From cells to atoms: Cryo-EM as an essential tool to investigate pathogen biology, host-pathogen interaction, and drug discovery.

Electron cryo-microscopy (cryo-EM) has lately emerged as a powerful method in structural biology and cell biology. While cryo-EM single-particle analysis (SPA) is now routinely delivering structures of purified proteins and protein complexes at near-atomic resolution, the use of electron cryo-tomography (cryo-ET), together with subtomogram averaging, is allowing visualization of macromolecular complexes in their native cellular environment, at unprecedented resolution. The unique ability of cryo-EM to provide information at many spatial resolution scales from ångströms to microns makes it an invaluable tool that bridges the classic "resolution-gap" between structural biology and cell biology domains. Like in many other fields of biology, in recent years, cryo-EM has revolutionized our understanding of pathogen biology, host-pathogen interaction and has made significant strides toward structure-based drug discovery. In a very recent example, during the ongoing coronavirus disease (COVID-19) pandemic, the structure of the stabilized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein was deciphered by SPA. This led to the development of multiple vaccines. Alongside, cryo-ET provided key insights into the structure of the native virion, mechanism of its entry, replication, and budding; demonstrating the unrivaled power of cryo-EM in investigating pathogen biology, host-pathogen interaction, and drug discovery. In this review, we showcase a few examples of how different imaging modalities within cryo-EM have enabled the study of microbiology and host-pathogen interaction.

Three-dimensional insights into human enveloped viruses in vitro and in situ.

Viruses can be enveloped or non-enveloped, and require a host cell to replicate and package their genomes into new virions to infect new cells. To accomplish this task, viruses hijack the host-cell machinery to facilitate their replication by subverting and manipulating normal host cell function. Enveloped viruses can have severe consequences for human health, causing various diseases such as acquired immunodeficiency syndrome (AIDS), seasonal influenza, COVID-19, and Ebola virus disease. The complex arrangement and pleomorphic architecture of many enveloped viruses pose a challenge for the more widely used structural biology techniques, such as X-ray crystallography. Cryo-electron tomography (cryo-ET), however, is a particularly well-suited tool for overcoming the limitations associated with visualizing the irregular shapes and morphology enveloped viruses possess at macromolecular resolution. The purpose of this review is to explore the latest structural insights that cryo-ET has revealed about enveloped viruses, with particular attention given to their architectures, mechanisms of entry, replication, assembly, maturation and egress during infection. Cryo-ET is unique in its ability to visualize cellular landscapes at 3-5 nanometer resolution. Therefore, it is the most suited technique to study asymmetric elements and structural rearrangements of enveloped viruses during infection in their native cellular context.

Cell-to-cell interactions revealed by cryo-tomography of a DPANN co-culture system.

DPANN is a widespread and diverse group of archaea characterized by their small size, reduced genome, limited metabolic pathways, and symbiotic existence. Known DPANN species are predominantly obligate ectosymbionts that depend on their host for proliferation. The structural and molecular details of host recognition, host-DPANN intercellular communication, and host adaptation in response to DPANN attachment remain unknown. Here, we use electron cryotomography (cryo-ET) to show that the Microcaldus variisymbioticus ARM-1 may interact with its host, Metallosphaera javensis AS-7 through intercellular proteinaceous nanotubes. Combining cryo-ET and sub-tomogram averaging, we show the in situ architectures of host and DPANN S-layers and the structures of the nanotubes in their primed and extended states. In addition, comparative proteomics and genomic analyses identified host proteomic changes in response to DPANN attachment. These results provide insights into the structural basis of host-DPANN communication and deepen our understanding of the host ectosymbiotic relationships.

Large attachment organelle mediates interaction between Nanobdellota archaeon YN1 and its host.

DPANN archaea are an enigmatic superphylum that are difficult to isolate and culture in the laboratory due to their specific culture conditions and apparent ectosymbiotic lifestyle. Here we successfully isolated and cultivated a co-culture system of a novel Nanobdellota archaeon YN1 and its host Sulfurisphaera ohwakuensis YN1HA. We characterised the co-culture system by complementary methods, including metagenomics and metabolic pathway analysis, fluorescence microscopy, and high-resolution electron cryo-tomography (cryoET). We show that YN1 is deficient in essential metabolic processes and requires host resources to proliferate. CryoET imaging revealed an enormous attachment organelle present in the YN1 envelope that forms a direct interaction with the host cytoplasm, bridging the two cells. Together our results unravel the molecular and structural basis of ectosymbiotic relationship between YN1 and YNHA. This research broadens our understanding of DPANN biology and the versatile nature of their ectosymbiotic relationships.

Morphological remodeling of Coxiella burnetii during its biphasic developmental cycle revealed by cryo-electron tomography.

Coxiella burnetii is an obligate zoonotic bacterium that targets macrophages causing a disease called Q fever. It has a biphasic developmental life cycle where the extracellular and metabolically inactive small cell variant (SCV) transforms inside the host into the vegetative large cell variant (LCV). However, details about the morphological and structural changes of this transition are still lacking. Here, we used cryo-electron tomography to image both SCV and LCV variants grown either under axenic conditions or purified directly from host cells. We show that SCVs are characterized by equidistant stacks of inner membrane that presumably facilitate the transition to LCV, a transition coupled with the expression of the Dot/Icm type IVB secretion system (T4BSS). A class of T4BSS particles were associated with extracellular densities possibly involved in host infection. Also, SCVs contained spherical multilayered membrane structures of different sizes and locations suggesting no connection to sporulation as once assumed.