic family, several studies have demonstrated that the bodonids, e.g. the biflagellate Bodo saltans, are modern-day representatives of free-living kinetoplastids.The diverse family of bodonids can be readily found in almost all marine and freshwater habitats and uses self-generated currents with one of their two flagella to filter feed on bacteria (Flegontova et al., 2018;Stevens, 2014).How this transition from bacterial predation to the parasitism we see in trypanosomatids today first occurred has been the source of contentious debate for almost a century, but with the discovery of Paratrypanosoma confusum as the earliest known basal branching monoxenous (one-host) trypanosomatid, it has become apparent that the initial transition to a parasitic lifestyle first began in arthropods and that dixenous parasitism likely arose independently on several occasions (Figure 2 adapted from Skalicky et al. 2017;Flegontov et al., 2013;Lukes et al., 2018;Simpson et al., 2002;Skalicky et al., 2017;Stevens, 2008).The colonization of the insect intestinal tract, it seems, proved advantageous as numerous examples of previously free-living organisms (including apicomplexans) are known to have taken up residence in the relatively stable ecological niche of the metazoan intestine (Harp, 2003;Sinha et al., 2012).It is in the arthropod intestine where these parasites presumably first perfected the fecal/oral route of transmission which, even today, is the dominant mode of monoxenous trypanosomatid dissemination.What has become clear from the analysis of the P. confusum genome is that during this first transition to a parasitic lifestyle, these organisms lost their 2nd flagellum and streamlined a number of metabolic pathways (Deschamps et al., 2011;Harmer et al., 2018;Opperdoes et al., 2016).Intriguingly, the fecal-transmitted parasites, like P. confusum and T. cruzi, retain many more ancestral genes and structures relative to the salivarian trypanosomatid clades and this includes the ancestral form of nutrient acquisition via the SPC endocytic organelle (Skalicky et al., 2017).It is worth noting that the presence of an SPC has been observed in all monoxenous trypanosomatids as well as in all the dixenous fecal-transmitted stercorarians studied to date, with only the salivarians (T.brucei and Leishmania spp.) completely discarding the SPC as an endocytic structure (Landfear & Ignatushchenko, 2001;Preston, 1969).Although we will discuss the question as to why the stercorarians may have selectively retained the SPC at the end of this review, it appears to correlate with some aspect of life within the insect vector intestinal tract itself.

WHAT DOES THE SPC LOOK LIKE? HISTORICAL OVERVIEW OF THE STRUCTURE AND DYNAMICS OF THE SPC IN TRYPANOSOMA CRUZI
Our current understanding of the SPC structure in kinetoplastids is the result of a 60+ years journey beginning with work published in 1960 by Steinert and Novikoff who, by examining TEM images of the frog trypanosome Trypanosoma mega, gave us our first direct look at the cytostome in this protozoan family (Steinert & Novikoff, 1960).In this era, there were already a number of excellent observational studies of the oral apparatus of many ciliates and flagellates using both light and electron microscopic techniques and, due to the clear structural similarities, the authors applied the same cytostome nomenclature to this structure in kinetoplastids (Boeck, 1921;Corliss, 1959).This seminal work was able to show that the trypanosome cytostome was a stable opening (Figure 3A blue lines) present at the parasite surface that connected to a tubular invagination that was seemingly lined with "pellicular fibrils", later shown to be microtubules.This study was also the first to note that tracer food, in this case, electron-dense ferritin, was curiously found only at the cytostomal mouth and nowhere else on the PM.This observation, it turns out, would be repeated numerous times over the subsequent decades and would lend support to the idea that surface receptors may also be present in these parasites, a topic we will discuss in greater detail in the next section.It was not, however, until the work of Milder and Deane in 1969 that we were able to get our first direct look at the SPC structure of Trypanosoma cruzi itself (Milder & Deane, 1969).This work put on full display the true complexity of this structure in what was the clearest view yet of the SPC's internal organization.This was an important finding, in part, because by this time, many salivarian trypanosomes had already been examined via EM and because they lacked an SPC, it was generally assumed that this apparatus was going to be universally absent in mammalian trypanosomatids (Vickerman, 1969).As seen with T. mega years prior, a number of cytoplasmic vesicles were observed lying adjacent to the cytopharynx in a seemingly organized fashion (Figure 3B orange outline) with associated microtubules prophetically suggested to act as "…a sort of skeleton for the whole organelle…" In an attempt to explain how the SPC might function, the authors put forth the intriguing hypothesis that these microtubules might be contractile in nature and thus could potentially operate as a rudimentary pump.This idea of an overtly mechanical SPC was met with skepticism in subsequent work by T. M. Preston that same year who, through careful examinations of the fish trypanosome Trypanosoma raiae, generated the most detailed analysis of the SPC apparatus yet seen (Preston, 1969).
