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- Al Adwani, Salma, et al.
(författare)
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Citrullination Alters the Antibacterial and Anti-Inflammatory Functions of the Host Defense Peptide Canine Cathelicidin K9CATH In Vitro
- 2021
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Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 207:3, s. 974-984
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Tidskriftsartikel (refereegranskat)abstract
- K9CATH is the sole cathelicidin in canines (dogs) and exhibits broad antimicrobial activity against both Gram-positive and Gram-negative bacteria. K9CATH also modulates inflammatory responses and binds to LPS. These activities depend on the secondary structure and a net-positive charge of the peptide. Peptidylarginine deiminases (PAD) convert cationic peptidyl arginine to neutral citrulline. Thus, we hypothesized that citrullination is a biologically relevant modification of the peptide that would reduce the antibacterial and LPS-binding activities of K9CATH. Recombinant PAD2 and PAD4 citrullinated K9CATH to various extents and circular dichroism spectroscopy revealed that both native and citrullinated K9CATH exhibited similar α-helical secondary structures. Notably, citrullination of K9CATH reduced its bactericidal activity, abolished its ability to permeabilize the membrane of Gram-negative bacteria and reduced the hemolytic capacity. Electron microscopy showed that citrullinated K9CATH did not cause any morphological changes of Gram-negative bacteria, whereas the native peptide caused clear alterations of membrane integrity, concordant with a rapid bactericidal effect. Finally, citrullination of K9CATH impaired its capacity to inhibit LPS-mediated release of proinflammatory molecules from mouse and canine macrophages. In conclusion, citrullination attenuates the antibacterial and the LPS-binding properties of K9CATH, demonstrating the importance of a net positive charge for antibacterial lysis of bacteria and LPS-binding effects and suggests that citrullination is a means to regulate cathelicidin activities.
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| 2. |
- Káradóttir, Harpa
(författare)
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Interactions between innate immune effectors and multidrug resistant bacteria
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibiotic resistance is an increasingly difficult problem in the clinic, where conventional antibiotics are failing, and new alternative solutions are in high demand. Infections caused by Gram-negative bacteria with multi drug resistance (MDR) mechanisms are increasing globally, and treatment options are limited. Plasmids encoding β-lactamases spread easily between bacteria, and the overuse of antibiotics select for MDR strains. β-lactamases are either serine-β-lactamases that are inhibited by certain β-lactamase inhibitors, and metallo-β-lactamases (MBLs), which are more difficult to inhibit with drugs. The current approach to fight MDR pathogens has mainly focused on finding β-lactamase inhibitors to use in combination with conventional antibiotics in the clinic. The overall aim of this thesis was to study the role of the cellular micro-environment and the importance of the innate immune system for antibiotic resistant bacterial infections. In Paper I we hypothesized that human cells secreted factors that could impair β-lactamase function and thus restore antibiotic susceptibility in resistant bacterial isolates. We found that thiols produced by the cells acted as zinc chelators that inhibited the degradation of cephalosporin antibiotics in VIM-1 producing K. pneumoniae. Notably, free thiols in urine samples had the same effect, suggesting that the environment at the site of infection can be highly important for antibiotic susceptibility and possibly also for the effect of antibiotic treatment in a clinical situation. In Paper II, we hypothesized that induction of innate effector molecules would reduce intracellular growth of MDR K. pneumoniae and exert synergistic effects with conventional antibiotics. We tested this by infecting human macrophages with MDR K. pneumoniae. Notably, induction of innate immunity in these cells resulted in improved intracellular killing of MDR K. pneumoniae. The inducers were combined with traditional antibiotics, which resulted in an additive killing effect. The data suggests that inducing innate immune effectors can be an effective alternative or addition to conventional treatments in infections caused by MDR K. pneumoniae. Finally, in Paper III, we tested the hypothesis that ESBL E. coli would be more susceptible to innate effectors compared to non-ESBL isolates. The ESBL producing isolates had lower survival in serum and whole blood than non-ESBL isolates, suggesting a biological cost for resistant isolates. In vivo studies with zebrafish embryos showed that the non-ESBL isolates killed the embryos more efficiently than ESBL isolates. The biological cost was not related to the ESBL plasmid per se as shown by experiments where the ESBL plasmid was transferred from a clinical isolate to a neutral background in non-resistant E. coli. Together, this thesis has highlighted the importance of considering the micro-environment at the site of infection, which may determine the effect of antibiotics. Next, I have shown that induction of innate immune effectors could be an alternative or additive treatment option for infections caused by MDR K. pneumoniae. Finally, I present data showing that ESBL E. coli are more susceptible to innate effectors than non-ESBL E. coli.
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