Pneumococcal cell envelope

Leader : Cécile Morlot

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 diseases such as otitis, pneumonia, bacteremia and meningitis, killing over 1 million people per year worldwide.

Cellules de pneumocoque à différentes étapes de division, observées 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 a D-iso-glutamine, which results from the amidation of a D-glutamate 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 cristallographique du complexe MurT-GatD du pneumocoque (panneau de gauche) ; modélisation des substrats dans le site actif de MurT (panneau du millieu) ; défauts morphologiques causés par la déplétion de MurT-GatD (panneau de droite)

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 hydrolases, N-deacetylation and O-acetylation of its glycan chains, and decoration with macromolecules such as TA.
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 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 is assembled and maturated with high spatial and temporal resolution, we investigate its biosynthesis using metabolic labeling, single-molecule localization microscopy and microbial genetics (Collab. C. Grangeasse, MMSB Lyon ; YS Wong, DPM). The labeling is achieved by the metabolic 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 video. Using this approach, we can localize PG 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 use our experimental data to simulate the morphogenesis of the ovoid cell in silico and test various hypotheses regarding the dynamics of PG synthesis. Our results have revealed that morphogenesis in ovococci relies on synthesis of sPG, which is then cleaved at its periphery to form 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 des composants de la paroi par incorporation de sondes cliquables dans les précurseurs du PG ou des TA. B. Localisation par dSTORM du PG marqué, observé juste après le marquage (expérience PULSE) ou après une période de maturation (PULSE-CHASE). C. Illustration 3D d’un stade de division intermédiaire montrant la synthèse de PG septal au niveau du front d’invagination de la membrane, le clivage du septum à sa périphérie et l’insertion de PG périphérique. Les nouveaux hémisphères qui en résultent contiennent un mélange de PG septal et périphérique.

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, our knowledge of the dynamics of teichoic acid (TA) synthesis and maturation is much more limited. 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, Choline-Binding Proteins (CBPs) at the cell surface (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 in S. pneumoniae, Cho decoration empowers TA with crucial roles in the physiology, division and virulence of the cell.
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). This approach has revealed that TA synthesis in ovococci is localized at midcell and is rather concomitant with PG synthesis. However, the mechanisms of assembly and maturation of TA in ovococci still remain largely unexplored.
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.

A. Localisation dSTORM des TA dans une souche de pneumocoque sauvage (gauche) ou délétée de tacL (droite). B. Analyse SDS-PAGE de preparations de TA marqués. C. Image CEMOVIS (cryo-EM of vitreous section) de l’enveloppe du pneumocoque, montrant la couche de TA dans l’espace périplasmique (PS), compris entre la couche de PG et la membrane cytoplasmique (CM).


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. (Calvez et al. Front. Microbiol., 2019).

Ségrégation de phases lipidiques L-alpha marquées par le composé DOPE (rouge) au niveau des anciens hémisphères dans des cellules de pneumocoque. FtsZ est marquée en vert et sert de référence pour la progression du cycle cellulaire.

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 toward 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 PBP variants produced by beta-lactam susceptible and resistant strains.
This work should provide 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 des mutations portées par PBP2b issue d’une souche de pneumocoque résistante à la pénicilline.

Innovative antibacterial strategies to fight the pneumococcus

Collaborations :

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

The fight against antibiotic 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 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 cell growth.

Identification de molécules cliquables qui inhibent la croissance du pneumocoque. La croissance cellulaire est évaluée par observation de la localisation de composants pariétaux marqués en microscopie de fluorescence (A), par analyse de démographes (B), et par suivi de la densité optique de cultures bactériennes (C).