García del Vello Moreno, María del Pilar (2021) Characterisation of the lipopolysaccharide and peptidoglycan and their structural determinants when bound to major proteins involved in its transport across the periplasm. [Tesi di dottorato]


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Item Type: Tesi di dottorato
Resource language: English
Title: Characterisation of the lipopolysaccharide and peptidoglycan and their structural determinants when bound to major proteins involved in its transport across the periplasm
García del Vello Moreno, María del
Date: 12 February 2021
Number of Pages: 240
Institution: Università degli Studi di Napoli Federico II
Department: Scienze Chimiche
Dottorato: Scienze chimiche
Ciclo di dottorato: 33
Coordinatore del Corso di dottorato:
De Castro, CristinaUNSPECIFIED
Date: 12 February 2021
Number of Pages: 240
Keywords: lipopolysaccharide, peptidoglycan, outer membrane, antibiotic resistance, microbiota, NMR, MALDI, mass spectrometry, immunology, TLR, NOD
Settori scientifico-disciplinari del MIUR: Area 03 - Scienze chimiche > CHIM/06 - Chimica organica
Date Deposited: 20 Feb 2021 23:28
Last Modified: 07 Jun 2023 10:30

Collection description

Gram-negative bacteria possess an outer membrane (OM), a lipidic bilayer that surrounds a thin peptidoglycan (PG) layer and the cytoplasmic membrane (CM). The OM is an asymmetric bilayer whose outer leaflet is mainly composed by lipopolysaccharides (LPSs). LPS and PG have a direct role in antibacterial resistance and in the communication bacteria-host. There are many open questions on how the bacterial surface of pathogens and commensals interacts with the host in order to escape immune recognition and produce harmful or beneficial effects as well as regarding the details on how the bacterial envelope is built. Therefore, the main focus of this Ph.D. thesis is to contribute to the characterisation of the LPS and PG to increase the knowledge on the interaction of pathogen and commensal Gram-negative bacteria with the host and to deepen the understanding of their structural determinants when bound to major proteins involved in its transport across the periplasm. With that aim the composition, structure and immune activities of the LPS and PG of Akkermansia muciniphila and Fusobacterium nucleatum is disclosed and the trans-envelope machineries of the model bacterium Escherichia coli studied. A. muciniphila is one of the few bacteria that successfully inhabits the mucus layer of humans and other mammals' intestines. Not only its presence is associated with a healthy intestine, but also it seems to improve insulin sensitivity, increase the mucosal barrier function, regulate glycemia levels, and reduce fat accumulation, insulinemia, cholesterol, body weight gain and inflammation in the intestine and body. The lipooligosaccharide (LOS or rough LPS) of A. muciniphila MucT is very complex: it includes more than the two canonical units of Kdo, is rich in fucose units and most of the fatty of the lipid A are branched at the penultimate carbon. The LOS seems to be a mild activator of TLR4, while it is a relevant activator of TLR2 which may play a role in the development of the beneficial effects of the bacterium. The PG of A. muciniphila MucT contains muropeptides with de-N-acetylated glucosamine, being the first time, such structure is described in a Gram-negative bacterium. Moreover, this modification of the PG has been linked to the avoidance of recognition by NOD-1 immune receptors and therefore bacterial clearance. F. nucleatum is an oral commensal that plays a crucial role in the formation of biofilms, being also involved in extra-oral disorders such as intrauterine infections and colorectal cancer, in which the subspecies animalis is the most-commonly isolated. The LPS of F. nucleatum spp. animalis ATCC 51191 has a trisaccharide repeating unit rich in amino- and aminuronic-monosaccharides, and a lipid A similar to that of Burkholderia cenocepacia. In addition, F. nucleatum ssp. polymorphum ATCC 10953, F. nucleatum ssp. animalis ATCC 51191 and F. nucleatum ssp. nucleatum ATCC 25586 full cells, outer membrane vesicles (OVMs), and LPSs stimulate monocyte-derived dendritic cells leading to an increased production of TNFa, IL-8 and IL-6, while in monocyte-derived macrophages the stimulation leads to the production of IL-10, IL-6 and IL-8 and to low levels of TNFa. These effects are measured in the three strains and seem to be mediated by Siglec-7, a sialic acid receptor, even though the O-antigen of only two of the strains tested (ATCC 10953 and 25586) expose this monosaccharide or the sialic acid-like molecule fusaminic acid. The PG of F. nucleatum spp. animalis ATCC 51191 presents an alteration of the most common stem peptide by substitution of the L-meso-diaminopimelic acid by the sulfur-containing diamino acid lanthionine or another amino acid. This may be crucial to avoid the recognition by NOD-1 immune receptors potentiating colorectal cancer development. The trans-envelope machineries were studied on E. coli, because of its importance as antibiotic-resistant "priority pathogen" of WHO and due to the fair amount of existing literature. The T5SS that transports, folds and insets b-barrel proteins in the OM is comprised of Skp, SurA, DegP and the BAM complex (that consists of four lipoproteins BamB, BamC, BamD, BamE and one OMP named BamA). In order to determine the extent of their influence on the composition and structure of LPS, the E. coli mutants delta-surA, delta-skp, delta-degP, delta-bamB, delta-bamC, and delta-bamE were produced and the structure of their LPS determined. The results suggested that the alterations on the BAM machinery do not significantly alter the composition nor the structure of the LPS, providing an insight on the mechanism by which the alteration of the BAM machinery may alter the integrity of the OM. The LPS transportation machinery (Lpt) deploys seven LPS transport proteins named Lpt A-G that extracts the LPS from the external leaflet of the CM, transports it across the periplasm and the PG, flips it across the OM and locates the LPS in its external face. All Lpt proteins are essential, which makes them candidates as targets for new antibiotics. There are many open questions on the working of this machinery, for instance the details of the interaction sites of the hydrophobic pocket and how the periplasmic bridge is formed. During this Ph.D., the development of a semi-synthetic lipid A with active nuclei instead of acyl chains was attempted in order to study the details of the interaction between LPS-Lpt proteins by NMR. The introduction of a paramagnetic group on the fully de-acylated lipid A failed, but the introduction of fluoropropanoyl chloride seemed to be partially successful. However, the product presents a high level of contamination that prevented the reliable determination of the product as well as the interaction studies. In addition, it is disclosed that LptA does not act as an amidase regulator for AmiA, AmiB nor AmiC nor as a ligand for the amidase activators YgeR or NlpD. Leaving unanswered the question on how the hole on the PG is open for the Lpt bridge. In conclusion, the architecture of bacterial envelope is crucial for the interaction with the host and the knowledge of the structure of its components is a fundamental prerequisite to proceed with functional studies, and to dissect the role of each component. In this frame, the knowledge of the molecular determinants of the bacteria of the microbiota is preliminary. Through the characterisation of LPS and PG, this Ph.D. thesis demonstrates that they have unexpected structures and activities. Likewise, their transport across the periplasm, dissected on model organisms, still presents many gaps to be filled. Thus, our understanding of the cell envelope and of its metabolism is still to an early stage, but it is mature enough to devise alive bacteria and/or synthetic analogues of their surface structures for clinical applications.


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