Interplay between Microbial Metal Regulation and Chemotaxis at the Host-Pathogen Interface Studied by Single-molecule Super-resolution Fluorescence Microscopy

Description

Antibiotic resistance has reached alarming levels in recent decades, with the identification of enzymes facilitating resistance growing exponentially, while approval of new antibiotics steadily declines. To pinpoint new potential antibiotic drug targets, better understanding of the intricate interplay between pathogens and host cells is required, especially during the onset of pathogenesis. Metal (especially trace transition metals) homeostasis, i.e., maintaining a steady physiological intracellular metal concentration, is crucial for bacterial pathogen survival and also plays a key role during host-pathogen interactions. For example, for bacterial pathogen Pseudomonas aeruginosa, Fe is an absolute requirement to turn on virulence. This makes metal homeostasis an attractive target for the development of novel antibiotic treatments.However, the involvement of metal homeostasis in host–pathogen interaction is not fully understood especially at the single-cell single-molecule level, such as whether there is some spatial heterogeneity of metal regulation within a bacteria community surrounding a host cell, what proteins are involved to help bacteria maximize their competition advantage over host cells to acquire metals, what other protein systems are involved to enhance pathogenesis and survival, is there any unique temporal and spatial patterns of the protein expression that can benefit the pathogen as a community during colonization process. Based on the previous work of our own and others, we recently hypothesize that metal homeostasis and chemotaxis (bacterial movement towards or away from chemical stimuli) may cooperatively govern pathogen’s mobility, metal regulation, colony initiation and development in response to a nearby host cell. Traditional approaches studying host-pathogen interactions mostly perform at the ensemble level (multiple cells) where the spatial and temporal resolutions are low and heterogeneity among cells are not resolved. To fully understand the underlying molecular level mechanisms behind these phenomena, high-resolution (single-cell) tools are needed.This proposed project aims to exploit the powerful live-cell single-molecule super-resolution optical imaging techniques, combined with microfluidic device design, to track single cell and single protein molecules with high spatiotemporal resolution, hence unravel the molecular basis underlying the complex relationship between bacterial metal homeostasis, chemotaxis, and host–pathogen interactions. With this tool, we can map the protein spatial distribution pattern, quantify fast protein dynamics and kinetics, and reveal protein-protein interaction mode in live bacteria and in real time. This project will start by investigating bacteria in a well-controlled artificial environment and progress to more complex host–pathogen interfaces, resembling native infection condition. By observing single-molecule dynamics in single cells, we are able to better understand how pathogens strategically orchestrate their metal homeostasis and chemotaxis systems to resist host defenses.Overall, the results from this project will provide important insights for understanding the critical roles of metal ions during host–pathogen interactions. By producing a comprehensive and highresolution picture of these processes, we will be able to precisely manipulate the pathogen metal homeostasis for preventative and therapeutic purposes, and eventually identify new drug targets for the development of next-generation antibiotics.