Checkpoints in the bacterial cell cycle: role of the cytokinetic Z-ring and implications for antibiotic resistance

Project Information

CheckBacZ

Grant agreement ID: 839596

DOI

10.3030/839596

Project closed

EC signature date 10 April 2019

Start date 1 September 2020

End date 31 August 2022

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 147 815,04

EU contribution

€ 147 815,04

Coordinated by

UNIVERSIDADE NOVA DE LISBOA
Portugal

Periodic Reporting for period 1 - CheckBacZ (Checkpoints in the bacterial cell cycle: role of the cytokinetic Z-ring and implications for antibiotic resistance)

Reporting period: 2020-09-01 to 2022-08-31

Summary of the context and overall objectives of the project

The occurrence of multiple-drug resistant bacteria constitutes an important threat to healthy lives. The clinically relevant pathogen Staphylococcus aureus is a major concern because of the emergence of methicillin-resistant S. aureus (MRSA) strains that cause life-threatening disease in humans. In 2019, MRSA was the second most common cause of global deaths associated with bacterial antimicrobial resistance. To efficiently combat difficult-to-treat infections in the future, the development of alternative strategies will be necessary, which require fundamental knowledge about essential processes in bacteria.

The project CheckBacZ addressed basic cell biology questions about regulatory mechanisms for the cell cycle and their implications for antibiotic resistance of the bacterial pathogen S. aureus. The bacterial cell cycle can be described as a series of coordinated events leading to cell proliferation. Despite overlapping cell cycle events in bacteria, several lines of evidence suggest the existence of so-called checkpoints that ensure an orderly progression through the cell cycle.

In particular, this project aimed to elucidate mechanisms for the initiation and control of the synthesis of the division septum, which starts with the assembly of the so-called Z ring, made of filaments of the bacterial tubulin homologue FtsZ and its membrane anchors. The Z ring then recruits later divisome proteins, including proteins involved in peptidoglycan biosynthesis, which will synthesize the cell wall, essential for cells to divide. Relatively little is known about what controls the timing of Z-ring assembly, the rate of cytokinesis or the organisation of the cell wall synthesis machinery to drive the coordinated synthesis of the septum.

The results of this project have contributed to our understanding of mechanisms underlying both the synthesis and splitting of the division septum and hence promoting cell cycle progression in S. aureus. In the long term, we hope these results contribute to uncover new targets for antibiotics to open new possibilities for counteracting difficult-to-treat bacterial infections.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The project CheckBacZ aimed to understand cellular processes driving cell cycle progression in the bacterial pathogen S. aureus. I was able to provide new insights into mechanisms underlying the synthesis and the remodeling of new cell wall, both of which are essential processes for S. aureus cells to divide. Techniques newly implemented in the lab in the framework of this project, such as single-molecule tracking (SMT) microscopy, together with the development of new image analysis software, have allowed for a quantitative analysis of the movement of the septum-specific peptidoglycan enzyme complex, composed of the two essential proteins FtsW and PBP1, in cells of S. aureus. Having used site-directed mutagenesis and antibiotic-induced perturbations, we found that the directed motion of FtsW-PBP1 depends on its two enzymatic activities (transglycosylation by FtsW and transpeptidation by PBP1) and, contrarily to rod-shaped bacterial models, is independent of FtsZ treadmilling (see attached image).

To advance in the establishment of a SMT analysis workflow, I initiated a collaboration and went for a three-week visit to the laboratory of Prof. Ethan Garner at Harvard University (USA, February 2020). In his group, I learned about computer-based analysis of SMT data which helped to develop a quantitative image analysis software in the host laboratory.

Following up on a microscopy screening of a mutant library previously performed in the host laboratory, I confirmed by super-resolution microscopy at least one S. aureus gene null mutant to be delayed in cell cycle progression, specifically at its final stage. The corresponding gene encodes a homolog of Structural Maintenance of Chromosomes (SMC) proteins with a new function in S. aureus. By combining various biochemical and molecular biology approaches with microscopy imaging, I obtained results which indicate this SMC-like integral membrane protein to bind DNA while post-translationally regulating levels of a major autolysin involved in the timely separation of daughter cells (see attached image).

Finally, the mutant delayed in cell cycle progression was tested against a panel of more than 240 antimicrobial compounds from various classes targeting different cellular processes. For none of the tested compounds the mutant showed an increased sensitivity relative to the parental wild-type strain. This result suggests that S. aureus cells arrested in the final stage of the cell cycle were not more susceptible to any of the applied antibiotics.

Two manuscripts on the work of this project, one about protein dynamics underlying cytokinesis and a second one about the SMC-like protein involved in cell cycle regulation, are currently in preparation and expected to be submitted to high-impact scientific journals in 2023.

Parts of the results obtained in this project were disseminated to a broader audience by holding oral presentations in the institutional seminar series SCAN (Portugal, May 2022) and at the international Young Microbiologists Symposium 2022 (UK, September 2022), as well as in a poster presentation at the Gordon Research Conference Bacterial Cell Surfaces (USA, June 2022).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The project CheckBacZ has contributed to better understand S. aureus cell cycle regulation with potential implications for antibiotic resistance, which is crucial for human health in future decades.

Cell division proteins have been proven a promising antibacterial target because most of the divisome components are essential for bacterial growth (and hence for causing infectious disease) and conserved in many bacterial pathogens. The study of Z-ring dynamics and the identification of new cell cycle regulators progressed our understanding of mechanisms underlying the coordination of key cell cycle events such as cytokinesis and, most importantly, in the long run will hopefully open new possibilities for counteracting difficult-to-treat bacterial infections. This may include the development of more accurate therapies using antibiotics with restored efficacy.

CheckBacZ combined a biochemical and cell biology approach with computer-based image analysis. The data generated has the potential for attracting the interest of researchers in various scientific fields. This project led to an advance in our understanding of the function of protein dynamics in bacterial cytokinesis and may therefore contribute to the development of artificial cells in physical sciences, i.e. liposomes that divide through the action of a simple bacterial division machinery. The identification of new bacterial cell cycle regulators could be of broad interest for synthetic biology projects following minimal genome approaches, by contributing to extend our knowledge on genetic determinants that ensure an orderly progression through the cell cycle and are responsible for maintaining cellular integrity upon cell division. Finally, genetically modified bacterial strains that have been constructed during this project may also be used for testing their infection competence in a pre-clinical mouse model of chronic MRSA infection.

Graphical summary of results obtained during the implementation phase of the project CheckBacZ