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postgraduate thesis: The investigation of antimicrobial peptides and siRNA therapeutics against mycobacterium tuberculosis

TitleThe investigation of antimicrobial peptides and siRNA therapeutics against mycobacterium tuberculosis
Authors
Issue Date2015
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Lan, Y. [蘭韻]. (2015). The investigation of antimicrobial peptides and siRNA therapeutics against mycobacterium tuberculosis. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5558980
AbstractTuberculosis (TB) is a severe infectious disease which leads to millions of deaths each year globally. It is caused by Mycobacterium tuberculosis (Mtb), an intracellular pathogen that infects alveolar macrophages. With the increasing cases of multiple drugresistant TB (MDR-TB), development of a safe and effective anti-TB therapy is urgently needed. In this study, two anti-TB strategies were investigated: (i) the use of antimicrobial peptides (AMPs) to kill Mtb; (ii) the use of small interfering RNA (siRNA) to target the host-dependent survival factors of Mtb in macrophages. In the first strategy, a novel class of AMPs, D-enantiomeric amphipathic AMPs (D-AMPs), was investigated as anti-TB agent. To examine the structure-activity relationships, the anti-TB activity of 13 D-AMPs with different charge angles, overall hydrophobicity and conformational flexibility was evaluated against H37Ra, an attenuated laboratory strain of Mtb. Hydrophilic D-AMPs with charge angle of 120 degree conferred the most potent anti-TB effect. From this, the six most potent D-AMPs were selected for further investigation on clinical Mtb isolates, including the drug-resistant strains. All six D-AMPs were able to inhibit the growth of Mtb in vitro and inside macrophages (differentiated THP-1 cells). D-AMPs displayed a ‘detergent-like’ property, which enabled them to reduce the hydrophobic interactions between the lipidrich cell walls of Mtb and prevent aggregation of mycobacteria. Furthermore, D-AMPs were effective as an adjunct agent at non-toxic concentration to potentiate the efficiency of conventional anti-TB drugs, isoniazid (INH) and rifampicin (RIF) against MDR-TB in vitro, possibly by facilitating the access of INH or RIF into the mycobacteria by increasing the surface permeability of the pathogen. Overall, D-LAK120-A was identified as the optimal candidate within the D-AMP family with a good balance between anti-TB potency and low cytotoxicity to mammalian cells. In the second strategy, siRNA was used to target the essential survival factors of Mtb in macrophages. Bfl-1/A1, a Bcl-2 family protein, was identified previously as one of the host-dependent survival factors of Mtb. Silencing Bfl-1/A1 expression could inhibit the growth of intracellular Mtb. Success of siRNA therapeutics relies on a safe and effective siRNA delivery agent. Six pH responsive peptides containing either histidine (LAH peptides) or ionizable 2, 3-diaminopropionic acid (Dap) (LADap peptides) were investigated here as siRNA delivery agents. Endosomal entrapment is a major barrier to siRNA delivery. These peptides could alter their conformation from α-helical to disordered structure with membrane-disturbing activity at low pH inside endosomes, thereby promoting endosomal escape of siRNA. Three peptides that demonstrated the highest transfection efficiency in mammalian cells were selected to deliver Bfl-1/A1 siRNA to macrophages (differentiated THP-1 cells), which were then challenged with drug-susceptible strains of Mtb. Significant reduction in bacterial colony forming unit (CFU) was observed in groups that were transfected with Bfl-1/A1 siRNA using LAH or LADap peptides compared with those transfected with negative control siRNA, confirming the bacterial inhibitory effect of Bfl-1/A1 siRNA on the intracellular Mtb. In conclusion, both AMPs and siRNA strategies demonstrated successful anti-TB activity in vitro and in macrophages. Further investigations on both strategies are warranted for future therapeutic development.
DegreeDoctor of Philosophy
SubjectTuberculosis - Treatment
Small interfering RNA - Therapeutic use
Peptide antibiotics
Dept/ProgramPharmacology and Pharmacy
Persistent Identifierhttp://hdl.handle.net/10722/216263

 

