| 1. |
- Alalam, Hanna, et al.
(författare)
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A Genetic Trap in Yeast for Inhibitors of SARS-CoV-2 Main Protease
- 2021
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Ingår i: MSYSTEMS. - 2379-5077. ; 6:6
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Tidskriftsartikel (refereegranskat)abstract
- The ongoing COVID-19 pandemic urges searches for antiviral agents that can block infection or ameliorate its symptoms. Using dissimilar search strategies for new antivirals will improve our overall chances of finding effective treatments. Here, we have established an experimental platform for screening of small molecule inhibitors of the SARS-CoV-2 main protease in Saccharomyces cerevisiae cells, genetically engineered to enhance cellular uptake of small molecules in the environment. The system consists of a fusion of the Escherichia coli toxin MazF and its antitoxin MazE, with insertion of a protease cleavage site in the linker peptide connecting the MazE and MazF moieties. Expression of the viral protease confers cleavage of the MazEF fusion, releasing the MazF toxin from its antitoxin, resulting in growth inhibition. In the presence of a small molecule inhibiting the protease, cleavage is blocked and the MazF toxin remains inhibited, promoting growth. The system thus allows positive selection for inhibitors. The engineered yeast strain is tagged with a fluorescent marker protein, allowing precise monitoring of its growth in the presence or absence of inhibitor. We detect an established main protease inhibitor by a robust growth increase, discernible down to 1 mM. The system is suitable for robotized large-scale screens. It allows in vivo evaluation of drug candidates and is rapidly adaptable for new variants of the protease with deviant site specificities. IMPORTANCE The COVID-19 pandemic may continue for several years before vaccination campaigns can put an end to it globally. Thus, the need for discovery of new antiviral drug candidates will remain. We have engineered a system in yeast cells for the detection of small molecule inhibitors of one attractive drug target of SARS-CoV-2, its main protease, which is required for viral replication. The ability to detect inhibitors in live cells brings the advantage that only compounds capable of entering the cell and remain stable there will score in the system. Moreover, because of its design in yeast cells, the system is rapidly adaptable for tuning the detection level and eventual modification of the protease cleavage site in the case of future mutant variants of the SARSCoV-2 main protease or even for other proteases.
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| 2. |
- Alalam, Hanna, et al.
(författare)
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A High-Throughput Method for Screening for Genes Controlling Bacterial Conjugation of Antibiotic Resistance.
- 2020
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Ingår i: mSystems. - 2379-5077. ; 5:6
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Tidskriftsartikel (refereegranskat)abstract
- The rapid horizontal transmission of antibiotic resistance genes on conjugative plasmids between bacterial host cells is a major cause of the accelerating antibiotic resistance crisis. There are currently no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by conjugation, which prevents understanding and targeting conjugation. We introduce a novel experimental framework to screen for conjugation-based horizontal transmission of antibiotic resistance between >60,000 pairs of cell populations in parallel. Plasmid-carrying donor strains are constructed in high-throughput. We then mix the resistance plasmid-carrying donors with recipients in a design where only transconjugants can reproduce, measure growth in dense intervals, and extract transmission times as the growth lag. As proof-of-principle, we exhaustively explore chromosomal genes controlling F-plasmid donation within Escherichia coli populations, by screening the Keio deletion collection in high replication. We recover all seven known chromosomal gene mutants affecting conjugation as donors and identify many novel mutants, all of which diminish antibiotic resistance transmission. We validate nine of the novel genes' effects in liquid mating assays and complement one of the novel genes' effect on conjugation (rseA). The new framework holds great potential for exhaustive disclosing of candidate targets for helper drugs that delay resistance development in patients and societies and improve the longevity of current and future antibiotics. Further, the platform can easily be adapted to explore interspecies conjugation, plasmid-borne factors, and experimental evolution and be used for rapid construction of strains.IMPORTANCE The rapid transmission of antibiotic resistance genes on conjugative plasmids between bacterial host cells is a major cause of the accelerating antibiotic resistance crisis. There are currently no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by conjugation, which prevents understanding and targeting conjugation. We introduce a novel experimental framework to screen for conjugation-based horizontal transmission of antibiotic resistance between >60,000 pairs of cell populations in parallel. As proof-of-principle, we exhaustively explore chromosomal genes controlling F-plasmid donation within E. coli populations. We recover all previously known and many novel chromosomal gene mutants that affect conjugation efficiency. The new framework holds great potential for rapid screening of compounds that decrease transmission. Further, the platform can easily be adapted to explore interspecies conjugation, plasmid-borne factors, and experimental evolution and be used for rapid construction of strains.
