Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System

The adaptation of organisms to a parasitic life style is often accompanied by the emergence of novel biochemical pathways absent in free-living organisms. As a result, the genomes of specialized parasitic organisms often code for a large number (>50%) of proteins with no detectable homology or predictable function. Although understanding the biochemical properties of these proteins and their roles in parasite biogenesis is the next challenge of molecular parasitology, analysis tools developed for free-living organisms are often inadequate for this purpose. Here we attempt to solve some of these problems by developing a methodology for the rapid production of expressed proteomes in cell-free systems based on parasitic organisms. To do so we take advantage of Species Independent Translational Sequences (SITS), which can efficiently mediate translation initiation in any organism. Using these sequences we developed a single-tube in vitro translation system based on the parasitic protozoan Leishmania tarentolae. We demonstrate that the system can be primed directly with SITS containing templates constructed by overlap extension PCR. To test the systems we simultaneously amplified 31 of L. tarentolae's putative translation initiation factors and phosphatases directly from the genomic DNA and subjected them to expression, purification and activity analysis. All of the amplified products produced soluble recombinant proteins, and putative phosphatases could be purified to at least 50% purity in one step. We further compared the ability of L. tarentolae and E. coli based cell-free systems to express a set of mammalian, L. tarentolae and Plasmodium falciparum Rab GTPases in functional form. We demonstrate that the L. tarentolae cell-free system consistently produced higher quality proteins than E. coli-based system. The differences were particularly pronounced in the case of open reading frames derived from P. falciparum. The implications of these developments are discussed

New Molecular Reporters for Rapid Protein Folding Assays

The GFP folding reporter assay [1] uses a C-terminal GFP fusion to report on the folding success of upstream fused polypeptides. The GFP folding assay is widely-used for screening protein variants with improved folding and solubility [2]–[8], but truncation artifacts may arise during evolution, i.e. from de novo internal ribosome entry sites [9]. One way to reduce such artifacts would be to insert target genes within the scaffolding of GFP circular permuted variants. Circular permutants of fluorescent proteins often misfold and are non-fluorescent, and do not readily tolerate fused polypeptides within the fluorescent protein scaffolding [10]–[12]. To overcome these limitations, and to increase the dynamic range for reporting on protein misfolding, we have created eight GFP insertion reporters with different sensitivities to protein misfolding using chimeras of two previously described GFP variants, the GFP folding reporter [1] and the robustly-folding “superfolder” GFP [13]. We applied this technology to engineer soluble variants of Rv0113, a protein from Mycobacterium tuberculosis initially expressed as inclusion bodies in Escherichia coli. Using GFP insertion reporters with increasing stringency for each cycle of mutagenesis and selection led to a variant that produced large amounts of soluble protein at 37°C in Escherichia coli. The new reporter constructs discriminate against truncation artifacts previously isolated during directed evolution of Rv0113 using the original C-terminal GFP folding reporter. Using GFP insertion reporters with variable stringency should prove useful for engineering protein variants with improved folding and solubility, while reducing the number of artifacts arising from internal cryptic ribosome initiation sites

Mobile DNA elements in T4 and related phages

Mobile genetic elements are common inhabitants of virtually every genome where they can exert profound influences on genome structure and function in addition to promoting their own spread within and between genomes. Phage T4 and related phage have long served as a model system for understanding the molecular mechanisms by which a certain class of mobile DNA, homing endonucleases, promote their spread. Homing endonucleases are site-specific DNA endonucleases that initiate mobility by introducing double-strand breaks at defined positions in genomes lacking the endonuclease gene, stimulating repair and recombination pathways that mobilize the endonuclease coding region. In phage T4, homing endonucleases were first discovered as encoded within the self-splicing td, nrdB and nrdD introns of T4. Genomic data has revealed that homing endonucleases are extremely widespread in T-even-like phage, as evidenced by the astounding fact that ~11% of the T4 genome encodes homing endonuclease genes, with most of them located outside of self-splicing introns. Detailed studies of the mobile td intron and its encoded endonuclease, I-TevI, have laid the foundation for genetic, biochemical and structural aspects that regulate the mobility process, and more recently have provided insights into regulation of homing endonuclease function. Here, we summarize the current state of knowledge regarding T4-encoded homing endonucleases, with particular emphasis on the td/I-TevI model system. We also discuss recent progress in the biology of free-standing endonucleases, and present areas of future research for this fascinating class of mobile genetic elements

Gold mining areas in Suriname: reservoirs of malaria resistance?

Malti R Adhin,1 Mergiory Labadie-Bracho,2 Stephen Vreden31Faculty of Medical Sciences, Department of Biochemistry, Anton de Kom Universiteit van Suriname, 2Prof Dr Paul C Flu Institute for Biomedical Sciences, 3Academic Hospital Paramaribo, Paramaribo, SurinameBackground: At present, malaria cases in Suriname occur predominantly in migrants and people living and/or working in areas with gold mining operations. A molecular survey was performed in Plasmodium falciparum isolates originating from persons from gold mining areas to assess the extent and role of mining areas as reservoirs of malaria resistance in Suriname.Methods: The status of 14 putative resistance-associated single nucleotide polymorphisms in the pfdhfr, pfcrt, pfmdr1, and pfATP6 genes was assessed for 28 samples from gold miners diagnosed with P. falciparum malaria using polymerase chain reaction amplification and restriction fragment length polymorphism analysis, and the results were compared with earlier data from nonmining villagers.Results: Isolates from miners showed a high degree of homogeneity, with a fixed pfdhfr Ile51/Asn108, pfmdr1 Phe184/Asp1042/Tyr1246, and pfcrt Thr76 mutant genotype, while an exclusively wild-type genotype was observed for pfmdr1 Asn86 and pfdhfr Ala16, Cys59, and Ile164, and for the pfATP6 positions Leu263/Ala623/Ser769. Small variations were observed for pfmdr1 S1034C. No statistically significant difference could be detected in allele frequencies between mining and nonmining villagers.Conclusion: Despite the increased risk of malaria infection in individuals working/living in gold mining areas, we did not detect an increase in mutation frequency at the 14 analyzed single nucleotide polymorphisms. Therefore, mining areas in Suriname cannot yet be considered as reservoirs for malaria resistance.Keywords: Plasmodium falciparum, gold mining, mutation frequency, Surinam

