Periodic Reporting for period 1 - NIOBMT (Nanomechanical intervention of bacterial mechanotransduction)
Période du rapport: 2021-08-01 au 2023-07-31
Pathogenic bacteria possess a vast array of resources that enable the colonization of host epithelial tissues. The continuous appearance of bacterial strains resistant to most antibiotic treatments—the so-called “superbugs”—is a pressing public health problem that requires urgent efforts to tackle the life-threatening infections caused by these pathogens.
One of the main events that initiate tissue colonization and the onset of an infection is the recognition of surfaces—host tissues—by the pathogenic bacteria. Bacteria cells swim free in liquid media and the proximity or direct contact with the host tissues (respiratory tract, urinary tract, etc.) provide mechanical cues that trigger cell responses that lead to tissue colonization and the onset of an infection process (Summary Image, 1 and 2). This mechanically induced detection of host tissues depends on proteins—mechanosensors—located on the surface of the bacteria cells. Understanding how these proteins respond to mechanical forces and how the bacterium processes this information to decide to start an infection offers an ideal target for developing therapeutic strategies that combat the first stages of bacterial colonization. For instance, drugs that alter the mechanical properties of these proteins responsible for surface detection would render the bacteria “blind” to the host tissue, abolishing colonization, and the subsequent infection.
With these premises, this project aimed to delve into the process of bacterial surface detection and adhesion by focusing on the nanomechanics of the protein PilY1, whose mechanical activation has been suggested as a key event in the process that leads to host tissue colonization (Summary Image, 3). We focused on the PilY1 protein of Pseudomonas aeruginosa, an opportunistic pathogen that causes recurrent infections in cystic fibrosis patients. PilY1 is a complex protein that possesses a mechanosensitive role (detection of surfaces), and an adhesive role (binding to host tissue proteins). To interrogate the role of mechanical forces on the functions of PilY1, we employed single-molecule force spectroscopy techniques. These techniques allow the manipulation with the force of single PilY1 proteins and determine their mechanical properties in the nanoscale—nanomechanics—which provides key information about proteins that carry out their functions under mechanical loads. Certain molecules bind to PilY1 and modulate the processes of surface recognition and adhesion, and conducting measurements in the absence and presence of these molecules can give insights into how they regulate PilY1 behavior under force and, more importantly, they provide knowledge for the development of strategies that interfere with colonization and pathogenesis (Summary Image, 4). Therefore, the objectives of this project aimed to identify and dissect the effect of mechanical forces on the functions of PilY1 and identify potential molecular strategies to interfere with them.
One of the main events that initiate tissue colonization and the onset of an infection is the recognition of surfaces—host tissues—by the pathogenic bacteria. Bacteria cells swim free in liquid media and the proximity or direct contact with the host tissues (respiratory tract, urinary tract, etc.) provide mechanical cues that trigger cell responses that lead to tissue colonization and the onset of an infection process (Summary Image, 1 and 2). This mechanically induced detection of host tissues depends on proteins—mechanosensors—located on the surface of the bacteria cells. Understanding how these proteins respond to mechanical forces and how the bacterium processes this information to decide to start an infection offers an ideal target for developing therapeutic strategies that combat the first stages of bacterial colonization. For instance, drugs that alter the mechanical properties of these proteins responsible for surface detection would render the bacteria “blind” to the host tissue, abolishing colonization, and the subsequent infection.
With these premises, this project aimed to delve into the process of bacterial surface detection and adhesion by focusing on the nanomechanics of the protein PilY1, whose mechanical activation has been suggested as a key event in the process that leads to host tissue colonization (Summary Image, 3). We focused on the PilY1 protein of Pseudomonas aeruginosa, an opportunistic pathogen that causes recurrent infections in cystic fibrosis patients. PilY1 is a complex protein that possesses a mechanosensitive role (detection of surfaces), and an adhesive role (binding to host tissue proteins). To interrogate the role of mechanical forces on the functions of PilY1, we employed single-molecule force spectroscopy techniques. These techniques allow the manipulation with the force of single PilY1 proteins and determine their mechanical properties in the nanoscale—nanomechanics—which provides key information about proteins that carry out their functions under mechanical loads. Certain molecules bind to PilY1 and modulate the processes of surface recognition and adhesion, and conducting measurements in the absence and presence of these molecules can give insights into how they regulate PilY1 behavior under force and, more importantly, they provide knowledge for the development of strategies that interfere with colonization and pathogenesis (Summary Image, 4). Therefore, the objectives of this project aimed to identify and dissect the effect of mechanical forces on the functions of PilY1 and identify potential molecular strategies to interfere with them.
