The Natural Antimicrobial Chromogranins/Secretogranins- Derived Peptides – Production, Lytic Activity and Processing by Bacterial Proteases

International audienc

Exploring the Pharmacological Potential of Promiscuous Host-Defense Peptides: From Natural Screenings to Biotechnological Applications

In the last few years, the number of bacteria with enhanced resistance to conventional antibiotics has dramatically increased. Most of such bacteria belong to regular microbial flora, becoming a real challenge, especially for immune-depressed patients. Since the treatment is sometimes extremely expensive, and in some circumstances completely inefficient for the most severe cases, researchers are still determined to discover novel compounds. Among them, host-defense peptides (HDPs) have been found as the first natural barrier against microorganisms in nearly all living groups. This molecular class has been gaining attention every day for multiple reasons. For decades, it was believed that these defense peptides had been involved only with the permeation of the lipid bilayer in pathogen membranes, their main target. Currently, it is known that these peptides can bind to numerous targets, as well as lipids including proteins and carbohydrates, from the surface to deep within the cell. Moreover, by using in vivo models, it was shown that HDPs could act both in pathogens and cognate hosts, improving immunological functions as well as acting through multiple pathways to control infections. This review focuses on structural and functional properties of HDP peptides and the additional strategies used to select them. Furthermore, strategies to avoid problems in large-scale manufacture by using molecular and biochemical techniques will also be explored. In summary, this review intends to construct a bridge between academic research and pharmaceutical industry, providing novel insights into the utilization of HDPs against resistant bacterial strains that cause infections in humans

Identification and characterization of three new human [beta]-defensins : hBD23, hBD27, and hBD29

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Analysis of two human gene clusters involved in innate immunity

Human body surfaces are defended by epithelia, which provide the initial physical barrier against potential harmful microorganisms. Epithelia also provide chemical barriers to microbial colonization including low pH, hydrolytic enzymes, and defense molecules such as antimicrobial peptides (AMPs). The number of reports demonstrating the presence and upregulation of AMPs in human skin is increasing and reflects the significance of these peptides in cutaneous innate immunity. This thesis has performed an exhaustive characterization of two coherent gene clusters, S100 fused-type proteins (SFTPs) and kazal type serine protease inhibitors (SPINKs), with a part of the effort to elucidate the molecular mechanism of innate immunity in the skin. Included is expression analyses of various novel genes identified in primary keratinocytes and generation of goat antisera against recombinant hornerin fragments and recombinant LEKTI-2, respectively

Analysis of two human gene clusters involved in innate immunity

Human body surfaces are defended by epithelia, which provide the initial physical barrier against potential harmful microorganisms. Epithelia also provide chemical barriers to microbial colonization including low pH, hydrolytic enzymes, and defense molecules such as antimicrobial peptides (AMPs). The number of reports demonstrating the presence and upregulation of AMPs in human skin is increasing and reflects the significance of these peptides in cutaneous innate immunity. This thesis has performed an exhaustive characterization of two coherent gene clusters, S100 fused-type proteins (SFTPs) and kazal type serine protease inhibitors (SPINKs), with a part of the effort to elucidate the molecular mechanism of innate immunity in the skin. Included is expression analyses of various novel genes identified in primary keratinocytes and generation of goat antisera against recombinant hornerin fragments and recombinant LEKTI-2, respectively

Rôle et impact des protéines associées à la lame basale spécialisée dans l’attache gingivale et la maladie parodontale

