Biology subjects

Huan, Y. W.

Publications and source records attributed to Huan, Y. W..

3 recordsLinked to original sources

bioRxiv2024

Deep mutational scanning of influenza A virus NEP reveals pleiotropic mutations in its N-terminal domain

The influenza A virus nuclear export protein (NEP) is a multifunctional protein that is essential for the viral life cycle and has very high sequence conservation. However, since the open reading frame of NEP largely overlaps with that of another influenza viral protein, non-structural protein 1, it is difficult to infer the functional constraints of NEP based on sequence conservation analysis. Besides, the N-terminal of NEP is structurally disordered, which further complicates the understanding of its function. Here, we systematically measured the replication fitness effects of >1,800 mutations of NEP. Our results show that the N-terminal domain has high mutational tolerance. Additional experiments demonstrate that N-terminal domain mutations pleiotropically affect viral transcription and replication dynamics, host cellular responses, and mammalian adaptation of avian influenza virus. Overall, our study not only advances the functional understanding of NEP, but also provides insights into its evolutionary constraints.

cell biology↗

bioRxiv2022

P1 bacteriophage-enabled delivery of CRISPR-Cas9 antimicrobial activity against Shigella flexneri

The discovery of clustered, regularly interspaced, short palindromic repeats (CRISPR) and the Cas9 RNA-guided nuclease provides unprecedented opportunities to selectively kill specific populations or species of bacteria. However, the use of CRISPR-Cas9 to clear bacterial infections in vivo is hampered by the inefficient delivery of cas9 genetic constructs into bacterial cells. Here, we use a broad-host-range P1-derived phagemid to deliver the CRISPR-Cas9 chromosomal-targeting system into Escherichia coli and the dysentery-causing Shigella flexneri to achieve DNA sequence-specific killing of targeted bacterial cells. We show that genetic modification of the helper P1 phage DNA packaging site (pac) significantly enhances the purity of packaged phagemid and improves the Cas9-mediated killing of S. flexneri cells. We further demonstrate that P1 phage particles can deliver chromosomal-targeting cas9 phagemids into S. flexneri in vivo using a zebrafish larvae infection model, where it significantly reduces the bacterial load and promotes host survival. Our study highlights the potential of combining a P1 bacteriophage-based delivery with the CRISPR chromosomal-targeting system to achieve DNA sequence-specific cell lethality and efficient clearance of bacterial infection.

microbiology↗

bioRxiv2022

The host range and the role of O-antigen in P1 transduction with its alternative S' tail fibre

Enterobacteria phage P1 expresses two types of tail fibre, S and S. Despite the wide usage of phage P1 for transduction, the host range and the receptor for its alternative S tail fibre was never determined. Here, a {Delta}S-cin {Delta}pac E. coli P1 lysogenic strain was generated to allow packaging of phagemid DNA into P1 phage having either S or S tail fibre. P1(S) could transduce phagemid DNA into Shigella flexneri 2a 2457O, Shigella flexneri 5a M90T and Escherichia coli O3 efficiently. Mutational analysis of the O-antigen assembly genes and LPS inhibition assays indicated that P1(S) transduction requires at least one O-antigen unit. E. coli O111:B4 LPS produced a high neutralising effect against P1(S) transduction, indicating that this E. coli strain could be a host for P1(S). Mutations in the O-antigen modification genes of S. flexneri 2a 2457O and S. flexneri 5a M90T did not cause significant changes to P1(S) transduction efficiency. A higher transduction efficiency of P1(S) improved the delivery of a cas9 antimicrobial phagemid into both S. flexneri 2457O and M90T. These findings provide novel insights into P1 tropism-switching, by identifying the host range of P1(S) and demonstrating its potential for delivering a sequence-specific Cas9 antimicrobial into clinically relevant S. flexneri.

microbiology↗