Biology subjects

Yee, W.-X.

Publications and source records attributed to Yee, W.-X..

7 recordsLinked to original sources

bioRxiv2026

A tripartite mechanism licenses RNA polymerase import into the phage nucleus

Nucleus-forming jumbo phages import a phage-encoded non-virion RNA polymerase (nvRNAP) into a proteinaceous nucleus, but how it is selected for import is unknown. Here, we identify an import pathway that couples nvRNAP subunit interactions with phage-encoded import factors. Maximal nvRNAP import requires a novel factor Imp7 (gp166) and the essential import factor Imp1. Imp7 is essential at environmental temperatures (20 C), where impaired nvRNAP import causes a profound loss of middle and late transcription and blocks phage DNA replication. Import of individual nvRNAP subunits also depends on other subunits, consistent with import licensing at the level of an assembled complex. In the absence of middle transcription and DNA replication, the phage nucleus surprisingly still assembles and segregates from the cytoplasm, demonstrating that nvRNAP nuclear localization and activity are not a critical checkpoint for nucleus assembly. Together, these findings define a specialized pathway for nuclear import of the multi-subunit RNAP and reveal that nuclear compartment assembly precedes the transcriptional and replicative programs required for its maturation.

microbiology↗

bioRxiv2026

Intracellular barriers and receptor masking limit success of a Pseudomonas aeruginosa clinical phage family

Bacteriophage therapy is needed to treat antibiotic resistant infections; however, when a clinical isolate resists a given phage, it is often unclear why. It is therefore currently unknown how to rationally fortify phage therapies to circumvent a priori resistance. Using a family of broad host range therapeutic Pseudomonas aeruginosa phages (Pbunaviruses), we show that cell surface receptor masking and intracellular defenses are both common barriers in distinct clinical isolates. In some cases these barriers can be bypassed by intrafamily phage engineering. Using unbiased genome-wide CRISPRi screens, we reveal that the broadly conserved L-Rhamnose in the core polysaccharide is the receptor for Pbunavirus family. This molecule is often masked by diverse O-antigen structures. In other isolates with the L-Rha receptor accessible, internal defense mechanisms commonly prevent Pbunavirus DNA replication. A single anti-defense locus often encoding 8-11 different genes within the Pbunavirus family is required for optimal host range, providing anti-defense genes that enable replication of both Pbunavirus phages and phages of other families. Our work demonstrates the importance of both internal and surface defense mechanisms in clinical isolates causally antagonizing a commonly used phage therapeutic and presents phage engineering strategies to circumvent a priori resistance.

microbiology↗

bioRxiv2026

Phase-variable restriction modification systems dynamically regulate plasmid transmission in Neisseria gonorrhoeae

Horizontal gene transfer (HGT) in bacteria is shaped by restriction-modification systems (RMSs) that define genetic barriers between species and lineages. Neisseria gonorrhoeae harbours numerous RMS, most of which are part of its core genome. Here, we show that phase variation in these RMSs can limit interspecies and intraspecies plasmid exchange. While NlaIV likely influenced the acquisition of pConj from the meningococcus, phase variation of the NgoAV RMS produces epigenetically heterogeneous populations that present variable bottlenecks to within-species plasmid transmission, influencing the spread of anti-microbial-resistance plasmids, pConj and pbla. A LEAT sequence extends or retracts a molecular ruler within the HsdS specificity subunit, altering spacing of recognition motifs, while truncation of the protein leads to a switch from non-palindromic to palindromic recognition motifs. Thus, while the repertoire of gonococcal RMSs is largely conserved, phase-variation dynamically modulates gene flow, shaping plasmid transmission, and the evolution and spread of antimicrobial resistance.

microbiology↗

bioRxiv2025

Bacteriophage genome-wide transposon mutagenesis

Bacteriophages have genomes that span a wide size range, are densely packed with coding sequences, and frequently encode genes of unknown function. Classical forward genetics has defined essential genes for phage replication in a few model systems but remains laborious and non-scalable. Unbiased functional genomics approaches are therefore needed for phages, particularly for large lytic phages. Here, we develop a phage transposon sequencing (TnSeq) platform that uses the mariner transposase to insert an anti-CRISPR selectable marker into phage genomes. CRISPR-Cas13a-based enrichment of transposed phages followed by pooled sequencing identifies both fitness-conferring and dispensable genes. Using the Pseudomonas aeruginosa-infecting nucleus-forming jumbo phage {Phi}KZ (280,334 bp; 371 predicted genes) as a model, we show that [~]110 genes are fitness-conferring via phage TnSeq. These include conserved essential genes involved in phage nucleus formation, protein trafficking, transcription, DNA replication, and virion assembly. We also isolate hundreds of individual phages with insertions in non-essential genes and reveal conditionally essential genes that are specifically required in clinical isolates, at environmental temperature, or in the presence of a defensive nuclease. Phage TnSeq is a facile, scalable technology that can define essential phage genes and generate knockouts in all non-essential genes in a single experiment, enabling conditional genetic screens in phages and providing a broadly applicable resource for phage functional genomics.

