Antimicrobial peptides and pathogenic Neissera : Experimental studies in mouse, man and rat

Abstract: Antimicrobial peptides are important effector molecules of innate immunity. In this thesis, the focus is on antimicrobial peptides of the cathelicidin family, i.e. LL-37 in man, CRAMP in mouse and rCRAMP in rat. Expression of these cathelicidins in tissues and cells has been analyzed, and their functional relevance has been studied in relation to the human bacterial pathogens Neisseria gonorrhoeae and Neisseria meningitidis. A peptidelprotein extract was made from human colon mucosa and several antimicrobial peptides and proteins were identified by HPLC purification. We propose that this complex mixture of antimicrobial peptides and proteins provides a functional barrier protecting the human colon against invading microorganisms. One of the identified peptides, the human cathelicidin LL-37, is also expressed in cervical epithelial cells. Infection of these cells with Neisseria gonorrhoeae (gonococci) resulted in down-regulation of LL-37. Further, this peptide exhibited potent bactericidal activity against Neisseria gonorrhoea, suggesting that down-regulation of LL-37 may facilitate invasion of gonococci in the female genital tract. The brain - on the other hand - is rarely infected, and the protective mechanisms remain to be fully elucidated. A peptidelprotein extract of rat brain was found to be active against bacteria. Depletion experiments showed that the cathelicidin rCRAMP accounted for a large portion of this activity. Using RT-PCR and Western blot analysis, rCRAMP was localized to distinct regions of the brain. In addition, rCRAMP was found to be a potent killer of the neuropathogenic bacterium Neisseria meningitidis. Meningococcal infection was studied in mice expressing the human complement regulator CD46. These mice were found to be highly susceptible to meningococcal infection. After meningococcal challenge, bacteria were found in cerebrospinal fluid of CD46 mice, but not in control mice, demonstrating that CD46 is crucial for establishing meningococcal infection. Finally, the role of mouse CRAMP in meningococcal infection was investigated. By immunohistochemistry, CRAMP was detected in the blood brain barrier and meninges after infection. CRAMP-KO mice were used to evaluate the role of CRAMP in vivo. Bacterial crossing of the blood brain barrier occurred both in CRAMP-KO mice and in control mice. However, CRAMP-KO mice exhibited higher bacterial counts in blood, liver and spleen six hours post infection, demonstrating a non-redundant and early effect of CRAMP in meningococcal sepsis. Considering the emerging bacterial resistance against conventional antibiotics, it is important to investigate novel ways of treating infections. Isolation of antimicrobial peptides from tissue extracts and a detailed understanding of their regulation, may lead to the development of novel strategies in the treatment of infectious diseases.