Pneumococcal cell envelope

Peptidoglycan assembly and architecture

Collaborations
C. Grangeasse, MMSB, Lyon
YS. Wong, DPM, Grenoble
L. Pasquina-Lemonche, Sheffield University, UK
C. Moriscot & B. Gallet (Team headed by G. Schoehn), IBS, Grenoble

Bacterial growth, division and survival are intimately linked to the synthesis and maturation of the cell wall. The cell wall provides mechanical resistance to external and internal stresses (like the turgor pressure), and it confers a specific cell shape. Importantly, it is also involved in interactions with neighboring organisms (prokaryotic and eukaryotic cells), in the recognition of bacteria by the host innate immune system, and its assembly is the target of numerous antibiotics. The cell wall is therefore a central pillar of the bacterial life.
In Gram-positive bacteria, the cell wall contains two main components, the peptidoglycan (PG, a highly resistant mesh of glycan chains crosslinked by short peptides) and teichoic acids (TA, repeats of complex polysaccharidic units). As an eminent target of our most efficient antibiotics (β-lactams and glycopeptides), the PG has been extensively studied, but the mechanisms through which it reaches its final composition and architecture remain unclear.
Our main model for the study of PG assembly and architecture is the opportunistic human pathogen Streptococcus pneumoniae (the pneumococcus). The pneumococcus is an ovoid Gram-positive commensal bacterium of the naso-pharyngeal flora of about 10% of the human population. When it invades other sites and tissues, it causes a variety of infectious diseases such as otitis, pneumonia, bacteremia and meningitis, killing over 1 million people per year worldwide.

Pneumocoques à différents stades de division, observés par microscopie électronique.

The PG is a three-dimensional mesh consisting of glycan chains cross-linked by peptide bridges. PG synthesis begins in the cytoplasm where a cascade of enzymes synthesize the PG precursor called lipid II, a lipid-linked disaccharide-pentapeptide positioned at the inner face of the cytoplasmic membrane. In many Gram-positive species, the second residue of the peptide is amidated from a D-glutamate into a D-iso-glutamine by the essential cytoplasmic complex MurT/GatD. Our structure-function study of MurT/GatD from S. pneumoniae has revealed the molecular interface and the residues involved in the amidotransferase enzymatic activity of the complex, providing fundamental insights for the development of new antibiotics (Morlot et al. Nat. Commun. 2018).

Structure du complexe MurT-GatD du pneumocoque ; modèle des substrats dans le site actif ; aberrations morphologiques en absence de MurT-GatD.

The last two residues of the pentapeptide are D-alanines (D-Ala), added by the MurF ligase as a D-Ala-D-Ala dipeptide (called DADA henceforth). Once formed, lipid II is flipped to the outer face of the membrane where it is polymerized and cross-linked into the existing PG network by Penicillin-Binding Proteins (PBPs) and SEDS proteins (Shape Elongation, Division and Sporulation). To reach its final properties (composition, shape, elasticity), the PG also requires partial cleavage by PG hydrolases, N-deacetylation and O-acetylation of its glycan chains, and decoration with macromolecules such as TA. Publications of our team related to these PG-associated processes can be found here.
Despite the importance of PG for cell proliferation and survival, we still poorly understand how it is assembled and remodeled in space and time to ensure cell division, shape and integrity. This is particularly true for ovoid-shaped bacteria such as streptococci and enterococci, in which two different modes of PG assembly, dedicated to cell division (septal PG synthesis, sPG) and elongation (peripheral PG synthesis, pPG), are confined to an annular region whose dimensions flirt with the diffraction-limited resolution (≈ 250 nm) of conventional fluorescence microscopy. As a matter of fact, the major division protein FtsZ, which recruits all the PG synthases at the beginning of the cell cycle, forms an annular structure (called the Z-ring) about a hundred nanometers wide, as reported in our super-resolution PALM (PhotoActivated Localization Microscopy) study of FtsZ (Jacq et al., mBio, 2015).
To understand how PG assembly at the nanometer scale shapes a cell of micrometric dimensions, we investigate its biosynthesis using metabolic labeling, single-molecule localization microscopy and microbial genetics (Collab. C. Grangeasse, MMSB Lyon ; YS Wong, DPM). Metabolic labeling is achieved by the incorporation of D-alanine derivatives into the growing PG. These probes carry chemical functions that allow their conjugation to fluorescent dyes using click chemistry (Trouve et al., STAR Protoc., 2021). Labeled cells are then observed by super-resolution dSTORM (direct STochastic Optical Reconstruction Microscopy) to obtain nanoscale details of PG assembly and remodeling along S. pneumoniae cell cycle (movie). Using this approach, we have been able to localize sPG and pPG synthesis sites, detect variations in the dimensions of the labeling patterns to 30 nm accuracy, and quantify the relative amounts of newly synthesized PG. We further used the experimental geometrical and kinetic parameters of PG assembly to simulate the morphogenesis of the ovoid cell in silico and test various hypotheses regarding the dynamics of sPG and pPG synthesis. Altogether, our results revealed that morphogenesis in ovococci relies on synthesis of sPG, which is then cleaved at its periphery to become lateral wall, into which pPG is inserted (Trouve et al., Curr. Biol., 2021).
We are now currently applying our approach to mutant strains, in order to dissect the function of genes involved in PG assembly and remodeling.

