Ccf4 am

Cell nuclei were fluorescently labelled using DAPI (Sigma Aldrich) or Hoechst 33342 (ThermoFisher). Lysotracker Green DND-26, CCF4-AM and LipidTox Deep Red were obtained from ThermoFisher.

HeLa cells (2 × 10⁴/well) were seeded into black, clear bottom 96-well plates (Greiner). Cell monolayers were infected at an MOI of 5 and effector-β-lactamase (BlaM) fusion protein expression was induced using 10 ng/ml aTc as previously described^{20 (link)}. At 24h post-infection (hpi), cells were washed three times with 1X PBS and loaded with CCF4-AM using the alternative loading protocol per the manufacturer’s instructions (ThermoFisher Scientific). Samples were incubated in the dark for 1h at room temperature and were then read on a Tecan Infinite M200 Pro plate reader. To quantify effector translocation, the background was subtracted, the ratio of 460nm to 535nm (blue:green) was determined, and expression relative to cells infected with C.t. pBomb4 BlaM (negative control vector) was calculated as previously described^{31 (link)}.

Cells (10 × 10⁶ cells/condition for ascites cells and 5 × 10⁶ cells/condition for in vitro-generated cultures) were incubated with 0.5 μg mL⁻¹ CCF4-AM (Invitrogen) for 30 min at room temperature at 10 × 10⁶ cells mL⁻¹ in loading buffer (120 mM NaCl, 7 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgCl₂, 5 mM glucose, 25 mM Hepes, pH 7.3) containing solution B (dilution 1/20, LiveBLAzer FRET-B/G loading kit, Invitrogen) and 1 mM probenecid (Invitrogen). After washing, cells were incubated at 10 × 10⁶ cells mL⁻¹ in loading buffer containing 1 mM probenecid in the presence or absence of 2 mg mL⁻¹ of β-lactamase (Penicillinase from Bacillus cereus, Sigma) for 3 h at 4 or 37 °C. Cell viability was assessed using Fixable Viability Dye eFluor780 (eBioscience). After washing, cells were stained for surface markers (CD11c-PeCy7 and CD1c-APC for ascites cells; CD1a-APC-Vio770 and CD16-APC for in vitro-generated monocyte-derived cells; CD1a-APC and CD14-APC-Vio770 for CD34⁺cells-derived cultures). Cells were analyzed on a FACSVerse Instrument (BD Biosciences). CCF4-AM fluorescence was assessed by measuring the 450 and 520 nm channels.

The pseudovirus fusion with target cells was measured using the BlaM assay, as described previously [10 (link), 83 (link)]. Briefly, cells cultured in 96-well black clear-bottom plates were pre-treated for 30 min, at 37 °C, 5 % CO₂ with growth medium containing 20 μM MG132. The viruses were bound to target cells by centrifugation at 4 °C for 30 min at 1550×g, in the absence of MG132. After the virus binding step, cells were washed once with cold PBS and incubated in live cell imaging buffer/2 % FBS/20 μM MG132 at 37 °C for 90 min. The fusion reaction was stopped by placing the plates on ice, and the media was replaced with the BlaM substrate, CCF4-AM (Invitrogen). Cells were incubated at 11 °C overnight, and the BlaM activity was determined from the ratio of coumarin (blue) and fluorescein (green) fluorescence signals, using a SpectraMaxi3 fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA).

Detection of phagosomal rupture was performed as described using a single-cell fluorescence resonance energy transfer (FRET)-based assay (Ray et al. 2010 (link); Simeone et al. 2012 (link)). Briefly, M. tuberculosis, M. tuberculosis:ΔRD1, and M. kansasii were used to infect phorbol-myristate-acetate (PMA)-differentiated THP-1 macrophages. THP-1 cells were incubated with CCF-4-AM (Invitrogen), an esterified, lipophilic form of the CCF-4 substrate that can readily enter into cells, where it is converted by endogenous cytoplasmic esterases into CCF-4 that is negatively charged. CCF-4 is retained in the cytosol and emits green fluorescence (535 nm) upon stimulation at 405 nm, due to FRET from the 7-hydroxycoumarin donor to the fluoroscein acceptor. When in contact with endogenous beta-lactamases produced by mycobacteria, as occurs when the phagosomal compartment is disrupted, CCF-4 is cleaved, emitting at 450 nm when excited at 405 nm. All experimental steps were performed in the presence of probenecid upon CCF-4-AM treatment to avoid the export of the fluorescent reporter. The 450/535 nm intensity ratio measurements were obtained and analyzed by algorithms provided by the Metamorph software, as previously reported (Ray et al. 2010 (link)).