In addition to again pointing out the preferential binding of protein ligands to the cytostome entrance, the proposed model of the SPC suggested that the previously observed cytostomal microtubules were not, in fact, part of the subpellicular array as originally suspected, but instead originated at the base of the flagellum (location of the basal body), wound up and around the flagellar pocket and then descended again into the parasite cytosol alongside the cytopharynx.Between five and six SPC-associated microtubules were seen and, due to their distinctive electron density, were noticeably different from the PM-associated subpellicular microtubules.At the conclusion of this work, the author also speculated on the central question of how the SPC might function by saying "The way in which fluid is circulated within the cytopharynx is not clear.In the absence of any evidence that the cytostome contracts and acts as a pump, it may be that the tip to base beating of the flagellum causes a current down one side of the flagellar canal, and that part of this current is deflected into the cytopharynx."However, later work by Meyer and De Souza showing immotile amastigotes taking up host cytosolic melanin granules, appeared to make the necessity of flagellar currents to drive endocytosis less likely (Meyer & Souza, 1973).The field would have to wait until 1976 in work presented by Martinez-Palomo et al. for the next major advance in our understanding of the SPC (Martinez-Palomo et al., 1976).Although we will expound on the findings and implications of this work in greater detail in the next section, it is worth pointing out that this extensive freeze-fracture-based EM study of the T. cruzi PM revealed for the first time just how dramatically different the membrane surface adjacent to the cytostome entrance, later termed the preoral ridge (POR), was from the rest of the PM and highlighted the SPC's intimate connection to the flagellar pocket membrane itself (Figure 4C green line).These freeze-fracture observations were, years later, nicely complemented in work by Nakamura et al. who, using newly improved techniques for SEM, provided the first direct look of the SPC from the parasite exterior (Figure 3C green outline), reinforcing the idea that the POR region was indeed a distinct membrane domain with a potentially important role in the endocytic process (Vatarunakamura et al., 2005).In addition to the structure of the SPC, follow-up studies by De Souza et al. released in 1978 sought to determine what the final destination was for endocytosed material.This analysis followed tracer protein, initially bound to the cytostome entrance, and observed movement down the cytopharynx and deposition into multivesicular structures, previously observed by Bretana and O'Daly in 1976, that later came to be known as the reservosomes (Bretana & O'Daly, 1976;Soares & De Souza, 1988;Souza et al., 1978).It is now generally accepted that the bulk of endocytosed material ends up in these prelysosomal reservosomes which serve as the main energy source for metacyclogenesis when parasites are faced with nutrient deprivation in the vector hindgut (Soares et al., 1989).
Research in the following decades provided a number of interesting insights into the nature of SPC function with debates persisting over what constituted receptor-mediated versus fluid-phase uptake and whether or not endocytosis occurs within the flagellar pocket of T. cruzi as well.The details of the SPC structure, however, would not be seriously revisited until 1999 when Okuda et al. examined the microtubule arrays previously shown to associate with the SPC (Okuda et al., 1999).By this point, extensive studies of the microtubule quartet (MTQ) of T. brucei, which is thought to template the location where the flagellum and cell body connect, had clearly shown them to originate at the basal body (Dong et al., 2020;Hoog et al., 2016;Vaughan & Gull, 2015).However, because T. cruzi also has an MTQ, it remained possible that the previously observed SPC microtubules which originate at the flagellar base were, in fact, simply the MTQ root fibers.In this report, EM was combined with flagellar isolation methodologies to produce whole mounts of the T. cruzi cytoskeleton revealing a stable, detergent-resistant association of the SPC and the flagellar/basal body structures.This connection was now conclusively shown to be based upon microtubules that were an independent array and distinct from the previously described MTQ.
