Biology subjects

Luong, P.

Publications and source records attributed to Luong, P..

8 recordsLinked to original sources

bioRxiv2026

Orally available designed miniproteins inhibit enterotoxigenic Bacteroides fragilis pathology by blocking toxin receptor binding

Toxigenic bacterial infections in the gut are a significant contributor to the global burden of disease. Advanced tools for protein and live biotherapeutic engineering offer potentially transformative strategies for treating such diseases, while avoiding the collateral effects of traditional antibacterials. Here we used de novo protein design to identify inhibitors of the metzincin family protease Bacteroides fragilis toxin (BFT). These inhibitors, which bind distal to the active site, interfere with toxin-mediated E-cadherin cleavage and downstream proinflammatory signaling by blocking claudin-4 receptor binding. We tested the inhibitors as disulfide-stabilized variants administered directly to the cecum or in drinking water, as well as through in situ secretion by an engineered live biotherapeutic. Across these delivery modalities, the inhibitors successfully neutralized the toxin and effectively prevented BFT-associated gut pathology, including tumor formation. These results highlight the potential of de novo designed proteins as precise, non-antibiotic interventions to mitigate bacterial toxin-driven disease in the gut.

microbiology↗

bioRxiv2026

RNAseq analysis reveals the recurrent loss of heterozygosity in lung cancer and associated transcription patterns

A key limitation in cancer transcriptomics is the lack of accompanying genomic profiling such as whole-genome sequencing (WGS) or copy number alteration (CNA) data. Here we address this by showing that RNA-seq alone can be used to infer chromosomal aberrations and identify biologically meaningful patterns in lung cancer. Through a large-scale meta-analysis of publicly available RNA-seq datasets from non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), and matched controls, we reconstructed large scale CNA profiles and identified deletions in 3p, 9p, and 17p as the most frequent genomic events. Validation against paired WGS data confirmed a high degree of accuracy for RNA-seq-based inference. Our analyses revealed that while deletion-associated transcriptional heterogeneity exists, approximately 25% of differentially expressed genes were shared across all three deletion classes, indicating a conserved oncogenic program in lung cancer. Enrichment analysis linked these shared genes to pathways governing cell division, DNA replication, and extracellular matrix organization, while deletion-specific effects reflected disruption of tumor suppressor pathways, notably p53 signaling in 17p-deleted tumors, leading to deregulation of NOS2 and PLOD2. Integrating gene-level expression data, we identified both shared and deletion-specific biomarkers: TPX2 was consistently overexpressed across all deletion groups, while PTPRZ1 and CLDN9 were uniquely associated with del3p and del9p, respectively. Experimental validation in lung cancer cell lines confirmed these predictions, particularly the upregulation of CLDN9 in del9p carriers. By demonstrating that RNA-seq data can capture large-scale chromosomal events and reveal their transcriptional consequences, this study establishes an efficient framework for genomic inference and biomarker discovery, introducing CLDN9 as a novel, deletion-specific marker with potential prognostic and therapeutic value for 9p-deleted lung cancers.

cancer biology↗

bioRxiv2023

Metabolic connections between folate and peptidoglycan pathways in Pseudomonas aeruginosa inform rational design of a dual-action inhibitor

Peptidoglycan is an important bacterial macromolecule that confers cell shape and structural integrity, and a key antibiotic target. The synthesis and turnover of peptidoglycan are carefully coordinated with other cellular processes and pathways. Although there are established connections between peptidoglycan and DNA replication or outer membrane biosynthesis, connections between peptidoglycan and folate metabolism are comparatively unexplored. Folate is an essential cofactor for bacterial growth and required for the synthesis of many important metabolites. Here we show that inhibition of folate synthesis in the important Gram-negative pathogen Pseudomonas aeruginosa has downstream effects on peptidoglycan metabolism and integrity. Folate inhibitors reduced expression of the AmpC {beta}-lactamase through perturbation of peptidoglycan recycling, potentiating the activity of {beta}-lactams normally cleaved by that resistance enzyme. Folate inhibitors also synergized with fosfomycin, which inhibits MurA - the first committed step in peptidoglycan synthesis - resulting in dose-dependent formation of round cells that underwent explosive lysis.The insights from this work were used to design a dual-active inhibitor that overcomes NDM-1-mediated meropenem resistance and synergizes with the folate inhibitor, trimethoprim. This work shows that folate and peptidoglycan metabolism are intimately connected and offers new opportunities to exploit this relationship in strategies to overcome antibiotic resistance in Gram-negative pathogens.

