Biology subjects

Treat, B. R.

Publications and source records attributed to Treat, B. R..

4 recordsLinked to original sources

bioRxiv2025

In Vivo Imaging and Tracking of VRE-Microbiota Interactions via Anaerobic Fluorescent Reporters in Extremely Drug-Resistant Bacteria

Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of healthcare-associated bloodstream infection and a dominant colonizer of the antibiotic-perturbed gut. Experimental interrogation of VREfm colonization dynamics has been constrained by three major barriers: widespread resistance to conventional selection markers in contemporary clinical isolates, instability of plasmid-based constructs, and failure of oxygen-dependent fluorescent reporters in the anaerobic intestinal environment. To address these limitations, we develop genetic tools optimized for VREfm that enable stable anaerobic fluorescent labeling and longitudinal tracking of colonization dynamics. We show that bile pigment-binding fluorescent proteins, including eUnaG2 and smURFP, generate robust signal under anaerobic conditions across diverse clinical backgrounds. Systematic analysis of resistance profiles revealed that conventional selection markers are frequently incompatible with modern isolates; accordingly, we identify puromycin as a broadly effective entry-point strategy and demonstrate that a puromycin-selectable plasmid enables reporter expression for otherwise genetically intractable extensively drug-resistant (XDR) strains. To support long-term studies without continuous selection, we establish an enhanced pheS** counterselection system for chromosomal integration at neutral loci and engineer a tandem 2xsmURFP construct to optimize single-copy fluorescence. Using these tools, we directly track competition between fluorescently labeled VREfm strains in a murine model via flow cytometric analysis of stool samples, enabling longitudinal strain-level quantification without antibiotic selection or selective plating. This work provides a unified framework for genetic manipulation and anaerobic strain tracking in XDR E. faecium, establishing a practical platform for dissecting ecological processes that govern persistence and competition in the gut. ImportanceVancomycin-resistant Enterococcus faecium (VREfm) is a dangerous and difficult-to-treat hospital pathogen. How VREfm establishes itself in the gastrointestinal tract and competes with other strains remains poorly understood, yet this knowledge is essential for preventing infection. Studying these processes has been impeded by widespread antibiotic resistance and by the failure of conventional fluorescent proteins in the oxygen-free gut environment. We describe tools that overcome these barriers and provide a practical foundation for mechanistic studies of VREfm gut colonization and persistence.

microbiology↗

bioRxiv2022

Potent Neutralization of Omicron and other SARS-CoV-2 Variants of Concern by Biparatopic Human VH Domains

The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics that are effective against a variety of strains of the virus. Herein, we characterize a human VH domain, F6, which we generated by sequentially panning large phage displayed VH libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized VH domain, resulted in a construct (F6-ab8-Fc) that neutralized Omicron pseudoviruses with a half-maximal neutralizing concentration (IC50) of 4.8 nM in vitro. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 VOCs - including the recently emerged Omicron variant - and highlight a vulnerable epitope within the spike protein RBD that may be exploited to achieve broad protection against circulating variants.

immunology↗

bioRxiv2021

Emerging SARS-CoV-2 Variants of Concern: Spike Protein Mutational Analysis and Epitope for Broad Neutralization

Mutations in the spike glycoproteins of SARS-CoV-2 variants of concern have independently been shown to enhance aspects of spike protein fitness. Here, we report the discovery of a novel antibody fragment (VH ab6) that neutralizes all major variants, with a unique mode of binding revealed by cryo-EM studies. Further, we provide a comparative analysis of the mutational effects within variant spikes and identify the structural role of mutations within the NTD and RBD in evading antibody neutralization. Our analysis shows that the highly mutated Gamma N-terminal domain exhibits considerable structural rearrangements, partially explaining its decreased neutralization by convalescent sera. Our results provide mechanistic insights into the structural, functional, and antigenic consequences of SARS-CoV-2 spike mutations and highlight a spike protein vulnerability that may be exploited to achieve broad protection against circulating variants.

biochemistry↗

bioRxiv2020

Generation of Xylose-inducible promoter tools for Pseudomonas species and their use in implicating a role for the Type II secretion system protein XcpQ in inhibition of corneal epithelial wound closure

Tunable control of gene expression is an invaluable tool for biological experiments. In this study, we describe a new xylose-inducible promoter system and evaluate it in both Pseudomonas aeruginosa and P. fluorescens. The Pxut promoter derived from the P. flurorescens xut operon was incorporated into a broad host-range pBBR1-based plasmid and compared to the Escherichia coli-derived PBAD promoter using gfp as a reporter. GFP-fluorescence from the Pxut promoter was inducible in both Pseudomonas species, but not in E. coli, which may facilitate cloning of toxic genes using E. coli to generate plasmids. The Pxut promoter was expressed at a lower inducer concentration than PBAD in P. fluorescens and higher gfp levels were achieved using Pxut. Flow cytometry analysis indicated that Pxut was more leaky than PBAD in the tested Pseudomonas species, but was expressed in a higher proportion of cells when induced. D-xylose did not support growth of P. aeruginosa or P. fluorescens as a sole carbon source and is less expensive than many other commonly used inducers which could facilitate large scale applications. The efficacy of this system aided in demonstrating a role for the P. aeruginosa type II secretion system gene from xcpQ in bacterial inhibition of corneal epithelial cell wound closure. This study introduces a new inducible promoter system for gene expression for use in Pseudomonas species. ImportancePseudomonas species are enormously important in human infections, biotechnology, and as a model system for interrogating basic science questions. In this study we have developed a xylose-inducible promoter system and evaluated it in P. aeruginosa and P. fluorescens and found it to be suitable for the strong induction of gene expression. Furthermore, we have demonstrated its efficacy in controlled gene expression to show that a type 2 secretion system protein from P. aeruginosa, XcpQ, is important for host-pathogen interactions in a corneal wound closure model.

microbiology↗