The Trypanosoma cruzi Vitamin C Dependent Peroxidase Confers Protection against Oxidative Stress but Is Not a Determinant of Virulence
Publication Date
April 13, 2015
Journal
PLOS Neglected Tropical Diseases
Authors
Martin C. Taylor, Michael D. Lewis, Amanda Fortes Francisco, Shane R. Wilkinson, et al
Volume
9
Issue
4
Pages
e0003707
DOI
https://dx.plos.org/10.1371/journal.pntd.0003707
Publisher URL
http://journals.plos.org/plosntds/article?id=10.1371%2Fjournal.pntd.0003707
PubMed
http://www.ncbi.nlm.nih.gov/pubmed/25875298
PubMed Central
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395405
Europe PMC
http://europepmc.org/abstract/MED/25875298
Web of Science
000354972200051
Scopus
84929484738
Mendeley
http://www.mendeley.com/research/trypanosoma-cruzi-vitamin-c-dependent-peroxidase-confers-protection-against-oxidative-stress-not-det
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Mendeley | Further Information

{"title"=>"The Trypanosoma cruzi Vitamin C Dependent Peroxidase Confers Protection against Oxidative Stress but Is Not a Determinant of Virulence", "type"=>"journal", "authors"=>[{"first_name"=>"Martin C.", "last_name"=>"Taylor", "scopus_author_id"=>"7406239432"}, {"first_name"=>"Michael D.", "last_name"=>"Lewis", "scopus_author_id"=>"55295787300"}, {"first_name"=>"Amanda Fortes", "last_name"=>"Francisco", "scopus_author_id"=>"56464850800"}, {"first_name"=>"Shane R.", "last_name"=>"Wilkinson", "scopus_author_id"=>"35568154200"}, {"first_name"=>"John M.", "last_name"=>"Kelly", "scopus_author_id"=>"55462305400"}], "year"=>2015, "source"=>"PLoS Neglected Tropical Diseases", "identifiers"=>{"issn"=>"19352735", "pui"=>"604135688", "doi"=>"10.1371/journal.pntd.0003707", "sgr"=>"84929484738", "pmid"=>"25875298", "scopus"=>"2-s2.0-84929484738"}, "id"=>"f43f655b-437f-3a2c-b646-0a72ff8ed538", "abstract"=>"BACKGROUND: The neglected parasitic infection Chagas disease is rapidly becoming a globalised public health issue due to migration. There are only two anti-parasitic drugs available to treat this disease, benznidazole and nifurtimox. Thus it is important to identify and validate new drug targets in Trypanosoma cruzi, the causative agent. T. cruzi expresses an ER-localised ascorbate-dependent peroxidase (TcAPx). This parasite-specific enzyme has attracted interest from the perspective of targeted chemotherapy.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: To assess the importance of TcAPx in protecting T. cruzi from oxidative stress and to determine if it is essential for virulence, we generated null mutants by targeted gene disruption. Loss of activity was associated with increased sensitivity to exogenous hydrogen peroxide, but had no effect on susceptibility to the front-line Chagas disease drug benznidazole. This suggests that increased oxidative stress in the ER does not play a significant role in its mechanism of action. Homozygous knockouts could proceed through the entire life-cycle in vitro, although they exhibited a significant decrease in their ability to infect mammalian cells. To investigate virulence, we exploited a highly sensitive bioluminescence imaging system which allows parasites to be monitored in real-time in the chronic stage of murine infections. This showed that depletion of enzyme activity had no effect on T. cruzi replication, dissemination or tissue tropism in vivo.\\n\\nCONCLUSIONS/SIGNIFICANCE: TcAPx is not essential for parasite viability within the mammalian host, does not have a significant role in establishment or maintenance of chronic infections, and should therefore not be considered a priority for drug design.", "link"=>"http://www.mendeley.com/research/trypanosoma-cruzi-vitamin-c-dependent-peroxidase-confers-protection-against-oxidative-stress-not-det", "reader_count"=>23, "reader_count_by_academic_status"=>{"Unspecified"=>3, "Professor > Associate Professor"=>1, "Student > Doctoral Student"=>1, "Researcher"=>3, "Student > Ph. D. Student"=>8, "Student > Postgraduate"=>1, "Student > Master"=>2, "Student > Bachelor"=>1, "Lecturer > Senior