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Tag: Drew Etheridge

Trypanosoma cruzi heme responsive gene (TcHRG) plays a central role in orchestrating heme uptake in epimastigotes

Trypanosoma cruzi, a heme auxotrophic parasite, can control intracellular heme content by modulating heme responsive gene (TcHRG) expression when a free heme source is added to an axenic culture. Herein, we explored the role of TcHRG protein in regulating the uptake of heme derived from hemoglobin in epimastigotes. We demonstrate that the endogenous TcHRG (protein and mRNA) responded similarly to bound (hemoglobin) and free (hemin) heme. Endogenous TcHRG was found in the flagellar pocket boundaries and partially overlapping with the mitochondrion. On the other hand, endocytic null parasites were able to develop and exhibited a similar heme content compared to wild type when fed with hemoglobin, indicating that endocytosis is not the main entrance pathway for hemoglobin-derived heme in this parasite. Moreover, the overexpression of TcHRG led to an increase in heme content when hemoglobin was used as the heme source. Taken together, 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 control heme homeostasis by modulating TcHRG expression independently of the available source of heme.

Evelyn Tevere, Cecilia Beatriz Di Capua, Nathan Michael Chasen, Ronald Drew Etheridge, Julia Alejandra Cricco. FEBS J. 2023 Dec 13. doi: 10.1111/febs.17030.

Massive invasion of organellar DNA drives nuclear genome evolution in Toxoplasma

Figure 1 Characteristics of NUMTs and NUPTs in T. gondii ME49
Fig. 1 Characteristics of NUMTs and NUPTs in T. gondii ME49

 

Toxoplasma gondii is a zoonotic protist pathogen that infects up to one third of the human population. This apicomplexan parasite contains three genome sequences: nuclear (65 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 integrants of mitochondrial DNA) and NUPTs (nuclear integrants of plastid DNA) 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 mya, revealed that the movement and fixation of five 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.

Sivaranjani Namasivayam, Cheng Sun, Assiatu B Bah, Jenna Oberstaller, Edwin Pierre-Louis, Ronald Drew Etheridge, Cedric Feschotte, Ellen J Pritham, Jessica C Kissinger. Proc Natl Acad Sci U S A. 2023 Nov 7;120(45):e2308569120. doi: 10.1073/pnas.2308569120.

Disruption of Toxoplasma gondii-Induced Host Cell DNA Replication Is Dependent on Contact Inhibition and Host Cell Type

The protozoan Toxoplasma gondii is a highly successful obligate intracellular parasite that, upon invasion of its host cell, releases an array of host-modulating protein effectors to counter host defenses and further its own replication and dissemination. Early studies investigating the impact of T. gondii infection on host cell function revealed that this parasite can force normally quiescent cells to activate their cell cycle program. Prior reports by two independent groups identified the dense granule protein effector HCE1/TEEGR as being solely responsible for driving host cell transcriptional changes through its direct interaction with the cyclin E regulatory complex DP1 and associated transcription factors. Our group independently identified HCE1/TEEGR through the presence of distinct repeated regions found in a number of host nuclear targeted parasite effectors and verified its central role in initiating host cell cycle changes. Additionally, we report here the time-resolved kinetics of host cell cycle transition in response to HCE1/TEEGR, using the fluorescence ubiquitination cell cycle indicator reporter line (FUCCI), and reveal the existence of a block in S-phase progression and host DNA synthesis in several cell lines commonly used in the study of T. gondii. Importantly, we have observed that this S-phase block is not due to additional dense granule effectors but rather is dependent on the host cell line background and contact inhibition status of the host monolayer in vitro. This work highlights intriguing differences in the host response to reprogramming by the parasite and raises interesting questions regarding how parasite effectors differentially manipulate the host cell depending on the in vitro or in vivo context.

IMPORTANCE Toxoplasma gondii chronically infects approximately one-third of the global population and can produce severe pathology in immunologically immature or compromised individuals. During infection, this parasite releases numerous host-targeted effector proteins that can dramatically alter the expression of a variety of host genes. A better understanding of parasite effectors and their host targets has the potential to not only provide ways to control infection but also inform us about our own basic biology. One host pathway that has been known to be altered by T. gondii infection is the cell cycle, and prior reports have identified a parasite effector, known as HCE1/TEEGR, as being responsible. In this report, we further our understanding of the kinetics of cell cycle transition induced by this effector and show that the capacity of HCE1/TEEGR to induce host cell DNA synthesis is dependent on both the cell type and the status of contact inhibition.

