Contribution of Bacillus subtilis cell envelope stress responses to antibiotic survival
Doctoral thesis, 2024
Antibiotic potentiators, molecules that increase the activity of antibiotics by inhibiting bacterial defenses, are an emerging strategy to combat antimicrobial resistance. Cell envelope (cell wall and membrane) stress responses are particularly promising as they are the first defense against antibiotics. This thesis focuses on four systems that appeared crucial to cell envelope stress: osmoadaptation (i.e., adaptation to changing water environments) through mechanosensitive channels (MSCs) (I), membrane fluidity adaptations (i.e., adaptation related to the mobility of membrane components) through the lipid desaturase Des (II), the putative ATP-binding cassette transporter YtrBCDEF (III), and reactive oxygen species-mediated killing by the ionophore valinomycin (IV). Using Bacillus subtilis as model, I studied how and to which extent these cell envelope stress responses contribute to bacterial survival during antibiotic exposure.
When B. subtilis is exposed to membrane-targeting antibiotics, it releases amino acids through MSCs, a process coined antibiotic-induced amino acid release (AIAAR). I found that AIAAR is a widely conserved osmoprotection mechanism that is important for survival of antibiotic-induced membrane stress. (I). Postulating that AIAAR is mediated by antibiotic-induced membrane stretch, I aimed to develop a membrane thickness sensor based on the Des system. This system senses membrane thickness to adjust membrane fluidity. While no suitable reporter was obtained, new insights related to its function and mechanism were proposed (II). I then investigated the role of YtrBCDEF transporter, that is induced by cell wall synthesis inhibitors. I could confirm that this transporter plays a role in cell wall synthesis and sporulation, but its contribution to antibiotic survival was limited (III). Characterizing how the potassium ionophore valinomycin kills non-growing cells, we found that depolarization of the cell membrane causes mislocalization of the respiratory chain protein QcrA, leading to lethal accumulation of superoxide radicals. This finding explains why depolarization often induces an oxidative stress response (IV).
Based on my results, I propose AIAAR as promising target for antibiotic potentiators and suggest that oxidative stress responses should be assessed for this purpose in future research. The development of molecules interfering with AIAAR and oxidative stress responses as new antibiotic potentiators will help tackle the problem of antimicrobial resistance.
When B. subtilis is exposed to membrane-targeting antibiotics, it releases amino acids through MSCs, a process coined antibiotic-induced amino acid release (AIAAR). I found that AIAAR is a widely conserved osmoprotection mechanism that is important for survival of antibiotic-induced membrane stress. (I). Postulating that AIAAR is mediated by antibiotic-induced membrane stretch, I aimed to develop a membrane thickness sensor based on the Des system. This system senses membrane thickness to adjust membrane fluidity. While no suitable reporter was obtained, new insights related to its function and mechanism were proposed (II). I then investigated the role of YtrBCDEF transporter, that is induced by cell wall synthesis inhibitors. I could confirm that this transporter plays a role in cell wall synthesis and sporulation, but its contribution to antibiotic survival was limited (III). Characterizing how the potassium ionophore valinomycin kills non-growing cells, we found that depolarization of the cell membrane causes mislocalization of the respiratory chain protein QcrA, leading to lethal accumulation of superoxide radicals. This finding explains why depolarization often induces an oxidative stress response (IV).
Based on my results, I propose AIAAR as promising target for antibiotic potentiators and suggest that oxidative stress responses should be assessed for this purpose in future research. The development of molecules interfering with AIAAR and oxidative stress responses as new antibiotic potentiators will help tackle the problem of antimicrobial resistance.
ROS
cell envelope stress responses
osmotic stress
mechanosensitive channels
Des system
membrane fluidity
membrane potential
ABC transporter
cell wall synthesis
VASA A, Vasa Building 2-3, Vera Sandbergs Allé 8, entrance floor, Chalmers Campus Johanneberg, Gothenburg
Opponent: Professor Jörg Stülke, Institute for Microbiology and Genetics, Department of General Microbiology, Göttingen, Germany.
Author
Margareth Sidarta
Chalmers, Life Sciences, Chemical Biology
Sidarta, M., Barlog, K., Dietze, P., May, C., Bandow, J.E., Wenzel, M. Antibiotic-induced amino acid release (AIAAR): A bacterial emergency response to membrane-targeting antibiotics.
Lipid phase separation impairs membrane thickness sensing by the <i>Bacillus subtilis</i> sensor kinase DesK
Microbiology spectrum,;Vol. 12(2024)
Journal article
Baruah, L.*, Sidarta, M.*, Hammer úr Skúoy, P., Johnsson, O., Frisk, E., Didelot, P., Schäfer, A., Arshad, A., Wenzel, M. The ytrGABCDEF operon is involved in growth arrest and cell lysis of distinct Bacillus subtilis subpopulations.