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| 3. |
- Alalam, Hanna, et al.
(författare)
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Conjugation factors controlling F-plasmid antibiotic resistance transmission
- 2018
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Ingår i: BioRxiv. - : Cold Spring Harbor Laboratory.
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The rapid horizontal transmission of many antibiotic resistance genes between bacterial host cells on conjugative plasmids is a major cause of the accelerating antibiotic resistance crisis. Preventing understanding and targeting conjugation, there currently are no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by conjugation. We introduce a novel experimental framework to screen for conjugation based horizontal transmission of antibiotic resistance between >60,000 pairs of cell populations in parallel. Plasmid-carrying donor strains are constructed in high throughput. We then mix the resistance plasmid carrying donors with recipients in a design where only transconjugants can reproduce, measure growth in dense intervals and extract transmission times as the growth lag. As proof-of-principle, we exhaustively explored chromosomal genes controlling F plasmid donation within E. coli populations, by screening the Keio deletion collection at high replication. We recover all six known chromosomal gene mutants affecting conjugation and identify >50 novel factors, all of which diminish antibiotic resistance transmission. We verify 10 of the novel genes' effects in a liquid mating assay. The new framework holds great potential for exhaustive disclosing of candidate targets for helper drugs that delay resistance development in patients and societies and improves the longevity of current and future antibiotics.
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| 4. |
- Alalam, Hanna, et al.
(författare)
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Global SLAM-Seq for accurate mRNA decay determination and identification of NMD targets.
- 2022
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1469-9001 .- 1355-8382. ; 28:7
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Tidskriftsartikel (refereegranskat)abstract
- Gene expression analysis requires accurate measurements of global RNA degradation rates, earlier problematic with methods disruptive to cell physiology. Recently, metabolic RNA labeling emerged as an efficient and minimally invasive technique applied in mammalian cells. Here, we have adapted SH-Linked Alkylation for the Metabolic Sequencing of RNA (SLAM-Seq) for a global mRNA stability study in yeast using 4-thiouracil pulse-chase labeling. We assign high-confidence half-life estimates for 67.5 % of expressed ORFs, and measure a median half-life of 9.4 min. For mRNAs where half-life estimates exist in the literature, their ranking order was in good agreement with previous data, indicating that SLAM-Seq efficiently classifies stable and unstable transcripts. We then leveraged our yeast protocol to identify targets of the Nonsense-mediated decay (NMD) pathway by measuring the change in RNA half-lives; instead of steady-state RNA level changes. With SLAM-Seq, we assign 580 transcripts as putative NMD targets, based on their measured half-lives in wild-type and upf3Δ mutants. We find 225 novel targets, and observe a strong agreement with previous reports of NMD targets, 61.2 % of our candidates being identified in previous studies. This indicates that SLAM-Seq is a simpler and more economic method for global quantification of mRNA half-lives. Our adaptation for yeast yielded global quantitative measures of the NMD effect on transcript half-lives, high correlation with RNA half-lives measured previously with more technically challenging protocols, and identification of novel NMD regulated transcripts that escaped prior detection.
