Figure 4. Neutralization of bacteria-bearing lysosomes triggers lysosome exocytosis.

(A) Immunofluorescence staining of UPEC (blue) or E.coli K-12 (blue)-containing lysosomes (green) and lysotracker (red) at 6 h.p.i.. Lysotracker⁺ populations of bacteria-containing lysosomes were quantified, and expressed as the percentage of total examined lysosome-enclosed bacteria. Scale bar: 5 µm. n=3 slides.

(B) P62 level dynamics in naïve BECs (lane1), BECs treated with 200 nM rapamycin alone (lane 2) or rapamycin combined with UPEC (lane 3), E. coli K-12 (lane 4) or 1 µM bafilomycin A1 (lane 5).

(C) Intracellular bacteria CFU at 8 h.p.i in BECs infected with either UPEC or E.coli K-12 and treated with either vehicle, 1 µM bafilomycinA1 or 50 mM NH₄ Cl. Error bars, SEM. n=18.

(D) Immunoblot quantification of CD63 or LAMP1 present in the EUCV purified from UPEC, or E.coli K-12 infected BECs, or E.coli K-12 infected BECs treated with bafilomycin A1. Comparable CD63 or LAMP1 in total cell lysate (TCL) suggest similar numbers of cells were used.

(E) Immunoblot quantification of CD63 in exosomes purified from naïve BECs, BECs treated with 200 nM rapamycin alone, or with 200 nM rapamycin for 4 hours followed by 1 µM bafilomycinA1 treatment for12 hours. Comparable CD63 level in total cell lysate suggest that similar number of cells were used.

(F) FACS analysis of cell surface LAMP1⁺ populations in naïve BECs, or BECs treated with rapamycin for 4 hours followed by bafilomycin A1 treatment for 12 hours, or infected with UPEC CI5 or E.coli K-12 for 12 hours.

(G) Bacterial expulsion levels at 6 h.p.i. in infected BECs expressing control shRNA, or SYT7 shRNA. Knockdown efficiency is indicated by the western blot alongside. Error bars, SEM. n=18.