Biology subjects

Nguyen, G. T.

Publications and source records attributed to Nguyen, G. T..

6 recordsLinked to original sources

bioRxiv2025

β-nicotinamide mononucleotide production in Vibrio natriegens: a preliminary study

BackgroundNicotinamide mononucleotide (NMN), is a promising nutraceutical attracting much attention for its pharmacological and anti-aging efficacies. However, NMN-containing commercial products are very high-priced due to the lack of efficient and facile methods for industrial-scale production. To date, various metabolic engineering strategies have been successfully applied to produce NMN in Escherichia coli. Recently, Vibrio natriegens has become a promising host in the bioindustry thanks to its rapid growth and capabilities of broad substrate utilization. This study aims to evaluate the NMN biosynthesis capability of V. natriegens. MethodsFirstly, a mutant V. natriegens strain ({Delta}dns::araC-T7RNAP-KanR{Delta} pncC::FtnadE-SmR{Delta} nadR) was generated via multiplex genome editing by natural transformation (MuGENT). Nampt genes encoding nicotinamide phosphoribosyltransferase from Chitinophaga pinensis, Sphingopyxis sp. C-1, Haemophilus ducreyi, and Vibrio phage KVP40 were codon-optimized and cloned into pACYCDuet-1 under the control of T7 promoter. The recombinant plasmids were electroporated into the mutant strain. The expression of recombinant NAMPTs in V. natriegens was evaluated by SDS-PAGE analysis and the intracellular NMN concentrations were quantified by HPLC. ResultsAfter two rounds of MuGENT, V. natriegens V54-33 strain ({Delta}dns::araC-T7RNAP-KanR{Delta} pncC::FtnadE-SmR{Delta} nadR) was successfully generated. SDS-PAGE analysis demonstrated that all NAMPTs were strongly expressed in the V54-33 strain. HPLC analysis revealed that the highest intracellular NMN concentration was obtained with NAMPT from Chitinophaga pinensis (44.5 M), followed by NAMPT from Vibrio phage KVP40 (23.3 M). ConclusionThis study demonstrated the feasibility of NMN biosynthesis in V. natriegens.

bioengineering↗

bioRxiv2024

CRISPR-Cas9 target-strand nicking provides phage resistance by inhibiting replication

Cas endonucleases, like Cas9 and Cas12a, are RNA-guided immune effectors that provide bacterial defense against bacteriophages. Cas endonucleases rely on divalent metal ions for their enzymatic activities and to facilitate conformational changes that are required for specific recognition and cleavage of target DNA. While Cas endonucleases typically produce double-strand breaks (DSBs) in DNA targets, reduced, physiologically relevant Mg2+ concentrations and target mismatches can result in incomplete second-strand cleavage, resulting in the production of a nicked DNA. It remains poorly understood whether nicking by Cas endonucleases is sufficient to provide protection against phage. To address this, we tested phage protection by Cas9 nickases, in which only one of two nuclease domains is catalytically active. By testing a large panel of guide RNAs, we find that target strand nicking can be sufficient to provide immunity, while non-target nicking does not provide any additional protection beyond Cas9 binding. Target-strand nicking inhibits phage replication and can reduce the susceptibility of Cas9 to viral escape when targeting non-essential regions of the genome. Cleavage of the non- target strand by the RuvC domain is strongly impaired at low Mg2+ concentrations. As a result, fluctuations in the concentration of other biomolecules that can compete for binding of free Mg2+ strongly influences the ability of Cas9 to form a DSB at targeted sites. Overall, our results suggest that Cas9 may only nick DNA during CRISPR-mediated immunity, especially under conditions of low Mg2+ availability in cells.

biochemistry↗

bioRxiv2023

CRISPR-Cas12a exhibits metal-dependent specificity switching

Cas12a is the immune effector of type V-A CRISPR-Cas systems and has been co-opted for genome editing and other biotechnology tools. The specificity of Cas12a has been the subject of extensive investigation both in vitro and in genome editing experiments. However, in vitro studies have often been performed at high magnesium ion concentrations that are inconsistent with the free Mg2+ concentrations that would be present in cells. By profiling the specificity of Cas12a orthologs at a range of Mg2+ concentrations, we find that Cas12a switches its specificity depending on metal ion concentration. Lowering Mg2+ concentration decreases cleavage defects caused by seed mismatches, while increasing the defects caused by PAM-distal mismatches. We show that Cas12a can bind seed mutant targets more rapidly at low Mg2+ concentrations, resulting in faster cleavage. In contrast, PAM-distal mismatches cause substantial defects in cleavage following formation of the Cas12a-target complex at low Mg2+ concentrations. We observe differences in Cas12a specificity switching between three orthologs that results in variations in the routes of phage escape from Cas12a-mediated immunity. Overall, our results reveal the importance of physiological metal ion conditions on the specificity of Cas effectors that are used in different cellular environments.

