Aquisition of a confocal cryo-light microscope for correlative light and electron microscopy

Texas A&M Agrilife Research, College Station TX

Investigators

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Abstract

Project Summary: The Principle Investigators (PIs) are seeking an administrative supplement to obtain a confocal cryo-correlative light/electron microscope (cryo-CLEM) for their research project, "RNA Penetrations into Bacteria" (R01GM141659). The study aims to understand the molecular mechanisms underlying the interaction between single-stranded RNA bacteriophages (ssRNA phages) and their host bacteria, with a focus on PP7 and AP205 phages, which infect Pseudomonas aeruginosa and Acinetobacter spp., respectively. The PIs hope to use this information to develop new approaches to combat multi-drug-resistant bacterial infections by mitigating the virulence of these pathogens. The team plans to investigate the in situ phage packaging process, the phage adsorption pathway, and the RNA entry/pili detachment events using multi-scale imaging tools, including fluorescence microscopy and cryo-electron microscopy/tomography (cryo-EM/ET). They have already obtained high-resolution cryo-EM structures of both phage PP7 and AP205 alone or in complex with the host receptors, the type IV pili, and have characterized molecular events associated with pili detachment upon phage infection. The PIs request a confocal cryo-CLEM, which would enhance their imaging capabilities and benefit their project in two main ways. Firstly, a cryo-CLEM can provide both high-resolution imaging and real-time observation capabilities, bridging the gap between traditional light microscopy and cryo-EM/ET. Secondly, the team aims to test their findings in the context of Pseudomonas biofilms, and confocal fluorescence microscopy is essential for understanding the spatial organization of bacterial cells and phages during infection. Drs. Zhang and Zeng, the PIs, have collaborated with Nikon and Linkam to create a cryo-CLEM setup tailored to their project. The instrument will be located in Dr. Zhang's newly renovated lab, which is right next to Dr. Zeng's lab in the Department of Biochemistry and Biophysics building. Dr. Zeng, who has over 15 years of experience with fluorescence light microscopy, will supervise the operation and maintenance of the confocal cryo-CLEM in coordination with Dr. Zhang. This instrument will not only support the PIs’ current research project but also have long-term value for future research in the Texas A&M community.