At the time, it was difficult to imagine that additional insight into the SPC could be made with the available technology, but this lull would be finally broken in 2012 when Girard-Dias et al., using high-pressure freezing combined with serial electron tomography and 3D reconstructions, created an impressive high-resolution map of the internal structures of T. cruzi.This technique allowed researchers to clearly show that four SPC-specific microtubules originate at the flagellar pocket.Importantly, however, when this array was followed into the parasite cytosol, several additional microtubules seemed to spontaneously appear.In the end, the exact origin of these extra microtubules was not settled in this work, but, nevertheless, they were proposed to be the same five or six SPC microtubules observed by T.M. Preston over 40 years prior (Preston, 1969).Even though this work was unable to see the SPC in its entirety, it did show that it was now possible to produce a high-resolution reconstruction of the internal structure of these parasites.Within 2 years, Alcantara et al. used these same methodologies to provide the first definitive high-resolution 3D view of the entire endocytic apparatus of T. cruzi and revealed, with stunning clarity, the true nature of this cell spanning tubular invagination from start to finish (Figure 3D pink tubule; Alcantara et al., 2014).These results also settled several debates including questions regarding the microtubule root fibers that had emerged from the fragmentary data accumulated over the decades.We could now see that parasites in fact have two distinct sets of microtubule root fibers supporting the SPC structure.The first set, which had been observed by several groups, is specifically composed of four microtubules, referred to here as the cytostomal quartet or CyQ.The CyQ originates from the basal body complex, much like the MTQ, and winds up the flagellar pocket to the PM where a sharp curve in the quartet occurs prior to descending abruptly and traveling deep into the parasite cytosol.Unlike the fragmentary hints from prior TEM/whole-mount cytoskeletal analyses, this work clearly showed that it is the inherent curvature of the CyQ root fibers, present even after membrane extraction (Figure 3E purple outline), that produces the curved shape of the surface POR plasma membrane region.Significantly, the second rootlet set was now seen to be a completely independent array of three microtubules, referred to as the cytostomal triplet or CyT, which originate directly adjacent to the cytostome opening and track beside the CyQ to form a "gutter" shape around the cytopharynx (Figure 3F blue CyQ and green CyT lines).Using transferrin as a tracer, Alcantara et al. were also able to show that there is active membrane trafficking along the naked side of the cytopharynx tubule and not just at the terminating end.This work likely put to rest the perennial debate over endocytosis in the flagellar pocket, as no tracer-labeled vesicles were ever seen emanating from this region in any of the 3D reconstructions.From this foundational work, several follow-up reports from this group dissected how the SPC fully breaks down both when parasites differentiate into metacyclic trypomastigotes (Vidal et al., 2016) as well as when they transit through the cell cycle (Alcantara et al., 2017).It appears that the SPC undergoes a massive restructuring during the cell cycle as the cytopharynx tubule first begins to break down in early G2, followed by loss of the CyT and finally a shortening of the CyQ.As a result, in late G2/M, nearly all cells with a duplicated kinetoplast (i.e.1N2K2F) no longer endocytose.After duplication of the flagellar pocket, however, the CyQ is reextended, followed by generation of the CyT and finally ending in invagination of the FP membrane to regenerate the entire SPC.Endocytic capacity is, therefore, only briefly stopped and quickly restored even before cytokinesis had been completed.Follow-up reconstructions of the endosomal system itself also provided a look at the endocytic network connecting the SPC to the final reservosome destination (Alcantara et al., 2018).Recently, Alcantara et al. completed the life cycle characterization of the SPC by focusing on intracellular amastigotes and were able to show that this structure was highly analogous to that seen in epimastigotes (Alcantara et al., 2021).In trying to understand the nature of endocytosis in T. cruzi, we are fortunate to be in possession of a wealth of structural data with which to begin developing testable models regarding how this organelle functions.Thanks to recent advances in molecular tools to modify T. cruzi genes, as well as increasingly well-annotated genomic databases, the field is well positioned to begin deciphering how this structure is built and regulated in order to hopefully answer the question as to why related parasites, such as T. brucei, were able to completely abandon the SPC as a mode of nutrient uptake (Lander et al., 2017;Peng et al., 2014;Wang et al., 2021).

HOW IS FOOD CAPTURED AT THE SURFACE? THE MYSTERY OF THE PREORAL RIDGE AND THE ELUSIVE T. CRUZI RECEPTOR
Does T. cruzi use surface receptors to capture food and trigger endocytosis?This has been a long-standing question in trying to understand endocytosis in not only T. cruzi, but also in many species of protozoa that employ an SPC.In indirect studies of various free-living bacterivorous protozoans, it has been observed that the area adjacent to their cytostome is uniquely coated with what appear to be lectin-like surface receptors as well as mannose-d ecorated structures recognized by the lectin Concanavalin A (ConA; Roberts et al., 2006).Although speculative, it has been proposed that these carbohydrate-recognizing receptors may facilitate binding to the bacterial cell wall and thus enhance the overall efficiency of prey capture and uptake (Martel, 2009;Wootton et al., 2007).It is, therefore, within reason to consider that T. cruzi may continue to use versions of these ancestral surface receptors to seize nutrients directly from its host.Although a putative C-type lectin receptor was identified in the