biochemistry↗

bioRxiv2022

CRISPR-Cas effector specificity and target mismatches determine phage escape outcomes

CRISPR-mediated interference relies on complementarity between a guiding CRISPR RNA (crRNA) and target nucleic acids to provide defense against bacteriophage. Phages escape CRISPR-based immunity mainly through mutations in the PAM and seed regions. However, previous specificity studies of Cas effectors, including the class 2 endonuclease Cas12a, have revealed a high degree of tolerance of single mismatches. The effect of this mismatch tolerance has not been extensively studied in the context of phage defense. Here, we tested defense against lambda phage provided by Cas12a-crRNAs containing pre-existing mismatches against the genomic targets in phage DNA. We observe a correlation between Cas12a mismatch tolerance in vitro and phage defense on solid media. However, in liquid media, we find that most pre-existing crRNA mismatches lead to phage escape and lysis, regardless of whether the mismatches ablate Cas12a cleavage in vitro. We used high-throughput sequencing to examine the target regions of phage genomes following CRISPR challenge. Mismatches at all locations in the target accelerated emergence of mutant phage, including mismatches that greatly slowed cleavage in vitro. Mutations arose near the existing mismatch, in some cases resulting in multiple PAM-distal mismatches allowing for phage escape. Similar experiments with Cas9 showed the location of emergent target mutations was unaffected by pre-existing crRNA-target mismatches. Expression of multiple mismatched crRNAs prevented new mutations from arising in multiple targeted locations, allowing Cas12a mismatch tolerance to provide stronger and longer term protection. These results demonstrate that Cas effector mismatch tolerance and existing target mismatches strongly influence phage evolution.

microbiology↗

bioRxiv2021

Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation

Signaling cascades that convert the recognition of pathogens to efficient inflammatory responses by immune cells, specifically neutrophils, are critical for host survival. SKAP2, an adaptor protein, is required for reactive oxygen species (ROS) generation following stimulation by integrins, formyl peptide receptors and gram-negative bacteria Klebsiella pneumoniae and Yersinia pseudotuberculosis in vitro (Nguyen et al., 2020, Shaban et al., 2020, Boras et al., 2017). SKAP2 is also required for the host defense against K. pneumoniae and{Delta} yopH Y. pseudotuberculosis infection in vivo in mouse models (Shaban et al., 2020, Nguyen et al., 2020). Another class of pattern recognition receptors (PRR) is the C-type lectin receptors (CLR), such as Dectin-1, Dectin-2 and Mincle, that are critical to trigger innate immune responses. Using neutrophils from murine HoxB8-immortalized progenitors, we show that SKAP2 is crucial for maximal ROS response to purified CLR agonists and to the fungal pathogens Candida glabrata and C. albicans, as well as for robust killing of C. glabrata. Skap2-/- murine neutrophils failed to generate ROS and exhibited reduced cellular adhesion in response to trehalose-6,6-dibehenate (TDB), furfurman, and curdlan, Mincle, Dectin-2, and Dectin-1 agonists, respectively. TDB, furfurman, and curdlan stimulation also led to SKAP2-independent integrin conformational changes, showing that inside-out signaling by these CLRs to integrin occurs in the absence of SKAP2. Pyk2 phosphorylation was significantly reduced after infection with C. glabrata in Skap2-/- neutrophils, while Syk phosphorylation was unaffected by the loss of SKAP2. These data strengthen the importance of SKAP2 in the activation of neutrophil ROS production by PRRs to include CLRs and extend the role of SKAP2 in host defense beyond antibacterial immunity to include Candida species.

immunology↗

bioRxiv2021

Host-directed therapies against early-lineage SARS-CoV-2 retain efficacy against B.1.1.7 variant

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths worldwide and massive societal and economic burden. Recently, a new variant of SARS-CoV-2, known as B.1.1.7, was first detected in the United Kingdom and is spreading in several other countries, heightening public health concern and raising questions as to the resulting effectiveness of vaccines and therapeutic interventions. We and others previously identified host-directed therapies with antiviral efficacy against SARS-CoV-2 infection. Less prone to the development of therapy resistance, host-directed drugs represent promising therapeutic options to combat emerging viral variants as host genes possess a lower propensity to mutate compared to viral genes. Here, in the first study of the full-length B.1.1.7 variant virus, we find two host-directed drugs, plitidepsin (aplidin; inhibits translation elongation factor eEF1A) and ralimetinib (inhibits p38 MAP kinase cascade), as well as remdesivir, to possess similar antiviral activity against both the early-lineage SARS-CoV-2 and the B.1.1.7 variant, evaluated in both human gastrointestinal and lung epithelial cell lines. We find that plitidepsin is over an order of magnitude more potent than remdesivir against both viruses. These results highlight the importance of continued development of host-directed therapeutics to combat current and future coronavirus variant outbreaks.

microbiology↗