Antimicrobial peptides in innate immunity : interactions with antibiotics and effects of post-translational modifications

Abstract: Cationic antimicrobial peptides (AMPs) are important innate immunity factors contributing to the clearance of invading pathogens and immunomodulating immune responses. In this thesis, the focus is on AMPs of the cathelicidin family, i.e. the human LL-37 and the canine K9CATH. These peptides are positively charged at neutral pH and amphipathic in nature, making them prone to interact with negatively charged bacterial membranes. In this thesis, I have studied i) how the interaction between AMPs and bacteria can be modified by alterations of the LPSstructure of the bacteria and ii) how the host can regulate the function of AMPs, via a posttranslational modification designated citrullination. In part 1 of the thesis, I studied the potential cross-resistance between colistin and LL-37 in K. pneumoniae. Colistin is a cationic polypeptide antibiotic that shares a similar membranebinding mechanism with LL-37; i.e. they both target the negatively charged components of the bacterial membrane. Resistance to colistin has appeared in bacteria due to modifications in the lipopolysaccharide (LPS) structure by reduction of the negative surface charge. The hypothesis of cross-resistance between colistin and LL-37 still remains debatable. In Paper I, we studied two clinical isolates of Klebsiella pneumoniae (Kpn), with similar genetic background but with different susceptibility to colistin. The colistin-resistant isolate (Col-R) had an insertion element in the mgrB gene, which caused the resistant phenotype. Interestingly, the Col-R isolate was more resistant to LL-37 in contrast to the colistin-susceptible isolate (Col-S) but only at concentrations above 50 μg/ml. However, there was no significant survival differences between Col-R and Col-S isolates in blood, serum nor in a zebrafish infection model. The findings of this study suggest that cross-resistance most likely plays a minor role during physiological conditions in vivo, where lower levels of LL-37 are present. In part 2 of the thesis, I studied how the host, via citrullination, could affect the function of AMPs in various contexts. Peptidyl arginine deiminases (PADs) catalyze the conversion of the positively charged arginine residues into neutral citrulline residues in a process called citrullination or deimination. Notably, citrullination reduces the net charge of proteins and peptides and could thus, affect the biological functions of AMPs. Citrullinated LL-37 has not been detected in human samples and the knowledge on the functional and biophysical consequences of citrullination is limited. Paper II, describes a series of experiments characterizing the presence of citrullinated LL-37 in human bronchoalveolar lavage (BAL) fluid from the airways of healthy donors after exposure of LPS. We identified both native LL- 37 and different variants of citrullinated LL-37 in the BAL samples. Citrullinated LL-37 had no antibacterial activity against Escherichia coli, interacted differently with LPS and had reduced affinity to anionic phospholipids. Finally, a net positive charge was shown to be essential for the antimicrobial activity of LL-37. Finally, I expanded the studies on citrullination to another AMP, the K9CATH peptide, which is the only cathelicidin found in dogs. It has a broad antimicrobial activity against both grampositive and gram-negative bacteria, it binds to LPS and has anti-inflammatory functions. Similarly, to LL-37, K9CATH has a positive net charge at neutral pH due to lysine and arginine residues. PAD enzymes are found in all organisms studied, including dogs. However, it is unknown whether K9CATH is a substrate to PAD enzymes. In Paper III we report the that recombinant PAD2 and PAD4 citrullinated K9CATH at different degrees. Citrullination abrogated the antibacterial activity against gram negative bacteria and reduced the peptide’s anti-inflammatory activity on LPS-induced macrophage stimulation. In conclusion, my studies provide information on the interactions of cathelicidin peptides with bacterial membranes and the different host/microbe mechanisms, regulating the activity of the peptides. This information provides a platform for future research on the role of citrullination in health and disease.

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