Multiple reaction monitoring-MS analysis of lipid extracts from human carotid endarterectomy and carotid artery samples from young individuals consistently demonstrated the presence of bacterial serine dipeptide lipid classes, including Lipid 654, an agonist for human and mouse Toll-like receptor (TLR)2, and Lipid 430, the deacylated product of Lipid 654. The relative levels of Lipid 654 and Lipid 430 were also determined in common oral and intestinal bacteria from the phylum Bacteroidetes and human serum and brain samples from healthy adults. The median Lipid 430/Lipid 654 ratio observed in carotid endarterectomy samples was significantly higher than the median ratio in lipid extracts of common oral and intestinal Bacteroidetes bacteria, and serum and brain samples from healthy subjects. More importantly, the median Lipid 430/Lipid 654 ratio was significantly elevated in carotid endarterectomies when compared with control artery samples. Our results indicate that deacylation of Lipid 654 to Lipid 430 likely occurs in diseased artery walls due to phospholipase A2 enzyme activity. These results suggest that commensal Bacteriodetes bacteria of the gut and the oral cavity may contribute to the pathogenesis of TLR2-dependent atherosclerosis through serine dipeptide lipid deposition and metabolism in artery walls.
The serine-glycine dipeptide lipid classes, including lipid 430 and lipid 654, are produced by the periodontal pathogen Porphyromonas gingivalis and can be detected in lipid extracts of diseased periodontal tissues and teeth of humans. Both serine-glycine lipid classes were previously shown to engage human and mouse Toll-like receptor 2 (TLR2) and to inhibit mouse osteoblast differentiation and function through engagement of TLR2. It is not clear if other lipids related to serine-glycine lipids are also produced by P. gingivalis. The goal of this investigation was to determine whether P. gingivalis produces additional lipid classes similar to the serine-glycine lipids that possess biological properties. P. gingivalis (ATCC 33277) was grown in broth culture, and lipids were extracted and fractionated by high-performance liquid chromatography (HPLC). Lipids were separated using semipreparative HPLC, and specific lipid classes were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-multiple reaction monitoring (LC-MRM) mass spectrometric approaches. Two glycine lipid classes were identified, termed lipid 567 and lipid 342, and these lipid classes are structurally related to the serine-glycine dipeptide lipids. Both glycine lipid classes were shown to promote TLR2-dependent tumor necrosis factor alpha (TNF-α) release from bone marrow macrophages, and both were shown to activate human embryonic kidney (HEK) cells through TLR2 and TLR6 but not TLR1. These results demonstrate that P. gingivalis synthesizes glycine lipids and that these lipids engage TLR2 similarly to the previously reported serine-glycine dipeptide lipids.
Copper (Cu) ions are critical in controlling bacterial infections, and successful pathogens like Mycobacterium tuberculosis (Mtb) possess multiple Cu resistance mechanisms. We report, as proof of concept, that a novel Cu hypersensitivity phenotype can be generated in mycobacteria, including Mtb, through a peptide, DAB-10, that is able to form reactive oxygen species (ROS) following Cu-binding. DAB-10 induces intramycobacterial oxidative stress in a Cu-dependent manner in vitro and during infection. DAB-10 penetrates murine macrophages and encounters intracellular mycobacteria. Significant intracellular Cu-dependent protection was observed when Mtb-infected macrophages were treated with DAB-10 alongside a cell-permeable Cu chelator. Treatment with the Cu chelator reversed the intramycobacterial oxidative shift induced by DAB-10. We conclude that DAB-10 utilizes the pool of phagosomal Cu ions in the host-Mtb interface to augment the mycobactericidal activity of macrophages while simultaneously exploiting the susceptibility of Mtb to ROS. DAB-10 serves as a model with which to develop next-generation, multifunctional antimicrobials.
Tumor hypoxia is associated with the rapid proliferation and growth of malignant tumors, and the ability to detect tumor hypoxia is important for predicting tumor response to anti-cancer treatments. We have developed a class of dye-conjugates that are related to indocyanine green (ICG, 1) to target tumor hypoxia, based on in vivo infrared fluorescence imaging using nitroimidazole moieties linked to indocyanine fluorescent dyes. We previously reported that linking 2-nitroimidazole to an indocyanine dicarboxylic acid dye derivative (2) using an ethanolamine linker (ethanolamine-2-nitroimidazole-ICG, 3), led to a dye-conjugate that gave promising results for targeting cancer hypoxia in vivo. Structural modification of the dye conjugate replaced the ethanolamine unit with a piperazineacetyl unit and led a second generation dye conjugate, piperzine-2-nitroimidazole-ICG (4). This second generation dye-conjugate showed improved targeting of tumor hypoxia when compared with 3. Based on the hypothesis that molecules with more planar and rigid structures have a higher fluorescence yield, as they could release less absorbed energy through molecular vibration or collision, we have developed a new 2-nitroimidazole ICG conjugate, 12, with two carbon atoms less in the polyene linker. Dye-conjugate 12 was prepared from our new dye (8), and coupled to 2-nitroimidazole using a piperazine linker to produce this third-generation dye-conjugate. Spectral measurements showed that the absorption/emission wavelengths of 657/670 were shifted ∼100 nm from the second-generation hypoxia dye of 755/780 nm. Its fluorescence quantum yield was measured to be 0.467, which is about 5 times higher than that of 4 (0.083). In vivo experiments were conducted with balb/c mice and 12 showed more than twice the average in vivo fluorescence intensity in the tumor beyond two hours post retro-orbital injection as compared with 4. These initial results suggest that 12 may significantly improve in vivo tumor hypoxia targeting.
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