A. Marquage métabolique de la paroi par incorporation de sondes clickables dans le PG ou les TA. B. Localization dSTORM de PG marqué après marquage (PULSE) ou après une courte période de maturation (PULSE-CHASE). C. Illustration d’un stade mi-division montrant la synthèse de PG septal synthesis au front de l’invagination de la membrane, le clivage du septum à sa péripherieet l’insertion de PG péripherique. Les nouveaux hémispheres contiennent un mix de PG septal et péripherique.

Trouve et al., STAR Protoc., 2021
Trouve et al., Curr. Biol., 2021
Bonnet et al. ACS Chem. Biol. 2018


Teichoic acid assembly and maturation

Collaborations
C. Grangeasse, MMSB, Lyon
YS. Wong, DPM, Grenoble
C. Laguri (Team headed by F. Fieschi), IBS, Grenoble
C. Moriscot & B. Gallet (Team headed by G. Schoehn), IBS, Grenoble

Compared to PG, much less is known about TA, both in terms of their assembly dynamics and their maturation at the cell surface. These gaps of knowledge are particularly dramatic given that TA are involved in a wide range of processes, including cell morphogenesis and division, autolysis, biofilm formation, host tissue adhesion and infection, ion homeostasis, susceptibility to antibiotics and cationic anti-microbial peptides. TA have a particularly important role in S. pneumoniae, in which they are decorated with phosphorylcholine (PCho) residues that retain, and in some cases activate, surface-exposed Choline-Binding Proteins (CBPs) (Frolet et al., BMC Microbiol., 2010). CBPs are involved in PG remodeling (insertion of new material, daughter-cell separation), autolysis, competence and host-cell interactions (Bonnet et al., Cell Surf., 2018). Therefore, Cho decoration empowers TA with crucial roles in the physiology, division and virulence of S. pneumoniae.
Teichoic acids (TA) are complex linear polysaccharides that are assembled from a common precursor, which can be transferred to the PG (WTA for Wall Teichoic Acids) or to the cytoplasmic membrane (LTA for LipoTeichoic Acids). TA synthesis and maturation have been poorly described in the literature because most bacterial strains lack specific TA constituents for unambiguous labeling. We have developed a pioneering labeling method for TA (Collab. YS Wong, DPM), based on the incorporation of a clickable Cho derivatives, further conjugated to a fluorophore carrying a matching clickable function Bonnet et al. ACS Chem. Biol. 2018.
We have recently optimized TA labeling for super-resolution dSTORM and established a biochemical method to isolate WTA and LTA and observe them by gel electrophoresis. Using this combination of techniques, together with NMR (Collab. C. Laguri, IBS), cellular electron microscopy (Collab. G. Schoehn, IBS) and microbial genetics (Collab. C. Grangeasse, MMSB, Lyon), we are investigating the mechanisms of TA synthesis and maturation in S. pneumoniae, as well as their interplay with PG-associated processes.

