Anti esat 6

Proteins from LIΔ-Rv3875 and LIΔ-Rv0129c were obtained by trichloroacetic acid precipitation as described previously^{17 (link)}. Proteins were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Following incubation with Tris-buffered saline containing 0.1% Tween-20 with 5% skim milk, the blots were probed with anti-ESAT6 (Abcam, USA) at a dilution of 1: 5 000 and anti-Mycobacterium tuberculosis Ag85 (Abcam, USA) at a dilution of 1: 500 overnight at 4 °C. Following horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody incubation (1:1000) (Beyotime Institute of Biotechnology, China), protein bands were visualized using Super Signal West Pico (Thermo Scientific, USA).

The bacterial cells were harvested, lysed and loaded onto an SDS-PAGE gel as previously described [16 ]. The Bradford assay (Bio-Rad Laboratories, Hercules, CA) was used to determine the protein concentration of the cell free lysates. Approximately 0.8 µg crude protein extract was loaded onto the SDS-PAGE gel. After electrophoresis, proteins were blotted onto a nitrocellulose membrane using an iBlot™ Transfer Device (Invitrogen, Waltham, MA). Following the protein transfer, the SNAP i.d. 2.0 kit (Sigma-Aldrich) was used for antibody hybridization to the H56 antigen according to the manufacturer’s protocol. The mouse monoclonal antibody anti-ESAT-6 (ab26246, Abcam Inc, Cambridge, United Kingdom) primary antibody was diluted 1:2 000, and the secondary antibody m-IgGκ BP-HRP (Santa Cruz Biotechnology, Dallas, TX) was diluted 1:15 000. The proteins were visualized using the SuperSignal West Pico PLUS Chemiluminescent substrate (ThermoFisher Scientific) and the signals were imaged with an Azure c400 system (Azure biosystems, Dublin, CA).

Bacterial cultures were grown and induced as described above. Flow cytometry analysis was performed to verify surface display of AgE6 in L. plantarum. Cells harvested from approximately 500 μl culture were washed once with PBS. The pellet was then resuspended in a 1:250 dilution of the primary antibody anti-ESAT-6 (Abcam) in PBS/2% (w/v) BSA followed by incubation for 30 min at room temperature. Subsequently, the bacteria were washed two times with 600 μl PBS/2% BSA. After the second washing step, the pellet was resuspended in a 1:170 dilution of FITC-conjugated anti-mouse IgG secondary antibody (Sigma-Aldrich) in PBS/2% BSA followed by incubation for 30 min at room temperature, protected from light. The cells were then washed three times with PBS/2% BSA before subsequent analysis using a MACSQuant analyzer (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The data were analyzed using FlowJo software. Bacterial samples used for fluorescence microscopy were prepared in the same manner and analyzed with a Zeiss LSM 700 Confocal Microscope using Zen software. Image analysis was performed using the ImageJ plugin MicrobeJ (Ducret et al., 2016 (link)). The phase contrast images were used for determination of the shape and size of the bacterial cell, and the FITC signal intensities were extracted from the corresponding fluorescent images.