Lecturer"=>1, "Professor"=>2}, "reader_count_by_user_role"=>{"Unspecified"=>3, "Professor > Associate Professor"=>1, "Student > Doctoral Student"=>1, "Researcher"=>3, "Student > Ph. D. Student"=>8, "Student > Postgraduate"=>1, "Student > Master"=>2, "Student > Bachelor"=>1, "Lecturer > Senior Lecturer"=>1, "Professor"=>2}, "reader_count_by_subject_area"=>{"Unspecified"=>4, "Biochemistry, Genetics and Molecular Biology"=>3, "Agricultural and Biological Sciences"=>13, "Medicine and Dentistry"=>1, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Chemistry"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Chemistry"=>{"Chemistry"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>13}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>3}, "Unspecified"=>{"Unspecified"=>4}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}}, "reader_count_by_country"=>{"United States"=>1, "United Kingdom"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/2017867"], "description"=>"<p>Epimastigotes seeded at 5 x 10<sup>5</sup> ml<sup>-1</sup> were exposed to various concentrations of <b>A</b> hydrogen peroxide and <b>B</b> benznidazole. The number of viable cells after 9 days was measured using resazurin fluorescence. TcΔAPx1comp refers to TcΔAPx1 cells retransformed with pTEX-APx to complement the null phenotype. Data were analysed by sigmoidal curve fitting using GraphPad Prism. The table below shows the EC<sub>50</sub> values (μM) for each compound against the various cell lines +/- standard deviation. Significance of differences in the EC<sub>50</sub> for H<sub>2</sub>O<sub>2</sub> was measured using the F-test. (ND—not done).</p>", "links"=>[], "tags"=>["TcAPx", "enzyme", "bioluminescence imaging system", "Chagas disease drug benznidazole", "parasite", "infection Chagas disease", "Trypanosoma cruzi Vitamin C Dependent Peroxidase Confers Protection", "er", "virulence", "oxidative stress", "exogenous hydrogen peroxide", "role"], "article_id"=>1377728, "categories"=>["Uncategorised"], "users"=>["Martin C. Taylor", "Michael D. Lewis", "Amanda Fortes Francisco", "Shane R. Wilkinson", "John M. Kelly"], "doi"=>"https://dx.doi.org/10.1371/journal.pntd.0003707.g003", "stats"=>{"downloads"=>0, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_In_vitro_sensitivity_of_null_mutants_to_oxidative_stress_and_benznidazole_/1377728", "title"=>"<i>In vitro</i> sensitivity of null mutants to oxidative stress and benznidazole.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-04-13 05:36:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/2017862"], "description"=>"<p><b>A</b> Growth rate of <i>T</i>. <i>cruzi</i> epimastigotes for wild type and null mutant (TcΔAPx1 and 2) clones. Triplicate cultures were followed for 10 days. There was no significant difference in growth rate. <b>B</b> Parasites lacking TcAPx can differentiate to amastigotes (AM) and trypomastigotes (TR). Examples shown are Giemsa stained wild type and null mutant (TcΔAPx2) cells. <b>C</b><i>In vitro</i> infectivity for L6 rat myoblast cells. Metacyclic trypomastigotes were used to infect L6 cells at a ratio of 5 trypanosomes per cell and left for 48 hours (Methods). Cells were Giemsa stained and the number of infected cells counted. Infections were carried out with seven replicates per parasite line. TcΔAPx1comp refers to TcΔAPx1 cells retransformed with pTEX-APx to complement the null phenotype. Data presented as mean <u>+</u> SD. Significance of difference between each pair was assessed by Student’s t-test, (**) corresponds to <i>P</i> = 0.007, (***) <i>P</i> = 0.0006. <i>P</i> values for wild type:TcΔAPx1 indicated by short horizontal line, wild type:TcΔAPx2 indicated by long horizontal line. The difference between the wild type and complemented lines was not significant. <b>D</b><i>In vitro</i> infectivity for Vero epithelial cells. Metacyclic trypomastigotes were used to infect Vero cells at a ratio of 5 trypanosomes per cell and left for 48 hours (Methods). Cells were Giemsa stained and the number of infected cells counted. Infections were carried out with seven replicates per parasite line. Data presented as mean <u>+</u> SD. Significance of difference was assessed by Student’s t-test, (***) corresponds to <i>P</i> = 0.0006. <i>P</i> values for wild type:TcΔAPx1 indicated by short horizontal line, wild type:TcΔAPx2 indicated by long horizontal line.