Membranes

Collaborations
J. Jouhet, IRIG, CEA Grenoble.

The enzymes that synthesize the cell wall and most morphogenetic proteins are membrane proteins. The PG and TA precursors are also membrane-bound. We have been investigating the role of the nature of the membrane lipids in morphogenesis. Using a combination of fluorescent lipid probes, we have uncovered the existence of different physical lipid phases localized according the cellular geometry. Lipid phases may in turn localize morphogenetic proteins.

Ségrégation de lipide DOPE marqué (rouge) en phase L-alpha sur les hemisphères parentaux de cellules pneumococcales. FtsZ est marqué en vert et sert de référence pour la progression du cycle cellulaire.

Calvez et al. J. Biol. Chem. 2017

Beta-lactam resistance

Collaborations
S. Fort, CERMAV, Grenoble
C. Contreras-Martel (Group headed by A. Dessen), IBS, Grenoble

Penicillin-binding proteins (PBPs) are the targets of beta-lactams, the most widely used antibiotics such as penicillins, cephalosporins or carbapenems. PBPs are transpeptidases that catalyze the cross-linking of the PG. Pathogens such as Staphylococcus aureus, enterococci, Neisseria ssp. and S. pneumoniae become resistant to penicillins by expressing PBPs with a low reactivity for beta-lactams. Since these drugs are structural mimics of the natural substrate, how PBPs from resistant strains have lost their reactivity with beta-lactams while retaining their enzymatic function ?
Although the reaction between PBPs and β-lactams is well understood kinetically and structurally, the enzymatic transpeptidation catalyzed by PBPs is poorly studied due to the difficulty to obtain the substrates. Progress in the enzymology of PBPs have recently uncovered some requirements of the substrates of the transpeptidation reaction but structural information about the PBP-substrate interaction is still lacking.
We have set up a chemo-enzymatic approach that allows to synthesize PG fragments of defined size (Collab. S. Fort, CERMAV), which we use to characterize the interaction between PBPs and their substrates, by combining X-ray crystallography and NMR with enzymology and binding studies. In particular, we study pneumococcal PBPs involved in the resistance to β-lactams and we investigate variants from susceptible and resistant strains.
This work should provide for the first time a detailed understanding of the important drug targets that are the PBPs, unravel the reason why some last generation beta-lactams are active against strains resistant to older drugs, and help design further improvements.

Distribution of amino acid substitutions in PBP2b from a penicillin-resistant strain of pneumococcus

Innovative antibacterial strategies to fight the pneumococcus

Collaborations
Y.S. Wong, DPM, Grenoble
I. Pelloux, CHU, Grenoble

The fight against bacterial resistance is a major public health issue and requires constantly finding new solutions to fight bacterial infections and stay one step ahead. We are exploring novel antibacterial strategies based on metabolic click labeling of the bacterial cell wall (Collab. YS Wong). From a chemical point of view, the creation of chemically controlled bonds in a living environment remains a major challenge. We have synthesized numerous clickable PG and TA probes, test their ability to be integrated into the pneumococcal cell wall and to cross-link its two main components, the PG and TA. This work has led to the development of new cell wall probes, providing tools for co-labeling of PG and TA. In addition, we have identified pairs of clickable molecules that artificially cross-link the pneumococcal cell wall, resulting in impaired S. pneumoniae growth.

Identification de molécules clickables qui inhibent la croissance du pneumocoque. La croissance a été évaluée par la chasse des composants de la paroi marqués observés par microscopie de fluorescence (A) et analysée par des démographes (B), et suivi de cultures (C).