DC FieldValueLanguage
dc.contributor.authorLan, Yun-
dc.contributor.author蘭韻-
dc.date.accessioned2015-09-08T23:11:34Z-
dc.date.available2015-09-08T23:11:34Z-
dc.date.issued2015-
dc.identifier.citationLan, Y. [蘭韻]. (2015). The investigation of antimicrobial peptides and siRNA therapeutics against mycobacterium tuberculosis. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5558980-
dc.identifier.urihttp://hdl.handle.net/10722/216263-
dc.description.abstractTuberculosis (TB) is a severe infectious disease which leads to millions of deaths each year globally. It is caused by Mycobacterium tuberculosis (Mtb), an intracellular pathogen that infects alveolar macrophages. With the increasing cases of multiple drugresistant TB (MDR-TB), development of a safe and effective anti-TB therapy is urgently needed. In this study, two anti-TB strategies were investigated: (i) the use of antimicrobial peptides (AMPs) to kill Mtb; (ii) the use of small interfering RNA (siRNA) to target the host-dependent survival factors of Mtb in macrophages. In the first strategy, a novel class of AMPs, D-enantiomeric amphipathic AMPs (D-AMPs), was investigated as anti-TB agent. To examine the structure-activity relationships, the anti-TB activity of 13 D-AMPs with different charge angles, overall hydrophobicity and conformational flexibility was evaluated against H37Ra, an attenuated laboratory strain of Mtb. Hydrophilic D-AMPs with charge angle of 120 degree conferred the most potent anti-TB effect. From this, the six most potent D-AMPs were selected for further investigation on clinical Mtb isolates, including the drug-resistant strains. All six D-AMPs were able to inhibit the growth of Mtb in vitro and inside macrophages (differentiated THP-1 cells). D-AMPs displayed a ‘detergent-like’ property, which enabled them to reduce the hydrophobic interactions between the lipidrich cell walls of Mtb and prevent aggregation of mycobacteria. Furthermore, D-AMPs were effective as an adjunct agent at non-toxic concentration to potentiate the efficiency of conventional anti-TB drugs, isoniazid (INH) and rifampicin (RIF) against MDR-TB in vitro, possibly by facilitating the access of INH or RIF into the mycobacteria by increasing the surface permeability of the pathogen. Overall, D-LAK120-A was identified as the optimal candidate within the D-AMP family with a good balance between anti-TB potency and low cytotoxicity to mammalian cells. In the second strategy, siRNA was used to target the essential survival factors of Mtb in macrophages. Bfl-1/A1, a Bcl-2 family protein, was identified previously as one of the host-dependent survival factors of Mtb. Silencing Bfl-1/A1 expression could inhibit the growth of intracellular Mtb. Success of siRNA therapeutics relies on a safe and effective siRNA delivery agent. Six pH responsive peptides containing either histidine (LAH peptides) or ionizable 2, 3-diaminopropionic acid (Dap) (LADap peptides) were investigated here as siRNA delivery agents. Endosomal entrapment is a major barrier to siRNA delivery. These peptides could alter their conformation from α-helical to disordered structure with membrane-disturbing activity at low pH inside endosomes, thereby promoting endosomal escape of siRNA. Three peptides that demonstrated the highest transfection efficiency in mammalian cells were selected to deliver Bfl-1/A1 siRNA to macrophages (differentiated THP-1 cells), which were then challenged with drug-susceptible strains of Mtb. Significant reduction in bacterial colony forming unit (CFU) was observed in groups that were transfected with Bfl-1/A1 siRNA using LAH or LADap peptides compared with those transfected with negative control siRNA, confirming the bacterial inhibitory effect of Bfl-1/A1 siRNA on the intracellular Mtb. In conclusion, both AMPs and siRNA strategies demonstrated successful anti-TB activity in vitro and in macrophages. Further investigations on both strategies are warranted for future therapeutic development.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.subject.lcshTuberculosis - Treatment-
dc.subject.lcshSmall interfering RNA - Therapeutic use-
dc.subject.lcshPeptide antibiotics-
dc.titleThe investigation of antimicrobial peptides and siRNA therapeutics against mycobacterium tuberculosis-
dc.typePG_Thesis-
dc.identifier.hkulb5558980-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplinePharmacology and Pharmacy-
dc.description.naturepublished_or_final_version-

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