microbiology↗

bioRxiv2023

Structural basis for recognition of unfolded proteins by the ER stress sensor ERN1/IRE1α

IRE1 is an endoplasmic reticulum sensor that recognizes misfolded proteins to activate the unfolded protein response (UPR). We used cholera toxin (CTx), which activates IRE1 in cells, to understand how unfolded proteins are recognized. In vitro, the A1 subunit of CTx (CTxA1) bound IRE1 lumenal domain (IRE1LD). Global unfolding was not required. Instead, IRE1LD recognized a 7-residue motif within a metastable region of CTxA1 that was also found in microbial and host proteins involved in IRE1 activation. Binding mapped to a pocket on IRE1LD normally occupied by a segment of the IRE1 C-terminal flexible loop implicated in IRE1 regulation. Mutation of the recognition motif blocked CTx-induced IRE1 activation in live cells. These findings describe a mechanism for substrate recognition by IRE1 that induces the UPR.

cell biology↗

bioRxiv2022

Targeted IS-element sequencing uncovers transposition dynamics during selective pressure in enterococci

Insertion sequences (IS) are simple transposons implicated in the genome evolution of diverse pathogenic bacterial species. Enterococci have emerged as important human intestinal pathogens with newly adapted virulence potential and antibiotic resistance. These genetic features arose in tandem with large-scale genome evolution mediated by mobile elements. Pathoadaptation in enterococci is thought to be mediated in part by the IS element IS256 through gene inactivation and recombination events. However, the regulation of IS256 and the mechanisms controlling its activation are not well understood. Here, we adapt an IS256-specfic deep sequencing method to describe how chronic lytic phage infection drives widespread diversification of IS256 in E. faecalis and how antibiotic exposure is associated with IS256 diversification in both E. faecalis and E. faecium during a clinical human infection. We show through comparative genomics that IS256 is primarily found in hospital-adapted enterococcal isolates. Analyses of IS256 transposase gene levels reveal that IS256 mobility is regulated at the transcriptional level by multiple mechanisms in E. faecalis, indicating tight control of IS256 activation in the absence of selective pressure. Our findings reveal that stressors such as phages and antibiotic exposure drives rapid genome-scale transposition in the enterococci. IS256 diversification can therefore explain how selective pressures mediate evolution of the enterococcal genome, ultimately leading to the emergence of dominant nosocomial lineages that threaten human health. Author SummaryInsertion sequence (IS) elements are simple transposons that are ubiquitous in bacteria. In the enterococci, which includes medically relevant species such as Enterococcus faecalis and Enterococcus faecium, the IS element IS256 is widespread and has been implicated in pathogenesis and antibiotic resistance. Despite the importance of IS256 to the biology of the enterococci, we know very about how this element is regulated and diversifies enterococcal genomes. Here, we show that IS256 is preferentially found in hospital-adapted and virulent strains of the enterococci. In E. faecalis V583, a vancomycin resistant blood isolate, IS256 is regulated by multiple transcriptional mechanisms. To understand how IS256 is mobilized, we adapted an Illumina-based deep sequencing method called IS-Seq to find novel IS256 insertions when applying the selective pressure of bacteriophage (phage) predation. Using this method, we found that chronic phage infection drives IS256 diversification of the E. faecalis V583 genome. Additionally, we tracked IS256 insertional activity during a human E. faecium infection and found that increased IS256 diversity was associated with specific antibiotic usage. Together, our results demonstrate that the enterococci control IS256 activity to diversify their genomes which may lead to the emergence of hospital-adapted strains that threaten human health.

