Effects of rTMD1 on bacterial agglutination and phagocytosis by THP-1 cells. (A) FITC-labeled K pneumoniae and E coli DH5α were incubated in buffer containing 5 mM CaCl2 and in the absence or presence of rTMD1 without or with 0.2 M mannose and 5 mM EDTA. The fluorescently labeled bacteria were observed using a fluorescence microscope to evaluate bacterial agglutination. Nontagged rTMD1 was prepared by incubation of rTMD1 with enterokinase and purified as described in Document S1. Mammalian expressed rTMD1 and an internal control with tagged rTMD23 were also included. Photomicrographs are representative of 3 independent experiments. (B) Representative photomicrograph showing the effect of rTMD1 on bacterial phagocytosis. Differentiated THP-1 cells were incubated with rTMD1-pretreated FITC-labeled K pneumoniae. The left panel shows the bright field image and the right panel shows the fluorescence photomicrographs (scale bars represent 200 μm). (C) FACS analysis of the samples from panel B. The graphs depict the level of FITC fluorescence (x-axis) versus the relative cell numbers (y-axis). These graphs represent the results from 3 independent experiments.