Assessing parasite clearance during uncomplicated Plasmodium falciparum infection treated with artesunate monotherapy in Suriname

Stephen GS Vreden,1 Rakesh D Bansie,2 Jeetendra K Jitan,3 Malti R Adhin4 1Foundation for Scientific Research Suriname (SWOS), 2Department of Internal Medicine, Academic Hospital Paramaribo, 3Department of Public Health, Ministry of Health, 4Department of Biochemistry, Anton de Kom University of Suriname, Paramaribo, Suriname Background: Artemisinin resistance in Plasmodium falciparum is suspected when the day 3 parasitemia is >10% when treated with artemisinin-based combination therapy or if >10% of patients treated with artemisinin-based combination therapy or artesunate monotherapy harbored parasites with half-lives ≥5 hours. Hence, a single-arm prospective efficacy trial was conducted in Suriname for uncomplicated P. falciparum infection treated with artesunate-based monotherapy for 3 days assessing day 3 parasitemia, treatment outcome after 28 days, and parasite half-life. Methods: The study was conducted in Paramaribo, the capital of Suriname, from July 2013 until July 2014. Patients with uncomplicated Plasmodium falciparum infection were included and received artesunate mono-therapy for three days. Day 3 parasitaemia, treatment outcome after 28 days and parasite half-life were determined. The latter was assessed with the parasite clearance estimator from the WorldWide Antimalarial Resistance Network (WWARN). Results: Thirty-nine patients were included from July 2013 until July 2014. The day 3 parasitemia was 10%. Eight patients (20.5%) could be followed up until day 28 and showed adequate clinical and parasitological response. Parasite half-life could only be determined from ten data series (25.7%). The median parasite half-life was 5.16 hours, and seven of these data series had a half-life ≥5 hours, still comprising 17.9% of the total data series. Conclusion: The low follow-up rate and the limited analyzable data series preclude clear conclusions about the efficacy of artesunate monotherapy in Suriname and the parasite half-life, respectively. The emergence of at least 17.9% of data series with a parasite half-life ≥5 hours supports the possible presence of artemisinin resistance. Keywords: malaria, falciparum, resistance, parasite half-lif

Primaquine double dose for 7 days is inferior to single-dose treatment for 14 days in preventing Plasmodium vivax recurrent episodes in Suriname

M Sigrid Mac Donald-Ottevanger,1 Malti R Adhin,2 Jeetendra Kumar Jitan,3 Gustavo Bretas,4 Stephen GS Vreden1 1Foundation for Scientific Research Suriname (SWOS), 2Department of Biochemistry, Anton de Kom University of Suriname, 3Department of Public Health, Ministry of Health, Paramaribo, Suriname; 4Independent consultant, Rio de Janeiro, Brazil Background: Recurrent episodes of Plasmodium vivax are caused by dormant liver stages of the parasite, which are not eradicated by choloroquine. Therefore, effective treatment also includes the use of primaquine (PQ). However, this secondary preventive therapy is often not effective, mostly due to poor adherence to the relatively long treatment course, justifying a comparative study of the efficacy of different durations of PQ treatment. Materials and methods: We included patients presenting with an acute and documented P. vivax infection from January 2006 to February 2008. All patients received chloroquine 25 mg/kg over a 3-day period. Subsequently, patients in group 7D received PQ 30 mg/day for 7 days, and patients in group 14D received standard PQ 15 mg/day for 14 days. All doses were given under supervision and patients were followed up for at least 6 months. The Kaplan–Meier method was used to estimate cumulative probability of recurrence up to 12 months after treatment initiation stratified by treatment group. Cox regression was used to assess possible determinants for recurrent parasitemia. Results: Forty-seven of the 79 included patients (59.5%) were allocated to group 7D and 32 patients (40.5%) were allocated to group 14D. Recurrent parasitemia was detected in 31.9% of the cases in group 7D compared to 12.5% of the cases in group 14D (hazard ratio [HR] =3.36, 95% CI 1.11–10.16). Cumulative probability for recurrent parasitemia at 3, 6, and 12 months was 0.201 (95% CI 0.106–0.362), 0.312 (95% CI 0.190–0.485), and 0.424 (95% CI 0.274– 0.615) for group 7D and 0.100 (95% CI 0.033–0.279), 0.100 (95% CI 0.033–0.279), and 0.138 (95% CI 0.054–0.327) for group 14D, respectively. When adjusted for possible confounders, differences in recurrent parasitemia remained significant between the two regimens in Cox regression analysis. Conclusion: More than 30% of the patients receiving shorter treatment course had recurrent parasitemia, suggesting that the standard dose of 15 mg/day PQ for 14 days is more efficacious than 30 mg for 7 days in preventing P. vivax recurrent episodes. Furthermore, we suggest that P. vivax treatment in Suriname should be changed to PQ 30 mg/day for 14 days, as per Center for Disease Control and Prevention recommendation, in light of a recurrence rate of over 10%, even in group 14D. Keywords: malaria, vivax, recurrent episode, primaquine, treatment duration, efficac