</p>", "links"=>[], "tags"=>["TcAPx", "enzyme", "bioluminescence imaging system", "Chagas disease drug benznidazole", "parasite", "infection Chagas disease", "Trypanosoma cruzi Vitamin C Dependent Peroxidase Confers Protection", "er", "virulence", "oxidative stress", "exogenous hydrogen peroxide", "role"], "article_id"=>1377723, "categories"=>["Uncategorised"], "users"=>["Martin C. Taylor", "Michael D. Lewis", "Amanda Fortes Francisco", "Shane R. Wilkinson", "John M. Kelly"], "doi"=>"https://dx.doi.org/10.1371/journal.pntd.0003707.g002", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Phenotypic_assessment_of_null_mutants_in_vitro_/1377723", "title"=>"Phenotypic assessment of null mutants <i>in vitro</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-04-13 05:36:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/2017874"], "description"=>"<p><i>Ex vivo</i> imaging of organs from two representative mice of each group (groups as in <a href=\"http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003707#pntd.0003707.g004\" target=\"_blank\">Fig 4</a>), one sacrificed at day 56 (<b>A</b>) and one at day 63 (<b>B</b>). Each of the images is on the same luminescence scale (right) for radiance where purple indicates low signal intensity and red indicates a high signal. The maximum (5.5x10<sup>5</sup>) and minimum (4.9 x10<sup>3</sup>) signals are indicated at the top and bottom of the scale bar respectively. The heart is indicated in each image by a white arrow. Foci of infection are visible in the stomach and colon in all cases. Occasional foci were detected in the heart, gut mesenteries and lungs. The schematic at the bottom of the TcΔAPx1 column indicates the layout of organs in each dish, abbreviations: Gut Mes: Gut mesenteries, SKM: skeletal muscle (right hind leg), vis fat: visceral fat, OES: oesophagus, STM: stomach.</p>", "links"=>[], "tags"=>["TcAPx", "enzyme", "bioluminescence imaging system", "Chagas disease drug benznidazole", "parasite", "infection Chagas disease", "Trypanosoma cruzi Vitamin C Dependent Peroxidase Confers Protection", "er", "virulence", "oxidative stress", "exogenous hydrogen peroxide", "role"], "article_id"=>1377731, "categories"=>["Uncategorised"], "users"=>["Martin C. Taylor", "Michael D. Lewis", "Amanda Fortes Francisco", "Shane R. Wilkinson", "John M. Kelly"], "doi"=>"https://dx.doi.org/10.1371/journal.pntd.0003707.g005", "stats"=>{"downloads"=>0, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_gastrointestinal_tract_is_the_major_site_of_T_cruzi_persistence_in_mice_infected_with_either_wild_type_or_null_mutant_parasites_/1377731", "title"=>"The gastrointestinal tract is the major site of <i>T</i>. <i>cruzi</i> persistence in mice infected with either wild type or null mutant parasites.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-04-13 05:36:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/2017855"], "description"=>"<p><b>A</b> Map of the <i>T</i>. <i>cruzi</i> Sylvio X10/6 <i>TcAPx</i> locus (1) indicating the derivation of constructs used for targeted integration. The location of the flanking <i>TcCLPTM1</i> (Cleft-lip and palate transmembrane 1–like protein) and <i>TcG6PDH</i> (glucose-6-phosphate dehydrogenase) genes are indicated and the hatched box represents a degenerate VIPER/SIRE element. The ‘first round’ gene disruption construct is shown in (2), with the ‘second round’ gene deletion vector represented by (3). Restriction sites shown are C: Cla I and S: Sma I. <b>B</b> Strategy used to generate TcAPx null mutants. Briefly, the first allele was disrupted by insertional integration of the <i>PAC</i> gene into the ORF (1). An episomal copy of TcAPx was introduced into the <i>TcAPX</i><sup>+/-</sup> heterozygote line (2). The second endogenous allele was then deleted by homologous recombination