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. 2019 May 27;9:7903. doi: 10.1038/s41598-019-44263-7

Figure 1.

Figure 1

Diverse bacterial strains promote VSV-Gpp entry into epithelial cells. (a) Schematic diagram outlining lentiviral pseudoparticle (pp) infection. Particles lacking a viral envelope protein (NEpp) enter cells via non-specific phagocytic uptake mechanisms. In contrast, VSV glycoprotein (VSV-Gpp) or measles virus glycoprotein (MeVpp) expressing pp enter cells through specific receptors (LDL receptor or SLAMF1, respectively). Following glycoprotein-dependent fusion the lentivirus capsid uncoats, the genome replicates and expresses the reporter gene. (b) A549 lung epithelial cells were exposed to diverse bacterial strains: B. subtilis; enteropathogenic (EP) E. coli; K. pneumoniae; STm or S.aureus at a MOI of 10 for 1 h before inoculating with VSV-Gpp and antibiotic chloramphenicol (34 ug/mL; or tobramycin, 40 μg/mL, for chloramphenicol-resistant P. aeruginosa) and cultured for 48 h. Cells were lysed and luciferase activity measured and data expressed relative to untreated cells (UT). (c) Non-polarized (Non-pol) or polarized A549 cells were exposed to STm (MOI 10) for 1 h prior to inoculating with VSV-Gpp and chloramphenicol (34 μg/mL) for 48 h and infection quantified. (d) Polarized A549 cells were exposed to STm (MOI 10) for 1 h prior to apical addition of 70 kDa FITC-dextran and basolateral media sampled at various times to measure fluorescence and permeability, as a control non-polarized cells were evaluated. Polarized A549 cells were exposed to STm (MOI 10) for 1 h prior or left untreated (UT), fixed and stained for tight junction occludin (FITC – open arrow) and ZO-1 (PE- closed arrow) expression and imaged by confocal microscopy. (e) A549 cells were inoculated with STm (MOI 10) for 1 h before infecting with NE or VSV-Gpp particles for 48 h. (f) A549 cells were transfected with the lentiviral genome reporter DNA (pNL4.3env−rev−luc) for 8 h and treated with STm (MOI 10) and chloramphenicol for 16 h. (g) A549 cells were exposed to STm (MOI 10) for 1 h prior to inoculating with VSV-Gpp for defined periods of time and infection assessed after 48 h. (h) A549 cells were treated with STm at differing MOI, from 1 bacterium per 100 A549 cells to 10 bacteria per A549 cell, for 1 h prior to VSV-Gpp infection and culturing for 48 h in the presence of chloramphenicol. Bars represent mean ± S.D. for n = 3. Statistical comparison by unpaired t test where: n.s. p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 and ****p ≤ 0.0001. All data sets are representative of at least two independent experiments.