T. cruzi endocytic proteome, to date, there have been no published reports characterizing even a single-surface receptor for T. cruzi involved in either binding to or supporting endocytosis of cargo (Brosson et al., 2017).On the other hand, what we do have are reams of observational data showing that protein targets ranging from ferritin, horseradish peroxidase (HRP), bovine serum albumin (BSA), transferrin, and immunoglobulin (IgG) are readily taken up by the SPC (Alcantara et al., 2014;Chasen et al., 2019Chasen et al., , 2020;;Correa et al., 2008;Scott et al., 1997).What's more, these proteins bind specifically to the specialized surface membrane of the preoral ridge (POR) with sufficient affinity to resist displacement following multiple washes.In fact, we have not identified a protein that cannot be bound by this region and it is this seemingly nonspecific binding or "sticky" quality of POR which makes the search for potential receptors so intriguing.To begin thinking seriously about how this specialized membrane area might bind cargo, it is worth considering the many unique qualities of the POR itself.As mentioned in the prior section, the first evidence which demonstrated just how different this POR membrane really was, came from several freeze-fracture and transmission EM experiments, carried out in 1976 by Martinez-Palomo et al.By freezing parasites and splitting them in such a way as to separate the plasma membrane bilayer, one could get a glimpse of the normally hidden internal face of both the extracellular (E face) and cytoplasmic (P face) leaflets.Using this technique, transmembrane (TM) proteins will appear as raised protuberances on the cytoplasmic P face and, due to being removed from the outer leaflet, as divots in the extracellular E face.Freeze fracture, therefore, revealed that the POR region connecting the cytostome to the flagellar pocket was so distinct that it had the appearance not unlike a "membrane river" flowing down into the SPC opening (Figure 4A green outline; Martinez-Palomo et al., 1976).Zooming in on this POR region (Figure 4B green outline), the authors highlighted two important phenomena; a clear paucity of TM domain-containing proteins within the POR membrane region, thus giving it a smooth appearance, and a linear "palisade" boundary of TM proteins lining the banks of this membrane river.This palisade is reminiscent of the cytoskeleton-driven "fence and picket" like structures that partition lipid raft domains in other eukaryotic systems to prohibit the free flow of TM proteins into this region (Ritchie et al., 2003;Saha et al., 2016).Using TEM and the polysaccharide stain Ruthenium red, the authors observed that the POR surface (underlined in green) was not only uniquely glycan rich when compared to the rest of the PM (Figure 4C and E red arrow), but it also extended deep into the normally unseen domain of the flagellar pocket itself (Figure 4C green line).Although not explicitly stated, this observation opened the possibility that the membrane being pulled into the SPC may originate from the flagellar pocket via vesicle fusion and thus provide a continuous source of endocytic membrane and surface receptor materials.The glycans observed by Ruthenium red staining also appear to be rich in mannose as the lectin ConA also preferentially labels the POR ridge not unlike what had been observed in free-living protozoa mentioned previously (Figure 4D blue arrow).This binding of ConA to the POR has since been observed using a variety of methods ranging from fracture-flip replica staining, SEM as well as fluorescent super-resolution microscopy (Chasen et al., 2019;Pimenta et al., 1989;Vatarunakamura et al., 2005).There was also a notable compositional difference in the cytoskeleton directly beneath the POR membrane (Figure 4E arrowheads), inferring a likely influence on the PM domain above it.So, despite an obvious dearth in TM domain-containing proteins in this area, there is a clear enrichment of mannose-rich glycans suggesting that whatever is present on the POR surface, it is highly decorated with carbohydrate moieties.The role of these carbohydrate modifications in SPC function has not been conclusively determined, although reports suggest that they also play a role in endocytic activity (Brosson et al., 2016).Apart from its protein and carbohydrate composition, fracture-flip-based EM has also highlighted the POR surface membrane as both thicker and more roughly textured than the surrounding PM (Figure 4F; Pimenta et al., 1989).This difference in thickness is intriguing because the presence of sterols, e.g.ergosterol and cholesterol, is known to increase the observed width of membranes and this is especially true for ordered membrane domains such as lipid rafts (Yang et al., 2016).An important role for these membrane sterols in endocytosis is also supported by the observation that agents which bind to or deplete cholesterol, such as filipin or methyl-β-cyclodextrin (MβCD), completely inhibit cargo uptake (Correa et al., 2008).The rough texture of the POR, mentioned previously, has also been seen multiple times with various techniques including the first SEM picture of this structure (Figure 3C; Vatarunakamura et al., 2005).This texture is thought to relate back to the inherent "stickiness" of the region with the "rugous" appearance being a byproduct of material having been bound to the POR.Taken together, the SPC has been suggested to operate as a "sticky", cholesterol-rich, detergent-resistant membrane with many of the hallmarks of a lipid raft domain (Correa et al., 2007).As we initiate a search for potential cargo receptors, it is reasonable to leverage these prior observations in order to establish a set of criteria for potential targets.The first and most obvious type of candidate receptor would be a glycosylphosphatidylinositol-anchored protein (GPI-AP).This is an attractive class of potential molecules for a number of reasons; first, we already know that the T. brucei transferrin receptor is a GPI-AP and so some precedent already exists in this parasite family even though no homologs of the ESAG6/7 receptor exists in the T. cruzi