microbiology↗

bioRxiv2025

END nucleases: Antiphage defense systems targeting multiple hypermodified phage genomes

Prokaryotes carry clusters of phage defense systems in "defense islands" that have been extensively exploited bioinformatically and experimentally for discovery of immune functions. However, little effort has been dedicated to determining which specific system(s) within defense islands limit lytic phage reproduction in clinical bacterial strains. Here, we employed the CRISPR-based Cascade-Cas3 system to delete defense islands in a Pseudomonas aeruginosa clinical isolate to identify mechanisms of lytic phage antagonism. Deletion of one island in a cystic fibrosis-derived clinical isolate sensitized the strain to phages from the Pbunavirus family, which are commonly used as therapeutics. The causal defense system is a Type IIS restriction endonuclease-like protein (ENDPaCF1), common in Pseudomonads, however it lacks an associated methyltransferase typical Type IIS R-M systems. ENDPaCF1 protects bacteria against phages with hypermodified DNA and is surprisingly agnostic to the specific structure of the modification, which is unlike typical type IV restriction endonucleases. In ENDPaCF1, the endonuclease domain is fused to a catalytically inactive Endonuclease III (iEndoIII), a domain that recognizes non-canonical bases to repair DNA in prokaryotes and eukaryotes. We therefore propose that nucleases containing an iEndoIII domain (END nucleases) can sense diverse DNA hypermodifications. Our findings reveal modularity of the sensing and cleavage domains, as expected of a modification-dependent endonucleases. We further show that some hypermodified phages, including Pbunavirus family members and Wrowclawvirus family (Pa5oct-like) of jumbo phages, encode END nuclease inhibitors that directly bind to the nuclease, likely via the iEndoIII domain. These inhibitors are necessary for Pbunavirus to plaque on clinical isolates and sufficient to enable other hypermodified phages to plaque in the presence of this defense system.

microbiology↗

bioRxiv2019

Novel differential linear B-cell epitopes to identify Zika and dengue virus infections in patients

BackgroundRecent Zika virus (ZIKV) outbreaks challenged existing laboratory diagnostic standards, especially for serology-based methods. Due to the genetic and structural similarity of ZIKV with other flaviviruses, this results in cross-reactive antibodies which confounds serological interpretations.\n\nMethodsPlasma from Singapore ZIKV patients was screened longitudinally for antibody responses and neutralizing capacities against ZIKV. Samples from healthy controls, ZIKV and DENV patients were further assessed using ZIKV and DENV peptides of precursor membrane (prM), envelope (E) or non-structural 1 (NS1) viral proteins in a peptide-based ELISA for epitope identification. Identified epitopes were re-validated and diagnostically evaluated using sera of patients with DENV, bacteria or unknown infections from Thailand.\n\nResultsLong-lasting ZIKV-neutralizing antibodies were elicited during ZIKV infection. Thirteen potential linear B-cell epitopes were identified and of these, four common flavivirus, three ZIKV-specific, and one DENV-specific differential epitopes had more than 50% sensitivities and specificities. Notably, ZIKV-specific peptide 26 on domain I/II of E protein (amino acid residues 271-288) presented 80% sensitivity and 85.7% specificity. Importantly, the differential epitopes also showed significance in differentiating non-flavivirus patient samples.\n\nConclusionsLinear B-cell epitope candidates to differentiate ZIKV and DENV infections were identified, providing the first step towards the design of a much-needed serology-based assay.

immunology↗

bioRxiv2017

Zika virus infection preferentially counterbalances human peripheral monocyte and/or NK-cell activity

Zika virus (ZIKV) has re-emerged in the population and caused unprecedented global outbreaks. Here, the transcriptomic consequences of ZIKV infection were studied systematically firstly in human peripheral blood CD14+ monocytes and monocyte-derived macrophages with high density RNA-sequencing. Analyses of the ZIKV genome revealed that the virus underwent genetic diversification and differential mRNA abundance was found in host cells during infection. Notably, there was a significant change in the cellular response with crosstalk between monocytes and natural killer (NK) cells as one of the highly identified pathway. Immune-phenotyping of peripheral blood from ZIKV-infected patients further confirmed the activation of NK cells during acute infection. ZIKV infection in peripheral blood cells isolated from healthy donors led to the induction of IFN{gamma} and CD107a -- two key markers of NK-cell function. Depletion of CD14+ monocytes from peripheral blood resulted in a reduction of these markers and reduced priming of NK cells during infection. This was complemented by the immunoproteomic changes observed. Mechanistically, ZIKV infection preferentially counterbalances monocyte and/or NK-cell activity, with implications for targeted cytokine immunotherapies.

immunology↗