# Structure and function of pathogenesis-associated bacterial structures by electron cryotomography

> **NIH NIH R01** · CALIFORNIA INSTITUTE OF TECHNOLOGY · 2020 · $410,750

## Abstract

Project Summary
Pathogenic bacteria use specialized “nanomachines” to identify and interact with host cells. These machines
are attractive drug targets because they are surface-exposed, widespread, and vital for pathogenicity. While
one of these machines, the Type III Secretion System, has been well studied, other systems remain relatively
poorly understood. Here, we propose to use electron cryotomography (ECT) to dissect the structures and
functions of multiple pathogenic nanomachines. ECT is a powerful technique to image intact structures with
macromolecular (2-5 nm) resolution inside cells. Previously, we applied ECT to the Type VI Secretion System
(T6SS), where our images immediately revealed its "spring-loaded" contractile mechanism. Going a step
further, we realized that we could combine ECT and high-resolution subtomogram averaging with available
knowledge from other techniques to produce a complete architectural model of the Myxococcus xanthus Type
IVa Pilus (T4aP). This produced a flood of new mechanistic insights and inspired us to apply the same
approach to pathogenic secretion systems. In this project, we propose to use ECT to reveal the structure and
function of pathogenic Type IV Pilus (T4P), Type VI Secretion (T6SS), and Type IV Secretion System (T4SS)
machineries. For each system, 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 in order to produce architectural
models of the structures. In cases where crystal structures 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 current study), we aim to identify adaptations
underlying virulence functions. We will also apply state-of-the-art cryogenic-focused ion beam milling and
correlated cryogenic fluorescence light microscopy and ECT to image secretion structures in action – in
bacterial cells infecting eukaryotic hosts. This will provide the first such images of the critical human
pathogens Helicobacter pylori and Legionella pneumophila, which we expect to provide invaluable insights into
the operation of their pathogenic machinery in vivo. Together, we expect this project to produce a detailed
mechanistic picture of the T4P, T6SS, and T4SS nanomachines that mediate pathogenesis, an important first
step in identifying therapeutic targets in the future.

## Key facts

- **NIH application ID:** 9991727
- **Project number:** 5R01AI127401-05
- **Recipient organization:** CALIFORNIA INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** GRANT J JENSEN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $410,750
- **Award type:** 5
- **Project period:** 2016-09-23 → 2022-04-05

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9991727

## Citation

> US National Institutes of Health, RePORTER application 9991727, Structure and function of pathogenesis-associated bacterial structures by electron cryotomography (5R01AI127401-05). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/9991727. Licensed CC0.

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