Biology subjects

Wang, A. L.

Publications and source records attributed to Wang, A. L..

5 recordsLinked to original sources

bioRxiv2026

Regulation of polarity during morphological transitions in Aureobasidium pullulans

Many fungi are able to switch between different morphologies by regulating the behavior of the cytoskeleton. In turn, the cytoskeleton is regulated by Rho-family GTPases, whose differing spatiotemporal patterns of activity lead to different modes of growth. The polymorphic fungus Aureobasidium pullulans alternates between multiple growth modes, including multibudding, isotropic, and hyphal growth. Here, we describe hyphal growth and the yeast-to-hyphal transition in this organism, with a focus on cell polarity. Yeast cells switched between cycles of budding and isotropic expansion in a variable manner prior to initiating hyphal extension. Initial hyphal extension proceeded in a stop-and-start manner with frequent pauses, before switching to continuous growth. Hyphal branching occurred from the sides of hyphae, and septation at multiple sites occurred in more mature hyphae. Previous work showed that either of two Rho-family GTPases, Rac1 and Cdc42, were sufficient promote budding. However, while Rac1 was also able to drive hyphal extension without Cdc42, Cdc42 could not promote hyphal growth without Rac1. Our findings provide the basis for an investigation of how polarity components are re-purposed for different fungal growth modalities.

cell biology↗

bioRxiv2026

A Rationally Designed Transgene Drives CAR T Functional Persistence and Durable Regression of Solid Tumors

The eradication of solid tumors by chimeric antigen receptor (CAR) T cells requires dynamic therapies capable of outlasting an immune suppressive tumor microenvironment (TME). However, biological barriers--including rapid exhaustion, poor expansion, and loss of stem-like memory--quickly neutralize these therapies. Because single-technology interventions often introduce unacceptable tradeoffs between efficacy and safety, durable remission demands a paradigm where multiple engineered solutions work in concert. To holistically address these mechanisms, we rationally designed a single-vector transgene that intrinsically drives CAR T functional persistence. The platform integrates four synergistic technologies: a high-avidity mesothelin (MSLN)-targeting CAR optimized to resist shed decoy antigens, a T-cell activation-responsive promoter (OUTLAST OP1) resisting exhaustion, a CD8-targeted designed IL-2 cytokine (OUTSMART dIL-2) driving intratumoral CAR-T expansion, and an EGFRopt safety switch. In lung and ovarian cancer models, this rational integration was required to drive antigen-dependent T cell expansion and eradicate established tumors at extremely low CAR T doses. Furthermore, engineered cells established a self-renewing pool of stem-like memory T cells capable of rejecting tumor rechallenge months later. Ultimately, this work demonstrates that intrinsic T cell dysfunction and extrinsic tumor-derived barriers can be simultaneously overcome by integrating synergistic technologies.

synthetic biology↗

bioRxiv2026

Identification of disease-specific alleles and gene duplications from 1,600 Haemophilus influenzae genomes using predicted protein analyses from an unsupervised language model and clinical metadata

Haemophilus influenzae, a Gram-negative bacterium that is an obligate commensal of the human nasopharynx, is associated with both acute and chronic infections of the ears, adenoids, sinuses, and lungs, as well as pelvic inflammatory disease, sepsis, and meningitis. This diverse array of clinical disease phenotypes is due to H. influenzaes natural competence providing for frequent distributed gene exchange among strains during polyclonal colonizations. We performed whole genome sequencing (WGS) on [~]1000 H. influenzae strains and combined these data with [~]600 publicly available genomes. Clinical metadata including isolation site and disease were available for all strains. Our goal was to identify variations in protein sequences inferred from the WGS that correlate with the clinical metadata. We employed the alpha-fold unsupervised machine-learning model to all protein sequences. For each unique AA sequence, a numerical vector was generated reflecting the proteins biochemical properties. Multidimensional clustering was used to group variants into clusters by distance. For gene groups with two or more clusters, clinical metadata from the strain of origin was overlayed and subsequently tested for correlation with the clustering. This allowed identification of multiple gene groups with clusters of variants that correlated significantly with the patients disease. COG category analysis showed that many of the significant gene groups were associated with antibiotic targets. The TbpA gene group type was found to be significantly correlated with disease type with three of five variant clusters having a 95% prevalence among strains that were isolated from the lungs of COPD patients or other lower pulmonary tract infections.