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| 5. |
- Alalam, Hanna
(författare)
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Simplified Nonsense: New Methods for Interrogating NMD
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Nonsense mediated decay (NMD) is a pathway that regulates RNA turnover. Since its discovery, this pathway has been implicated in a variety of cellular processes ranging from differentiation to the restriction of viral replication. While NMD has been heavily studied since its discovery, the understanding of how the pathway carries out its function has been a long and convoluted process, where the current cornerstones that establish our present understanding of the regulatory mechanisms are continuously challenged. In this thesis, new methods were explored with the goal to provide tools that would simplify investigating the NMD pathway and potentially other pathways regulating RNA. We studied the use of nucleotide conversion methods and their applicability to yeast. Additionally, we designed a set of reporters that allow in vivo monitoring of NMD with an easy-to-read phenotype as an output. Moreover, we modified a reporter that was developed during the construction of the NMD reporters to also be applicable for alternative studies. In this particular case, we adapted one of our reporters to the study of the SARS-CoV-2 major protease (NSP5). Overall, simplified methods to interrogate cellular NMD were successfully constructed, in addition to establishing a sensitive yeast based system for the detection of anti-viral compounds.
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| 6. |
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| 7. |
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| 8. |
- Park, Kyong-Su, et al.
(författare)
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Detoxified synthetic bacterial membrane vesicles as a vaccine platform against bacteria and SARS-CoV-2
- 2023
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Ingår i: Journal of Nanobiotechnology. ; 21:1
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Tidskriftsartikel (refereegranskat)abstract
- The development of vaccines based on outer membrane vesicles (OMV) that naturally bud off from bacteria is an evolving field in infectious diseases. However, the inherent inflammatory nature of OMV limits their use as human vaccines. This study employed an engineered vesicle technology to develop synthetic bacterial vesicles (SyBV) that activate the immune system without the severe immunotoxicity of OMV. SyBV were generated from bacterial membranes through treatment with detergent and ionic stress. SyBV induced less inflammatory responses in macrophages and in mice compared to natural OMV. Immunization with SyBV or OMV induced comparable antigen-specific adaptive immunity. Specifically, immunization with Pseudomonas aeruginosa-derived SyBV protected mice against bacterial challenge, and this was accompanied by significant reduction in lung cell infiltration and inflammatory cytokines. Further, immunization with Escherichia coli-derived SyBV protected mice against E. coli sepsis, comparable to OMV-immunized group. The protective activity of SyBV was driven by the stimulation of B-cell and T-cell immunity. Also, SyBV were engineered to display the SARS-CoV-2 S1 protein on their surface, and these vesicles induced specific S1 protein antibody and T-cell responses. Collectively, these results demonstrate that SyBV may be a safe and efficient vaccine platform for the prevention of bacterial and viral infections
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| 9. |
- Peri, Kameshwara Venkata Ramana, 1990, et al.
(författare)
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Regulation of lactose and galactose growth: Insights from a unique metabolic gene cluster in Candida intermedia
- 2023
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Lactose assimilation is a relatively rare trait in yeasts, and Kluyveromyces yeast species have long served as model organisms for studying lactose metabolism. Meanwhile, the metabolic strategies of most other lactose-assimilating yeasts remain unknown. In this work, we have elucidated the genetic determinants of the superior lactose-growing yeast Candida intermedia. Through genomic and transcriptomic analyses and deletion mutant phenotyping, we identified three interdependent gene clusters responsible for the metabolism of lactose and its hydrolysis product galactose: the conserved LAC cluster (LAC12, LAC4) for lactose uptake and hydrolysis, the conserved GAL cluster (GAL1, GAL7, GAL10) for galactose catabolism, and a unique “GALLAC” cluster. This novel GALLAC cluster, which has evolved through gene duplication and divergence, proved indispensable for C. intermedia’s growth on lactose and galactose. The cluster contains the transcriptional activator gene LAC9, second copies of GAL1 and GAL10 and the XYL1 gene encoding an aldose reductase involved in carbon overflow metabolism. Notably, the regulatory network in C. intermedia, governed by Lac9 and Gal1 from the GALLAC cluster, differs significantly from the (ga)lactose regulons in Saccharomyces cerevisiae, Kluyveromyces lactis and Candida albicans. Moreover, although lactose and galactose metabolism are closely linked in C. intermedia, our results also point to important regulatory differences. This study paves the way to a better understanding of lactose and galactose metabolism in C. intermedia and provides new evolutionary insights into yeast metabolic pathways and regulatory networks. In extension, the results will facilitate future development and use of C. intermedia as a cell-factory for conversion of lactose-rich whey into value-added products.
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