Antibacterial activity of a Tribolium castaneum defensin in an in vitro infection model of Moraxella Catarrhalis

Introduction: Bacterial multidrug-resistance is a growing global health problem. This includes pathogens that cause severe lung disease, such as Moraxella Catarrhalis (M. c.).

M. c. is a gram-negative, human-restricted diplococcus that colonizes the respiratory tract. It is the predominant cause of Otitis Media in children and also causes exacerbations in COPD and chronic bronchitis. Gram-negative bacteria secrete outer membrane vesicles (OMVs), which might be involved in the process of bacterial infection. In this study we investigated three insect-derived antimicrobial peptides (AMP) of known antibacterial properties as an alternative for antibiotics against M. c. infection in an in vitro model with blood-derived macrophages (BDM). We also confirmed the secretion of M. c. OMVs and tested their immunologic properties.

Methods: The effect of AMPs on M. c. replication was tested by Colony-Forming-Unit (CFU) Assays. BDMs were infected with M. c. for 24 h, with onset of treatment with AMPs at 1 h or 5 h post infection. Direct cytotoxicity of AMPs on BDMs was tested by Lactate Dehydrogenase Assay (LDH). M. c. OMVs were isolated by ultracentrifugation and used in an in vitro 24 h BDM stimulation model in comparison to M. c. infection. Results were analysed by qPCR.

Results: No cytotoxicity of AMPs on BDMs could be detected. M. c. CFU data show that especially the AMP Defensin TCA1 strongly reduced bacterial growth, hinting at a bacteriostatic or bactericidal effect. Furthermore, infected cells produced less IL-8 transcript when treated with AMPs in comparison to untreated cells.

Stimulation of MEF-cells with M. c.-derived OMVs showed that IL-6 induction was comparable to the induction observed upon M. c. infection.

Conclusion: The chosen AMPs, especially TCA1, might be potential antibacterial agents, as they cause a significant decrease in bacterial growth and a decreased immune response. As M. c. OMVs also induce proinflammatory cytokines, the effect of AMPs on OMV secretion might also be worth investigating.