Periodic Reporting for period 4 - ChronosAntibiotics (Exploring the bacterial cell cycle to re-sensitize antibiotic-resistant bacteria)
Periodo di rendicontazione: 2022-09-01 al 2024-03-31
Over the next 35 years, antibiotic resistant bacteria are projected to cause over 300 million deaths. Finding alternative strategies for antimicrobial therapies is a global challenge, hindered by several obstacles in the antibiotic discovery process. To address this challenge, we must thoroughly understand the biology of bacterial pathogens, and use that knowledge to develop new antimicrobial agents. These two objectives form the core aims of this project.
More specifically, we want to
(i) find new pathways to combat resistance: Bacteria change shape during their cell cycle, and we hypothesize these changes create moments when they are more vulnerable to antibiotics. We aim to identify key regulators of the cell cycle to better understand fundamental cellular processes required for bacteria to divide inside infected hosts and/or in the presence of antibiotics.
(ii) develop new tools to screen for new antibiotics: We will construct new fluorescence-based reporter strains that allow the identification of new antibacterial compounds. These strains respond to the inhibition of specific, essential cellular pathways, and therefore can also be used to study the mode of action of new antibiotics.
More specifically, we want to
(i) find new pathways to combat resistance: Bacteria change shape during their cell cycle, and we hypothesize these changes create moments when they are more vulnerable to antibiotics. We aim to identify key regulators of the cell cycle to better understand fundamental cellular processes required for bacteria to divide inside infected hosts and/or in the presence of antibiotics.
(ii) develop new tools to screen for new antibiotics: We will construct new fluorescence-based reporter strains that allow the identification of new antibacterial compounds. These strains respond to the inhibition of specific, essential cellular pathways, and therefore can also be used to study the mode of action of new antibiotics.
In this project, we have used Staphylococcus aureus, the second most common cause of death by antibiotic-resistant infections in the world, and an excellent model organism for cell division in cocci, to study the bacterial cell cycle. We started by screening 2000 mutants in non-essential S. aureus genes by fluorescence microscopy and training an artificial neural network to automatically classify the cell cycle phase of individual S. aureus cells. This allowed us to automatically classify the cell cycle stage of tens of thousands of cells, as well as to measure various morphological parameters, and identify specific mutants that that are impaired in cell cycle progression, in chromosome segregation and in cell morphology. Study of the biological role of the proteins that are missing in each mutant uncovered new mechanisms of regulation of cell cycle progression and morphogenesis, as well as previously unknown proteins required for correct chromosome segregation, which are critical for bacteria to survive inside an infected host.
At the same time, we constructed a library of S. aureus reporter strains for the identification of new antimicrobial compounds. These strains encode fluorescent proteins under the control of promoters that respond to the inhibition of specific metabolic pathways. We currently have reporter strains that become fluorescent upon inhibition of cell wall synthesis, fatty acids biosynthesis, DNA damage, loss of membrane potential etc. These strains are useful for whole-cell screens for new antibiotics, as well as to study the mode of action of new compounds with antimicrobial activity.
Results from this project have resulted in 14 publications in peer-reviewed journals and have been presented in 20 lectures at international conferences and 12 seminars in various universities. Six PhD degrees have been awarded to students working in this project.
At the same time, we constructed a library of S. aureus reporter strains for the identification of new antimicrobial compounds. These strains encode fluorescent proteins under the control of promoters that respond to the inhibition of specific metabolic pathways. We currently have reporter strains that become fluorescent upon inhibition of cell wall synthesis, fatty acids biosynthesis, DNA damage, loss of membrane potential etc. These strains are useful for whole-cell screens for new antibiotics, as well as to study the mode of action of new compounds with antimicrobial activity.
Results from this project have resulted in 14 publications in peer-reviewed journals and have been presented in 20 lectures at international conferences and 12 seminars in various universities. Six PhD degrees have been awarded to students working in this project.
In this project we have characterized in detail the mode of division and the morphological changes that occur during the cell cycle of the important pathogen Staphylococcus aureus. We have corrected statements present in the literature for the last five decades by demonstrating that, contrary to what was previously thought, S. aureus cells (i) do not necessarily divide in three orthogonal planes over three consecutive division cycle and (ii) are capable of slight cell elongation, which may be important for invading certain infection niches.
We have also uncovered new, surprising, levels of regulation during cell cycle progression. The best example links a cytoplasmic protein involved in chromosome segregation, FtsK, and a protein anchored at the outer layer of the cell (the cell wall), required for splitting of the division septum, Sle1. We found that FtsK regulates the levels of Sle1 so that the cell can delay septum splitting while chromosome segregation is taking place, or when DNA damage occurs. Tight regulation of the timing of septum splitting during the cell cycle is essential to avoid exposure of an immature cell surface, which would be easily recognized by the infected host, leading to clearance of the bacterial cells by the innate immune system.
A second example of progress beyond the state of the art was the imaging of single molecules of peptidoglycan synthases in live, dividing S. aureus cells, and the demonstration that their processive movement is driven by the enzymatic activity of the proteins (and not by the cytoskeleton, as previously proposed) and therefore can be stopped with cell wall-targeting antibiotics, such as beta-lactams or vancomycin.
Overall, this project allowed significant progress in our understanding of the cell cycle of an important bacterial pathogen, as well as the development of useful tools for the study and screening of antibiotics.
We have also uncovered new, surprising, levels of regulation during cell cycle progression. The best example links a cytoplasmic protein involved in chromosome segregation, FtsK, and a protein anchored at the outer layer of the cell (the cell wall), required for splitting of the division septum, Sle1. We found that FtsK regulates the levels of Sle1 so that the cell can delay septum splitting while chromosome segregation is taking place, or when DNA damage occurs. Tight regulation of the timing of septum splitting during the cell cycle is essential to avoid exposure of an immature cell surface, which would be easily recognized by the infected host, leading to clearance of the bacterial cells by the innate immune system.
A second example of progress beyond the state of the art was the imaging of single molecules of peptidoglycan synthases in live, dividing S. aureus cells, and the demonstration that their processive movement is driven by the enzymatic activity of the proteins (and not by the cytoskeleton, as previously proposed) and therefore can be stopped with cell wall-targeting antibiotics, such as beta-lactams or vancomycin.
Overall, this project allowed significant progress in our understanding of the cell cycle of an important bacterial pathogen, as well as the development of useful tools for the study and screening of antibiotics.