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Category: publications

Genome Editing by CRISPR/Cas9 in Trypanosoma cruzi

Donna Huber on March 15, 2019

The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 system for genome editing in this organism. The system was initially used for gene knockout in T. cruzi, later on for endogenous gene tagging and more recently for gene complementation. Mutant cell lines obtained by CRISPR/Cas9 have been used for the functional characterization of proteins in different stages of this parasite’s life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter we describe the methodology to achieve genome editing by CRISPR/Cas9 in T. cruzi. Our method involves the utilization of a template cassette (donor DNA) to promote double-strand break repair by homologous directed repair (HDR). In this way, we have generated homogeneous populations of genetically modified parasites in 4–5 weeks without the need of cell sorting, selection of clonal populations, or insertion of more than one resistance marker to modify both alleles of the gene. The methodology has been organized according to three main genetic purposes: gene knockout, gene complementation of knockout cell lines generated by CRISPR/Cas9, and C-terminal tagging of endogenous genes in T. cruzi. In addition, we refer to the specific results that have been published using each one of these strategies.

 

Noelia Lander, Miguel A. Chiurillo, Roberto Docampo. 2019. Methods Mol Biol. 2019;1955:61-76. doi: 10.1007/978-1-4939-9148-8_5

Serological proteomic screening and evaluation of a recombinant egg antigen for the diagnosis of low-intensity Schistosoma mansoni infections in endemic area in Brazil

Donna Huber on March 14, 2019

BACKGROUND:

Despite decades of use of control programs, schistosomiasis remains a global public health problem. To further reduce prevalence and intensity of infection, or to achieve the goal of elimination in low-endemic areas, there needs to be better diagnostic tools to detect low-intensity infections in low-endemic areas in Brazil. The rationale for development of new diagnostic tools is that the current standard test Kato-Katz (KK) is not sensitive enough to detect low-intensity infections in low-endemic areas. In order to develop new diagnostic tools, we employed a proteomics approach to identify biomarkers associated with schistosome-specific immune responses in hopes of developing sensitive and specific new methods for immunodiagnosis.

 

METHODS AND FINDINGS:

Immunoproteomic analyses were performed on egg extracts of Schistosoma mansoni using pooled sera from infected or non-infected individuals from a low-endemic area of Brazil. Cross reactivity with other soil-transmitted helminths (STH) was determined using pooled sera from individuals uniquely infected with different helminths. Using this approach, we identified 23 targets recognized by schistosome acute and chronic sera samples. To identify immunoreactive targets that were likely glycan epitopes, we compared these targets to the immunoreactivity of spots treated with sodium metaperiodate oxidation of egg extract. This treatment yielded 12/23 spots maintaining immunoreactivity, suggesting that they were protein epitopes. From these 12 spots, 11 spots cross-reacted with sera from individuals infected with other STH and 10 spots cross-reacted with the negative control group. Spot number 5 was exclusively immunoreactive with sera from S. mansoni-infected groups in native and deglycosylated conditions and corresponds to Major Egg Antigen (MEA). We expressed MEA as a recombinant protein and showed a similar recognition pattern to that of the native protein via western blot. IgG-ELISA gave a sensitivity of 87.10% and specificity of 89.09% represented by area under the ROC curve of 0.95. IgG-ELISA performed better than the conventional KK (2 slides), identifying 56/64 cases harboring 1-10 eggs per gram of feces that were undiagnosed by KK parasitological technique.

 

CONCLUSIONS:

The serological proteome approach was able to identify a new diagnostic candidate. The recombinant egg antigen provided good performance in IgG-ELISA to detect individuals with extreme low-intensity infections (1 egg per gram of feces). Therefore, the IgG-ELISA using this newly identified recombinant MEA can be a useful tool combined with other techniques in low-endemic areas to determine the true prevalence of schistosome infection that is underestimated by the KK method. Further, to overcome the complexity of ELISA in the field, a second generation of antibody-based rapid diagnostic tests (RDT) can be developed.

