MSACL 2022 Abstract

INTRODUCTION: Imaging mass spectrometry is a powerful technology that enables the visualization of biochemical processes directly in tissues by combining the molecular specificity of mass spectrometry with the spatial fidelity of microscopic imaging. This label-free technology has proven exceptionally useful in areas of study such as cancer diagnosis, diabetes, and infectious disease. However, these types of modern imaging mass spectrometry studies are already stressing the limits of current analytical technologies and improvements are crucial in order to answer increasingly complicated biological and clinical questions. Research in our lab develops analytical instrumentation and methodologies in order to enable novel insights into disease mechanisms. For example, Clostriodioles difficile is an emerging human nosocomial pathogen and poses an urgent public health threat worldwide. During colonization in the gastrointestinal tract, C. difficile interacts with a widely diverse polymicrobial environment, including commensally found Enterococcus faecalis, which has been demonstrated to alter C. difficile growth morphology and virulence. However, little is known about the molecular mechanisms underlying these interactions.

OBJECTIVES: In order to study the role of metabolic cross-communication during C. difficile infection in the presence of E. faecalis, we have performed spatial mapping of metabolites using matrix-assisted laser/desorption/ionization (MALDI) imaging mass spectrometry in bacterial colonies and mouse models of infection.

METHODS: Specifically, we have leveraged continuous accumulation of selected ions (CASI) technology during MALDI imaging to enable the study of arginine metabolism as an important metabolic modulator in host immune responses. All experiments were performed on a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (7T solariX, Bruker Daltonics).

RESULTS: Our studies indicate that E. faecalis uptakes arginine through the cell’s ArcD antiporter and concomitantly exports high levels of ornithine during arginine metabolism, which we hypothesize can be used by C. difficile as an energy resource to promote growth and virulence. Our animal studies involve mice harboring commensal E. faecalis that were infected with C. difficile following cefoperazone treatment, reveal significantly increased ornithine and significantly decreased arginine in co-infected ceca tissues. Subsequent imaging experiments have been conducted in germ-free mice, including C. difficile mono-infected and C. difficile + E. faecalis co-infected mouse models. These studies again reveal increased ornithine and decreased arginine. Finally, imaging of bacterial co-cultures and mouse ceca tissues have been performed using an E. faecalis ArcD knock out, which no longer possess the ability to import arginine or export ornithine. Arginine and ornithine distributions and abundances observed using the C. difficile + E. faecalis with the ArcD knock out again resemble the C. difficile mono-infected model (i.e., increased ornithine and decreased arginine levels).

CONCLUSIONS: These models demonstrate a dynamic relationship between arginine and ornithine production and C. difficile pathogenesis. Future work will involve extending these studies to human clinical samples.