Edwin Pierre-Louis, Menna G Etheridge, Rodrigo de Paula Baptista, Asis Khan, Nathan M Chasen, Ronald D Etheridge. mSphere. 2022 May 19;e0016022. doi: 10.1128/msphere.00160-22.

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.

Ronald Drew Etheridge. J Eukaryot Microbiol. 2022 Feb 17;e12896. doi: 10.1111/jeu.12896.

Trainee Spotlight: Nathan Chasen

Nathan Chasen is a post-doctoral fellow in Drew Etheridge’s laboratory (submitted photo)

Nathan Chasen, a postdoctoral fellow in Drew Etheridge’s laboratory, is originally from Richmond, Virginia. After receiving his undergraduate degree from Emory University, he worked as a research technician at UGA. He then decided to attend UGA for graduate school. Under the mentorship of Silvia Moreno, Chasen received two American Heart Association Predoctoral Fellowship Awards and earned his Ph.D. in December of 2017.

Why did you choose UGA? 

I chose UGA because it is one of the best places in the world to study parasites for both the quality of the work and the collaborative research environment.

What is your research focus/project and why are you interested in the topic? 

My current research focus is the poorly understood endocytic organelle of the parasite Trypanosoma cruzi, which is the causal agent of Chagas disease.

What are your future professional plans?  

I plan to establish an academic lab that continues to unravel the nature of this neglected parasite, using state-of-the-art molecular tools and microscopy methods.

What is your favorite thing about UGA and Athens? 

The area is a great low-cost living area, with little traffic and essentially everything you need within a 15-minute drive, including great food and a lively downtown area. The ability to live affordably within a short bike ride of campus is also a plus.

 

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Trainee Spotlight: Edwin Pierre Louis

Trainee Edwin Pierre Louis

 

Edwin Pierre Louis is a pre-doctoral trainee in the laboratory of Dr. Drew Etheridge. Originally from Haiti, he immigrated to the US to attend the University of Florida (UF), where he graduated with a Bachelor of Science in Biochemistry Molecular Biology. After earning his degree at UF, Edwin accepted a position as a biological scientist in the UF Center of Excellence for Regenerative Health Biotechnology, with a focus on gene and cell based therapeutic development, where he worked for three years. There, he first discovered his love of host-pathogen interactions as a biological scientist working under the supervision of Dr. Richard Snyder for the component Florida Biologix at this center and later merged to create Brammer Bio which was subsequently acquired by Thermo Fisher Scientific. During this time in industry, he realized that to improve his scientific capacities he would need to continue his studies by pursuing a graduate degree. As part of his preparations to apply to a graduate program, he joined the UGA post-baccalaureate PREP program whose mission is to prepare students interested in a graduate degree for the application process. During this time, he was granted the opportunity to join Dr. Michael Terns’ laboratory for a year where he investigated the molecular mechanism of CRISPR-Cas based viral defense in Streptococcus thermophilus as well as prime adaptation events in the type II-A CRISPR-Cas system.

Since attending UGA, Edwin has been awarded both the Gateway to Graduate School Bridge Program and the Graduate Scholars Leadership, Engagement and Development Program (GS LEAD) scholarships sponsored by the National Science Foundation (NSF).

What is your research focus and why are you interested in the topic?

Broadly, my key research interests center around how organisms like viruses and parasites manipulate their host cell in order to grow and propagate. My current project is focused on elucidating how the protozoan pathogen Toxoplasma gondii is able to use secreted protein effectors to manipulate its host cells functions.

Why did you choose UGA?

I chose to study at the University of Georgia, in part, because of my excellent post-baccalaureate experience in the PREP program. It was evident from my interactions that UGA excels at fostering a productive relationship between students and faculty. Regardless of any faculty member’s relationship to the students, there was a sustained willingness for faculty to give of their time in order to see the students succeed.  I also decided to pursue my PhD at UGA because of the cutting-edge research and in particular the collection of outstanding parasitologists that is uniquely found in the Center for Tropical and Emerging Global Diseases (CTEGD).

What are your future professional plans?

As I continue my graduate studies on host pathogen interaction, I plan to do some post-doctoral trainings to augment my apprenticeship and ultimately become an independent scientist to lead my own research group.  I also hope to be able to give back to the local community that has contributed so much to my own personal success by donating my time and knowledge to mentor young budding scientists especially those from underprivileged homes and/or underdeveloped countries.

 

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Identification and Localization of the First Known Proteins of the Trypanosoma cruzi Cytostome Cytopharynx Endocytic Complex

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.

Nathan Michael Chasen, Isabelle Coppens and Ronald Drew Etheridge. Front Cell Infect Microbiol. 2020 Jan 17;9:445. doi: 10.3389/fcimb.2019.00445. eCollection 2019.