 

Vanessa Silva-Moraes, Lisa Marie Shollenberger, William Castro-Borges, Ana Lucia Teles Rabello, Donald A. Harn, Lia Carolina Soares Medeiros, Wander de Jesus Jeremias, Liliane Maria Vidal Siqueira, Caroline Stephane Salviano Pereira, Maria Luysa Camargos Pedrosa, Nathalie Bonatti Franco Almeida, Aureo Almeida, Jose Roberto Lambertucci, Nídia Francisca de Figueiredo Carneiro, Paulo Marcos Zech Coelho, Rafaella Fortini Queiroz Grenfell. 2019. PLOS Neglected Tropical Diseases.
https://doi.org/10.1371/journal.pntd.0006974

Further insights of selenium-containing analogues of WC-9 against Trypanosoma cruzi

Donna Huber on February 28, 2019

Graphical abstract

As a continuation of our project aimed at searching for new chemotherapeutic agents against American trypanosomiasis (Chagas disease), new selenocyanate derivatives were designed, synthesized and biologically evaluated against the clinically more relevant dividing form of Trypanosoma cruzi, the etiologic agent of this illness. In addition, in order to establish the role of each part of the selenocyanate moiety, different derivatives, in which the selenium atom or the cyano group were absent, were conceived, synthesized and biologically evaluated. In addition, in order to study the optimal position of the terminal phenoxy group, new regioisomers of WC-9 were synthesized and evaluated against T. cruzi. Finally, the resolution of a racemic mixture of a very potent conformationally rigid analogue of WC-9 was accomplished and further tested as growth inhibitors of T. cruzi proliferation. The results provide further insight into the role of the selenocyanate group in its antiparasitic activity.

 

María N. Chao, María V. Lorenzo-Ocampo, Sergio H. Szajnman, Roberto Docampo, Juan B. Rodriguez. 2019. Bioorganic & Medicinal Chemistry. https://doi.org/10.1016/j.bmc.2019.02.039

Prime-Boost Vaccine Regimen for SjTPI and SjC23 Schistosome Vaccines, Increases Efficacy in Water Buffalo in a Field Trial in China

Donna Huber on February 20, 2019

Schistosomiasis remains a serious zoonotic disease in China and the Philippines. Water buffalo and cattle account for the majority of transmission. Vaccination of water buffalo is considered a key strategy to reduce disease prevalence. Previously, we showed that vaccination of water buffalo with SjC23 or SjCTPI plasmid DNA vaccines, induced 50% efficacy to challenge infection. Here, we evaluated several parameters to determine if we can develop a two dose vaccine that maintains the efficacy of the three dose vaccine. We performed four trials evaluating: (1) lab produced vs. GLP grade vaccines, (2) varying the time between prime and boost, (3) the influence of an IL-12 adjuvant, and (4) a two dose heterologous (DNA-protein) prime-boost. We found the source of the DNA vaccines did not matter, nor did increasing the interval between prime and boost. Elimination of the IL-12 plasmid lowered homologous DNA-DNA vaccine efficacy. A major finding was that the heterologous prime boost improved vaccine efficacy, with the prime-boost regimen incorporating both antigens providing a 55% reduction in adult worms and 53% reduction in liver eggs. Vaccinated buffalo produced vaccine-specific antibody responses. These trials suggest that highly effective vaccination against schistosomes can be achieved using a two dose regimen. No adjuvants were used with the protein boost, and the potential that addition of adjuvant to the protein boost to further increase efficacy should be evaluated. These results suggest that use of these two schistosome vaccines can be part of an integrated control strategy to reduce transmission of schistosomiasis in Asia.