genome (Kariuki et al., 2019) and, second, it is the fact that GPI-APs are known to cluster in cholesterol-rich lipid raft domains similar to what we see in the POR.The POR itself could, therefore, function as an organizing platform in order to aggregate this class of membrane receptor (Denny et al., 2001;Edidin, 2003).The enforced clustering of GPI-APs in this region could also explain the preferential binding of ConA lectins to the POR which may be labeling the mannose-rich portion of the GPI anchor itself (Paulick & Bertozzi, 2008).However, it is worth noting that the ubiquitous labeling of other GPI-APs on the surface of T. cruzi in all life cycle stages makes it conceptually difficult to reason how partitioning of different types of GPI-APs at the PM would actually be accomplished in practice (Borges et al., 2021;Buscaglia et al., 2006;Freire-de-Lima et al., 2012).The freeze-fracture observations highlighting the low abundance of TM domain-containing proteins in the POR do not discount a potential role for this type of protein acting as a cargo receptor either.It has been observed that lipid rafts often have specific TM domain proteins associated with them, yet how these proteins selectively associate with lipid-ordered regions has itself been the subject of intense study.Numerous reports have shown that retention within lipid rafts often relies on a simple physical organizing principle; thicker lipid raft membranes can accommodate longer hydrophobic TM domains while simultaneously repelling shorter ones (Sharpe et al., 2010).Through a combination of biochemical techniques to enrich for detergent-resistant membranes, followed by selective enrichment of POR components using ConA pull-downs and mass spectrometry, it should be possible to begin identifying, in an unbiased way, potential receptor proteins associated with these areas of the PM.A bioinformatic search as well for proteins conserved in all SPC-containing organisms which retain; lectin domains, long (≥24 amino acids) hydrophobic TM domains and high expression in the replicative (i.e.active SPC) stages could provide additional criteria to identify cargo receptors from these analyses.In thinking about the capture of food to be endocytosed by this endocytic structure, it is enticing to imagine that this POR region may function as a lipid-raft platform of GPIanchored and/or long TM domain receptor-like proteins that bind and signal internally to activate the endocytic machinery facilitating cargo uptake.Although there have been, as of yet, no cell surface cargo receptors characterized in any SPC-containing heterotroph, indirect observations support their existence (Alcantara et al., 2017) and the identification of the first feeding cargo receptor in T. cruzi will, no doubt, be an important step in understanding how this organism directly interacts with its host to acquire necessary nutrients.

HOW IS CARGO PROPELLED DOWN THE SPC? SPECULATIONS ON THE IDENTITY AND ORGANIZATION OF SPC MACHINERY
Much has been made of the structure of the SPC in this review, but how does it actually work?At the moment we clearly do not have a full answer to this question, however, a good place to start may be in proposing a potential model that can serve as a tool to develop testable hypotheses.In this section, we will take the limited experimental data we currently have in order to propose a functional model for the SPC and highlight both important hints from the data and major gaps in our understanding that the field can now address.First, however, it is important that we briefly revisit findings from our recently published work on cytostomal myosins in order to make the case that a full molecular dissection of the SPC is indeed possible (Chasen et al., 2020).
As part of our analysis of the cytopharynx-targeted myosin motor MyoF, we generated parasite lines overexpressing a dominant-negative "rigor" mutant of this enzyme that, due to an inability to hydrolyze ATP and complete its power stroke, is unable to let go of actin filaments.Importantly, the overexpression of this rigor mutant made parasites completely defective in measurable endocytosis, suggesting that the inactive MyoF enzyme had poisoned the myosin motor network, thus stopping this process entirely.What was striking to us was that this endocytic block was not only not lethal, it had no significant effect at all on parasite growth or viability in in vitro culture.Following this observation, we generated a full deletion of the MyoF gene and, while not leading to an endocytic-null phenotype, reduced the endocytic rate by 86%.This ΔMyoF mutant again showed no measurable decrease in parasite growth or viability.We do not know how these parasites are able to survive in the absence of endocytosis but, as stated in the introduction, it may be due to surface membrane transporters that import simple nutrients that are provided in excess in the standard epimastigote liver infusion tryptose (LIT) media (Costa et al., 2020).This ability to make endocytic-null mutants, it turns out, may be extremely fortuitous, as the lack of a conditional knockdown system in T. cruzi continues to make the study of essential gene function exceedingly difficult (Chiurillo & Lander, 2021).This, therefore, opens up the possibility of using CRISPR/Cas9 gene-editing tools to begin ablating SPC components to directly assess their role in the endocytic process without compromising parasite fitness (Lander et al., 2017(Lander et al., , 2019;;Peng et al., 2014).While we have yet to thoroughly examine how the lack of endocytosis affects in vivo infection of T. cruzi's vertebrate and invertebrate hosts, it is difficult to fathom that such a sophisticated system would have been retained across such long stretches of evolutionary time if it did not provide a clear benefit for the organism.We simply have not yet determined when, or under what conditions, the endocytic process indeed becomes essential.Importantly, this newfound capacity to interrogate SPC function has the unique potential of converting T. cruzi into a valuable model to study protozoan phagotrophy about which we also know surprisingly little.