Le corps est entouré d’une enveloppe épithéliale afin de se protéger de l’environnement agressif qui l’entoure. La dent est la seule structure dans le corps qui crée une brèche de cette barrière épithéliale. Elle est de plus constamment exposée à un environnement humide, chaud et riche en nutriment qui est un milieu parfait pour la croissance de micro-organismes. Pour prévenir et contrôler l’intrusion de corps étrangers tels que des bactéries, le corps a élaboré une jonction dite dento-gingivale. Celle-ci sépare les tissus du parodonte sous-jacent du milieu buccal septique en créant un scellement autour de la dent formant ainsi une véritable barrière contre les bactéries. Ces dernières sont reconnues comme les agents causatifs dans l’initiation et le développement de la maladie parodontale. En effet, par leurs activités enzymatiques, les bactéries orales affectent le scellement de la jonction dento-gingivale exposant ainsi les tissus sous-jacents aux toxines et micro-organismes menant à la maladie parodontale. Les maladies parodontales affectent une grande partie de la population et leurs gravités augmentent avec l'âge. Les formes légères à modérées touchent plus de 80% des adultes, tandis qu’environ 15% souffrent de parodontite. Dans les cas les plus sévères, la maladie parodontale mène éventuellement à la perte de dent et d’os. Au-delà des graves conséquences de cette maladie sur la cavité buccale, il y a de plus en plus de preuves que la maladie parodontale est reliée à différentes maladies systémiques telles que les maladies cardio-vasculaires et respiratoires mais aussi le diabète. La maladie parodontale a ainsi une incidence sur la qualité de vie mais représente également un lourd fardeau financier et sanitaire pour le patient et la société. Il n’y a actuellement aucun remède pour cette maladie répandue et le traitement repose sur une gestion continue et chirurgicale. Il est alors urgent et nécessaire de développer de nouveaux traitements. Il est crucial de comprendre l’architecture et la composition de la matrice extracellulaire permettant le scellement dento-gingival afin d’ouvrir de nouveaux horizons à la lutte contre la maladie parodontale et pour le développement de traitement préventif et/ou thérapeutique.The body is surrounded by an epithelial envelope to protect itself from the aggressive environment around it. The tooth is the only structure in the body that creates a breach of this epithelial barrier. It is also constantly exposed to a humid, warm and nutrient-rich environment that is a perfect medium for the growth of micro-organisms. To prevent and control the intrusion of external organisms such as bacteria, the body has developed the so-called dento-gingival junction. This epithelial attachment separates the underlying periodontal tissues from the septic oral medium by creating a seal around the tooth, thus forming a true barrier against bacteria. These are recognized as causative agents in the initiation and development of periodontal disease. Indeed, by their enzymatic activities, oral bacteria break the seal of the dento-gingival junction to expose underlying tissues to toxins and microorganisms leading to periodontal diseases. Periodontal diseases affect a large part of the population and their severity increases with age. Mild to moderate forms affect more than 80% of adults, while about 15% suffer from periodontitis. In the most severe cases, periodontal disease eventually leads to loss of tooth and bone. Beyond the serious consequences of this disease on the oral cavity, there is growing evidence that periodontal disease is related to various systemic diseases such as cardiovascular and respiratory diseases but also diabetes. Periodontal disease affects the quality of life but also represents a heavy financial and health burden for the patient and society. There is currently no cure for this widespread disease and the treatment is based on continuous and surgical management. It is then urgent and necessary to develop new treatments. It is crucial to understand precisely the architecture and composition of the extracellular matrix allowing sealing to open up new horizons for the fight against periodontal disease and for the development of preventive and / or therapeutic treatment

Characterization of antimicrobial peptides deriving from insects and their application in the biomedical field