 

Akram A. Da’Dara, Changlin Li, Xinling Yu, Mao Zheng, Jie Zhou, Lisa M. Shollenberger, Yue-sheng Li and Donald A. Harn. 2019. Front. Immunol. https://doi.org/10.3389/fimmu.2019.00284

Targeted Inhibition of Plasmodium falciparum Calcium-Dependent Protein Kinase 1 with a Constrained J Domain-Derived Disruptor Peptide

Donna Huber on February 19, 2019

graphical abstract

To explore the possibility of constrained peptides to target Plasmodium-infected cells, we designed a J domain mimetic derived from Plasmodium falciparum calcium-dependent protein kinase 1 ( PfCDPK1) as a strategy to disrupt J domain binding and inhibit PfCDPK1 activity. The J domain disruptor (JDD) peptide was conformationally constrained using a hydrocarbon staple and was found to selectively permeate segmented schizonts and colocalize with intracellular merozoites in late-stage parasites. In vitro analyses demonstrated that JDD could effectively inhibit the catalytic activity of recombinant PfCDPK1 in the low micromolar range. Treatment of late-stage parasites with JDD resulted in a significant decrease in parasite viability mediated by a blockage of merozoite invasion, consistent with a primary effect of PfCDPK1 inhibition. To the best of our knowledge, this marks the first use of stapled peptides designed to specifically target a Plasmodium falciparum protein and demonstrates that stapled peptides may serve as useful tools for exploring potential antimalarial agents.

Briana R. Flaherty, Tienhuei G. Ho, Sven H. Schmidt, Friedrich W. Herberg, David S. Peterson, and Eileen J. Kennedy. 2019. ACS Infectious Diseases. DOI: 10.1021/acsinfecdis.8b0034

Priority use cases for antibody-detecting assays of recent malaria exposure as tools to achieve and sustain malaria elimination

Donna Huber on February 12, 2019

Measurement of malaria specific antibody responses represents a practical and informative method for malaria control programs to assess recent exposure to infection. Technical advances in recombinant antigen production, serological screening platforms, and analytical methods have enabled the identification of several target antigens for laboratory based and point-of-contact tests. Questions remain as to how these serological assays can best be integrated into malaria surveillance activities to inform programmatic decision-making. This report synthesizes discussions from a convening at Institut Pasteur in Paris in June 2017 aimed at defining practical and informative use cases for serology applications and highlights five programmatic uses for serological assays including: documenting the absence of transmission; stratification of transmission; measuring the effect of interventions; informing a decentralized immediate response;  and testing and treating P. vivax hypnozoite carriers.

Greenhouse B, Daily J, Guinovart C, Goncalves B, Beeson J, Bell D, Chang MA, Cohen JM, Ding X, Domingo G, Eisele TP, Lammie PJ, Mayor A, Merienne N, Monteiro W, Painter J, Rodriguez I, White M, Drakeley C, Mueller I, Malaria Serology Convening. 2019. Gates Open Res.; doi: 10.12688/gatesopenres.12897.1. eCollection 2019.

Mitochondrial genome sequence variation as a useful marker for assessing genetic heterogeneity among Cyclospora cayetanensis isolates and source-tracking

Donna Huber on January 21, 2019

Abstract

BACKGROUND: Cyclospora cayetanensis is an important enteric pathogen, causing diarrhea and food-borne cyclosporiasis outbreaks. For effective outbreak identification and investigation, it is essential to rapidly assess the genetic heterogeneity of C. cayetanensis specimens from cluster cases and identify the likely occurrence of outbreaks.

METHODS: In this study, we developed a quantitative PCR (qPCR) targeting the polymorphic link region between copies of the mitochondrial genome of C. cayetanensis, and evaluated the genetic heterogeneity among 36 specimens from six countries using melt curve, gel electrophoresis, and sequence analyses of the qPCR products.

RESULTS: All specimens were amplified successfully in the qPCR and produced melt peaks with different Tm values in the melt curve analysis. In gel electrophoresis of the qPCR products, the specimens yielded bands of variable sizes. Nine genotypes were identified by DNA sequencing of the qPCR products. Geographical segregation of genotypes was observed among specimens analyzed, which could be useful in geographical source-tracking.

CONCLUSIONS: The length and nucleotide sequence variations in the mitochondrial genome marker allow rapid assessment of the genetic heterogeneity among C. cayetanensis specimens by melt curve, gel electrophoresis, or DNA sequence analysis of qPCR products. The sequence data generated could be helpful in the initial source-tracking of the pathogen.