microbiology↗

bioRxiv2019

IRE1β negatively regulates IRE1α signaling in response to endoplasmic reticulum stress

IRE1{beta} is an ER stress sensor uniquely expressed in epithelial cells lining mucosal surfaces. Here, we show that intestinal epithelial cells expressing IRE1{beta} have an attenuated response to ER stress. IRE1{beta} assembles with and blocks activation of the closely related and most evolutionarily ancient stress-sensor IRE1 to suppress stress-induced xbp1 splicing, a key mediator of the unfolded protein response. In comparison, IRE1{beta} has weak xbp1 splicing activity, largely explained by a non-conserved amino acid in the kinase domain that impairs its phosphorylation and restricts oligomerization. This enables IRE1{beta} to act as a dominant negative suppressor of IRE1. The inhibitory effect is amplified in cells by disrupting an XBP1-dependent feedback loop regulating stress-induced expression of IRE1. Thus IRE1{beta} functions to negatively regulate IRE1 signaling, perhaps enabling intestinal epithelial cells to manage the response to chronic stress stimuli at the host-environment interface.

cell biology↗

bioRxiv2019

Bacteriophage resistance alters antibiotic mediated intestinal expansion of enterococci

Enterococcus faecalis is a human intestinal pathobiont with intrinsic and acquired resistance to many antibiotics, including vancomycin. Nature provides a diverse and virtually untapped repertoire of bacterial viruses, or bacteriophages (phages), that could be harnessed to combat multi-drug resistant enterococcal infections. Bacterial phage resistance represents a potential barrier to the implementation of phage therapy, emphasizing the importance of investigating the molecular mechanisms underlying the emergence of phage resistance. Using a cohort of 19 environmental lytic phages with tropism against E. faecalis, we found that these phages require the enterococcal polysaccharide antigen (Epa) for productive infection. Epa is a surface-exposed heteroglycan synthesized by enzymes encoded by both conserved and strain specific genes. We discovered that exposure to phage selective pressure favors mutation in non-conserved epa genes both in culture and in a mouse model of intestinal colonization. Despite gaining phage resistance, epa mutant strains exhibited a loss of resistance to the cell wall targeting antibiotics, vancomycin and daptomycin. Finally, we show that an E. faecalis epa mutant strain is deficient in intestinal colonization, cannot expand its population upon antibiotic-driven intestinal dysbiosis and fails to be efficiently transmitted to juvenile mice following birth. This study demonstrates that phage therapy could be used in combination with antibiotics to target enterococci within a dysbiotic microbiota. Enterococci that evade phage therapy by developing resistance may be less fit at colonizing the intestine and sensitized to vancomycin preventing their overgrowth during antibiotic treatment.

microbiology↗

bioRxiv2018

A fungal ribonuclease-like effector protein inhibits plant host ribosomal RNA degradation

The biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals and grasses. Proteins with a predicted ribonuclease (RNase)-like fold (termed RALPHs) comprise the largest set of secreted effector candidates within the B. graminis f. sp. hordei genome. Their exceptional abundance suggests they play crucial functions during pathogenesis. We show that transgenic expression of RALPH CSEP0064/BEC1054 increases susceptibility to infection in monocotyledenous and dicotyledonous plants. CSEP0064/BEC1054 interacts in planta with five host proteins: two translation elongation factors (eEF1 and eEF1{gamma}), two pathogenesis-related proteins (PR5 and PR10) and a glutathione-S-transferase. We present the first crystal structure of a RALPH, CSEP0064/BEC1054, demonstrating it has an RNase-like fold. The protein interacts with total RNA and weakly with DNA. Methyl jasmonate levels modulate susceptibility to aniline-induced host RNA fragmentation. In planta expression of CSEP0064/BEC1054 reduces the formation of this RNA fragment. We propose that CSEP0064/BEC1054 is a pseudoenzyme that binds to host ribosomes, thereby inhibiting the action of plant ribosome-inactivating proteins that would otherwise lead to host cell death, an unviable interaction and demise of the fungus.

plant biology↗