Thrust 1: Molecular Precision Mechanisms

To develop efficient pathogen detection techniques, as well as effective prophylactic and treatment strategies, it is important to understand the fundamental mechanisms of infection by studying interactions between pathogens and host cells at the single molecule level. Pathogens have numerous mechanisms by which they infect a host cell. Current single cell techniques have provided a deep understanding of pathogen-host interactions, which are otherwise averaged out in bulk measurements of cell populations.

However, within an individual cell, heterogeneity of multiple molecules operating through coordinated expression and biophysical interactions can be analyzed to gain a deeper understanding of underlying mechanisms. Sub-diffraction imaging techniques such as super-resolution optical imaging, plasmonic tweezers, atomic force microscopy, cryogenic electron microscopy (cryo-EM), and molecular engineering can observe pathogen-induced biomolecular behavior within cells with nanometer spatial resolution, piconewton force sensitivity, and molecular precision at the single molecule level.

We will apply these tools within the context of the CPD to gain a deeper understanding of pathogen-host interactions at the molecular level, and thus provide targets for new pathogen detection platforms. In this Aim, we will focus on studying fundamental principles of pathogen infection through three novel techniques.

Study cellular-protein-pathogen-interactions with cryo-EM

One of the first identified viruses, tobacco mosaic virus, was visualized by transmission electron microscopy in the 1930s. With the advancement of cryo-EM, the 3D structure of the whole protein shell of a virus can be determined with atomic resolution. Recently the structures of the RNA replication system (nsp12-nsp7-nsp8) as well as the trimeric spike glycoprotein of SARS-CoV-2 have been determined by cryo-EM40,41. These results
fundamentally illustrate how viruses infect cells and how they replicate. The same is true for bacteria secretion systems that inject toxic molecules into cells, where cryo-EM has revealed the 3D molecular architecture of protein machinery with unprecedented detail. While cryo-EM studies mostly focus on large protein complexes with molecular weight greater than 300 kDa, another important structure biology tool, X-ray crystallography, usually focuses on proteins smaller than 150 kDa and was the main method used to study biomolecule structures before the recent resolution revolution of cryo-EM. Over the years, numerous virus, bacteria, fungi protein crystal structures, e.g., HIV reverse transcriptase, influenza spike proteins, SARS-CoV-2 spike-ACE2 complex, E. coli shiga toxins, S. Typhimurium typhoid toxin, have been resolved by X-ray crystallography and these structures greatly enrich our knowledge of fundamental biology, including their pathogenicity.

In this sub-Aim, we will focus on studying how cellular proteins facilitate SARS-CoV-2 entry. It is shown that S glycoprotein of both SARS-CoV and SARS-CoV-2 binds to human ACE2 receptor and undergoes proteolytic processing to mediate fusion of the viral and cellular membranes43. However, for SARS-CoV and SARS-CoV-2, direct release of the viral genome into cells through the plasma membrane was not observed, subsequent viral entry and genome release is carried out by cellular endocytosis processes. One key enzyme, PIKfyve, which phosphorylates the D-5 position in phosphatidylinositol-3-phosphate (PI3P) to yield phosphatidylinositol 3,5-bisphosphate, has been implicated in various trafficking events associated with the endocytic pathway. Since PI(3,5)P2 is important for endosome homeostasis, knockdown or inhibition of PIKfyve causes endosomal swelling and vacuolation of late endosomes and endolysosomes44. It was reported that inhibition of PIKfyve disrupts intracellular replication of Salmonella. Importantly, one inhibitor of PIKfyve, apilimod, has been shown to block Ebola virus, Marburgvirus, and SARS-Cov2 entry into host cells. Apilimod was tested in phase 2 clinical trials for treatment of Crohn’s disease, psoriasis, and rheumatoid arthritis and considered to be well tolerated in humans, and is currently proposed as a potential drug for the treatment of COVID-1946. This research will shed light on how virus entry is linked to cellular processes and inform the development of better drugs to inhibit this process.