using the flanking DNA external to the ORF to insert the <i>BLA</i> gene (3). The parasites were then removed from G418 selective pressure and passaged for up to 125 generations (4). Clones were then isolated and characterised (5). <b>C</b> The pTEX-APx episome is unstable in both wild type and <i>TcAPx</i> null backgrounds. The autoradiographs show Southern blots containing genomic DNA from wild type and null mutant cells isolated before (lanes 0) and after removal of G418 from the growth medium. Generations without G418 selection are indicated above the blot. The blot was probed with the <i>Neo</i><sup>R</sup> ORF. <b>D</b> Western blot showing expression level of TcAPx in parasite populations 125 generations after removal of G418 selection. WT: wild type Sylvio X10/6 lysate; SK: lysate from cells with a single copy of <i>TcAPX</i> disrupted; TcΔAPx: lysate from cells with both genes ablated. The panel below shows the blot probed with anti-TbBiP (a kind gift from Jay Bangs, University of Wisconsin-Madison) to control for loading. <b>E</b> Southern blot of genomic DNA digested with Cla I and Sma I, showing that <i>TcAPx</i> is absent from cloned null mutant cells. The left hand panel was probed with the <i>TcAPx</i> ORF and shows the endogenous gene (lane WT, 2.8 kb Cla I-Sma I fragment) and the gene disrupted by <i>PAC</i> insertion (lanes TcΔAPx 1 and 2, 3.1 kb Cla I fragment). <b>F</b> Western blot indicating that the null mutant clones do not express TcAPx. The wild type population show a single band of ~30kDa (lane WT: wild type) which is absent from the null mutant clones (lanes TcΔAPx 1 and 2). Equivalent loading is indicated by the Coomassie stained gel below.</p>", "links"=>[], "tags"=>["TcAPx", "enzyme", "bioluminescence imaging system", "Chagas disease drug benznidazole", "parasite", "infection Chagas disease", "Trypanosoma cruzi Vitamin C Dependent Peroxidase Confers Protection", "er", "virulence", "oxidative stress", "exogenous hydrogen peroxide", "role"], "article_id"=>1377716, "categories"=>["Uncategorised"], "users"=>["Martin C. Taylor", "Michael D. Lewis", "Amanda Fortes Francisco", "Shane R. Wilkinson", "John M. Kelly"], "doi"=>"https://dx.doi.org/10.1371/journal.pntd.0003707.g001", "stats"=>{"downloads"=>0, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Generation_of_TcAPx_null_mutants_/1377716", "title"=>"Generation of <i>TcAPx</i> null mutants.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-04-13 05:36:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/2017910", "https://ndownloader.figshare.com/files/2017911", "https://ndownloader.figshare.com/files/2017912", "https://ndownloader.figshare.com/files/2017913"], "description"=>"<div><p>Background</p><p>The neglected parasitic infection Chagas disease is rapidly becoming a globalised public health issue due to migration. There are only two anti-parasitic drugs available to treat this disease, benznidazole and nifurtimox. Thus it is important to identify and validate new drug targets in <i>Trypanosoma cruzi</i>, the causative agent. <i>T</i>. <i>cruzi</i> expresses an ER-localised ascorbate-dependent peroxidase (TcAPx). This parasite-specific enzyme has attracted interest from the perspective of targeted chemotherapy.</p><p>Methodology/Principal Findings</p><p>To assess the importance of TcAPx in protecting <i>T</i>. <i>cruzi</i> from oxidative stress and to determine if it is essential for virulence, we generated null mutants by targeted gene disruption. Loss of activity was associated with increased sensitivity to exogenous hydrogen peroxide, but had no effect on susceptibility to the front-line Chagas disease drug benznidazole. This suggests that increased oxidative stress in the ER does not play a significant role in its mechanism of action. Homozygous knockouts could proceed through the entire life-cycle <i>in vitro</i>, although they exhibited a significant decrease in their ability to infect mammalian cells. To investigate virulence, we exploited a highly sensitive bioluminescence imaging system which allows parasites to be monitored in real-time in the chronic stage of murine infections. This showed that depletion of enzyme activity had no effect on <i>T</i>. <i>cruzi</i> replication, dissemination or tissue tropism <i>in vivo</i>.