Figure 5
Biochemical characterization of bacteria-like forms isolated from human blood. Human blood was incubated for seven days and bacteria-like forms were isolated by centrifugation, before processing for (A) dark-field microscopy, (B) thin-section TEM, (C) annexin V staining, (D) DiR staining, (E) Western blotting, (F) proteomics analysis and (G) immunofluorescence as described in Methods. In (A), the isolated bacteria-like forms show homogenous composition (see inset). In (B), the isolated vesicles show round and bacillus-like forms delineated by a membrane. In (C), bacteria-like forms observed under TEM were stained with annexin V conjugated with gold nanoparticles. In (D), bacteria-like forms were stained with DiR as did THP-1 cells used as positive control while HEPES buffer used as a negative control showed no reaction. The star symbol denotes p < 0.01 vs. control HEPES. In (E), proteins isolated from bacteria-like forms consist of common blood cell proteins commonly found in blood and exosomes. Human lysosomal protein Lamp-2 or bacterial RNA polymerase sigma S factor was not detected. Cropped Western blots showing full lanes are shown here; full, uncropped blots are provided in Supplementary Figure 1. In (F), the bacteria-like structures contain various blood proteins (for a complete list, see Supplementary Table 1). Proteins were ranked by the number of matching spectra. Exponentially modified protein abundance index (emPAI) values are also shown. In (G), yellow protein particles containing albumin and fetuin-A are shown (inset). Scale bars: 8 μm (A); 200 nm (B,C); (G) 500 nm.
