Biology subjects

Etheridge, R. D.

Publications and source records attributed to Etheridge, R. D..

3 recordsLinked to original sources

bioRxiv2023

Massive invasion of organellar DNA drives nuclear genome evolution in Toxoplasma

Toxoplasma gondii is a zoonotic protist pathogen that infects up to 1/3 of the human population. This apicomplexan parasite contains three genome sequences: nuclear (63 Mb); plastid organellar, ptDNA (35 kb); and mitochondrial organellar, mtDNA (5.9 kb of non-repetitive sequence). We find that the nuclear genome contains a significant amount of NUMTs (nuclear DNA of mitochondrial origin) and NUPTs (nuclear DNA of plastid origin) that are continuously acquired and represent a significant source of intraspecific genetic variation. NUOT (nuclear DNA of organellar origin) accretion has generated 1.6% of the extant T. gondii ME49 nuclear genome; the highest fraction ever reported in any organism. NUOTs are primarily found in organisms that retain the non-homologous end-joining repair pathway. Significant movement of organellar DNA was experimentally captured via amplicon sequencing of a CRISPR-induced double-strand break in non-homologous end-joining repair competent, but not ku80 mutant, Toxoplasma parasites. Comparisons with Neospora caninum, a species that diverged from Toxoplasma [~]28 MY ago, revealed that the movement and fixation of 5 NUMTs predates the split of the two genera. This unexpected level of NUMT conservation suggests evolutionary constraint for cellular function. Most NUMT insertions reside within (60%) or nearby genes (23% within 1.5 kb) and reporter assays indicate that some NUMTs have the ability to function as cis-regulatory elements modulating gene expression. Together these findings portray a role for organellar sequence insertion in dynamically shaping the genomic architecture and likely contributing to adaptation and phenotypic changes in this important human pathogen. Significance StatementThis study reveals how DNA located in cellular compartments called organelles can be transferred to the nucleus of the cell and inserted into the nuclear genome of apicomplexan parasite Toxoplasma. Insertions alter the DNA sequence and may lead to significant changes in how genes function. Unexpectedly, we found that the human protist pathogen, Toxoplasma gondii and closely-related species have the largest observed organellar genome fragment content (>11,000 insertion comprising over 1 Mb of DNA) inserted into their nuclear genome sequence despite their compact 65 Mb nuclear genome. Insertions are occurring at a rate that makes them a significant mutational force that deserves further investigation when examining causes of adaptation and virulence of these parasites.

evolutionary biology↗

bioRxiv2023

TcHRG plays a central role in orchestrating heme uptake in Trypanosoma cruzi epimastigotes

Trypanosoma cruzi, a heme auxotrophic parasite, can control intracellular heme content by modulating TcHRG expression when a free heme source is added to axenic culture. Herein, we explore the role of TcHRG protein in regulating the uptake of heme derived from hemoglobin in epimastigotes. It was found that the parasites endogenous TcHRG (protein and mRNA) responds similarly to bound (hemoglobin) and free (hemin) heme. Additionally, the overexpression of TcHRG leads to an increase in intracellular heme content. The localization of TcHRG is also not affected in parasites supplemented with hemoglobin as the sole heme source. Endocytic null epimastigotes do not show a significant difference in growth profile, intracellular heme content and TcHRG protein accumulation compared to WT when feeding with hemoglobin or hemin as a source of heme. These results suggest that the uptake of hemoglobin-derived heme likely occurs through extracellular proteolysis of hemoglobin via the flagellar pocket, and this process is governed by TcHRG. In sum, T. cruzi epimastigotes controls heme homeostasis by modulating TcHRG expression independently of the source of available heme.

biochemistry↗

bioRxiv2022

Endocytosis in Trypanosoma cruzi Depends on Proper Recruitment and Regulation of Functionally Redundant Myosin Motors

Utilized by the free-living kinetoplastid Bodo saltans to feed on bacterial prey, the cytostome-cytopharynx complex (SPC) is an endocytic organelle absent from all human trypanosomatid pathogens save Trypanosoma cruzi. Building upon our previous work identifying the myosin motor MyoF as the first enzymatic component of the T. cruzi SPC, we sought to expand our understanding of this distinct organelle by identifying additional protein machinery which contribute to the endocytic process. While deletion of MyoF alone did not fully ablate endocytosis, we found that deletion of both MyoF and the similarly localized MyoC produced an endocytic-null phenotype that was rescued upon complementation. To identify potential regulatory components of this motor complex, we pulled down MyoF and identified an SPC-targeted protein that contained an annotated EF-hand calcium-binding motif that was conserved across a wide range of protozoan lineages. Surprisingly, deletion of this myosin associated protein (MyAP) alone was sufficient to produce an endocytic-null phenotype, which we were able to fully rescue via complementation. The deletion of MyAP also caused the mis-localization of both cytopharynx myosins to the cytosol. While MyAP lacking the EF-hand domain was unable to complement endocytosis, it was sufficient to restore proper myosin localization. This suggested that MyAP plays two distinct roles, one in targeting myosins to the SPC and a second in regulating myosin motor activity. Transmission electron microscopy also revealed that endocytic-null mutants lacked the electron lucent lipid inclusions typically seen in the pre-lysosomal reservosomes of T. cruzi epimastigotes. Mass spectrometry based lipidomic analysis subsequently revealed a dramatic reduction in the scavenged cholesterol content in the endocytic-null mutants, which can be attributed to an inability to endocytose exogenous lipid-protein complexes for storage in the reservosomes. Overall, this work showcases the first viable endocytic-null mutants generated in T. cruzi through specific gene deletion and highlights the feasibility of leveraging this strategy towards a full dissection of the endocytic machinery and biogenesis of the SPC. ImportanceTrypanosoma cruzi chronically infects over 7 million people in the Americas and current therapeutics are insufficient to effectively cure infection. The lack of progress in developing effective vaccines or drug treatments is due, in part, to longstanding technical limitations in studying this parasite and a lack of resources committed to support research and eradication efforts. As part of its parasitic lifestyle, T. cruzi is forced to obtain basic nutrients directly from its host environment, making the development of methods to block nutrient uptake an attractive strategy to control parasite growth and transmission. While the bulk uptake of complex nutrients by T. cruzi occurs via an endocytic structure, often referred to as the cytostome-cytopharynx complex (SPC), how exactly this tubular endocytic organelle functions at a mechanistic level has remained a mystery. In this work, we investigated the contribution of several SPC targeted myosin motors and an associated protein factor to endocytic activity. By identifying and characterizing the molecular machinery responsible for nutrient uptake, we hope to both expand our basic understanding of how this deadly pathogen acquires essential nutrients from its host, while also revealing new potential therapeutic targets to impede nutrient uptake.

microbiology↗