genomics↗

bioRxiv2024

Evolutionary and functional divergence of Sfx, a plasmid-encoded H-NS homolog, underlies the regulation of IncX plasmid conjugation

Conjugative plasmids are widespread among prokaryotes, highlighting their evolutionary success. Despite being an important route for plasmid transmission, most natural plasmids do not constitutively conjugate and are conjugatively repressed by default. In the F plasmid, the negative regulation of conjugation is partially mediated by the chromosomal nucleoid structuring protein (H-NS). Recent bioinformatic analyses have revealed that plasmid-encoded H-NS homologs are widespread and exhibit high sequence diversity. However, the functional roles of most of these homologs and the selective forces driving their phylogenetic diversification remain unclear. In this study, we characterized the functionality and evolution of Sfx, a H-NS homolog encoded by the model IncX2 plasmid R6K. We demonstrate that Sfx, but not chromosomal H-NS, can repress R6K conjugation. Notably, we find evidence of positive selection acting on the ancestral Sfx lineage. These positively selected sites are located in the dimerization, oligomerization, and DNA-binding interfaces, many of which contribute to R6K repression activity - indicating that adaptive evolution drove the functional divergence of Sfx. We additionally show that Sfx can physically interact with various chromosomally encoded proteins, including H-NS, StpA, and Hha. Hha enhances the ability of Sfx to regulate R6K conjugation, suggesting that Sfx retained functionally important interactions with chromosomal silencing proteins. Surprisingly, the loss of Sfx does not negatively affect the stability or dissemination of R6K in laboratory conditions, reflecting the complexity of selective pressures favoring conjugation repression. Overall, our study sheds light on the functional and evolutionary divergence of a plasmid-borne H-NS-like protein, highlighting how these loosely specific DNA-binding proteins evolved to specifically regulate different plasmid functions. IMPORTANCEConjugative plasmids play a crucial role in spreading antimicrobial resistance and virulence genes. Most natural conjugative plasmids conjugate only under specific conditions. Therefore, studying the molecular mechanisms underlying conjugation regulation is essential for understanding antimicrobial resistance and pathogen evolution. In this study, we characterized the conjugation regulation of the model IncX plasmid R6K. We discovered that Sfx, a H-NS homolog carried by the plasmid, represses conjugation. Molecular evolutionary analyses combined with gain-of-function experiments indicate that positive selection underlies the conjugation repression activity of Sfx. Additionally, we demonstrate that the loss of Sfx does not adversely affect R6K maintenance under laboratory conditions, suggesting additional selective forces favoring Sfx carriage. Overall, this work underscores the impact of protein diversification on plasmid biology, enhancing our understanding of how molecular evolution affects broader plasmid ecology.

microbiology↗

bioRxiv2020

Genetically encoded ratiometric biosensor for probing lysosomal pH in mammalian cells and C. elegans

Lysosomes are important sites for macromolecular degradation, defined by an acidic lumenal pH of [~]4.5. To better understand lysosomal pH, we designed a novel, genetically encoded, fluorescent protein (FP) based pH biosensor called FIRE-pHLy (Fluorescence Indicator REporting pH in Lysosomes). This biosensor was targeted to lysosomes with lysosomal-associated membrane protein 1 (LAMP1) and reported lumenal pH between 3.5 and 6.0 with monomeric teal fluorescent protein 1 (mTFP1), a bright cyan pH sensitive FP variant with a pKa of 4.3. Ratiometric quantification was enabled with cytosolically oriented mCherry using high-content quantitative imaging. We expressed FIRE-pHLy in several cellular models and quantified the alkalinizing response to bafilomycin A1, a specific V-ATPase inhibitor. In summary, we have engineered FIRE-pHLy, a specific, robust and versatile lysosomal pH biosensor that has broad applications for investigating pH dynamics in aging and lysosome-related diseases, as well as in lysosome-based drug discovery.

cell biology↗