Caenorhabditis elegans shifting assay

Wild-type Bristol (N2) and UL1447: leEx1447 [hif-1::GFP +unc-119(+)] Caenorhabditis elegans were used in this study (Caenorhabditis Genetics Center, MN, USA). P. aeruginosa and Escherichia coli OP50 lawns were created on 100-mm plates using 500 µL of a 15.25 mg/mL (based on cell pellet mass) suspension diluted from an overnight culture, incubated at 37°C for 1 h, then returned to room temperature prior to the addition of C. elegans. C. elegans were synchronized using a bleaching protocol and grown until the L4 stage at 20°C (62). The worms were transferred onto the P. aeruginosa lawns to feed for 24 h at 20°C. Following the 24-h infection feeding, the worms were gravity washed briefly in M9 buffer containing 200 µg/mL neomycin to kill any P. aeruginosa cells that were not ingested (63) and again gravity washed three times in M9 to remove any residual neomycin before loading the worms into the microfluidic chips (Infinity Chips; NemaLife, Inc., TX, USA) (64, 65). Before use, the interiors of the microfluidic chips were prepared according to the manufacturer’s protocol. The worms in the chips were flushed and fed daily with 15.25 mg/mL OP50. The chips were incubated in humidified chambers at 20°C until all animals perished (66). Videos were acquired each day after flushing and before feeding fresh OP50 to determine live counts. Live counts were processed by a beta version of NemaStudio.ai, a cloud-based data annotation tool, followed by manual annotation (NemaLife, Inc., TX, USA). Infection assays were conducted at 20°C for the indicated time intervals. Statistical analyses were calculated using GraphPad Prism version 9.5.0 (GraphPad Software, San Diego, CA, USA), and P values were estimated using the log-rank (Mantel-Cox) test. Statistical tests were performed for each strain/condition compared to the wild-type (PA14) control. For all cases, P-values of <0.001 were considered significant.