To begin constructing a potential model for SPC function, we should start with the first enzymatic component characterized; MyoF.Our initial localization of this molecular motor, using super-resolution microscopy, revealed MyoF to follow the corkscrew path of the SPC but, unfortunately, this technique did not allow us to see exactly where this protein was binding (Figure 5A).Likely scenarios involve either myosin tail attachment to cargo-laden vesicles and motor head movement along stable actin tracks or, conversely, tail binding to the SPC microtubule rootlets and the inward pulling of actin coated vesicles.Tail-based attachment to the microtubules was a favored model as we were able to show that the tail portions of these myosins were sufficient to send the fluorescent protein mNeon to the SPC structure, hence negating the need for the actin-binding motor head for SPC targeting.Cytochalasin treatment also appears to have no effect on MyoF localization at the SPC, again suggesting its association is actin independent.Direct evidence, however, has recently come from a follow-up study of MyoF by Alves et al. who, using immuno-EM labeling of tagged MyoF on whole-mount cytoskeletons of T. cruzi, showed that this motor protein directly associates with the rootlet microtubules (likely the CyQ although unclear) of the SPC (Figure 5B purple line; Alves et al., 2022).This observation lends credence to the idea that MyoF may, in fact, be fixed in place on the rootlets and act as a sort of "conveyer belt" for actin-coated endocytosed membrane.This might also explain how flooding the system with rigor mutants of MyoF could have impeded endocytosis with interspersed mutant enzymes irreversibly binding to and halting the inward flow of the endocytosed membrane.With MyoF fixed at the CyQ/CyT, the question then returns to the actin substrate of the SPC.The ability to visualize actin filaments has been extremely difficult in T. cruzi whether using EM or immunofluorescence microscopy (Souza et al., 1983).The majority of what we do know has come from a number of well-executed experimental studies and bioinformatic analyses of actin proteins in trypanosomatids which have shown that there are four different actin isoforms in T. cruzi while the related T. brucei has only a single isoform (Cevallos et al., 2011;Gupta et al., 2020;Vizcaino-Castillo et al., 2020;Vizcaíno-Castillo et al., 2019).A phylogenetic analysis revealed that, as compared to free-living B. saltans, P. confusum or Leishmania spp., isoforms 2 and 3 are consistently present in the genome of SPC-containing kinetoplastids.As a result, these seem like promising candidates although prior localization studies of Act2 did not show any clear localization to structures resembling the SPC within T. cruzi (Vizcaíno-Castillo et al., 2019).This could be for a variety of reasons including the short length or unbundled nature of actin microfilaments making visualization relative to the monomeric pools difficult.Since numerous studies using cytochalasin inhibitors of actin polymerization have shown this molecule to be essential for endocytosis, the identification of the actin isoform responsible will be an important step in constructing a likely model (Bogitsh et al., 1995;Chasen et al., 2019;Correa et al., 2008).
In thinking about the role of actin in the SPC, it should be reiterated that actin polymers are polar filaments with one end being referred to as barbed or plus (+) and the opposite end as pointed or minus (−; Figure 5 central schematic; Dominguez & Holmes, 2011).Polymerization of these filaments preferentially occurs at the plus end, while disassembly happens primarily at the minus end, resulting in a "treadmilling" effect that can be used to power a wide variety of cellular processes (Theriot, 2000).This filament polarity also facilitates molecular motors, such as myosins, moving in a single direction along the filament (Hartman & Spudich, 2012).Although the directionality of the myosin motors targeted to the SPC has not been experimentally determined, they are likely plus-end directed myosins based on structure and the extremely small number of minus-end directed myosins (type VI) which have been identified.With this constraint in mind, we can presume a necessity for the polarity of the actin network of the SPC in order to achieve the unidirectional flow of endocytosed membrane.With a fixed microtubule platform decorated with myosins in place (Figure 5 schematic), inward pulling of the cytopharynx membrane would require the plus ends (dark red) of the microfilament network coating the membrane to be oriented towards the cytostome opening and the minus end (light red) aiming toward the interior of the parasite.This orientation would, in many ways, be analogous to the actin network thought to support endocytosis in other vesicle trafficking systems as well (Kaksonen et al., 2006).Since, as stated previously, actin filaments in cells polymerize preferentially at the plus end and disassemble at the minus end, regulation of the location and timing of actin polymerization is likely an important point of control in the SPC-mediated endocytic process as well.One potential way of achieving this setup would be to target plus-end polymerizing enzymes to the SPC opening itself.In a recent bioinformatics analysis of actin and actin-regulating proteins, it was shown that all the human-infecting trypanosomatids have the ARP2/3 actin nucleating complex which supports branched actin networks as well as two plus-end polymerizing formin domain-containing proteins.Uniquely, however, T. cruzi and all other SPC-containing kinetoplastids retain a single additional formin isoform (TcCLB.511393.30)making it a promising candidate for this role (Gupta et al., 2020;Vizcaino-Castillo et al., 2020).This same analysis also noted that T. brucei and Leishmania spp.had lost both components (α and β subunits) of the plus-end capping complex CapZ.These capping proteins have the potential to regulate filament length or stability and therefore impact SPC function as well.