The work carried out has consisted in characterizing the nanomechanics of the PilY1 protein in the presence and absence of ligand molecules that naturally bind to its structure. The first goal was to determine if PilY1 could behave as a force sensor, as it has been suggested. This property can be inferred by exposing the protein to low mechanical loads and observing if it experiences conformational changes in narrow force ranges. The observations revealed that PilY1 behaves as a mechanosensor, confirming its alleged role for the first time. In very small force ranges, PilY1 dynamically changes its conformation, enabling the activation of other parts of its structure involved in adhesion to host tissue ligands. This discovery is key to understanding which force magnitudes trigger its activation, which is intimately linked to the mechanical perturbations that bacteria sense and interpret to decide substrate colonization.
The next goal was to test how small compounds alter PilY1 nanomechanics. These molecules are known to modulate protein mechanical stability, and they increase it in the case of PilY1. These compounds are crucial for binding to host tissue ligands and for bacteria cell motility. This information can be used to design molecules that compete with these compounds for binding, to disrupt or alter the mechanical stability of PilY1. The therapeutic goal would be to shut down the mechanosensing or adhesive functions of PilY1, which could render the bacteria unable to detect surfaces and therefore unable to start an infection process.
The final achievement of this project was to test how the host tissue ligands affect the nanomechanics of PilY1. In the presence of these ligands, PilY1 tightly binds to them and experiences an increase in its mechanical stability, which could be directly related to the process by which P. aeruginosa and other pathogens establish a strong interaction with the host´s tissues. In the respiratory tract, mechanical perturbations arise from the flow of mucus or coughing, and the tight anchoring of the bacterium to the tissue through PilY1 would prevent its dislodgement by these clearance mechanisms.
These results confirmed the key role of mechanical forces in the activity of PilY1. This is the first time this protein has been studied directly under force, the perturbation under which it naturally operates, and the results obtained shed light on the force-related mechanisms that underlie its functions. Given the new insights it provides into the poorly understood role of forces in bacterial pathogenesis and the potential therapeutic targets that can be derived from this work, the findings of this research will be disseminated to the scientific community by means of publications and presentations at conferences, and outreach events for wider audiences.
The next goal was to test how small compounds alter PilY1 nanomechanics. These molecules are known to modulate protein mechanical stability, and they increase it in the case of PilY1. These compounds are crucial for binding to host tissue ligands and for bacteria cell motility. This information can be used to design molecules that compete with these compounds for binding, to disrupt or alter the mechanical stability of PilY1. The therapeutic goal would be to shut down the mechanosensing or adhesive functions of PilY1, which could render the bacteria unable to detect surfaces and therefore unable to start an infection process.
The final achievement of this project was to test how the host tissue ligands affect the nanomechanics of PilY1. In the presence of these ligands, PilY1 tightly binds to them and experiences an increase in its mechanical stability, which could be directly related to the process by which P. aeruginosa and other pathogens establish a strong interaction with the host´s tissues. In the respiratory tract, mechanical perturbations arise from the flow of mucus or coughing, and the tight anchoring of the bacterium to the tissue through PilY1 would prevent its dislodgement by these clearance mechanisms.
These results confirmed the key role of mechanical forces in the activity of PilY1. This is the first time this protein has been studied directly under force, the perturbation under which it naturally operates, and the results obtained shed light on the force-related mechanisms that underlie its functions. Given the new insights it provides into the poorly understood role of forces in bacterial pathogenesis and the potential therapeutic targets that can be derived from this work, the findings of this research will be disseminated to the scientific community by means of publications and presentations at conferences, and outreach events for wider audiences.
The findings of this project shed light on the effect of mechanical forces on a bacterial protein directly involved in detecting surfaces susceptible to colonization. Surface recognition and colonization are essential steps for many bacteria that cause disease in humans. This project focused on dissecting the modulatory role of mechanical forces and the binding of molecules on a protein known to mediate the first steps involved in surface recognition. At the current pace, it is expected that by 2050 up to ten million people could die annually because of antibiotic-resistant infections. With the increasing appearance of recalcitrant infections that are unresponsive to treatment, it is an urgent endeavor to identify new therapeutic targets. Novel and multidisciplinary efforts must be implemented to find alternatives to antibiotic treatments. Targeting the first stages of tissue colonization by disrupting the ability of bacteria to sense and respond to the proximity of the tissue or its molecular features could provide the means to prevent further spreading and abolishing infection. The continuation of this project is oriented to design molecular strategies that “disarm” the recognition abilities of pathogenic bacteria by altering the proteins that they use to sense surfaces.