Saline solution

C57BL/6 mice were immunized subcutaneously in the flank with 10µg of each PE/PPE protein and 1µg Quil-A (Invivogen) or a matched volume of saline solution (Sigma) as a control. Quil-A, known for inducing a robust adjuvant response, promotes Th1-biased immune responses and potentiates the response to mucosal antigens (29 (link)). A second subcutaneous immunization was administered three weeks later in the opposing flank, followed by a final intranasal boosting under isoflurane anaesthesia (using 0.1µg Quil-A per animal in this case) three weeks after. BCG vaccinated mice were given 5×10⁵ CFU BCG Pasteur in 0.1mL subcutaneously in the first week. Three weeks after the last immunization, mice from each group (n=3) were humanely sacrificed for immunogenicity analysis by overdose of pentobarbital given intraperitoneally, and death was verified by severing femoral artery. At the same time, remaining mice (n=6 from each group for PE/PPE and n=7 for PBS and BCG) were used for an Mtb challenge experiment. Further details of the immunization and dosing regimen are given in the corresponding figure legends.

The tetrachloroauric acid (HAuCl₄·H₂O) (99.99%) was purchased from Thermo Fisher Scientific (Thermo Fisher Scientific, Waltham, MA, USA). The sodium citrate (HOC(COONa)(CH₂COONa)₂·2H₂O) (≥99.00%), cetyltrimethylammonium bromide (CTAB) (99.00%), sodium borohydride (NaBH₄) (98.00%), silver nitrate (AgNO₃) (99.99%), ascorbic acid (AA) (99.00%), O-(2-mercaptoethyl)-O′-methylpolyethylene glycol (PEG-SH Mw ≈ 2000) (99.99%), and saline solution were purchased from Sigma-Aldrich (Sigma-Aldrich, St Louis, MO, USA).

Under sterile conditions, 20 g of fruit salad was homogenized with a saline solution (0.9% NaCl) (Sigma, Milan, Italy). Decimal dilutions of the homogenate sample were made using the same diluent and plated on selective media to determine the specific microbial groups. Lactic acid bacteria were plated into de Man Rogosa and Sharpe (MRS) agar supplemented with cycloheximide (0.17 g/L) (Sigma, Milan, Italy) and incubated under anaerobic conditions at 37 °C for 48 h. Plate Count Agar (PCA) was used to enumerate the total mesophilic bacterial count incubated at 30 °C for 48 h, and the total psychrotrophic bacteria were incubated for 10 days at 4 °C. Yeasts and molds were determined in Sabouraud Dextrose Agar (SAB), supplemented with chloramphenicol (0.1 g/L) with incubation at 25 °C for 48 h and 5 days, respectively. Violet Red Bile Glucose Agar (VRBGA) incubated at 37 °C for 24 h was instead used for Enterobacteriaceae. All of the cultured media and supplements were obtained from Oxoid (Milan, Italy). The analyses were performed in duplicate on different samples, and the results were expressed as log colony-forming units/gram of fruit salad (CFU/g).

Tryptic
soy broth (TSB), tryptic soy agar (TSA), Tween 20, saline solution
(0.85 wt %), and hydrogen peroxide were obtained from Sigma-Aldrich
and used as received. Phosphate buffer solution (PBS) was prepared in situ, and the pH was adjusted with hydrochloric acid
or sodium hydroxide to the chosen value.

SHED were obtained using a modified Miura's protocol [12 (link)], with collagenase type I for pulp digestion (3 mg/mL). SHED were carefully prepared to animal infusion. After trypsinization, cells were washed twice and counted and 1 × 10⁴ cells were ressuspended in saline solution (Sigma-Aldrich, MO, USA) in a very low volume (20 microliters).

Immediately after sacrifice, eyes were enucleated and transferred to a saline solution with a pH of 7.4 (Sigma Aldrich). Retinas were rapidly dissected by making a small incision with a scalpel blade at 1 mm behind the limbus. The incision was then extended 360° around the globe using fine ophthalmic scissors. Anterior segment structures (cornea, iris, and lens) were removed and the retina was separated from the RPE-choroidal complex. Retinas were homogenized using an Ultra-Turrax (Ika Werke GmbH & Co. KG, Staufen, Germany) to prepare them for posterior reverse transcription polymerase chain reaction (RT-PCR) analyses. The RPE-choroid samples were also homogenized, but with a Teflon pestle (Thomas Scientific, Swedesboro, NJ, USA) in 75 µL of phosphate buffer (Sigma Aldrich). The RPE-choroid samples were then centrifuged (13,000 rpm) for 20 min at 4 °C. The resulting supernatant was collected and protein concentration was determined using a slightly modified Bradford assay (Bio-Rad, Hercules, CA, USA) [28 (link)].