Background: We have found that serum KD-specific molecules possess molecular structures common to microbe-associated molecular patterns (MAMPs) from several bacteria by liquid chromatography-mass spectrometry (LC-MS) analysis. In the present study, we have extensively searched for the culture conditions that reproducibly produce MAMPs common to serum KD specific molecules.
Methods: Yersinia pseudotuberculosis, spore-forming bacteria and pathogenic bacteria were obtained from KD patients. Others were purchased from American Type Culture Collection. Microbes were cultured in a variety of conditions (medium, temperature, duration, shaking and biofilm formation). For biofilm formation, a various kind of oil/butter was added to the culture medium. Extraction from in vitro samples (culture supernatants, bacteria cells, and biofilms) as well as in vivo biofilm samples (teeth, tongue, nasal cavity, or rectum) was performed with ethyl acetate. Samples were analyzed by HPLC and MS (Esquire 6000 electrospray ionization). Human coronary artery endothelial cells (HCAECs) were employed for cytokine assay. The concentrations of cytokines were measured by EC800 cell analyzer with a BD™ Cytometric Bead Array.
Results: Serum KD-specific molecules showed m/z and MS/MS fragmentation patterns almost identical to those of MAMPs obtained from the biofilms formed in vitro (Bacillus cereus, Bacillus subtilis, Yersinia pseudotuberculosis, Staphylococcus aureus) or in vivo. Not culture supernatants but biofilm extracts from these bacteria, especially cultured in the presence of butter, induced proinflammatory cytokines by HCAECs.
Conclusions: Extensive analysis by LC-MS/MS revealed that serum KD-specific molecules common to MAMPs were mostly derived from biofilms in vitro and in vivo. This report offers novel insight into the pathogenesis of KD.