Evaluation and Development of Ligand-Dependent Fluorescent Reporters for Anaerobic Bacterial Imaging

Abstract

Fluorescence-based investigations of living cells and biological phenomena have been largely aided by the development of fluorescent proteins (FPs) to span a wide range of different colors and specialized photophysical properties. The most commonly used families of FPs are derived from Green Fluorescent Protein (GFP) and DsRed, which require oxidative post-translational modifications to produce mature chromophores. This oxidation step precludes their use in anaerobic conditions. Nevertheless, there are countless oxygen-sensitive biological systems and mechanisms that have yet to be explored using otherwise ubiquitous techniques such as fluorescence microscopy.

This dissertation aims to bridge this gap by evaluating and developing a set of ligand- dependent reporters, Bilin-based Fluorescent Proteins (BBFPs), as oxygen-independent fluorescent probes for applications in anaerobic bacterial systems such as the gut microbiome. Chapter I provides background on the importance of the gut microbiome and how investigations of this medically relevant polymicrobial community can be aided by fluorescence-based investigations. It also provides an overview of the development of both oxygen-dependent and -independent FPs and the latters’ respective advantages and dis- advantages in oxygen-free imaging. BBFPs are adapted from eel and bacterial phytochromes and bind the tetrapyrroles bilirubin (br) or biliverdin (bv) to fluoresce. These reporters are oxygen-independent due to fluorescence resulting from ligand binding, and the fluorogenic nature of these ligands enables an improved signal to noise ratio over other fluorescent dyes. Chapter II describes the first implementation of BBFPs in anaerobic gut bacteria using the blue-green UnaG and the far-red IFP2.0. These FPs are used to label the commensal gut bacteria Bacteroides thetaiotaomicron (B. theta) in monoculture in the common GFP and Cy5 microscopy channels as well as used in mixed-species two-color imaging.

To broaden the utility of the UnaG FP, I set out to diversify the color palette of BBFPs in Chapters III and IV. In Chapter III, high-throughput screening (HTS) has been employed to identify new fluorogenic ligands that could bind UnaG and fluoresce in different wavelengths outside of the native UnaG-br pair’s blue 488-nm channel. The new resulting UnaG-ligand pair can be used in the common green 532-nm excitation channel and again, could be used in anaerobic bacterial imaging and as an orthogonal label with IFP2.0-bv. As UnaG is a more desirable than other BBFPs in terms of size and monomeric form, Chapter IV describes efforts to engineer a bv-binding UnaG variant for a red-shifted variant that could be excited by blue or red fluorescence. These results provide insight into the br binding pocket and provide a blueprint for future engineering efforts in this BBFP.

Finally in Chapter V, future directions for oxygen-independent reporters are discussed that range from adjustments to engineering ligand recognition in UnaG to alternative ligand-dependent reporter systems that may also be implemented in anaerobic systems. This dissertation collectively validates and expands the fluorescent toolbox for probing anaerobic bacterial systems and extends fluorescence applications to previously inacces sible biological systems.