Structural insight into the mechanism of Clostridium difficile surface formation

The cell wall of Gram-positive bacteria is composed of proteins and peptidoglycan with the protruding secondary polysaccharides, which differ in the chemical composition and structure among various species. The cell wall secondary polysaccharides are covalently attached to the peptidoglycan and can account for more than a half of the total cell wall mass. They form a dense network of negative charges and impact cation homeostasis, membrane permeability, antibiotic susceptibility and survival in the host. In many species they also act as the anchors to which the outermost paracrystalline protein surface layer, called the S-layer, is non-covalently attached. We have already begun to elucidate the S-layer structure of Clostridium difficile. However, the mechanisms responsible for the organization of the S-layer structure and its attachment to the peptidoglycan remain poorly understood.

C. difficile is a dangerous nosocomial pathogen. When the normal gut microbiota of a patient is compromised, C. difficile spores germinate and the infection, even when treated, can reoccur and lead to life-threatening complications. The wide-spread use of broad-spectrum antibiotics in treatment of C. difficile infections has already resulted in a number of (multiple) antibiotic resistant strains. Considering that the contact with the host takes place at the surface of the bacterium and that the S-layer of C. difficile is an essential virulence factor, it is worthwhile to study the associated processes.

Gene manipulation studies of enzymes involved in biosynthesis of C. difficile secondary polysaccharides showed that their impairment leads to bacterial growth defects, diffused cell wall, defective anchoring, altered shedding and deposition of secondary polysaccharides, as well as defects in morphology and assembly of the S-layer, changes in biofilm formation and finally, changes in virulence. This prompts us to study the mechanisms of C. difficile S-layer assembly and in particular address the enzymes involved in the biosynthesis of secondary polysaccharides. Moreover, characterization of the respective enzymes might expand our understanding in the biogenesis of other bacterial cell wall polymers.

With this proposal we plan to gain novel insight into the mechanisms underlying the biosynthesis of secondary polymers and their role in the S-layer assembly. We will study three groups of enzymes involved in biosynthesis of secondary polymers (enzymes involved in mannose conversion, glycosyltransferases, and enzymes attaching the secondary polysaccharides to the peptidoglycan). We will use complementary approaches of molecular biology, crystal structure analysis, enzyme activity measurements, structure-based virtual screening of ligands and classical inhibitor synthesis, biochemical and mass spectroscopy analysis, microscopic (optical, electronic and CryoEM) observations of bacteria or their fragments originating from wild type bacteria and those impaired by gene manipulations and inhibitors.

We believe that research systematically addressing the whole group of the respective enzymes has the potential to deliver fundamental discoveries about their roles in the biosynthesis of secondary polymers and S-layer assembly. Hence, a successful outcome of this project will lay foundations for novel drug discovery programs that have the potential to improve human healthcare in life-threatening situations of C. difficile infections in a species-specific manner and thereby reduce risks of widely spreading antibiotic resistance.

ARRS project no. J1-1709, Dušan Turk

Duration: 1. 7. 2019 – 30. 6. 2022

Role of legumain in infection and inflammation

Legumain or asparaginyl endopeptidase (AEP) is a member of the CD clan of cysteine proteases and cleaves protein substrates exclusively after asparagine or (to a minor extent) aspartic acid residues. It is a highly conserved protein which is present in a large variety of animal species. It was shown that legumain has possible roles in normal lysosomal functions, antigen presentation, immune response and immune signalling, but also in apoptosis and osteoclast remodelling.

The most reliable experimental evidence for major physiological roles of legumain was obtained from animal models, where legumain null mice exhibited hemophagocytic syndrome, impaired kidney function and accumulation of macromolecules in the lysosomes which is characteristic for lysosomal storage diseases.

Recently, several reports linked legumain to various pathological conditions such as cancer and Alzheimer’s disease. However, one of the most important physiological roles of mammalian legumain is in the immune response, where it was originally thought to participate solely in the processing of foreign proteins for presentation on the MHCII complex. In the last decade it was also shown to activate TLR receptors of the innate immune system and influence signalling pathways through the processing of other membrane receptors. Contrary to many other proteases, physiological substrates of legumain were never studied on a system wide level and all known substrates were identified on a case-by-case basis. Although legumain is emerging as physiologically and clinically relevant target, due to the lack of experimental data on its substrates the majority of legumain physiological functions, especially the ones related to immune response, still remain largely unknown. This emphasizes the need to address its physiological functions by identification of its substrates, which has the potential for a major scientific breakthrough and could therefore significantly improve our understanding of legumain-dependent mechanisms behind immunity.

We recently performed a pilot proteomic analysis of macrophages from legumain null mice and our preliminary results showed that legumain ablation in macrophages caused extremely high upregulation of two peroxidases related to the antimicrobial immune response (myeloperoxidase and eosinophil peroxidase). Both peroxidases are known to produce hypochlorous acid which is a potent antimicrobial agent involved in elimination of infectous organisms. However, hypohalous acids were also reported to damage the host tissue during the inflammatory conditions such as asthma and hypereosinophilic syndrome which means that legumain could be directly involved in regulation of those processes, but the molecular mechanisms remain unknown.

The data obtained in our preliminary experiments demonstrated that the proposed research can provide an explanation of legumain-null phenotype at the molecular level. Moreover, this project enables a novel and unique insight into the role of legumain in immune response and inflammation, as well as on the regulation of immune response and inflammation in general. In this proposal, we plan to expand our research by performing a systematic proteomic analysis of several tissue types of young and aged mice in order to obtain a detailed understanding of how legumain regulates physiological processes at the molecular level. The obtained results will be supplemented with quantitative PCR and antibody array data and will be used for biochemical and biological validations of the identified molecular processes and pathways. Finally, the in vivo role of legumain in defense against pathogen infection will be examined in animal models. Legumain dependant effect on antimicrobial action of immune cells through the generation of hypochlorous acid could open new venues for development of therapeutic approaches to fight infection.

ARRS project: J7-9435, Marko Fonović

Duration: 1. 7. 2018 – 30. 6. 2021