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Phagosomal Copper Triggers a Peptidomimetic's Oxidative Activity and Enables Eradication of Intracellular Mycobacterium tuberculosis
Author(s) -
Libardo Mark Daben Javate,
Anand Kushi,
Krishnamoorthy Gopinath,
Kaufmann Stefan H.E.,
Singh Amit,
AngelesBoza Alfredo
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.939.11
Subject(s) - phagosome , isothermal titration calorimetry , chemistry , reactive oxygen species , ascorbic acid , mycobacterium smegmatis , biochemistry , mycobacterium tuberculosis , oxidative stress , in vitro , intracellular , copper , microbiology and biotechnology , biology , tuberculosis , medicine , food science , organic chemistry , pathology
Copper, due to its inherent antimicrobial properties is enriched at sites of infection, particularly in mycobacteria‐containing phagosomes. However, recent studies show that M tuberculosis ( Mtb ) has multiple copper detoxification pathways allowing it to persist inside macrophages. A thorough in vitro investigation also showed that Mtb is highly susceptible to Ascorbic acid‐induced Reactive Oxygen Species (ROS). Using these information, we designed an all‐D‐amino acid antimicrobial peptide (DL‐10, D VIHRAGLQFPVGRVHRLLRK‐NH 2 ) containing the Amino Terminal Copper and Nickel (ATCUN) Binding Unit Val‐Ile‐His. The ATCUN Motif binds strongly to cupric ions to form a metallopeptide that directly generates ROS under physiological conditions. We find that DL‐10 displays high activity against Mtb both in vitro and inside THP‐1 macrophages. This antimycobacterial activity was found to be linked to levels of free copper and molecular oxygen. Using a genetically encoded fluorescent sensor, we found that DL‐10 generates oxidative stress in M smegmatis ( Msm ), leading to efficient DNA cleavage. Incidentally, the oxidative burst coincides with the onset of Msm killing, further implicating ROS in its bactericidal action. DL‐10 is internalized by RAW264.7 macrophages via COP1‐mediated endocytosis. Using strategically positioned fluorophores along the DL‐10 primary structure, we were able to confirm DL‐10 copper binding inside macrophages, while, Isothermal Titration Calorimetry revealed a 500 nM K d of binding. Moreover, by confocal microscopy, we found that DL‐10 effectively co‐localizes with intracellular Msm . This suggests that DL‐10 likely utilizes the endogenous phagosomal copper pool to kill intracellular mycobacteria. Finally, DL‐10 exhibits low cytotoxicity against RAW264.7 macrophages up to 128 uM. The high potency and the fact that DL‐10 circumvents proteolytic cleavage suggests that it is a good drug candidate. Our study also reveals an efficient way of sterilizing mycobacterial infections using host‐derived metal ions.