Proteomic examination of <i>Leishmania chagasi</i> plasma membrane proteins: Contrast between avirulent and virulent (metacyclic) parasite forms

Yao, Chaoqun; Li, Yalan; Donelson, John E.; Wilson, Mary E.

doi:10.1002/prca.200900050

Cited by 28 publications

(36 citation statements)

References 43 publications

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“…in the genomes of A. deanei and S. culicis , respectively. Proteins belonging to this group of zinc metalloproteases, also known as major surface protease (MSP) or leishmanolysin, have been characterized in various species of Leishmania and Trypanosoma [111]. Extensive studies on the role of this family in Leishmania indicate that they are involved in several aspects of host-parasite interaction including resistance to complement-mediated lysis, cell attachment, entry, and survival in macrophages [112].…”

Section: Resultsmentioning

confidence: 99%

Predicting the Proteins of Angomonas deanei, Strigomonas culicis and Their Respective Endosymbionts Reveals New Aspects of the Trypanosomatidae Family

et al. 2013

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Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.

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Section: Resultsmentioning

confidence: 99%

Predicting the Proteins of Angomonas deanei, Strigomonas culicis and Their Respective Endosymbionts Reveals New Aspects of the Trypanosomatidae Family

et al. 2013

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“…Furthermore, the temperature and speed of freezing seem to be key factors that influence its effectiveness. In a study where cells were suspended in hypotonic buffer and processed by five cycles of freezing in −80°C and thawing in a RT water bath, microscopy revealed that about 10% of the cells were not disrupted . In another study, a dry ice/ethanol bath was used to freeze red blood cells in hypotonic lysis buffer .…”

Section: Resultsmentioning

confidence: 99%

Reproducible method to enrich membrane proteins with high purity and high yield for an LC‐MS/MS approach in quantitative membrane proteomics

Lai

2013

Electrophoresis

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The proportionately low abundance of membrane proteins hampers their proteomic analysis, especially for a quantitative LC-MS/MS approach. To overcome this limitation, a method was developed that consists of one cell disruption step in a hypotonic reagent using liquid nitrogen, one isolation step using a low speed centrifugation, and three wash steps using high speed centrifugation. Pellets contained plasma, nuclear, and mitochondrial membranes, including their integral, peripheral, and anchored membrane proteins. The reproducibility of this method was verified by protein assay of four separate experiments with a CV of 7.7%, and by comparative LC-MS/MS label-free quantification of individual proteins between two experiments with 99% of the quantified proteins having a CV ≤ 30%. Western blot and LC-MS/MS results of markers for cytoplasm, nucleus, mitochondria, and their membranes indicated that the enriched membrane fraction was highly pure by the absence of, or presence of trace amounts of, non-membrane marker proteins. The average yield of membrane proteins was 237 μg/10 million HT29-MTX cells. LC-MS/MS analysis of the membrane enriched sample resulted in the identification of 2,597 protein groups. In summary, the developed method is reproducible, produces a highly pure membrane fraction, and generates a high yield of membrane proteins.

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“…Finally, Leishmania has been mainly studied by LC‐MS [], including the mode of action of antimonial drugs []. This will afford the comparison with Leishmania fingerprinting by CE‐MS, which to the best of our knowledge has not been previously carried out.…”

Section: Introductionmentioning

confidence: 99%

CE‐ESI‐MS metabolic fingerprinting of Leishmania resistance to antimony treatment

et al. 2012

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Metabolomics has become an invaluable tool to unveil biology of pathogens, with immediate application to chemotherapy. It is currently accepted that there is not one single technique capable of obtaining the whole metabolic fingerprint of a biological system either due to their different physical-chemical properties or concentrations. In this work, we have explored the capability of capillary electrophoresis mass spectrometry with a sheathless interface with electrospray ionization (CE-ESI-TOF-MS) to separate metabolites in order to be used as a complementary technique to LC. As proof of concept, we have compared the metabolome of Leishmania infantum promastigotes BCN 150 (Sb (III) IC(50) = 20.9 μM) and its variation when treated with 120 μM of Sb(III) potassium tartrate for 12 h, as well as with its Sb(III) resistant counterpart obtained by growth of the parasites under increasing Sb(III) in a step-wise manner up to 180 μM. The number of metabolites compared were of 264 for BCN150 Sb(III) treated versus nontreated and of 195 for Sb(III) resistant versus susceptible parasites. After successive data filtering, differences in seven metabolites identified in databases for Leishmania pathways, showed the highest significant differences, corresponding mainly to amino acids or their metabolite surrogates. Most of them were assigned to sulfur containing amino acids and polyamine biosynthetic pathways, of special relevance considering the deterioration of the thiol-dependent redox metabolism in Leishmania by Sb(III). Given the low concentrations typical for most of these metabolites, the assay can be considered a success that should be explored for new biological questions.

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Proteomic examination of Leishmania chagasi plasma membrane proteins: Contrast between avirulent and virulent (metacyclic) parasite forms

Cited by 28 publications

References 43 publications

Predicting the Proteins of Angomonas deanei, Strigomonas culicis and Their Respective Endosymbionts Reveals New Aspects of the Trypanosomatidae Family

Predicting the Proteins of Angomonas deanei, Strigomonas culicis and Their Respective Endosymbionts Reveals New Aspects of the Trypanosomatidae Family

Reproducible method to enrich membrane proteins with high purity and high yield for an LC‐MS/MS approach in quantitative membrane proteomics

CE‐ESI‐MS metabolic fingerprinting of Leishmania resistance to antimony treatment

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