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MapZ: Defining a new regulatory mechanism of bacterial cell division – Map-CellDiv

Deciphering a cellular guidance system for the control of pneumococcal infections

Understand how the MapZ protein localizes at the center of the cell by interacting with the cell wall and recruits and controls the positioning and the functioning of cell division machinery.

Characterization of bacterial cell division mechanisms

The aims of the project are to characterize at the molecular and cellular levels the mechanisms by which the MapZ protein governs the positioning of the cell division site at the center of the bacterial cell using the bacterium Streptococcus pneumoniae as a model. They are organized around three research axes concerning the structure-function analysis i) of the extracellular domain of MapZ, ii) the cytoplasmic domain of MapZ and finally iii) the phosphorylation of MapZ. The experiments carried out will allow to understand how MapZ is positioned at the center of the cell by interacting with the cell wall in order to recruit and control the installation and operation of the cell division machinery. Finally, the study of the phosphorylation of MapZ will allow deciphering the mode of regulation of this process in the course of the cell cycle. This work paves the way toward future applied projects aiming at using MapZ as a potential therapeutic target to combat some bacterial infections.

We have implemented a methodological approach based on different expertise and technologies in order to characterize the structure of the MapZ protein (NMR), to study the biochemical properties of its interaction with the cell wall and its cellular partners (protein biochemistry) and to elucidate its biological role and the process of regulation by phosphorylation using different approaches of fluorescence microscopy.

The structural characterization of the extracellular domain of MapZ by Nuclear Magnetic Resonance (NMR) has demonstrated the existence of two sub-domains structured and separated by a flexible linker region. Using live cell microscopy and protein biochemistry approaches, we demonstrated that one of the two sub-domains served as a pedestal for the latter so that it could be correctly positioned with the flexible linker and thus interact with the peptidoglycan, the major component of the cell wall responsible for cellular elongation. A more precise analysis of the second subdomain has allowed to characterize a conserved signature and comprising the amino acids essential for the interaction with the peptidoglycan. MapZ is thus shuttled by the peptidoglycan produced during cellular elongation. When the elongation of the mother cell is sufficient to generate two daughter cells, MapZ is thus mechanically positioned in the center of the future daughter cell and can then act as a molecular beacon to position the cell division machinery.

This work will likely provide very interesting benefits for the fight against infectious diseases of bacterial origin. Indeed, this work will serve as a fundamental basis for the rational design of specific molecules preventing MapZ from interacting with the peptidoglycan and thus limiting the multiplication of bacteria. Knowing the urgency to discover new antibiotics, the development of molecules blocking MapZ would represent a major step towards the discovery of new antibiotics and an example of valorization of fundamental research.

Rewiring the pneumococcal cell cycle with Serine/threonine-and tyrosine-kinases Grangeasse, C. Trends Microbiol., (2016), 24, 713-24. Structure-function analysis of the extracellular domain of the pneumococcal cell division site positioning protein MapZ Manuse, S., Jean, N.L., Guinot, M., Lavergne, JP., Laguri, C., Bougault, C.M., VanNieuwenhze, M.S., Grangeasse, C*. and Simorre, JP. Nat. Commun. (2016), 7, 12071 * co-dernier auteurs. Cell division of Streptococcus pneumoniae: think positive! Garcia, P.S., Simorre, JP., Brochier-Armanet, C. and Grangeasse, C. Curr. Opin. Microbiol. (2016), 34, 18-23. Wrapping the cell in a CozE shell Ducret, A. and Grangeasse, C. Nat. Microbiol. (2017), 2, 16262.

A major challenge in microbiology concerns the nature of the mechanisms of cell division. This process requires a myriad of protein–protein interactions, the coordination of a remarkable suite of strategies and competing biochemical reactions. In most bacteria, cell division results in the formation of two genetically and morphologically identical daughter cells. Such binary fission process first requires the identification of the cell middle followed by the recruitment of the division machinery, which first component is the highly conserved tubulin-like protein FtsZ. FtsZ forms a ring that eventually constricts to give rise to two newborn cells. A long-standing question was thus how bacterial find their middle to select the site of cell division. To date, most of our knowledge of bacterial cell division and morphogenesis comes from studies on a few bacterial models and notably Escherichia coli and Bacillus subtilis. However, these two rod-shaped bacteria are actually not representative of the diversity of existing bacterial cell shape, mode of growth and developmental behavior. In addition, the systems identified in Escherichia coli and Bacillus subtilis, that prevent the assembly of the division machinery near the cell pole or over the chromosome are not found in a large array of bacteria. Last, there are several lines of evidence that these systems are not sufficient per se for the identification of the cell center. Collectively, it came thus has no surprised that other proteins have been found to participate in the positioning of the division site at mid-cell. This is the case in some proteobacteria and actinobacteria. In the ongoing efforts to determine what does identify the division site, we have recently uncovered an unprecedented mechanism in the bacterium Streptococcus pneumoniae. The key player of this system is a protein of unknown function that we named MapZ and that is conserved among streptococci and lactococci and most enterococci. We have shown that MapZ localizes at the division site before FtsZ to guide septum positioning. We found that MapZ moves apart as the cell elongates, therefore behaving as a permanent beacon of division sites. MapZ positioning at the future division site relies on the synthesis of the cell wall that mechanically pushes MapZ rings. MapZ then positions the FtsZ ring through direct protein-protein interactions. Further, we have found that MapZ phosphorylation is required for proper FtsZ ring formation and dynamics. The aim of this project is to determine at the molecular and cellular level how MapZ positions at midcell and how it controls the constriction of the FtsZ ring. To achieve our goals, we will use multidisciplinary and complementary approaches combining molecular genetics, proteomics, structural biology and cutting-edge live cell imaging techniques of different resolution and scale. This strategy will allow to track and measure the dynamic localization of MapZ and its partners and to give and integrated vision of the regulatory mechanism mediated by MapZ. The work is divided in three main tasks dedicated to (i) the structural features of the MapZ extracellular domain and mode of interaction with the cell wall, (ii) the characterization of MapZ partners in the cell division and cell wall synthesis machineries and the impact of MapZ on their localization and iii) the understanding of how MapZ phosphorylation influences the closure of the FtsZ ring. The outcomes of this project will allow deciphering a mechanism that is at odds with model regulatory systems of bacterial cell division and will contribute to the fundamental knowledge in life sciences. Streptococcus pneumoniae being an important human pathogens, one can even anticipate that our data could serve as a fundamental basis for future studies aiming at developing strategies to combat bacterial infectious diseases.

Project coordination

Christophe Grangeasse (Bases Moléculaires et Structurales des Systèmes Infectieux)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

BMSSI CNRS Bases Moléculaires et Structurales des Systèmes Infectieux
IBS CNRS/CEA/UJF Institut de Biologie Structurale

Help of the ANR 416,999 euros
Beginning and duration of the scientific project: November 2015 - 36 Months

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