Study of the metal ions transport phenomena involved in the expression of pathogenic virulence

The necessity of new antimicrobial agents is unarguable, since current therapeutic treatments are not always effective: drug-resistant pathogenic microorganisms have significantly increased over the last decades and their associated mortality rate still remains a global concern. Several studies have shown that metal acquisition and regulation greatly contribute to virulence and physiology of pathogens. In particular, to prevent infections, humans restrict the access to essential micronutrients by means of an innate immune response termed "nutritional immunity”; on the contrary, pathogens rely on sophisticated systems (e.g. siderophores) to overcome the scarce metal bioavailability. From this perspective, a deeper insight into the mechanism of metal trafficking in pathogens and host nutritional immune response can provide crucial information to design new effective antibiotic therapies. Furthermore, metal complexes are not only a promising tool in antimicrobial treatments, but they can also find application against pathologies which, in general, involve metal ions (e.g. neurodegenerative diseases or cancer). The first essential step to develop novel metal-based antimicrobials is the elucidation of thermodynamics and coordination chemistry of the metal chelators involved in pathogenic events. The main aim of this work is to provide insight into the correlation between metal transport, homeostasis and virulence in pathogens. The research work is mostly focused on Zn(II), which is crucial for the survival of human and pathogen cells. Its assimilation by pathogens is extremely challenging: free zinc concentration is normally subnanomolar, and many other divalent endogenous metal ions, such as Cu(II) (or Ni(II) in some organisms), can compete for the same protein binding sites, thus requiring a more extensive study of the phenomenon. For this purpose, the interaction of metal ions with human antimicrobial peptides, metal transporters, natural metallophores and biomimetic molecular systems are investigated, with particular emphasis on the thermodynamic properties of the obtained complexes and the elucidation of their speciation in solution. The choice of the unstructured peptide fragments as protein models, that simulate the coordination and transport of metals, is the first essential step of this research. Consultation of specific databases and scientific literature provides information about evolutionarily conserved sequences with metal binding function. The further characterization of the metal complexes required several experimental techniques. Mass spectrometry and potentiometric titrations allow to evaluate the stoichiometry, coordination modes and stability of the formed complexes in aqueous solution, varying the experimental conditions like metal/ligand ratio and the pH value. Furthermore, competition diagrams obtained from calculated partial and overall stability constants provide a deeper understanding of the metal binding affinity. Spectroscopic techniques, such as UV-Vis spectrophotometry, circular dichroism (CD), electron paramagnetic resonance and nuclear magnetic resonance ensure a complementary study to understand the coordination geometries and to identify the precise binding sites. CD measurements in the far-UV region also give indications on the presence of specific conformations (α-helices, random coils, etc.). Biological studies in vitro of potential antimicrobial agents involved in the process of nutritional immunity can complete the information about the way of action of some systems and the role of metal ions. Moreover, the comparison between different peptide analogues helps clarifying the role of some residues of the sequence, not always directly involved in complexation but rather influencing the complex geometry and contributing to its stability.