scholarly journals Conducting genetic interaction analysis with CRISPR-Cas9-based strategies in Candida albicans

2021 ◽  
Vol 3 (12) ◽  
Author(s):  
Viola Halder ◽  
Brianna McDonnell ◽  
Rebecca Shapiro
Keyword(s):  
Candida Albicans ◽  
Stress Response ◽  
Genetic Interaction ◽  
Interaction Analysis ◽  
Relative Fitness ◽  
Vaginal Mucosa ◽  
Response Gene ◽  
Fungal Genes ◽  
Genetic Interaction Analysis ◽  
Stress Response Gene

Candida albicans is an opportunistic fungal pathogen found in the oral mucosa, the gut, the vaginal mucosa, and humans' skin. While C. albicans can cause superficial infections, severe invasive infections can occur in immunocompromised individuals. Understanding the survival mechanisms and pathogenesis of C. albicans is critical for novel antifungal drug discovery. Determining the relationships between different genes can create a genetic interaction map, which can identify complementary gene sets, central to C. albicans survival, as potential drug targets in combination therapy. A genetic approach using the CRISPR-Cas9-based genome editing platform will focus on genetic interaction analysis of C. albicans stress response genes. The ultimate goal is to create a stress response gene deletion library to study its pathogen survival role. This library of single and double stress response gene mutants will be screened under diverse growth conditions to assess their relative fitness. Genetic interaction analysis will help map out epistatic interactions between fungal genes involved in growth, survival, and pathogenesis and uncover putative targets for combination antifungal therapy based on negative or synthetic lethal genetic interactions.

scholarly journals The Candida albicans stress response gene Stomatin-Like Protein 3 is implicated in ROS-induced apoptotic-like death of yeast phase cells

PLoS ONE ◽  
2018 ◽  
Vol 13 (2) ◽  
pp. e0192250 ◽  
Author(s):  
Karen A. Conrad ◽  
Ronald Rodriguez ◽  
Eugenia C. Salcedo ◽  
Jason M. Rauceo
Keyword(s):  
Candida Albicans ◽  
Stress Response ◽  
Response Gene ◽  
Yeast Phase ◽  
Stress Response Gene

scholarly journals A CRISPR–Cas9-based gene drive platform for genetic interaction analysis in Candida albicans

2017 ◽  
Vol 3 (1) ◽  
pp. 73-82 ◽  
Author(s):  
Rebecca S. Shapiro ◽  
Alejandro Chavez ◽  
Caroline B. M. Porter ◽  
Meagan Hamblin ◽  
Christian S. Kaas ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Genetic Interaction ◽  
Interaction Analysis ◽  
Gene Drive ◽  
Genetic Interaction Analysis

scholarly journals Deuterium Oxide (D2O) Induces Early Stress Response Gene Expression and Impairs Growth and Metastasis of Experimental Malignant Melanoma

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 605
Author(s):  
Jana Jandova ◽  
Anh B. Hua ◽  
Jocelyn Fimbres ◽  
Georg T. Wondrak
Keyword(s):  
Gene Expression ◽  
Stress Response ◽  
Malignant Melanoma ◽  
Tumor Growth ◽  
Deuterium Oxide ◽  
Human Melanoma ◽  
Pharmacological Intervention ◽  
Melanoma Metastasis ◽  
Response Gene ◽  
Stress Response Gene

There are two stable isotopes of hydrogen, protium (1H) and deuterium (2H; D). Cellular stress response dysregulation in cancer represents both a major pathological driving force and a promising therapeutic target, but the molecular consequences and potential therapeutic impact of deuterium (2H)-stress on cancer cells remain largely unexplored. We have examined the anti-proliferative and apoptogenic effects of deuterium oxide (D2O; ‘heavy water’) together with stress response gene expression profiling in panels of malignant melanoma (A375V600E, A375NRAS, G361, LOX-IMVI), and pancreatic ductal adenocarcinoma (PANC-1, Capan-2, or MIA PaCa-2) cells with inclusion of human diploid Hs27 skin fibroblasts. Moreover, we have examined the efficacy of D2O-based pharmacological intervention in murine models of human melanoma tumor growth and metastasis. D2O-induction of apoptosis was substantiated by AV-PI flow cytometry, immunodetection of PARP-1, and pro-caspase 3 cleavage, and rescue by pan-caspase inhibition. Differential array analysis revealed early modulation of stress response gene expression in both A375 melanoma and PANC-1 adenocarcinoma cells elicited by D2O (90%; ≤6 h) (upregulated: CDKN1A, DDIT3, EGR1, GADD45A, HMOX1, NFKBIA, or SOD2 (up to 9-fold; p < 0.01)) confirmed by independent RT-qPCR analysis. Immunoblot analysis revealed rapid onset of D2O-induced stress response phospho-protein activation (p-ERK, p-JNK, p-eIF2α, or p-H2AX) or attenuation (p-AKT). Feasibility of D2O-based chemotherapeutic intervention (drinking water (30% w/w)) was demonstrated in a severe combined immunodeficiency (SCID) mouse melanoma metastasis model using luciferase-expressing A375-Luc2 cells. Lung tumor burden (visualized by bioluminescence imaging) was attenuated by D2O, and inhibition of invasiveness was also confirmed in an in vitro Matrigel transwell invasion assay. D2O supplementation also suppressed tumor growth in a murine xenograft model of human melanoma, and median survival was significantly increased without causing adverse effects. These data demonstrate for the first time that systemic D2O administration impairs growth and metastasis of malignant melanoma through the pharmacological induction of deuterium (2H)-stress.

