Novel D-LAK peptides combinations against mycobacteria : bioefficacy and mechanistic studies

Abstract

Emergence of multidrug-resistant tuberculosis (MDR-TB) renders the two most powerful and commonly used TB antibiotics, rifampicin and isoniazid, ineffective. There is an urgent need for the development of safe and effective strategies against drug-resistant TB. Based on previous studies, two novel antimicrobial peptides, namely D-LAK120-A and D LAK120 HP13, both consist of all D-amino acid residues, have demonstrated inhibitory effect on MDR Mycobacterium tuberculosis (Mtb) strains when cultured in macrophages and potentiated the antimycobacterial activity of isoniazid in vitro. Here, the antimycobacterial activity of D-LAK peptides in combination with anti-TB drugs against MDR-TB clinical isolates was investigated. Furthermore, the mechanisms of action of the proline-containing and proline-free D-LAK peptides, anti-TB drugs alone or in combinations were studied using two mycobacterial models Mycobacterium smegmatis and the severely attenuated Mtb Bleupan. Finally, the applicability of these two microorganisms for mycobacterial studies was discussed. In vitro and ex vivo antimycobacterial assays revealed the synergy between D-LAK peptides and rifampicin or isoniazid against MDR Mtb strain. These findings suggested D-LAK peptide can facilitate the re sensitization of MDR-TB clinical isolates towards rifampicin and isoniazid possibly through their surface activity. To gain further understanding on their mechanisms on mycobacteria, M. smegmatis was employed due to its fast-growing nature and widespread usage in TB studies. With confocal and transmission electron microscopy, the surface-active action of D-LAK peptides were observed which led to visible morphological changes in M. smegmatis. Using high-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) metabolomics, trans-1,6-diphenyl-1,3,5-hexatriene (DPH) and Laurdan fluorescence spectroscopy, it was shown that bacterial metabolism was substantially altered when challenged with anti TB agents, specifically by the remodelling of mycobacterial membrane. The proline-containing D LAK120 HP13 and proline-free D-LAK120-A peptides demonstrated disparate behaviours in the initiation of membrane remodelling which provides possible rationale for their synergistic interaction with anti-TB agents. These techniques were then applied to the study of another potential mycobacterial model Mtb Bleupan which was selected for its safer nature as a Mtb surrogate. Changes in membrane components triggered by anti-TB agents were observed but the effect was subtler than that observed in M. smegmatis. These findings suggested the potential of using these biophysical techniques to study the action of anti-TB drugs on mycobacteria and to elucidate the underlying mechanism of synergy demonstrated in the combinations of D-LAK peptides with anti-TB agents. Finally, the applicability of M. smegmatis and Mtb Bleupan to be exploited as a Mtb surrogate was evaluated. Some key differences in response to anti-TB agents were detected between Mtb Bleupan and M. smegmatis in terms of the changes in metabolism and membrane properties. These findings demonstrated Mtb Bleupan as a safe and desirable model for the generation of translatable findings for TB studies. Further optimization of the growth response study using Mtb Bleupan is essential for a more comprehensive understanding of anti-TB agent mechanism. Overall, this thesis concluded that the combinations of D-LAK peptides with rifampicin or isoniazid were effective against MDR clinical isolates and paved the next step to elucidate the mechanisms using a more reliable surrogate, Mtb Bleupan.