Proximity Labeling To Map Host-Pathogen Interactions at the Membrane of a Bacterium-Containing Vacuole in Chlamydia trachomatis-Infected Human Cells

Many intracellular bacteria, including the obligate intracellular pathogen Chlamydia trachomatis, grow within a membrane-bound bacterium-containing vacuole (BCV). Secreted cytosolic effectors modulate host activity, but an understanding of the host-pathogen interactions that occur at the BCV membrane is limited by the difficulty in purifying membrane fractions from infected host cells.

The effect of LRRF1 -GFP and FLII-GFP overexpression and LRRF1 knockdown on C. trachomatis progeny production.
trachomatis L2 wild-type. Inclusion area is reported for non-transfected, LRRF1-GFP Total (the inclusions from both high and low LRRF1-GFP expressing cells) and broken into LRRF1-GFP high and low expression only (see inset). Two independent experiments were performed. Inclusion area was graphed in GraphPad Prism 7 and a one-way ANOVA with Tukey's multiple comparisons post-hoc test was performed to determine statistical significance. There was no significant difference in inclusion area.
(D) siRNA knockdown of LRRF1 in HeLa cells. 20 nM of non-targeting (NT), GAPDH, LRRF1 single siRNA or 3 pooled siRNAs were reverse transfected as indicated into HeLa cells that were then infected with C. trachomatis L2 wild-type at 48 hours post-transfection and collected 24 hours later. Lysates from siRNA treated, C. trachomatis L2 wild-type infected cells were collected in 2x Laemmli sample buffer, electrophoresed, transferred to PVDF and blotted to confirm siRNA knockdown efficiency of LRRF1 and GAPDH.

Fig S5. T18-IncE is expressed in E. coli
DH5α lacI q E. coli were transformed with pUT18C-IncE and grown overnight. Expression of T18-IncE was induced or not using 0.5 mM IPTG and with or without the presence of 0.4% glucose, then grown for 4 hours at 30°C. Bacterial lysates were collected, separated by SDS-PAGE, and transferred to a PVDF membrane. The membrane was blotted for expression of T18-IncE using an antibody against T18 (T18-IncE 34 kDa). Fig S6. Overexpression of CT226FLAG from C. trachomatis L2 CT226FLAG transformants results in increased LRRF1 and FLII at the inclusion membrane HeLa cells seeded on glass coverslips were infected with C. trachomatis L2 CT226FLAG transformants and either not induced or induced for expression at 7 hpi using 5 nM or 20nM aTc. At 24 hpi, coverslips were fixed with 3% formaldehyde and 0.022% glutaraldehyde, permeabilized with methanol, and stained for immunofluorescence to visualize construct expression (FLAG; red), Chlamydiae (MOMP; gray), DNA (DAPI; blue), and A) LRRF1 (green) or B) FLII (green). Coverslips were imaged using a Zeiss confocal LSM 800 with 63x magnification and 2x zoom. Scale bar = 5 μm. Images were captured using the same exposure time (set for 20 nM aTc images) for uninduced and 5 nM aTc samples. trachomatis L2 CT226FLAG infected HeLa cells using normal exposure levels HeLa cells seeded on glass coverslips were infected with C. trachomatis L2 CT226FLAG transformants and either not induced or induced for expression at 7 hpi using 5 nM. At 24 hpi, coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.5% triton X-100, and stained for immunofluorescence to visualize construct expression (FLAG; red), LRRF1 (green), GFP expressing Chlamydiae (pseudo-color blue), and DNA (DAPI; blue).

CT226FLAG
HeLa cells seeded in a 6-well plate with glass coverslips were infected with C. trachomatis L2 CT226FLAG or IncFFLAG and either not induced or induced for expression at 7 hpi with 5 nM aTc (CT226FLAG) or 1 nM aTc (IncFFLAG). At 24 hpi, cells were collected, solubilized, normalized, and affinity purified using FLAG beads. The clarified lysates (soluble), unbound fractions and eluates were probed for construct expression (FLAG; CT226FLAG 19.2 kDa; IncFFLAG 11.3 kDa monomer and 22.6 kDa dimer) and LRRF1 (dimer 160 kDa).
(A) and (B) are representative of two biological replicates. A total of three independent experiments were performed.