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    • Ten strains of lactobacilli TMW isogenic

    2023-03-15

    Ten strains of lactobacilli: TMW 1.1434 (isogenic with strain F19 ), TMW 1.1733 (isolated from fermented food), TMW 1.1628 (isolated from baby feces), TMW 1.1609 (isolated from baby feces), TMW 1.1734 (isolated from fermented food), TMW 1.313 (isolated from non-pasteurized Heineken beer), TMW 1.465 (isolated from soft drink), TMW 1.317 (isolated from beer), TMW 1.485 (isolated from beer), and TMW 1.240 (isolated from beer) were found to display diverse translocation through murine epithelial cells monolayer along with our former study, probably due to different ability to bind to epithelium . The ten bacterial strains were therefore evaluated for their ability to adhere to actin. Performance of bacterial adhesion to actin was conducted according to Boone et al. with modifications . Briefly, 100 μL of actin solution (1 μg/μL, Sigma Aldrich) was immobilized into micro-wells (96-well plate, Sarstedt, Newton, NC, USA) by incubation at 37 °C for 2 h then at 4 °C overnight. The unbound sites were blocked by 1% BSA in PBS for 2 h at 37 °C. Plates were then washed 3 times with PBS. Lactobacilli grown overnight in MRS were washed 3 times with PBS to get rid of media and 100 μL of bacterial suspension with an OD of 0.4 was added to each well, followed by incubation at 37 °C for 2 h. Subsequently, plates were washed 3 times to remove unbound bacteria, followed by crystal violet (0.5%) staining at room temperature for 1 min. Crystal violet was then dissolved by 5% acetic A-674563 synthesis and Abs of bacteria was measured on a microplate reader. One blocked micro-well without addition of bacteria was used as the negative control. showed that TMW 1.1434 displayed highly significant adhesion (p < 0.01), followed by TMW 1.1734 and TMW 1.485 with significant adhesion (p < 0.05), comparing with BSA control (ANOVA-LSD test). The three strains were therefore further used to isolate actin-binding proteins. Supernatants and crude extracts of TMW 1.1434, TMW 1.1734, and TMW 1.485 were obtained by filtration (0.45 μm) and ultra-sonication (70 W, 10 min), respectively. Isolation of actin-binding proteins was performed following Boone and Sánchez with modifications , . Actin was immobilized to 24-well plates as described above. Unbound sites were blocked by 1% BSA at 37 °C for 2 h. Subsequently, 200 μL of bacterial supernatants or crude extracts were added into each micro-well and incubated at 37 °C for 2 h. One blocked micro-well without addition of bacterial supernatants or crude extracts was used as the negative control. SDS buffer (1%, W/V) was added into each micro-well after plates were washed twice with PBS buffer. Plates were then incubated at 37 °C for 2 h with gentle agitation (120 rpm). Subsequently, wells were dried and adherent proteins were dissociated from wells by gentle incubation with 100 μL of Laemmli buffer for 1.5 h. Eluted solutions were analyzed by duplicate SDS-PAGE followed by silver staining. No proteins from supernatants were isolated (data not shown). Due to low concentration preventing from sequencing, only 6 of the bands from gel with bacterial crude extracts were cut and sent for sequencing via LC- MS/MS (ZfP Zentrallabor für Proteinanalytik, München, Germany). The actin-binding proteins were characterized as (): pyruvate kinase (MW: 65 kDa, band E), glucose-6-phosphate isomerase (MW: 49 kDa, band F), and phosphoglycerate kinase (MW: 44 kDa, band G) from TMW 1.1434; pyruvate kinase (MW: 65 kDa, band B), chaperonin GroEL (MW: 60 kDa, band C), and EF-Tu (MW: 44 kDa, band D) from TMW 1.1734; chaperonin GroEL (MW: 60 kDa, band A) from TMW 1.485. Of the isolated proteins, pyruvate kinase (PK) and EF-Tu are mainly cytoplasmic protein, glucose-6-phosphate isomerase (PGI) and GroEL are both cytoplasmic and secreting protein, and phosphoglycerate kinase (PGK) is a membrane protein, all of which could display on the cell surface. It is worth noting that, BSA could also be isolated in this strategy even though it displayed very weak adhesion to actin in the microplate assay. We argued that this highly purified protein with a high concentration promoted its adhesion. This phenomenon was also seen in Sanchez's work .