# Structure and Function of Pathogenesis-Associated Bacterial Structures by Electron Cryotomography

> **NIH NIH R01** · BRIGHAM YOUNG UNIVERSITY · 2022 · $369,480

## Abstract

Project Summary
Pathogenic bacteria employ specialized secretion systems to identify and interact with host cells and to
exchange genetic information through horizontal gene transfer. These machines are attractive drug targets
because they are surface-exposed, widely conserved, and specific for pathogenicity. Unfortunately, however,
the structures of many of these critical systems remain poorly understood. Here we describe how we will
continue to use electron cryotomography (cryoET) to dissect the structures and functions of pathogenic
nanomachines. CryoET is a revolutionary imaging technique with the power to reveal native structures inside
intact cells in 3D with macromolecular (2-5 nm) resolution. Subtomogram averaging of identical structures from
one or more cryotomograms can push this resolution to better than 1 nm in the most favorable cases, enabling
components to be placed in their context in the complete machine. My group has pioneered the development
of this revolutionary imaging technology, and in just under four years of our first award period, we have used
cryoET to produce tens of new structures of pathogenic secretion systems and build architectural models of
key systems belonging to the type IV pilus (T4P), type VI secretion system (T6SS) and type IV secretion
system (T4SS) families, producing a flood of new mechanistic insights. By exploiting new cryoET technologies
we have just developed in the past couple years, here we propose to extend our work in the next award period
to different functional states of these complexes, key related systems, and a new target: the pathogenic type IX
secretion system (T9SS). In addition, we will push the whole body of work to higher resolution. For each target,
we will image the entire, intact structure in situ. In most cases, this will be the first high-resolution imaging of
these structures. We will then combine subtomogram averaging with difference analysis of mutants in which
individual components are knocked out or tagged with additional density in order to produce architectural
models of the complexes. In cases where atomic models of components (or homologs) are available, we will
dock them into our maps to produce pseudo-atomic models of each machine. By comparing these structures
with those of non-pathogenic relatives (solved previously or in the proposed work), we aim to identify
adaptations underlying virulence functions. We will also apply state-of-the-art cryogenic correlated light and
electron microscopy (cryo-CLEM) to guide cryogenic focused ion beam (FIB) milling to enable us to image
pathogenic secretion systems in action: in bacterial cells infecting eukaryotic hosts. This will provide the first
such images of critical human pathogens, which we expect to provide invaluable insights into the operation of
their virulence machinery in vivo. Together, we expect this project to produce a detailed mechanistic picture of
the T4SS, T4P, and T9SS nanomachines that mediate pathogenesis, an...

## Key facts

- **NIH application ID:** 10212062
- **Project number:** 2R01AI127401-06
- **Recipient organization:** BRIGHAM YOUNG UNIVERSITY
- **Principal Investigator:** GRANT J JENSEN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $369,480
- **Award type:** 2
- **Project period:** 2016-09-23 → 2027-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10212062

## Citation

> US National Institutes of Health, RePORTER application 10212062, Structure and Function of Pathogenesis-Associated Bacterial Structures by Electron Cryotomography (2R01AI127401-06). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10212062. Licensed CC0.

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