Antimicrobial peptides and proteins in host-microbe interaction and immediate defense

Abstract: Antimicrobial peptides and proteins (AMPs) are effector molecules of innate immunity and are capable to kill a broad spectrum of microbes, i.e. bacteria, fungi and viruses. They are widespread in nature and have been found in almost all species of the animal kingdom, as well as in plants. The mammalian repertoire of antimicrobial peptides includes the defensins and the cathelicidins. Furthermore, several of the antimicrobial proteins are members of the S100 family. AMPs are predominantly expressed at exposed surfaces and in specific white blood cells. In the first part of this thesis, the presence of AMPs in material derived from the skin and gut of the fetus/neonate was investigated. Interestingly, the levels of AMPs of the gut were found to be induced after birth. Apart from being antimicrobial, AMPs are important in immunity by exhibiting additional functions, such as chemotactic, mitogenic and wound healing activities. Hence, in the second part of this thesis, novel immunological functions of several AMPs were discovered, further supporting their multifunctional role in host defense. Vernix caseosa (vernix) is a lipid-rich material covering the skin of newborn babies and is suggested to serve as an anti-infective substance, both for the fetus during the last trimester of gestation, and for the neonate during the first days of life. In paper I, protein extracts of vernix were found to exhibit antimicrobial activity against three bacterial strains and one fungal strain. In a proteomic approach, we identified approximately 20 proteins possessing diverse effects implicated in innate immunity, such as opsonizing, anti-parasitic and anti-protease activities. In paper II, we reported that protein extracts of meconium and neonatal feces exhibit antimicrobial activity against gram-positive and gram-negative bacteria. We also identified several AMPs in both these extracts that most likely contribute to the observed antimicrobial activities. Interestingly, the levels of some AMPs were found at higher levels in feces than in meconium, suggesting an induction or release of AMPs after birth upon colonization and breast feeding. As demonstrated in paper III, physiological concentrations of hydrophilic components of breast milk induce the expression of the cathelicidin LL-37 in colonic epithelial cells after 48 h of stimulation. This novel role of breast milk may contribute to the protection against infections in the gut of breast-fed babies. In paper IV of this thesis, LL-37 was found to inhibit biofilm formation of uropathogenic Escherichia coli. This inhibition is likely mediated by binding of LL-37 to the major curli subunit CsgA, preventing polymerization of CsgA to curli fibers. We demonstrated that biofilm of E. coli increased the resistance to cathelicidins, indicating that the inhibition of biofilm is a crucial anti-infective mechanism, making the bacteria more susceptible to killing. Furthermore, in paper V, we reported that neutrophil defensins 1-3 (HNP1-3) and heparin binding protein (HBP) enhanced macrophage phagocytosis of IgG-opsonized bacteria. The mechanism mediating this effect was found to be the release of IFNgamma and TNFalpha, activating the macrophages in an autocrine manner, ultimately resulting in upregulation of Fc-receptors. The anti-biofilm effect and the enhanced phagocytosis mediated by AMPs are novel effects, demonstrating that AMPs contribute to additional defense strategies that most likely are important in combating infections.