KEYWORDS: Cyclospora cayetanensis; Genotyping; Mitochondrion; Source-tracking; qPCR

Yaqiong Guo, Yuanfei Wang, Xiaolan Wang, Longxian Zhang, Ynes Ortega, and Yaoyu Feng. Parasite Vectors 2019 Jan 21; 12(1):47. doi.1186/s13071-019-3294-1

Trypanosoma cruzi 13C-labeled O-Glycan standards for mass spectrometry

Donna Huber on January 12, 2019

Abstract

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, a debilitating condition that affects over 10 million humans in the American continents. In addition to its traditional mode of human entry via the ‘kissing bug’ in endemic areas, the infection can also be spread in non-endemic countries through blood transfusion, organ transplantation, eating food contaminated with the parasites, and from mother to fetus. Previous NMR-based studies established that the parasite expresses a variety of strain-specific and developmentally-regulated O-glycans that may contribute to virulence. In this report, we describe five synthetic O-glycan analytical standards and show their potential to enable a more facile analysis of native O-glycan isomers based on mass spectrometry.

M. Osman Sheikh, Elisabet Gas-Pascual, John N. Glushka, Juan M. Bustamante, Lance Wells, Christopher M. West. 2019. Glycobiology. https://doi.org/10.1093/glycob/cwy111

Kinetoplast Division Factors in a Trypanosome

Donna Huber on January 10, 2019

Trypanosoma brucei

Highlights

  • Kinetoplasts (mitochondrial genome nucleoids) are important in bloodstream trypanosomes for the establishment of mitochondrial membrane potential.
  • Many proteins involved in segregation of kinetoplasts have been identified.
  • A region between a kinetoplast and basal bodies is described as a tripartite attachment complex (TAC).
  • A set of TAC-associated proteins (TACAPs) has been proposed as the machinery for kinetoplast segregation.
  • Subcomplexes of TACAPs that form in vivo have been described.
  • Several proteins that do not associate with TAC are involved in the maintenance of the kinetoplast.
  • New kinetoplast-associated proteins have been identified.
  • We are approaching an exciting period in the field when a molecular understanding of how all aspects of kinetoplast biogenesis are executed seems achievable.

Kojo Mensa-Wilmot, Benjamin Hoffman, Justin Wiedman, Catherine Sullenberger, Amrita Sharma. 2019. Trends in Parasitology. https://doi.org/10.1016/j.pt.2018.11.002

Survival and Internalization of Salmonella and Escherichia coli O157:H7 Sprayed onto Different Cabbage Cultivars during Cultivation in Growth Chambers

Donna Huber on January 9, 2019

cabbage

ABSTRACT

BACKGROUND: Cabbage may become contaminated with enteric pathogens during cultivation. Using multiple cabbage cultivars at two maturity stages (small plants or plants with small heads) in growth chamber studies, the fate (internalization or surface survival) of Salmonella and Escherichia coli O157:H7 (0157) were examined in conjunction with any potential relationships to the plant’s antimicrobial content.

RESULTS: Internalized Salmonella was detected in cabbage within 24 hours with prevalence ranging from 62% (16 of 26) for the ‘Super Red 80’ cultivar to 92% (24 of 26) for the ‘Red Dynasty’ cultivar. The fate of Salmonella and O157 on small cabbage plants over nine days was significantly affected by cultivar with both these pathogens surviving the least and most on the ‘Capture’ and ‘Farao’ cultivars, respectively (P < 0.05). Survival of O157 was slightly higher on cabbage heads for O157 than small plants suggesting that the maturity stage may affect this pathogen’s fate. An inverse relationship existed between antimicrobial levels and a pathogen’s fate on cabbage heads (P < 0.05).

CONCLUSIONS: The fate of pathogens varied with the cabbage cultivar in growth chamber studies highlighting the potential to explore cultivar in field studies to reduce the risk of microbiological contamination in this crop.

Marilyn C. Erickson, Jye-Yin Liao, Alison S. Payton, Peter W. Cook, Ynes R. Ortega. 2019. Journal of the Science of Food and Agriculture.
https://doi.org/10.1002/jsfa.9573