Abstract
The etiological agent of Chagas disease, Trypanosoma cruzi, is an obligate intracellular parasite that infects an estimated 7 million people in the Americas, with an at-risk population of 70 million. Despite its recognition as the highest impact parasitic infection of the Americas, Chagas disease continues to receive insufficient attention and resources in order to be effectively combatted. Unlike the other parasitic trypanosomatids that infect humans (Trypanosoma brucei and Leishmania spp.), T. cruzi retains an ancestral mode of phagotrophic feeding via an endocytic organelle known as the cytostome-cytopharynx complex (SPC). How this tubular invagination of the plasma membrane functions to bring in nutrients is poorly understood at a mechanistic level, partially due to a lack of knowledge of the protein machinery specifically targeted to this structure. Using a combination of CRISPR/Cas9 mediated endogenous tagging, fluorescently labeled overexpression constructs and endocytic assays, we have identified the first known SPC targeted protein (CP1). The CP1 labeled structure co-localizes with endocytosed protein and undergoes disassembly in infectious forms and reconstitution in replicative forms. Additionally, through the use of immunoprecipitation and mass spectrometry techniques, we have identified two additional CP1-associated proteins (CP2 and CP3) that also target to this endocytic organelle. Our localization studies using fluorescently tagged proteins and surface lectin staining have also allowed us, for the first time, to specifically define the location of the intriguing pre-oral ridge (POR) surface prominence at the SPC entrance through the use of super-resolution light microscopy. This work is a first glimpse into the proteome of the SPC and provides the tools for further characterization of this enigmatic endocytic organelle. A better understanding of how this deadly pathogen acquires nutrients from its host will potentially direct us toward new therapeutic targets to combat infection.
Highlights
The causal agent of Chagas disease, Trypanosoma cruzi, is an obligate intracellular parasite of the kinetoplastid family that infects upwards of 7 million people in the Americas with ∼30% developing life-threatening clinical disease (WHO, 2015; PerezMolina and Molina, 2018)
With the recent identification of the earliest branching monoxenous trypanosomatid, Paratrypanosoma confusum, as the “missing link” between B. saltans and the dixenous trypanosomatids (Simpson et al, 2002; Stevens, 2008), it has become clear that obligatory parasitism first began as an association with the arthropod lineage and that the dixenous parasitism we see in the pathogenic trypanosomatids likely arose independently on several occasions
In images from immunofluorescence assays (IFA) taken using super-resolution structured illumination microscopy (SR-SIM), we noted the localization of CP1-Ty to a distinct linear structure that was present in both of the replicating stages, the epimastigote and amastigote (Figures 1D,F), but was absent in the non-replicating trypomastigote stage (Figure 1E)
Summary
The causal agent of Chagas disease, Trypanosoma cruzi, is an obligate intracellular parasite of the kinetoplastid family that infects upwards of 7 million people in the Americas with ∼30% developing life-threatening clinical disease (WHO, 2015; PerezMolina and Molina, 2018). The most heavily studied kinetoplastids are the disease causing parasites of humans and domesticated animals (Trypanosoma spp. and Leishmania spp.), these organisms originally evolved from environmentally ubiquitous free-living, flagellated protozoans of the Excavata supergroup (phylum Euglenozoa) (Simpson et al, 2002; Lukes et al, 2014) These early branching eukaryotes comprise a diverse family of flagellates characterized primarily by the presence of an “excavated” feeding groove used to funnel food into the mouth of the cell, a plasma membrane pore known as the cytostome. It is notable that the dixenous stercorarian trypanosomes (which includes T. cruzi) still rely on fecal transmission from the insect to their vertebrate host This initial transition of freeliving kinetoplastids into arthropod parasites coincided with an acute reduction in the size of their genome (∼50% gene loss), which included the loss of many metabolic pathways (Opperdoes et al, 2016). In the second major transition of the pathogenic trypanosomatids to a dixenous lifestyle, T. cruzi alone retained the SPC whereas the salivarian lineages (Trypanosoma brucei and Leishmania spp.) abandoned this endocytic structure entirely (Porto-Carreiro et al, 2000; Field and Carrington, 2009)
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