Membrane depolarization kills dormant Bacillus subtilis cells by generating a lethal dose of ROS
Nature Communications,;Vol. 15(2024)
Journal article
Bacteria are everywhere. At least 30,000 types of bacteria have been discovered around the world. While only a few of them are harmful to us, they can cause serious diseases such as diarrhea, typhus, and tuberculosis. To treat such infections, we take antibiotics, life-saving essential medicines that kill bacteria. Unfortunately, many bacteria have become resistant to antibiotics (superbugs) and many of these treatments have become ineffective. Antibiotic drug development is a costly and slow process that simply cannot keep up with the fast evolution of these superbugs. This problem, called the antimicrobial resistance crisis, has become a global health threat and is predicted to cause 10 million deaths per year by 2050. Thus, finding new antibiotics and alternative strategies to tackle this problem is pivotal for our future.
Bacteria possess a complicated outer layer, the cell envelope. It can be imagined as a fortress wall that keeps out intruders, in this case antibiotics. It is the bacteria’s first line of defense and harbors a range of defense mechanisms against antibiotic attacks. Breaking down this wall or disabling its defense mechanisms will leave bacteria vulnerable to antibiotic treatment. This approach can restore the effectiveness of antibiotics against superbugs. Compounds that break through bacterial defense mechanisms are called antibiotic potentiators, because they potentiate (increase) antibiotic activity. Combination therapies of antibiotics with a suitable potentiator are a new strategy to tackle the antimicrobial resistance crisis.
In my thesis, I have studied four different bacterial defense mechanisms that are rooted in their cell envelope. My goals were to (i) better understand how these mechanisms work and (ii) assess whether they have the potential to be exploited as new drug targets for antibiotic potentiators. Two of the studied defense mechanisms turned out to be well-suited for this purpose, mechanosensitive channels and an enzyme called superoxide dismutase. Mechanosensitive channels maintain the integrity of the cell envelope under stress. I could show that they play a crucial role for bacteria to survive treatment with antibiotics that target the cell envelope, decreasing the effectiveness of antibiotics up to complete inactivity. Thereby, they affect a range of antibiotics including some of the most important drugs to date. Moreover, they are important for a broad range of bacteria. These properties make mechanosensitive channels very promising drug targets as they promise (i) a high ability to increase antibiotic effect (i.e., potentiation), (ii) broad usability with a range of drugs, and (ii) a broad activity spectrum against many diseases. I found similarly promising results for superoxide dismutase, an enzyme that helps cells to inactivate harmful chemicals. Overall, my thesis demonstrates that bacterial defense mechanisms are promising targets for antibiotic ‘helper drugs’ (potentiators) in new combination therapy approaches.
Bacteria possess a complicated outer layer, the cell envelope. It can be imagined as a fortress wall that keeps out intruders, in this case antibiotics. It is the bacteria’s first line of defense and harbors a range of defense mechanisms against antibiotic attacks. Breaking down this wall or disabling its defense mechanisms will leave bacteria vulnerable to antibiotic treatment. This approach can restore the effectiveness of antibiotics against superbugs. Compounds that break through bacterial defense mechanisms are called antibiotic potentiators, because they potentiate (increase) antibiotic activity. Combination therapies of antibiotics with a suitable potentiator are a new strategy to tackle the antimicrobial resistance crisis.
In my thesis, I have studied four different bacterial defense mechanisms that are rooted in their cell envelope. My goals were to (i) better understand how these mechanisms work and (ii) assess whether they have the potential to be exploited as new drug targets for antibiotic potentiators. Two of the studied defense mechanisms turned out to be well-suited for this purpose, mechanosensitive channels and an enzyme called superoxide dismutase. Mechanosensitive channels maintain the integrity of the cell envelope under stress. I could show that they play a crucial role for bacteria to survive treatment with antibiotics that target the cell envelope, decreasing the effectiveness of antibiotics up to complete inactivity. Thereby, they affect a range of antibiotics including some of the most important drugs to date. Moreover, they are important for a broad range of bacteria. These properties make mechanosensitive channels very promising drug targets as they promise (i) a high ability to increase antibiotic effect (i.e., potentiation), (ii) broad usability with a range of drugs, and (ii) a broad activity spectrum against many diseases. I found similarly promising results for superoxide dismutase, an enzyme that helps cells to inactivate harmful chemicals. Overall, my thesis demonstrates that bacterial defense mechanisms are promising targets for antibiotic ‘helper drugs’ (potentiators) in new combination therapy approaches.
Antibiotic-induced amino acid release - A new antimicrobial strategy?
Swedish Research Council (VR) (2019-04521), 2020-01-01 -- 2023-12-31.
Subject Categories
Biological Sciences
Roots
Basic sciences
Areas of Advance
Health Engineering
ISBN
978-91-8103-107-2
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5565
Publisher
Chalmers
VASA A, Vasa Building 2-3, Vera Sandbergs Allé 8, entrance floor, Chalmers Campus Johanneberg, Gothenburg
Opponent: Professor Jörg Stülke, Institute for Microbiology and Genetics, Department of General Microbiology, Göttingen, Germany.