There are clearly a host of other components which are open to speculation regarding SPC function including the identity of the vesicular/tubular coat proteins, Rab GTPases and associated GAPs and GEFs, and various kinases or phosphatases that could regulate this process.With so many gaps in our understanding of SPC function, a good place to start might be to focus on the limited data we have regarding signaling events that initiate endocytosis in the first place.We can reasonably assume that once a ligand is bound to its receptor at the surface of T. cruzi, there would be a need to communicate this information across the plasma membrane to activate signaling cascades and initiate endocytosis.Prior work in T. cruzi has shown that the use of chemical inhibitors of both protein kinases and phosphatidylinositol-3 (PI3) kinases significantly diminish the endocytic rate and even cause overt surface membrane changes in the POR structure of parasites (Figure 5C; Correa et al., 2008;Schoijet et al., 2008).Kinases, it seems, could be central to this process through either modulating the activity of protein components directly or impacting the phosphatidylinositol phosphate makeup of the inner membrane which often serve as recruitment platforms for endocytic machinery (Wallroth & Haucke, 2018).It is worth pointing out that the details of how this, or any signaling pathway for that matter, operates in protozoans to sense the extracellular environment remain generally poorly understood (Subramanya & Mensa-Wilmot, 2010).This is due, in part, to the fact that standard mammalian signaling components are highly divergent or not present at all in these organisms.Kinetoplastids, for example, lack G-protein-coupled receptors entirely, have no equivalents of class I adenylyl cyclases, and no receptor ligands, agonists or antagonists have yet been identified (Gould & Koning, 2011).Even for the well-studied process of transferrin endocytosis in T. brucei, the full identity of signaling components involved remain a mystery, although the data clearly point to a role for kinases in this process as well (Horejsi et al., 1999;Joshi et al., 2007;Kariuki et al., 2019).Additionally, classical signaling is often carried out by tyrosine kinase modifications, yet the kinome of kinetoplastids is devoid of tyrosine kinase and tyrosine kinase-like groups (Bahia et al., 2009;Parsons et al., 2005).Despite this, the phosphoproteome of T. cruzi has revealed the presence of phosphorylated tyrosine, suggesting that this modification is, most likely, being carried out by either atypical tyrosine kinases or dual-specificity kinases that can phosphorylate serine, threonine, and tyrosine (Amorim et al., 2017;Marchini et al., 2011).The identity of a true tyrosine kinase in T. cruzi, however, remains to be demonstrated.The previously mentioned analysis of the kinetoplastid kinome has also revealed the existence of several putative kinases, either present only in T. cruzi or SPC-containing kinetoplastids that are promising targets for gene deletion studies that could be an important step forward in dissecting signaling networks regulating endocytosis.There are, no doubt, additional dimensions of this process that merit speculation and we suspect that there is a high probability that many of the hypotheses we propose here will not stand up to empirical testing.Only through experimentation and refinement of this model, we will arrive at a topological and mechanistic understanding of the molecular machinery driving this process.

