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Category: publications

Inorganic Polyphosphate Interacts with Nucleolar and Glycosomal Proteins in Trypanosomatids

Summary

Inorganic polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high‐energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over‐representation of nucleolar and glycosomal proteins. Using the polyphosphate‐binding domain (PPBD) of Escherichia coliexopolyphosphatase (PPX), we localized long‐chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre‐incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration‐dependent and chain length‐dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress.

Raquel S. Negreiros, Noelia Lander, Guozhong Huang, Ciro D. Cordeiro, Stephanie A. Smith, James H. Morrissey, Roberto Docampo. 2018. Molecular Microbiology; 110(6):973-994. https://doi.org/10.1111/mmi.14131

Calcium-sensitive pyruvate dehydrogenase phosphatase is required for energy metabolism, growth, differentiation, and infectivity of Trypanosoma cruzi

Abstract

In vertebrate cells, mitochondrial Ca2+ uptake by the mitochondrial calcium uniporter (MCU) leads to Ca2+-mediated stimulation of an intramitochondrial pyruvate dehydrogenase phosphatase (PDP). This enzyme dephosphorylates serine residues in the E1α subunit of pyruvate dehydrogenase (PDH), thereby activating PDH and resulting in increased ATP production. Although a phosphorylation–dephosphorylation cycle for the E1α subunit of PDH from non-vertebrate organisms has been described, the Ca2+-mediated PDP activation has not been studied. In this work we investigated the Ca2+ sensitivity of two recombinant PDPs from the protozoan human parasites Trypanosoma cruzi (TcPDP) and Trypanosoma brucei (TbPDP) and generated a TcPDP-KO cell line to establish TcPDP’s role in cell bioenergetics and survival. Moreover, the mitochondrial localization of the TcPDP was studied by CRISPR/Cas9-mediated endogenous tagging. Our results indicate that TcPDP and TbPDP both are Ca2+-sensitive phosphatases. Of note, TcPDP-KO epimastigotes exhibited increased levels of phosphorylated TcPDH, slower growth and lower oxygen consumption rates than control cells, an increased AMP:ATP ratio and autophagy under starvation conditions, and reduced differentiation into infective metacyclic forms. Furthermore, TcPDP-KO trypomastigotes were impaired in infecting culture host cells. We conclude that TcPDP is a Ca2+-stimulated mitochondrial phosphatase that dephosphorylates TcPDH and is required for normal growth, differentiation, infectivity and energy metabolism in T. cruzi.  Our results support the view that one of the main roles of the MCU is linked to the regulation of intramitochondrial dehydrogenases.

Noelia Lander, Miguel A. Chiurillo, Mayara S. Bertolini, Melissa Storey, Anibal E. Vercesi and Roberto Docampo. 2018. Journal of Biological Chemistry; 293(45):17402-17417. doi: 10.1074/jbc.RA118.004498

The Mitochondrial Ca2+ Uniporter Complex (MCUC) of Trypanosoma brucei Is a Hetero-oligomer That Contains Novel Subunits Essential for Ca2+ Uptake

ABSTRACT

The mitochondrial calcium uniporter complex (MCUC) is a highly selective channel that conducts calcium ions across the organelle inner membrane. We previously characterized Trypanosoma brucei’s MCU (TbMCU) as an essential component of the MCUC required for parasite viability and infectivity. In this study, we characterize its paralog T. brucei MCUb (TbMCUb) and report the identification of two novel components of the complex that we named TbMCUc and TbMCUd. These new MCUC proteins are unique and conserved only in trypanosomatids. In situ tagging and immunofluorescence microscopy revealed that they colocalize with TbMCU and TbMCUb to the mitochondria of T. brucei. Blue Native PAGE and immunodetection analyses indicated that the MCUC proteins exist in a large protein complex with a molecular weight of approximately 380 kDa. RNA interference (RNAi) or overexpression of the TbMCUc and TbMCUd genes significantly reduced or enhanced mitochondrial Ca2+uptake in T. brucei, respectively, without affecting the mitochondrial membrane potential, indicating that they are essential components of the MCUC of this parasite. The specific interactions of TbMCU with TbMCUb, TbMCUc, or TbMCUd were confirmed by coimmunoprecipitation and split-ubiquitin membrane-based yeast two-hybrid (MYTH) assays. Furthermore, combining mutagenesis analysis with MYTH assays revealed that transmembrane helices (TMHs) were determinant of the interactions between TbMCUC subunits. In summary, our study has identified two novel essential components of the MCUC of T. brucei and defined their direct physical interactions with the other subunits that result in a hetero-oligomeric MCUC.

