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

Abstract

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.