Mycoplasma removal from cell culture using antimicrobial photodynamic therapy.

OBJECTIVE: The objective of this research was to determine the effectiveness of antimicrobial photodynamic therapy (aPDT) in the removal of mycoplasmas from contaminated cells. BACKGROUND DATA: Mycoplasmas often contaminate cell cultures. The cell-contaminating mycoplasmas are removed by antibiotics, but the use of antibiotics usually induces antibiotic-resistant bacteria. aPDT is expected to be a possible alternative to antibiotic treatments for suppressing infections. MATERIALS AND METHODS: Mycoplasma salivarium (Ms)-infected human embryonic kidney (HEK) 293 cells were irradiated using a red light-emitting diode (LED) in the presence of methylene blue (MB) as a photosensitizer. The Ms viable count was determined using culture on agar plates or using a mycoplasma detection kit. RESULTS: aPDT performed using red LED irradiation was effective in decreasing live Ms in the presence of MB without damaging the HEK293 cells. aPDT removed live Ms from the infected cells after washing the cells with sterilized phosphate-buffered saline (PBS) to decrease the initial number of live Ms before aPDT. CONCLUSIONS: This study suggests that aPDT could remove mycoplasmas from contaminated cells.

The Toll-like receptor 2 (TLR2) ligand FSL-1 is internalized via the clathrin-dependent endocytic pathway triggered by CD14 and CD36 but not by TLR2.

Little is known of how Toll-like receptor (TLR) ligands are processed after recognition by TLRs. This study was therefore designed to investigate how the TLR2 ligand FSL-1 is processed in macrophages after recognition by TLR2. FSL-1 was internalized into the murine macrophage cell line, RAW264.7. Both chlorpromazine and methyl-beta-cyclodextrin, which inhibit clathrin-dependent endocytosis, reduced FSL-1 uptake by RAW264.7 cells in a dose-dependent manner but nystatin, which inhibits caveolae- and lipid raft-dependent endocytosis, did not. FSL-1 was co-localized with clathrin but not with TLR2 in the cytosol of RAW264.7 cells. These results suggest that internalization of FSL-1 is clathrin dependent. In addition, FSL-1 was internalized by peritoneal macrophages from TLR2-deficient mice. FSL-1 was internalized by human embryonic kidney 293 cells transfected with CD14 or CD36 but not by the non-transfected cells. Also, knockdown of CD14 or CD36 in the transfectants reduced FSL-1 uptake. In this study, we suggest that (i) FSL-1 is internalized into macrophages via a clathrin-dependent endocytic pathway, (ii) the FSL-1 uptake by macrophages occurs irrespective of the presence of TLR2, and (iii) CD14 and CD36 are responsible for the internalization of FSL-1.