Guozhong Huang, Roberto Docampo. 2018. Molecular Biology and Physiology. DOI: 10.1128/mBio.01700-18

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

ABSTRACT

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

 

Kudyba, H. M., Cobb, D. W., Florentin, A., Krakowiak, M., Muralidharan, V. 2018. J. Vis. Exp. (139), e57747, doi:10.3791/57747

 

Young Adults in Endemic Areas: An Untreated Group in Need of School-Based Preventive Chemotherapy for Schistosomiasis Control and Elimination

Abstract

Parasitologic surveys of young adults in college and university settings are not commonly done, even in areas known to be endemic for schistosomiasis and soil-transmitted helminths. We have done a survey of 291 students and staff at the Kisumu National Polytechnic in Kisumu, Kenya, using the stool microscopy Kato-Katz (KK) method and the urine point-of-care circulating cathodic antigen (POC-CCA) test. Based on three stools/two KK slides each, in the 208 participants for whom three consecutive stools were obtained, Schistosoma mansoni prevalence was 17.8%. When all 291 individuals were analyzed based on the first stool, as done by the national neglected tropical disease (NTD) program, and one urine POC-CCA assay (n = 276), the prevalence was 13.7% by KK and 23.2% by POC-CCA. Based on three stools, 2.5% of 208 participants had heavy S. mansoni infections (≥400 eggs/gram feces), with heavy S. mansoni infections making up 13.5% of the S. mansoni cases. The prevalence of the soil-transmitted helminths (STH: Ascaris lumbricoidesTrichuris trichiura and hookworm) by three stools was 1.4%, 3.1%, and 4.1%, respectively, and by the first stool was 1.4%, 2.4% and 1.4%, respectively. This prevalence and intensity of infection with S. mansoni in a college setting warrants mass drug administration with praziquantel. This population of young adults is ‘in school’ and is both approachable and worthy of inclusion in national schistosomiasis control and elimination programs.

Harrison K. Korir, Diana K. Riner, Emmy Kavere, Amos Omondi, Jasmine Landry, Nupur Kittur, Eric M. Ndombi, Bartholomew N. Ondigo, W. Evan Secor, Diana M. S. Karanja and Daniel G. Colley. 2018. Trop. Med. Infect. Dis.; 3(3):100. https://doi.org/10.3390/tropicalmed3030100

Antiplasmodial flavanones and a stilbene from Carpha glomerata

graphical abstract

Abstract

Bioassay-guided fractionation of an extract of Carpha glomerata (Cyperaceae) led to the isolation of seven compounds. Compounds 1 (carphorin A), 3 (carphorin C), 4(carphorin D), and 5 (carphabene) are new compounds, and compound 2 (8-(3″-hydroxyisoamyl)-naringenin) was isolated for the first time as a natural product. All structures were elucidated based on analyses of their HR-ESIMS and 1D and 2D NMR data. Compounds 12, and 6, which have prenyl or hydroxyprenyl side chains, exhibited antiplasmodial activities with IC50 values of 5.2 ± 0.6, 3.4 ± 0.4, and 6.7 ± 0.8 µM against the drug-resistant Dd2 strain of Plasmodium falciparum. In addition the prenylated stilbene 5 also showed good activity, with IC50 5.8 ± 0.7 µM.

Namki Cho, Ana Lisa Valenciano, Yongle Du, Jason Clement, Maria B. Cassera, Michael Goetz, David G. I. Kingston. 2018. Bioorganic & Medicinal Chemistry Letters; 28(20):3368-3371. https://doi.org/10.1016/j.bmcl.2018.09.003

Spatio-temporal spillover risk of yellow fever in Brazil

Abstract

Background: Yellow fever virus is a mosquito-borne flavivirus that persists in an enzoonotic cycle in non-human primates (NHPs) in Brazil, causing disease in humans through spillover events. Yellow fever (YF) re-emerged in the early 2000s, spreading from the Amazon River basin towards the previously considered low-risk, southeastern region of the country. Previous methods mapping YF spillover risk do not incorporate the temporal dynamics and ecological context of the disease, and are therefore unable to predict seasonality in spatial risk across Brazil. We present the results of a bagged logistic regression predicting the propensity for YF spillover per municipality (administrative sub-district) in Brazil from environmental and demographic covariates aggregated by month. Ecological context was incorporated by creating National and Regional models of spillover dynamics, where the Regional model consisted of two separate models determined by the regions’ NHP reservoir species richness (high vs low).

Results: Of the 5560 municipalities, 82 reported YF cases from 2001 to 2013. Model accuracy was high for the National and low reservoir richness (LRR) models (AUC = 0.80), while the high reservoir richness (HRR) model accuracy was lower (AUC = 0.63). The National model predicted consistently high spillover risk in the Amazon, while the Regional model predicted strong seasonality in spillover risk. Within the Regional model, seasonality of spillover risk in the HRR region was asynchronous to the LRR region. However, the observed seasonality of spillover risk in the LRR Regional model mirrored the national model predictions.

Conclusions: The predicted risk of YF spillover varies with space and time. Seasonal trends differ between regions indicating, at times, spillover risk can be higher in the urban coastal regions than the Amazon River basin which is counterintuitive based on current YF risk maps. Understanding the spatio-temporal patterns of YF spillover risk could better inform allocation of public health services.