Study genetically diverse pathogen-host interactions with high efficiency super-resolution microscopy

Super-resolution imaging techniques overcome the diffraction limit of light microscopy and can achieve spatial resolution down to a few nanometers. These techniques enable a detailed view of biological structures and dynamics at the subcellular and molecular level. With the advantages of high molecular specificity and live-cell imaging compatibility, super-resolution imaging offers an ideal solution to the problem of visualizing the infection process of bacteria and viruses. During infection, pathogens interact with host cells to trigger a wide range of cellular processes in order to survive and self-replicate. Elucidating the molecular mechanisms of these interactions provides helpful insights to novel diagnostic and treatment methods.

One focus of pathogen-host interaction investigation is the influence of the genetic diversity and variability on pathogenicity. The large population of pathogens displays enormous genetic diversity; and while their rapid replication enables efficient selection of beneficial genetic variations that adapt to new host cell environments. One of the consequences of host-pathogen coevolution is bacterial antibiotic resistance. In-depth study of bacterial genetic diversity and antibiotic resistance requires imaging a large number of biological samples under various conditions, which challenges the efficiency of current superresolution imaging workflow. To improve the efficiency of imaging pathogen-host interaction, in this sub-Aim, we plan to integrate super-resolution techniques with microfluidic devices. The microfluidic device will capture and release host cells in a controlled manner. After the cells are captured, by varying the timing of introduction of pathogens and/or drugs, one can effectively differentiate the time points at which different cells are infected, and thereby carry out multiplexed imaging experiments under a variety of biochemical conditions on a single microfluidic device.

Study biophysical pathogen-host interactions with plasmonic tweezers and atomic force microscopy

The binding between pathogen and host cells marks the first step of infection. Viruses, for example, use the protein on the viral envelope to “unlock” the host cells. Studies have suggested that this first step engages a series of cell receptors from low-affinity and high-avidity to high-affinity interactions. The initial low-affinity interaction involves non-specific forces such as van der Waals force. This seemingly redundant step is found to be common in viruses50. The non-specific and specific interactions can be differentiated with different levels of force. The ability to precisely quantify these forces
can provide essential knowledge to more clearly understanding how viruses initiate cellular attachment, mediate entry, and trigger subsequent signaling pathways that promote infection.

To improve the dynamic range of force measurement while maintaining the precision of plasmonic tweezers, in this sub-Aim, we will combine plasmonic tweezers with atomic force microscopy to directly control and quantify molecular-level interaction forces between pathogen and host cells. Our preliminary results have shown that when combing plasmonic tweezers with atomic force microscopy, the system is able to measure forces in the piconewton regime with nanometer spatial resolution in three dimensions. We will implement an environmental plasmon force microscopy system that enables measurement of the molecular-level forces under physiological conditions. This force quantification tool can be extended to identify molecular targets that selectively bind to the pathogen for developing novel diagnostic tools, and to study small molecule drugs that selectively interact with the pathogen for developing novel therapeutic strategies. To strengthen and enhance the precision of optical forces, we design plasmonic tweezers that offer a promising route to directly manipulate dielectric nano-specimens down to two nanometers without tethering large, wavelength-scale particles or probes. Importantly, we have recently developed plasmonic tweezers that can selectively exert attractive or repulsive forces on magnetic dipoles, which are important in studying proteins that are generally optically active. Our new design offers control of the exerted forces in three dimensions with nanometer spatial precision and piconewton force sensitivity. To study single molecular level forces, another limitation of current techniques is the dynamic range of forces. While plasmonic tweezers provides piconewton range forces, adhesion forces oftentimes go beyond the piconewton range. Hu’s group recently developed a new methodology for fabricating nano-spherical atomic force microscopic probes with a monolithic nanosphere that is precisely positioned (<10 nm alignment error) on a micro-cantilever. The technology holds promise for measuring antibody-antigen interaction forces for efficient antibody selection for virus detection.