Antibiotics are the current drugs used to treat pathogenic bacteria, but their prolonged use contributes to the development and spread of drug-resistant microorganisms. The antibiotic resistance issue led to the need to find new alternative molecules, which should be less prone to bacterial resistance. Antimicrobial peptides (AMPs) aroused great interest as potential next-generation antibiotics. AMPs are involved in several defence-related processes such as the binding and neutralization of endotoxins, the modulation of the immune responses to infection and the killing of pathogens. Antimicrobial peptides are small molecules with an amino acid composition ranging from 10 to 100 residues and are biosynthesized by all living organisms but it is known that the class of insects represents the largest source of these molecules. This aspect is related to insect’s biodiversity and their ability to live in hostile environments rich of pathogens. Most insect AMPs are cationic molecules due to the presence of basic residues and according to their amino acid sequences and structures, they can be classified in four different groups: cysteine-rich peptides (e.g., defensins), the α-helical peptides (e.g., cecropins), glycine-rich proteins (e.g., attacins) and proline-rich peptides (e.g., drosocins). Insect AMPs have demonstrated to be useful in several applications concerning the pharmaceutical as well as the agricultural fields. Moreover, insect AMPs aroused great interest for their biomedical application thanks to the increasing number of peptides that can inhibit human pathogens. For this reason, this Ph.D. project aimed to the identification of antimicrobial peptides deriving from insects, particularly from the Black Soldier Fly Hermetia illucens (L.) (Diptera: Stratiomyidae). Through a combination of transcriptomics and bioinformatics analysis, 57 antimicrobial peptides have been identified from H. illucens insect. Through an in silico analysis, the biological activity have been predicted and the physio-chemical properties have been calculated for all the identified peptides. Based on the bioinformatics results, the in vitro production of the most promising sequences has been performed through molecular cloning strategies in order to evaluate the antibacterial activity in vitro. Particularly, some of the identified peptides (C16571, C46948, C16634, and C7985) showed the ability to inhibit E. coli growth at a concentration value of 3 μM. For the C15867 peptide, recombinantly produced and expressed, a MIC (Minimum Inhibitory Concentration) value of 18 μM has been determined. Moreover, an in vivo approach was carried out for the identification of antimicrobial peptides by extracting the hemolymph from the H. illucens larvae, recovering then the peptides fraction from the larvae’s plasma and its antibacterial activity has been evaluated against both Gram-positive and Gram-negative bacteria. The performed analysis showed that a small amount (7.5/15 μL) of the peptide fraction recovered from the larvae’s plasma was able to inhibit the cell growth of different bacterial strains

Crytic antimicrobial peptides hidden in protein precursors: identification of novel bioactive molecules

Antibiotics are the mainstay in treatment of bacterial infections. However, resistance to antibacterial treatments has been rising since the 1970s, causing serious problems in the treatment and control of infectious diseases. Antibiotic resistance is now considered as one of the major global health threats of the 21st century in that the worldwide use of antibiotics is predicted to increase by more than 65% in the coming decades due to the increasing demand for meat and shift in agriculture practices in developing countries. New antibacterial drugs are urgently needed, but only three antibacterial drugs have been brought to the market since 1999 and very few new antibiotics are currently in development. This has prompted the search for alternatives to conventional antibiotics. Multiple alternative anti-infective strategies are being investigated, including vaccines, probiotics and phage therapy. Other promising alternatives to antibiotics are host defense peptides (HDPs), an important component of the first line of defense against infection, found in all multicellular organisms. HDPs are seen as true multifunctional peptides with activities as diverse as chemotaxis, inhibition of LPS-induced inflammation, modulation of leukocyte differentiation and promotion of wound healing. Interestingly, novel functions of these peptides are still being described to date. Many human proteins with functions not necessarily related to host defense behave as sources of HDPs. Some examples are lactoferrin, lysozyme and thrombin. Since these peptides are hidden in large proteins, they can be defined as “cryptic”. In order to identify by a rational approach further human proteins carrying cryptic HDPs, we recently developed an in-silico screening method to localize antimicrobial regions hidden inside the primary structure of precursor proteins. A wide list of potential new antimicrobial peptides was obtained by applying this method to about 4,000 human extracellular proteins. The main aim of this PhD project was the identification of interesting potential HDPs, to develop novel bioactive peptides. Firstly, we developed a novel and cost-effective method to produce recombinant HDPs, based on the use a cheap and efficient medium to be employed in an auto-inducing fermentation process. Furthermore, to avoid HDPs toxicity towards bacterial host, a novel fusion system based on a carrier protein (derived from a Rana pipiens ribonuclease) was used. Once optimized the production system, a broad characterization of two novel recombinant peptides, previously identified in human Apolipoprotein B (ApoB), was performed. We demonstrated that both peptides are endowed with a significant antimicrobial activity towards Gram-negative and Gram-positive strains, and are able to prevent biofilm formation in several strains at concentrations lower than those required to directly kill planktonic bacterial cells. Moreover, ApoB-derived peptides were found to be endowed with anti-inflammatory properties as well as the ability to promote wound healing in keratinocytes. In addition, two further cryptic HDPs have been structurally and functionally characterized. One of these HDPs has been identified in human 11-hydroxysteroid dehydrogenase-1 β-like, the other represents the first HDP from an archaeal protein, the transcription factor Stf76 encoded by the hybrid plasmid-virus pSSVx from Sulfolobus islandicus. By means of a multidisciplinary approach including biochemical, cellular biology and spectroscopic techniques, the action mechanism of both peptides has been elucidated, and intriguing results have been obtained by testing their immunomodulatory and anti-cancer activities. Hence, the in silico-derived panel of potential HDPs is a rich source of peptides with pharmacologically relevant properties