CONCLUDING REMARKS
We would like to end this review by noting that until now, we have focused primarily on the basic mechanics of the SPC and how it might operate.This fixation on the "how" of the SPC in the end fails to address an equally important and nevertheless often neglected "why" question; Why were the salivarians able to abandon the SPC while the majority of trypanosomatids were compelled to retain it?What exactly can a cytostomecytopharynx do that a flagellar pocket cannot?In thinking about other intestinal parasites which are fecal-oral transmitted, the monoxenous apicomplexans, for example, one quickly realizes that these organisms interact with their hosts in a fundamentally different way from the trypanosomatids.The apicomplexans do not rely on food ingested by their host for sustenance, instead they employ a type of sophisticated myzocytosis or "cellular vampirism" that directly extracts cytosolic nutrients from the host's intestinal cells (Gubbels & Duraisingh, 2012).The trypanosomatids, on the other hand, operate more like members of the arthropod microbiome as they never directly target or consume their host's tissues (Schaub, 2021).Assuming that ancestral kinetoplastids were likely free-living and consumed bacteria, it is tempting to speculate that the early association between these proto-parasites and the intestinal milieu of arthropods may have been originally based on the consumption of the resident microflora itself.This mirrors what can be seen today with our own resident protozoan excavate Chilomastix mesnili which is nonpathogenic and feeds on our intestinal bacteria (Zaman et al., 2000).The establishment of a replicative niche could have then allowed the subsequent diversification of feeding targets to also include food ingested by the bug itself, thus opening the door to these kinetoplastids abandoning bacterial predation entirely.This change in food source would have, in turn, promoted a reliance on the vector's own eating habits, thus setting them in direct competition for nutrients and space not only with their host's digestive system but also with other bacterial or fungal constituents of the microbiome.Is it possible then to consider that trypanosomatids may never have fully given up this ability to phagocytose microbes?Could a reversion back to an ancestral state of consuming prokaryotic prey be a potential mechanism to explain how T. cruzi is able to survive such long periods of time between vector blood meals (Kollien & Schaub, 1998)?This is clearly an unorthodox proposition that lacks any supporting data, but it would, in the end, be one of the simplest hypotheses for why this expansive group of organisms kept this complex structure.This is an intriguing premise and with the ability to generate endocytic-null mutants, we can now begin directly testing what effect a loss of endocytosis may have on T. cruzi's ability to colonize its insect vector.
In conclusion, our overarching goal with this review has been simply to show both how much progress the field has made in dissecting the structure of the T. cruzi endocytic organelle and also illustrate how much further we still have to go to fully understand this process.Using the many seminal contributions which have shaped our perception of the SPC, we have ended this review by presenting a testable model with which to begin interrogating how this oral apparatus both captures, activates, and pulls material in.We believe that the continued investigation into this fascinatingly complex oral apparatus has the potential to not only impact our ability to combat agents of human disease, but also shed light on the evolutionary past and ecological presence of these important phagotrophic protozoans.With our recently published studies of the SPC myosin motors, we have only just begun to decipher how it is that a single-celled protozoan is able to spatially and temporally construct and control such a diverse array of components in order to engage in this unique mode of endocytosis.By combining the wealth of genomic and structural data already in hand, with the recent advances in available molecular tools to manipulate the genome of T. cruzi, the field is well positioned to begin elucidating the mechanistic basis of this ancient protozoal feeding apparatus with the goal of providing insight into fundamental processes ranging from global microbial food webs to parasitic diseases.Life cycle of Trypanosoma cruzi and dynamics of the cytostome-cytopharynx (SPC) endocytic organelle.Actively dividing epimastigotes (green) endocytose via the SPC and colonize the insect vector gastrointestinal tract.In the vector hindgut, parasites transform into metacyclic trypomastigotes (blue) break down the SPC and are excreted onto mammalian hosts.These infectious and nondividing forms of the parasite invade host cells and replicate cytosolically as amastigotes (red).The amastigotes phagocytose via the SPC before regenerating their flagellum and converting into infectious trypomastigotes (blue).These forms are able to reinvade additional host cells or are taken up by the vector during a blood meal to complete the life cycle Structure of the cytostome-cytopharynx complex (SPC).Center: A schematic reference for the endocytic apparatus of T. cruzi: Preoral ridge (green), cytostome pore (blue), cytopharynx (orange), CyQ (purple), CyT (red).(A) First transmission EM (TEM) of a trypanosomatid (T.mega) SPC entrance (blue).Adapted from Steinert and Novikoff (1960).(B) TEM of the T. cruzi SPC cytopharynx (orange).Adapted from Milder and Deane (1969) FIGURE 1.

FIGURE 4 .
FIGURE 4. Composition of the T. cruzi preoral ridge (POR).Center: schematic focus on POR region in green.(A) Freeze-fracture EM of P-face of POR (green).(B) Higher magnification of freeze-fracture EM of P-face.POR outlined in green.(C) TEM and ruthenium red stain (red arrow) of T. cruzi POR (green).SPC (arrowheads).(D) TEM and ConA staining (blue arrow) of POR (green line).(E) TEM and ruthenium red stain (red arrow) of POR (green line).(F) Freeze-fracture flip EM of POR (green outline).Figures (A-E) adapted from Martinez-Palomo et al. 1976.Figure (F) adapted fromPimenta et al. (1989)