RajReni B. Kaul, Michelle V. Evans, Courtney C. Murdock and John M. Drake. 2018. Parasites & Vectors; 11:488. https://doi.org/10.1186/s13071-018-3063-6

Estimating the effects of variation in viremia on mosquito susceptibility, infectiousness, and R0 of Zika in Aedes aegypti

Abstract

Zika virus (ZIKV) is an arbovirus primarily transmitted by Aedes mosquitoes. Like most viral infections, ZIKV viremia varies over several orders of magnitude, with unknown consequences for transmission. To determine the effect of viral concentration on ZIKV transmission risk, we exposed field-derived Aeaegypti mosquitoes to four doses (103, 104, 105, 106 PFU/mL) representative of potential variation in the field. We demonstrate that increasing ZIKV dose in the blood-meal significantly increases the probability of mosquitoes becoming infected, and consequently disseminating virus and becoming infectious. Additionally, we observed significant interactions between dose and days post-infection on dissemination and overall transmission efficiency, suggesting that variation in ZIKV dose affects the rates of midgut escape and salivary gland invasion. We did not find significant effects of dose on mosquito mortality. We also demonstrate that detecting virus using RT-qPCR approaches rather than plaque assays potentially over-estimates key transmission parameters, including the time at which mosquitoes become infectious and viral burden. Finally, using these data to parameterize an R0 model, we showed that increasing viremia from 104 to 106 PFU/mL increased relative R0 3.8-fold, demonstrating that variation in viremia substantially affects transmission risk.

Blanka Tesla, Leah R. Demakovsky, Hannah S. Packiam, Erin A. Mordecai, Américo D. Rodríguez, Matthew H. Bonds, Melinda A. Brindley, Courtney C. Murdock. 2018. PLOS Neglected Tropical Diseases; 12(8): e0006733. https://doi.org/10.1371/journal.pntd.0006733

Temperature drives Zika virus transmission: evidence from empirical and mathematical models

Abstract

Temperature is a strong driver of vector-borne disease transmission. Yet, for emerging arboviruses we lack fundamental knowledge on the relationship between transmission and temperature. Current models rely on the untested assumption that Zika virus responds similarly to dengue virus, potentially limiting our ability to accurately predict the spread of Zika. We conducted experiments to estimate the thermal performance of Zika virus (ZIKV) in field-derived Aedes aegypti across eight constant temperatures. We observed strong, unimodal effects of temperature on vector competence, extrinsic incubation period and mosquito survival. We used thermal responses of these traits to update an existing temperature-dependent model to infer temperature effects on ZIKV transmission. ZIKV transmission was optimized at 29°C, and had a thermal range of 22.7°C–34.7°C. Thus, as temperatures move towards the predicted thermal optimum (29°C) owing to climate change, urbanization or seasonality, Zika could expand north and into longer seasons. By contrast, areas that are near the thermal optimum were predicted to experience a decrease in overall environmental suitability. We also demonstrate that the predicted thermal minimum for Zika transmission is 5°C warmer than that of dengue, and current global estimates on the environmental suitability for Zika are greatly over-predicting its possible range.

Blanka Tesla, Leah R. Demakovsky, Erin A. Mordecai, Sadie J. Ryan, Matthew H. Bonds, Calistus N. Ngonghala, Melinda A. Brindley, and Courtney C. Murdock. 2018. Proceedings of the Royal Society B; 285(1884):0962-8452. https://doi.org/10.1098/rspb.2018.0795

When Should the Emphasis on Schistosomiasis Control Move to Elimination?

Abstract

The stated goal of the World Health Organization’s program on schistosomiasis is paraphrased as follows: to control morbidity and eliminate transmission where feasible. Switching from a goal of controlling morbidity to interrupting transmission may well be currently feasible in some countries in the Caribbean, some areas in South America, northern Africa, and selected endemic areas in sub-Saharan Africa where there have been improvements in sanitation and access to clean water. However, in most of sub-Saharan Africa, where programmatic interventions still consist solely of annual mass drug administration, such a switch in strategies remains premature. There is a continued need for operational research on how best to reduce transmission to a point where interruption of transmission may be achievable. The level of infection at which it is feasible to transition from control to elimination must also be defined. In parallel, there is also a need to develop and evaluate approaches for achieving and validating elimination. There are currently neither evidence-based methods nor tools for breaking transmission or verifying that it has been accomplished. The basis for these statements stems from numerous studies that will be reviewed and summarized in this article; many, but not all of which were undertaken as part of SCORE, the Schistosomiasis Consortium for Operational Research and Evaluation

W. Evan Secor and Daniel G. Colley. 2018. Trop. Med. Infect. Dis.; 3(3):85. https://doi.org/10.3390/tropicalmed3030085