Use of Bacteroides thetaiotaomicron derived extracellular vesicles as vaccine delivery vehicles for mucosal vaccines against respiratory pathogens

Most infectious pathogens enter the body via mucosal sites, yet very few mucosal vaccines have been licensed. Major hurdles in mucosal vaccine development, such as stability, mucosal barriers and immune tolerance, hinder delivery of antigens to mucosa-immune cells. New vaccine delivery systems are therefore needed to provide broad and long-term protection against respiratory viruses. To address these issues Bacteroides thetaiotaomicron (Bt), a human commensal gut bacterium, has been engineered to export into their bacterial extracellular vesicles (BEVs) Yersinia pestis, influenza virus (IV), or SARS-CoV-2 antigens. In addition, two means of decorating Bt BEVs with antigens have also been explored, using highly expressed vitamin B12 receptors and chemical conjugation. Pre-clinical studies using non-human primates (Y. pestis) or murine models (IAV and SARS-CoV-2) were used to determine the immunogenicity of Bt BEV vaccines and their ability to induce protective immune responses. Native Bt BEVs displayed inherent adjuvanticity after intranasal administration, as shown by their ability to elicit mobilisation of immune cells and the development of organised lymphoid structures in the upper and lower respiratory tract. BEV vaccines were safe with no signs of any adverse effects in immunised animals. Bt BEVs vaccine formulations induced antigen-specific local and systemic humoral (IgA/IgG) and cellular (IFN- and/or TNF- producing CD4/8 T cells) immune responses. For Y. pestis BEV vaccines correlates of protection were obtained from serum antibody mediated neutralisation and cytotoxicity assays using live plague bacteria. BEV-IAV vaccines provided heterotypic protection against a lethal dose of H1N1 IAV. Initial pre-clinical studies of SARS-CoV-2 BEV vaccines showed them to be capable of inducing low levels of antigen-specific mucosal and systemic IgA and IgG antibodies. Additional studies to optimise antigen expression, dose and frequency refinements are needed. The results obtained showed that Bt BEV could be used as a platform to produce mucosal vaccines against respiratory pathogens

The Characterization of a Putative Virulence Factor Expressed By Sneathia amnii

Preterm birth, defined at birth before 37 weeks gestation, affects millions of newborns worldwide every year. Preterm birth is a leading cause of infant morbidity and mortality. One major cause of preterm birth is preterm premature rupture of membranes (PPROM), which can be triggered by bacterial infection and inflammation. A bacterial species that has been implicated in preterm birth and other obstetric complications is Sneathia amnii. The goals of this study were to observe cytopathogenic effects caused by S. amnii strain Sn35 and identify putative virulence factors causing those effects. Sn35 was able to adhere to, invade, and damage/kill various host cell lines. We characterized these virulence attributes. A putative virulence determinant was identified, and a fragment of the protein was expressed for polyclonal antiserum production. Antiserum was used to characterize the expression and subcellular localization of the protein in Sn35. However, antiserum was unable to prevent cytopathogenic effects