scholarly journals Activation of stress response gene SIRT1 by BCR-ABL promotes leukemogenesis

Blood ◽  
2012 ◽  
Vol 119 (8) ◽  
pp. 1904-1914 ◽  
Author(s):  
Hongfeng Yuan ◽  
Zhiqiang Wang ◽  
Ling Li ◽  
Hao Zhang ◽  
Hardik Modi ◽  
...  
Keyword(s):  
Stress Response ◽  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Gene Knockout ◽  
Myelogenous Leukemia ◽  
Hematopoietic Progenitor Cells ◽  
Imatinib Treatment ◽  
Hematopoietic Progenitor ◽  
Response Gene ◽  
Stress Response Gene

Abstract The tyrosine kinase inhibitor imatinib is highly effective in the treatment of chronic myelogenous leukemia (CML), but primary and acquired resistance of CML cells to the drug offset its efficacy. Molecular mechanisms for resistance of CML to tyrosine kinase inhibitors are not fully understood. In the present study, we show that BCR-ABL activates the expression of the mammalian stress response gene SIRT1 in hematopoietic progenitor cells and that this involves STAT5 signaling. SIRT1 activation promotes CML cell survival and proliferation associated with deacetylation of multiple SIRT1 substrates, including FOXO1, p53, and Ku70. Imatinib-mediated inhibition of BCR-ABL kinase activity partially reduces SIRT1 expression and SIRT1 inhibition further sensitizes CML cells to imatinib-induced apoptosis. Knockout of SIRT1 suppresses BCR-ABL transformation of mouse BM cells and the development of a CML-like myeloproliferative disease, and treatment of mice with the SIRT1 inhibitor tenovin-6 deters disease progression. The combination of SIRT1 gene knockout and imatinib treatment further extends the survival of CML mice. Our results suggest that SIRT1 is a novel survival pathway activated by BCR-ABL expression in hematopoietic progenitor cells, which promotes oncogenic transformation and leukemogenesis. Our findings suggest further exploration of SIRT1 as a therapeutic target for CML treatment to overcome resistance.

scholarly journals Integration of two RAB5 groups during endosomal transport in plants

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Emi Ito ◽  
Kazuo Ebine ◽  
Seung-won Choi ◽  
Sakura Ichinose ◽  
Tomohiro Uemura ◽  
...  
Keyword(s):  
Genetic Interaction ◽  
Interaction Analysis ◽  
Plant Cells ◽  
Eukaryotic Cells ◽  
Endosomal Trafficking ◽  
Effector Molecule ◽  
Endosomal Transport ◽  
Vacuolar Transport ◽  
Rab Effector ◽  
Genetic Interaction Analysis

RAB5 is a key regulator of endosomal functions in eukaryotic cells. Plants possess two different RAB5 groups, canonical and plant-unique types, which act via unknown counteracting mechanisms. Here, we identified an effector molecule of the plant-unique RAB5 in Arabidopsis thaliana, ARA6, which we designated PLANT-UNIQUE RAB5 EFFECTOR 2 (PUF2). Preferential colocalization with canonical RAB5 on endosomes and genetic interaction analysis indicated that PUF2 coordinates vacuolar transport with canonical RAB5, although PUF2 was identified as an effector of ARA6. Competitive binding of PUF2 with GTP-bound ARA6 and GDP-bound canonical RAB5, together interacting with the shared activating factor VPS9a, showed that ARA6 negatively regulates canonical RAB5-mediated vacuolar transport by titrating PUF2 and VPS9a. These results suggest a unique and unprecedented function for a RAB effector involving the integration of two RAB groups to orchestrate endosomal trafficking in plant cells.

