FIG. 3.

Sca2 mediates invasion of mammalian cells. (A) Scanning electron micrographs examining the surface interaction of Sca2-expressing E. coli with HeLa cells. HeLa cell monolayers on glass coverslips were infected with induced bacteria for 20 min and then processed for SEM. White arrowheads highlight possible Sca2-mediated cellular membrane rearrangements. Scale bars, 1 μm. (B) CFU-based quantification of bacterial invasion of HeLa, Vero, EAHY 926, and HLMV cells. Confluent cell monolayers were infected for 1 h with E. coli BL21(DE3) expressing the empty vector (pET-22b) or Sca2 from R. conorii (pSca2-200) and assessed for invasion by gentamicin protection assay. Invasion is presented as the percentage of bacteria recovered after the gentamicin challenge out of the inoculums. Actual percentages varied from assay to assay (ranging from 0.0007 to 0.07%), depending on the passage number of the cells used and the expression of Sca2 at the E. coli outer membrane. *, P < 0.05. The data presented are representative of at least two independent experiments for each individual cell line. Error bars represent the standard deviation of each data set. (C) Transmission electron micrographs of HeLa cells infected with Sca2-expressing E. coli demonstrate different steps of the uptake process, including the initial attachment event (left panel), the induction of changes in the plasma membrane (arrows in middle panel), and the presence of bacteria within membrane bound vacuoles (right panel). Scale bars are indicated in each panel.