*3.5. Isolation of IBs and GFP*

PldA-GFP IBs and PldA IBs were isolated as described in previous work [33]. Briefly, wet bacterial biomass was lysed by lysozyme and exposed to ultrasound. IBs were sedimented from the cell biomass lysate by centrifugation at 4500× *g* for 30 min. IBs pellets was sequentially treated with 10 mM Tris-HCl buffer (pH 8.0) containing 0.1% *N*-Lauroylsarcosine (Sarcosyl) and with the same buffer containing 1% sodium deoxycholate and 1 M urea for 15 min, with the aim to achieve a more thorough purification from contaminants (proteins, LPS, and nucleic acids). The protease inhibitor PMSF (1 mM) was

used at all stages of IBs isolation. IBs obtained by this scheme were stored at −70 ◦C for a maximum of 30 days.

*E. coli* cells expressing GFP were lysed by lysozyme and exposed to ultrasound under the conditions described in previous work [33] and centrifugated at 4500× *g* for 30 min. GFP was isolated from supernatant by size-exclusion chromatography on a Superdex 200HR column in PBS with an elution rate of 0.5 mL/min using a FPLC system (Amersham Pharmacia Biotech).

#### *3.6. Determination of PldA-GFP and GFP Oligomeric Structure*

The molecular weight of GFP (in PBS) and PldA-GFP (in 0.1% SDS, 50 mM Tris-HCl buffer, pH 8.0) was estimated by size-exclusion chromatography on a Superdex 200 column 10/300 GL using a FPLC system (Amersham Pharmacia Biotech) with an elution rate of 0.5 mL/min. The column was calibrated using proteins with known molecular weights ("ICN Biomedicals", Costa Mesa, CA, USA): apoferritin, bovine serum albumin, egg albumin, chymotrypsinogen, myoglobin, and cytochrome *c* (480, 67, 45, 24, 18, and 13 kDa, respectively). The relative error of the determination of the molecular weight was 5%.

#### *3.7. Fluorescence Confocal Microscopy*

Bacterial cells were pre-diluted in PBS to a concentration of 1 × <sup>10</sup><sup>6</sup> cells/mL and 20 μL of this solution was placed between two circle cover slips (Thermo Scientific) with a thickness of 0.08 to 0.12 mm. GFP fluorescence was visualized using a confocal microscope assembled based on Olympus FV1200 system equipped with a 488 nm argon laser with EX DM 405/488 and gallium arsenide phosphide (GaAsP) detector with filter set (DM 570, BA 505–540) and a 100× objective lens (UAPON100XOTIRF, Olympus). Transmission channel was visualized using 635 nm diode laser and transmission light detector. All images were obtained using Olympus FLUOVIEW software v.4.1.a with 4 us/pix exposure, 1600 × 1600 resolution and 4 kalman for digital noise elimination.

The murine neuroblastoma cell line Neuro-2a (CCL-131TM) was purchased from American Type Culture Collection ATCC (ATCC). A cell suspension in DMEM medium (BioloT, Russia) containing 10% fetal bovine serum and 1% penicillin/streptomycin solution (BioloT, Russia) was introduced into the wells of chamber for confocal microscopy at a concentration of 1 × <sup>10</sup><sup>3</sup> cells per well and left for adhesion for 24 h in a CO2-incubator (5% CO2, 37 ◦C). After that, a suspension of PldA-GFP IBs in PBS (20 μg/mL) was added to the cells and the chambers were additionally incubated for 0, 1, 4, and 24 h. The accumulation of IBs in Neuro-2a cells was studied using laser scanning confocal microscopy and scanning electron microscopy. The fluorescent images of 150 randomly selected cells for each time point were inspected and the amount of fluorescent IBs penetrating the cell surface was calculated and expressed as number of PldA-GFP IBs per cell.

Fluorescence images of PldA-GFP IBs and neuroblastoma Neuro-2a cells were obtained using an LSM 710 LIVE AxioObserver laser scanning confocal microscope (Carl Zeiss GmbH, Jena, Germany). Cell nuclei were stained using a Hoechst 33,342 fluorescent dye (Invitrogen, USA) at a concentration of 5 μM. Fluorescence was excited at 488 nm, and emission was recorded at 493–652 nm. Processing and subsequent analysis of cell images was performed using the ZEN 2011 software (Carl Zeiss GmbH, Jena, Germany).

#### *3.8. Scanning Electron Microscopy*

Neuroblastoma cells on cover slides were fixed in 4% (*v/v*) glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h at room temperature. After dehydrating the cells by passing them through an ethanol series, the ethanol was replaced by isoamyl acetate. Then, parts of the cover glass with cells were dried under Critical point dryers K850 equipment (Quorum Technologies, London, UK), mounted on stubs, and coated with carbon. The samples were examined at the Instrumental Centre of Biotechnology and Gene Engineering of FSCEATB FEB RAS using a Scanning electron microscope EVO (Carl Zeiss, Jena, Germany).
