Antimicrobial polypeptides and lipids as a part of innate defense mechanism of fish and human fetus

University dissertation from Stockholm : Karolinska Institutet, Department of Medical Biochemistry and Biophysics

Abstract: Animals are constantly threatened by vast numbers of microbes and have consequently evolved an array of strategies to prevent microbial invasion. In this thesis, I have studied natural antimicrobial lipids and polypeptides associated with immediate host defense. The antibacterial effects of fatty acids and monoglycerides were studied by incubation of bacteria with lipid solutions and consequent measurement of bacterial reduction. The susceptibility varied between bacterial species, where saturated medium-chain fatty acids, corresponding 1-monoglycerides and monounsaturated long-chain fatty acids were the most active. The mode of action against Chlamydia trachomatis and group B streptococci was studied by electron microscopy and indicated that the bacteria were killed by disintegration of the cell membrane(s) by the lipids, but leaving the bacterial cell wall intact. The rapid and extensive killing of bacteria suggests that natural lipids may be used as microbicidal agents in prevention and treatment of infections. Vernix caseosa (vernix) collected from the skin of 88 neonates exhibited antimicrobial activities. Here, 20 polypeptides plus additional variant peptide forms were identified and several of them were found to contribute to host defense. The lipids of vernix were separated and lipid classes were screened for antibacterial activity. The free fatty acids, which was the only lipid class found to exhibit antimicrobial activity, was further characterized and found to be rich in fatty acids able to kill microbes. Finally, a higher antimicrobial effect was visualized in samples of the á-helical peptide LL-37 incubated with vernix lipids than in samples containing LL-37 alone. Fecal and meconium extracts of neonates were collected. The fecal extracts exhibited higher antimicrobial activity than extracts of meconium. All activities were salt-sensitive. Utilizing high performance liquid chromatography (HPLC), mass spectrometry, sequence analysis, and immuno-blotting, eight antimicrobial polypeptides were identified in meconium and five in fecal samples. Higher levels of lysozyme and azurocidin were detected in feces than in meconium. An extract from the epidermal mucus of the Atlantic cod exhibited salt-dependent antimicrobial activities, which were eliminated by pepsin digestion. No lysozyme activity was detecte d. Four antimicrobial polypeptides were isolated from the mucus with HPLC. Utilizing amino acid sequence analyses, together with mass spectrometry, these polypeptides were identified as a histone and ribosomal proteins. Our findings reveal that a number of antimicrobial lipids and polypeptides are present in skin, epithelia and integumental tissues of fish and human fetus. These lipids and polypeptides kill potential pathogens of the skin and mucosa of animals. They may be implicated in the control of microbial colonization and contribute to the innate defense mechanism.

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