</p><p>Conclusions/Significance</p><p>TcAPx is not essential for parasite viability within the mammalian host, does not have a significant role in establishment or maintenance of chronic infections, and should therefore not be considered a priority for drug design.</p></div>", "links"=>[], "tags"=>["TcAPx", "enzyme", "bioluminescence imaging system", "Chagas disease drug benznidazole", "parasite", "infection Chagas disease", "Trypanosoma cruzi Vitamin C Dependent Peroxidase Confers Protection", "er", "virulence", "oxidative stress", "exogenous hydrogen peroxide", "role"], "article_id"=>1377756, "categories"=>["Uncategorised"], "users"=>["Martin C. Taylor", "Michael D. Lewis", "Amanda Fortes Francisco", "Shane R. Wilkinson", "John M. Kelly"], "doi"=>["https://dx.doi.org/10.1371/journal.pntd.0003707.s001", "https://dx.doi.org/10.1371/journal.pntd.0003707.s002", "https://dx.doi.org/10.1371/journal.pntd.0003707.s003", "https://dx.doi.org/10.1371/journal.pntd.0003707.s004"], "stats"=>{"downloads"=>0, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/The_Trypanosoma_cruzi_Vitamin_C_Dependent_Peroxidase_Confers_Protection_against_Oxidative_Stress_but_Is_Not_a_Determinant_of_Virulence/1377756", "title"=>"The <i>Trypanosoma cruzi</i> Vitamin C Dependent Peroxidase Confers Protection against Oxidative Stress but Is Not a Determinant of Virulence", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-04-13 05:36:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/2017868"], "description"=>"<p><b>A</b> Female BALB/c mice were infected with 2 x 10<sup>5</sup> culture-derived bloodstream trypomastigotes modified to express a red-shifted luciferase gene (Methods, [<a href=\"http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003707#pntd.0003707.ref019\" target=\"_blank\">19</a>]). Mice were imaged at the time points shown using an IVIS Illumina II system (Caliper Life Sciences). Images were analysed using the same signal intensity scale for radiance (right) where purple indicates low signal intensity and red indicates a high signal. The maximum (5x10<sup>7</sup>) and minimum (6.5 x10<sup>3</sup>) signals are indicated at the top and bottom of the scale bar respectively. Three representative mice are shown from each group of animals (<i>n</i> = 6 per group). DPI: days post infection, time 0 represents image taken one hour after infection. <b>B</b> Graph showing the mean total body flux measured in each group of animals throughout the experiment. The grey line indicates the wild type infection, blue is TcΔAPx1 and red is TcΔAPx2. Data are plotted as mean values, error bars indicate standard deviation. The black lines indicate the mean (solid line), and mean +2SD (dotted line) of background luminescence of control uninfected mice. All data were acquired and analysed using Living Image software (Caliper Life Sciences).</p>", "links"=>[], "tags"=>["TcAPx", "enzyme", "bioluminescence imaging system", "Chagas disease drug benznidazole", "parasite", "infection Chagas disease", "Trypanosoma cruzi Vitamin C Dependent Peroxidase Confers Protection", "er", "virulence", "oxidative stress", "exogenous hydrogen peroxide", "role"], "article_id"=>1377729, "categories"=>["Uncategorised"], "users"=>["Martin C. Taylor", "Michael D. Lewis", "Amanda Fortes Francisco", "Shane R. Wilkinson", "John M. Kelly"], "doi"=>"https://dx.doi.org/10.1371/journal.pntd.0003707.g004", "stats"=>{"downloads"=>0, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Course_of_infection_in_a_murine_model_monitored_by_bioluminescence_imaging_/1377729", "title"=>"Course of infection in a murine model monitored by bioluminescence imaging.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-04-13 05:36:24"}

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{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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