# Understanding type IV pili on the singe-molecule level

> **NIH NIH R35** · TEXAS A&M UNIVERSITY · 2024 · $370,086

## Abstract

Type IV pili (TFP) are captivating bacterial nanomachines that are broadly conserved and exhibit a diverse array
of physiological functions. In the opportunistic pathogen Pseudomonas aeruginosa (PA) for example, TFP are
implied in numerous virulence associated factors such as biofilm formation, mechanosensing, and motility. The
physiological function of TFP relies on repetitive cycles of extending and retracting of a short polymeric fiber into
the extracellular space, for example to adhere to a substrate and pull the cell forward. These dynamic cycles are
facilitated by a complex molecular machinery consisting of dozens of different proteins that work together in a
concerted manner. Although the constituents of TFP are known, the intricate molecular processes governing
their interaction and how these interactions enable the physiological function of TFP remain largely elusive.
Current limitations of observing the molecular interactions that drive the functions of TFP are intrinsic to the
experimental techniques that have been used to far: these techniques cannot resolve individual proteins or TFP
complexes. Single molecule techniques have the potential to fill this gap and to gain invaluable insight into the
interactions among the proteins of the TFP system and how they function together as multi-molecular complexes.
The power of single molecule studies is to resolve and observe individual TFP complexes, their constituents,
and to probe the rapid interactions between TFP and their effector molecules, which happens on the ten to
hundreds of milliseconds timescale. The long-term vision of my research program is to leverage the potential
that single molecule light-microscopy based techniques offer to understand TFP and their physiological function
at the molecular level: we will reveal how TFP machines are assembled molecule-by-molecule, how individual
molecular motors and regulatory proteins interact with the static components of TFP, and how these interaction
kinetics are changed and trigger downstream signaling pathways during the physiological function of TFP.
Specifically, firstly, we will use super-resolution microscopy to resolve individual TFP complexes and their
components and map their precise locations and assembly states molecule-by-molecule. Combined with
timelapse fluorescence microscopy to track the formation of new TFP components through the cell cycle, this
will reveal the first step to the physiological function of TFP: when, where, and how the different components of
new TFP are assembled and controlled. Secondly, we will use single-molecule Forster resonance energy
transfer (smFRET) to investigate the dynamic interactions between specific pairs of proteins of the TFP system.
This will reveal how molecular effectors enable and tune TFP dynamics to regulate the physiological functions
of TFP. Long term, we will couple these experiments with single-molecule force microscopy techniques to reveal
how the physiological functions of TFP...

## Key facts

- **NIH application ID:** 10939550
- **Project number:** 1R35GM155280-01
- **Recipient organization:** TEXAS A&M UNIVERSITY
- **Principal Investigator:** Matthias Daniel Koch
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $370,086
- **Award type:** 1
- **Project period:** 2024-07-01 → 2029-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10939550

## Citation

> US National Institutes of Health, RePORTER application 10939550, Understanding type IV pili on the singe-molecule level (1R35GM155280-01). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10939550. Licensed CC0.

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