Investigating the <i>Leishmania donovani sacp</i> Gene and Its Role in Macrophage Infection and Survival in Mice
The protozoan parasite <i>Leishmania donovani</i> is a causative agent of the neglected tropical disease known as visceral leishmaniasis, which can be lethal when untreated. Studying <i>Leishmania</i> viru-lence factors is crucial in determining how the parasite causes diseas...
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MDPI AG
2022-11-01
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| Series: | Tropical Medicine and Infectious Disease |
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| Online Access: | https://www.mdpi.com/2414-6366/7/11/384 |
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| author | Kayla Paulini Patrick Lypaczewski Wen-Wei Zhang Dilhan J. Perera Momar Ndao Greg Matlashewski |
| author_facet | Kayla Paulini Patrick Lypaczewski Wen-Wei Zhang Dilhan J. Perera Momar Ndao Greg Matlashewski |
| author_sort | Kayla Paulini |
| collection | DOAJ |
| description | The protozoan parasite <i>Leishmania donovani</i> is a causative agent of the neglected tropical disease known as visceral leishmaniasis, which can be lethal when untreated. Studying <i>Leishmania</i> viru-lence factors is crucial in determining how the parasite causes disease and identifying new targets for treatment. One potential virulence factor is <i>L. donovani’s</i> abundantly secreted protein: secreted acid phosphatase (SAcP). Whole-genome analysis revealed that the <i>sacp</i> gene was present in three copies in wild type <i>L. donovani</i>. Using CRISPR-Cas9 gene editing; we generated a <i>sacp</i> gene knockout termed LdΔSAcP, which demonstrated a loss of both the SAcP protein and an associated reduction in secreted acid phosphatase activity. Genome sequencing confirmed the precise dele-tion of the <i>sacp</i> gene in LdΔSAcP and identified several changes in the genome. LdΔSAcP demonstrated no significant changes in promastigote proliferation or its ability to infect and survive in macrophages compared to the wildtype strain. LdΔSAcP also demonstrated no change in murine liver infection; however, survival was impaired in the spleen. Taken together these results show that SAcP is not necessary for the survival of promastigotes in culture but may support long-term survival in the spleen. These observations also show that the use of CRISPR gene editing and WGS together are effective to investigate the function and phenotype of complex potential drug targets such as multicopy genes. |
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| issn | 2414-6366 |
| language | English |
| last_indexed | 2024-03-09T17:57:27Z |
| publishDate | 2022-11-01 |
| publisher | MDPI AG |
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| series | Tropical Medicine and Infectious Disease |
| spelling | doaj.art-b61955ee6eac430987519339564078892023-11-24T10:15:55ZengMDPI AGTropical Medicine and Infectious Disease2414-63662022-11-0171138410.3390/tropicalmed7110384Investigating the <i>Leishmania donovani sacp</i> Gene and Its Role in Macrophage Infection and Survival in MiceKayla Paulini0Patrick Lypaczewski1Wen-Wei Zhang2Dilhan J. Perera3Momar Ndao4Greg Matlashewski5Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 2B4, CanadaDepartment of Microbiology and Immunology, McGill University, Montreal, QC H3A 2B4, CanadaDepartment of Microbiology and Immunology, McGill University, Montreal, QC H3A 2B4, CanadaDivision of Experimental Medicine, McGill University, Montreal, QC H4A 3J1, CanadaDepartment of Microbiology and Immunology, McGill University, Montreal, QC H3A 2B4, CanadaDepartment of Microbiology and Immunology, McGill University, Montreal, QC H3A 2B4, CanadaThe protozoan parasite <i>Leishmania donovani</i> is a causative agent of the neglected tropical disease known as visceral leishmaniasis, which can be lethal when untreated. Studying <i>Leishmania</i> viru-lence factors is crucial in determining how the parasite causes disease and identifying new targets for treatment. One potential virulence factor is <i>L. donovani’s</i> abundantly secreted protein: secreted acid phosphatase (SAcP). Whole-genome analysis revealed that the <i>sacp</i> gene was present in three copies in wild type <i>L. donovani</i>. Using CRISPR-Cas9 gene editing; we generated a <i>sacp</i> gene knockout termed LdΔSAcP, which demonstrated a loss of both the SAcP protein and an associated reduction in secreted acid phosphatase activity. Genome sequencing confirmed the precise dele-tion of the <i>sacp</i> gene in LdΔSAcP and identified several changes in the genome. LdΔSAcP demonstrated no significant changes in promastigote proliferation or its ability to infect and survive in macrophages compared to the wildtype strain. LdΔSAcP also demonstrated no change in murine liver infection; however, survival was impaired in the spleen. Taken together these results show that SAcP is not necessary for the survival of promastigotes in culture but may support long-term survival in the spleen. These observations also show that the use of CRISPR gene editing and WGS together are effective to investigate the function and phenotype of complex potential drug targets such as multicopy genes.https://www.mdpi.com/2414-6366/7/11/384visceral leishmaniasisCRISPR-Cas9whole-genome sequencingvirulencedrug target |
| spellingShingle | Kayla Paulini Patrick Lypaczewski Wen-Wei Zhang Dilhan J. Perera Momar Ndao Greg Matlashewski Investigating the <i>Leishmania donovani sacp</i> Gene and Its Role in Macrophage Infection and Survival in Mice Tropical Medicine and Infectious Disease visceral leishmaniasis CRISPR-Cas9 whole-genome sequencing virulence drug target |
| title | Investigating the <i>Leishmania donovani sacp</i> Gene and Its Role in Macrophage Infection and Survival in Mice |
| title_full | Investigating the <i>Leishmania donovani sacp</i> Gene and Its Role in Macrophage Infection and Survival in Mice |
| title_fullStr | Investigating the <i>Leishmania donovani sacp</i> Gene and Its Role in Macrophage Infection and Survival in Mice |
| title_full_unstemmed | Investigating the <i>Leishmania donovani sacp</i> Gene and Its Role in Macrophage Infection and Survival in Mice |
| title_short | Investigating the <i>Leishmania donovani sacp</i> Gene and Its Role in Macrophage Infection and Survival in Mice |
| title_sort | investigating the i leishmania donovani sacp i gene and its role in macrophage infection and survival in mice |
| topic | visceral leishmaniasis CRISPR-Cas9 whole-genome sequencing virulence drug target |
| url | https://www.mdpi.com/2414-6366/7/11/384 |
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