Identification of Leishmania proteins preferentially released in infected cells using change mediated antigen technology (CMAT).
Although Leishmania parasites have been shown to modulate their host cell's responses to multiple stimuli, there is limited evidence that parasite molecules are released into infected cells. In this study, we present an implementation of the change mediated antigen technology (CMAT) to identify...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2010-10-01
|
| Series: | PLoS Neglected Tropical Diseases |
| Online Access: | http://europepmc.org/articles/PMC2950143?pdf=render |
| _version_ | 1819076934413320192 |
|---|---|
| author | Peter E Kima J Alfredo Bonilla Eumin Cho Blaise Ndjamen Johnathan Canton Nicole Leal Martin Handfield |
| author_facet | Peter E Kima J Alfredo Bonilla Eumin Cho Blaise Ndjamen Johnathan Canton Nicole Leal Martin Handfield |
| author_sort | Peter E Kima |
| collection | DOAJ |
| description | Although Leishmania parasites have been shown to modulate their host cell's responses to multiple stimuli, there is limited evidence that parasite molecules are released into infected cells. In this study, we present an implementation of the change mediated antigen technology (CMAT) to identify parasite molecules that are preferentially expressed in infected cells. Sera from mice immunized with cell lysates prepared from L. donovani or L. pifanoi-infected macrophages were adsorbed with lysates of axenically grown amastigotes of L. donovani or L. pifanoi, respectively, as well as uninfected macrophages. The sera were then used to screen inducible parasite expression libraries constructed with genomic DNA. Eleven clones from the L. pifanoi and the L. donovani screen were selected to evaluate the characteristics of the molecules identified by this approach. The CMAT screen identified genes whose homologs encode molecules with unknown function as well as genes that had previously been shown to be preferentially expressed in the amastigote form of the parasite. In addition a variant of Tryparedoxin peroxidase that is preferentially expressed within infected cells was identified. Antisera that were then raised to recombinant products of the clones were used to validate that the endogenous molecules are preferentially expressed in infected cells. Evaluation of the distribution of the endogenous molecules in infected cells showed that some of these molecules are secreted into parasitophorous vacuoles (PVs) and that they then traffic out of PVs in vesicles with distinct morphologies. This study is a proof of concept study that the CMAT approach can be applied to identify putative Leishmania parasite effectors molecules that are preferentially expressed in infected cells. In addition we provide evidence that Leishmania molecules traffic out of the PV into the host cell cytosol and nucleus. |
| first_indexed | 2024-12-21T18:49:11Z |
| format | Article |
| id | doaj.art-7e966181a4a34f76b9a22b5d255ffe02 |
| institution | Directory Open Access Journal |
| issn | 1935-2727 1935-2735 |
| language | English |
| last_indexed | 2024-12-21T18:49:11Z |
| publishDate | 2010-10-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Neglected Tropical Diseases |
| spelling | doaj.art-7e966181a4a34f76b9a22b5d255ffe022022-12-21T18:53:47ZengPublic Library of Science (PLoS)PLoS Neglected Tropical Diseases1935-27271935-27352010-10-0141010.1371/journal.pntd.0000842Identification of Leishmania proteins preferentially released in infected cells using change mediated antigen technology (CMAT).Peter E KimaJ Alfredo BonillaEumin ChoBlaise NdjamenJohnathan CantonNicole LealMartin HandfieldAlthough Leishmania parasites have been shown to modulate their host cell's responses to multiple stimuli, there is limited evidence that parasite molecules are released into infected cells. In this study, we present an implementation of the change mediated antigen technology (CMAT) to identify parasite molecules that are preferentially expressed in infected cells. Sera from mice immunized with cell lysates prepared from L. donovani or L. pifanoi-infected macrophages were adsorbed with lysates of axenically grown amastigotes of L. donovani or L. pifanoi, respectively, as well as uninfected macrophages. The sera were then used to screen inducible parasite expression libraries constructed with genomic DNA. Eleven clones from the L. pifanoi and the L. donovani screen were selected to evaluate the characteristics of the molecules identified by this approach. The CMAT screen identified genes whose homologs encode molecules with unknown function as well as genes that had previously been shown to be preferentially expressed in the amastigote form of the parasite. In addition a variant of Tryparedoxin peroxidase that is preferentially expressed within infected cells was identified. Antisera that were then raised to recombinant products of the clones were used to validate that the endogenous molecules are preferentially expressed in infected cells. Evaluation of the distribution of the endogenous molecules in infected cells showed that some of these molecules are secreted into parasitophorous vacuoles (PVs) and that they then traffic out of PVs in vesicles with distinct morphologies. This study is a proof of concept study that the CMAT approach can be applied to identify putative Leishmania parasite effectors molecules that are preferentially expressed in infected cells. In addition we provide evidence that Leishmania molecules traffic out of the PV into the host cell cytosol and nucleus.http://europepmc.org/articles/PMC2950143?pdf=render |
| spellingShingle | Peter E Kima J Alfredo Bonilla Eumin Cho Blaise Ndjamen Johnathan Canton Nicole Leal Martin Handfield Identification of Leishmania proteins preferentially released in infected cells using change mediated antigen technology (CMAT). PLoS Neglected Tropical Diseases |
| title | Identification of Leishmania proteins preferentially released in infected cells using change mediated antigen technology (CMAT). |
| title_full | Identification of Leishmania proteins preferentially released in infected cells using change mediated antigen technology (CMAT). |
| title_fullStr | Identification of Leishmania proteins preferentially released in infected cells using change mediated antigen technology (CMAT). |
| title_full_unstemmed | Identification of Leishmania proteins preferentially released in infected cells using change mediated antigen technology (CMAT). |
| title_short | Identification of Leishmania proteins preferentially released in infected cells using change mediated antigen technology (CMAT). |
| title_sort | identification of leishmania proteins preferentially released in infected cells using change mediated antigen technology cmat |
| url | http://europepmc.org/articles/PMC2950143?pdf=render |
| work_keys_str_mv | AT peterekima identificationofleishmaniaproteinspreferentiallyreleasedininfectedcellsusingchangemediatedantigentechnologycmat AT jalfredobonilla identificationofleishmaniaproteinspreferentiallyreleasedininfectedcellsusingchangemediatedantigentechnologycmat AT eumincho identificationofleishmaniaproteinspreferentiallyreleasedininfectedcellsusingchangemediatedantigentechnologycmat AT blaisendjamen identificationofleishmaniaproteinspreferentiallyreleasedininfectedcellsusingchangemediatedantigentechnologycmat AT johnathancanton identificationofleishmaniaproteinspreferentiallyreleasedininfectedcellsusingchangemediatedantigentechnologycmat AT nicoleleal identificationofleishmaniaproteinspreferentiallyreleasedininfectedcellsusingchangemediatedantigentechnologycmat AT martinhandfield identificationofleishmaniaproteinspreferentiallyreleasedininfectedcellsusingchangemediatedantigentechnologycmat |