scholarly journals Systematic characterization and genetic interaction analysis of adhesins in Candida albicans virulence

2020 ◽  
Author(s):  
Sierra Rosiana ◽  
Liyang Zhang ◽  
Grace H. Kim ◽  
Alexey V. Revtovich ◽  
Arjun Sukumaran ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Genetic Interaction ◽  
Interaction Analysis ◽  
Genetic Interactions ◽  
Fungal Virulence ◽  
C Elegans ◽  
Adhesin Proteins ◽  
Insight Into

AbstractCandida albicans is a microbial fungus that exists as a commensal member of the human microbiome and an opportunistic pathogen. Cell surface-associated adhesin proteins play a crucial role in C. albicans’ ability to undergo cellular morphogenesis, develop robust biofilms, colonize, and cause infection in a host. However, a comprehensive analysis of the role and relationships between these adhesins has not been explored. We previously established a CRISPR-based platform for efficient generation of single- and double-gene deletions in C. albicans, which was used to construct a library of 144 mutants, comprising 12 unique adhesin genes deleted singly, or in every possible combination of double deletions. Here, we exploit this adhesin mutant library to explore the role of adhesin proteins in C. albicans virulence. We perform a comprehensive, high-throughput screen of this library, using Caenorhabditis elegans as a simplified model host system, which identified mutants critical for virulence and significant genetic interactions. We perform follow-up analysis to assess the ability of high- and low-virulence strains to undergo cellular morphogenesis and form biofilms in vitro, as well as to colonize the C. elegans host. We further perform genetic interaction analysis to identify novel significant negative genetic interactions between adhesin mutants, whereby combinatorial perturbation of these genes significantly impairs virulence, more than expected based on virulence of the single mutant constituent strains. Together, this yields important new insight into the role of adhesins, singly and in combinations, in mediating diverse facets of virulence of this critical fungal pathogen.SummaryCandida albicans is a human fungal pathogen and cause of life-threatening systemic infections. Cell surface-associated adhesins play a central role in this pathogen’s ability to establish infection. Here, we provide a comprehensive analysis of adhesin factors, and their role in fungal virulence. Exploiting a high-throughput workflow, we screened an adhesin mutant library using C. elegans as a simple model host, and identified mutants and genetic interactions involved in virulence. We found that adhesin mutants are impaired in in vitro pathogenicity, irrespective of their virulence. Together, this work provides new insight into the role of adhesin factors in mediating fungal virulence.

scholarly journals Solar simulated ultraviolet radiation inactivates HCoV-NL63 and SARS-CoV-2 coronaviruses at environmentally relevant doses

2021 ◽  
Author(s):  
Georg T. Wondrak ◽  
Jana Jandova ◽  
Spencer J. Williams ◽  
Dominik Schenten
Keyword(s):  
Gene Expression ◽  
Stress Response ◽  
Uv Light ◽  
Gene Expression Array ◽  
Array Analysis ◽  
Expression Array ◽  
Response Gene ◽  
Full Spectrum ◽  
Solar Uv ◽  
Stress Response Gene

The germicidal properties of short wavelength ultraviolet C (UVC) light are well established and used to inactivate many viruses and other microbes. However, much less is known about germicidal effects of terrestrial solar UV light, confined exclusively to wavelengths in the UVA and UVB regions. Here, we have explored the sensitivity of the human coronaviruses HCoV-NL63 and SARS-CoV-2 to solar-simulated full spectrum ultraviolet light (sUV) delivered at environmentally relevant doses. First, HCoV-NL63 coronavirus inactivation by sUV-exposure was confirmed employing (i) viral plaque assays, (ii) RT-qPCR detection of viral genome replication, and (iii) infection-induced stress response gene expression array analysis. Next, a detailed dose-response relationship of SARS-CoV-2 coronavirus inactivation by sUV was elucidated, suggesting a half maximal suppression of viral infectivity at low sUV doses. Likewise, extended sUV exposure of SARS-CoV-2 blocked cellular infection as revealed by plaque assay and stress response gene expression array analysis. Moreover, comparative (HCoV-NL63 versus SARS-CoV-2) single gene expression analysis by RT-qPCR confirmed that sUV exposure blocks coronavirus-induced redox, inflammatory, and proteotoxic stress responses. Based on our findings, we estimate that solar ground level full spectrum UV light impairs coronavirus infectivity at environmentally relevant doses. Given the urgency and global scale of the unfolding SARS-CoV-2 pandemic, these prototype data suggest feasibility of solar UV-induced viral inactivation, an observation deserving further molecular exploration in more relevant exposure models.

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