Combining super-resolution & force microscopy, genetics, molecular biology, and theoretical modeling to understand how bacteria interact with their physical environment.
Bacteria strive in almost every niche on our planet and are exposed to a tremendous variety of physical properties of the environment. From changes in temperate to fluid flow, bacteria adapt to ever changing environments to ensure survival, spread, and colonization. A much overlooked environmental property is its mechanical rigidity. The stiffness of different tissue sites in the human body for example vary tremendously and can be as rigid as bone or as soft as mucus. P. aeruginosa is a multi drug resistant human pathogen that infect many mechanically different body sites and is able to tune its virulence to mechanical properties of the environment. This ability is mediated by so called type IV pili (TFP).
TFP are external bacterial appendages that dynamically extend and retract a polymeric filament to the environment (Fig. 1). Often referred to as the bacterial Swiss army knife, these broadly conserved structures are used for various physiological tasks such as DNA uptake, surface sensing, phage infection, and are important virulence factors. One of the most visually striking functions is surface motility called twitching. Here, entire groups of cells (see above) or even single cells (Fig. 2) migrate either in a "walking" style along the surface or crawl over the surface lying flat down.
Fig. 2. Individual P. aeruginosa cells twitching at a solid-liquid interface. LEFT: Right after cell division, one cell remains stationary while the other cell takes off and transitions multiple times between "walking"upright and crawling lying down. Why dows this happen? RIGHT: A cell with fluorescently labeled TFP walking accross the surface. At one point, the cells makes a "hand over hand" type of flip and switches from walking on one cell pole to the other pole.
Research in the Koch lab is currently focused on three major Aims/Questions:
1) How does the type IV pilus (TFP) function from the perspective of a molecular machine?
2) How do TFP facilitate substrate stiffness sensing?
3) What is the physiological function of stiffness sensing for the pathogen Pseudomonas aeruginosa?
1. Understanding TFP as a molecular machine:
Through the eyes of an engineer, TFP appear like a carefully crafted and well-oiled machine. About half a dozen molecular components are aligned together to form the core machine complex that spans the periplasm. In P. aeruginosa, three independent motor proteins interact with this machine to drive the extension and retraction of individual pili. Cryogenic electron microscopy (cryo EM) has given us detailed information about the static blueprint of this machine (Fig. 3). However, TFP are highly dynamic structure and we know little about how these dynamics are controlled, mainly due to a lack of experimental techniques capable of observing the single molecule dynamics of TFP.
Fig. 3. Cryo EM image of a single TFP machine (left) spanning form the inner to the outer membrane and several machines with extended pili (right). Individual machines are comprised of an outer membrane pore, periplasmic rings, a cytoplasmic dome, ring, and disc. Adapted from Chang et al. (2016) Science.
Using a click-chemistry approach and tailored point mutations of the major pilin that comprises the TFP fiber, we were recently able to fluorescently label and observe the dynamics of the TFP of P. aeruginosa (Figs. 1, 2, 4) (Koch et al. (2021) PNAS). Based on this new ability to observe TFP dynamics, we now leverage the strength of different multi-color super-resolution fluorescence microscopy techniques to watch the dynamics of TFP and their individual parts. Our goal is to unravel the secrets of how these machines are being build on the single molecule level and how this gives rise to TFP function.
Fig. 4. LEFT: The TFP of P. aeruginosa are highly dynamic as shown in this fluorescence movie. Pilus generation is a stochastic process and most cells make few (< 10 per minute) and only short (< 1 um) pili that immediately retract after extension come to an end. MIDDLE: High speed microscopy reveals rare intermittent events where a pilus extends, halts, and continuous extension. RIGHT: Pius retraction is triggered by the binding of the retraction motor (red).. Koch et al. (2021) PNAS
2. How are TFP dynamics controlled to facilitate stiffness sensing?
Our sense of touch is a crucial ability for humans. It allows us to distinguish ripe fruit or the “squishiness” of a surface. We recently discovered that bacteria have a similar ability: the clinically important human pathogen P. aeruginosa uses TFP as molecular scale ‘arms’ to tune the activity of its key transcription factor Vfr to substrate stiffness (Fig. 5 left). Importantly, Vfr controls over 100 virulence related genes, suggesting that P. aeruginosa is able to distinguish its broad spectrum of infection sites by substrate mechanics and modulate virulence specifically to each site. This novel connection between pathogenicity and mechanical properties of the infection site thus presents tremendous potential for both clinical applications and new biophysical insights into mechanosensing.
Fig. 5. LEFT: P. aeruginosa tunes the activity of the transcription factor Vfr to stiffness (experimental data points). Using biophysical modeling, we were able to show that the sensing mechanism relies on concentration changes of the major pilin PilA (yellow shaded line). RIGHT: The Pil-Chp pathway connects PilA concentration to Vfr activity via the second messenger cyclic AMP (cAMP).
Fig. 6. We use a variety of experimental biophysical tools to investigate the mechanisms and regulators of stiffness sensing. LEFT: Optical trapping allows to trap and hold a micron sized plastic bead. TFP readily adhere to plastic. Once a pilus adheres and retracts, it pulls on the bead against the optical forces. This process allows to measure the retraction force of individual pili. MIDDLE: Example of a Traction Force Microscopy (TFM) experiment. Small densely embedded fluorescent beads are displaced along with the gel when cells pull on the gel to deform it (areas indicated by red arrows). RIGHT: Experiments using Fluorescence Recovery After Photobleaching (FRAP) allow to measure the diffusivity of individual PilA proteins in the inner membrane. This is used to model concentration changes during pilus extension/retraction to understand the mechanism behind stiffness sensing. Koch et al. (2022) PNAS.
Stiffness sensing relies on an intricate chemotaxis-like signalling network, the so called Pil-Chp system (Fig.5 right). We are currently exploring how this system controls the dynamics of TFP and ultimately how this governs and tunes stiffness sensing. To tackle this problem, we employ a unique strategy combining tailored mutations in the Pil-Chp pathway with diverse force-probing and other biophysical techniques.
For example, optical tweezers (Fig. 6. left) use light to hold and move micron-sized particles (so called beads) in a science fiction tractor beam like fashion and can be used the measure the retraction force of individual TFP. Changes in the retraction force would result in changes in the cell's ability to deform the substrate and measure it's stiffness. This deformation of the substrate on the other hand we measure using a technique called Traction Force Microscopy (TFM). Here, small fluorescent beads are densely embedded in a transparent hydrogel. A localized displacement of beads thus indicates a deformation of the gel which can be measured (Fig. 6 middle).
3. What is the physiological function of stiffness sensing?
Bacteria use TFP to interact with and sense the environment. A particular interesting environment are biofilms, multicellular aggregates of bacteria, typically reinforced by a dense matrix of extracellular protein, polysaccharides, and DNA. Biofilms confer many advantages to individual cells like increased resistance to antibiotics. TFP are important for the development of biofilms, yet we know little about how the actual dynamics shape this process. Using the fluorescent TFP label that we developed, we are now able to look at TFP inside the biofilm as it develops (Fig. 7 left).
Secondly, as pointed out above, P. aeruginosa infects a mechanically broad range of host sites and tunes its virulence to mechanical properties of the substrate. To investigate this exciting connection, we develop single cell infection models that allow to study TFP, virulence, and the host response simultaneously over time.
Fig. 7. LEFT: The dynamics of fluorescently labeled TFP during biofilm development. RIGHT: P. aeruginosa use TFP (green) to invade epithelial cells (gray).