Lb media

A total of 54 urine samples were collected from Meridian Health Care Center (Maharajgunj, Kathmandu) and Abhiyan Pathology Lab (Sitapaila, Kathmandu) by the hospital staffs from patients visiting the hospitals with suspected urinary tract infection. Samples were stored in a refrigerator at -20°C until used. Isolation of plasmid DNA was carried out using a boiling method [22 ]. In brief, a single colony of E. coli isolates were taken and cultured in 5 mL LB media (Thermofisher Scientific, USA) and grown overnight at 37°C at 200 rpm. Overnight grown cultures were transferred to a 1.5 mL microcentrifuge tube and centrifuged at 10,000 rpm for 8 minutes [23 (link)]. Supernatant was removed and 50 μL of Tris-EDTA (TE) buffer was added to the pellet. Centrifuge tubes were then vortexed until the pellets completely dissolved. Tubes were then kept in a water bath maintained at 100°C for 10 minutes. Afterwards, tubes were cooled on ice and then subjected for centrifugation at about 10000 rpm for 5 minutes. Supernatant was then transferred to another tube and stored at -20°C until use.

E. coli strain ClearColi^TM BL21 (DE3) (Lucigen) production hosts harboring pMCS69 plasmid containing the BP precursor synthesis and the respective pET14b plasmids encoding PhaC and PhaC-fusion proteins were grown in LB media (Thermo Fisher Scientific) at 37 °C and 200 rpm as previously described [35 (link),36 (link)]. Bioreactor vessels (Eppendorf BioFlo 320, Waterloo Rd, Macquarie Park NSW, Australia) were used and prepared according to the manufacturer’s instructions. The main cultures were prepared using animal component-free synthetic mineral media using glucose as the sole carbon source.
The cultures were grown until optical density 600 (OD₆₀₀) reached 0.5−0.8, and fusion protein production was induced by adding 1 mM isopropyl β-D-1- thiogalactopyranoside (Sigma-Aldrich, St. Louis, MO, USA). Cells were harvested by centrifugation and mechanically disrupted, and BPs were isolated as previously described [21 (link),22 (link),23 (link),37 (link),38 ]. The sterile BP vaccines were stored at 4 °C in tris buffered saline (TBS, pH 7.5) until formulation for animal trials and analysis.

C. albicans strains were grown in YPD media (1% yeast extract, 2% peptone, 2% dextrose)(Thermo Fisher Scientific) while shaking at 225rpm at 30°C [78 (link)]. In conditions where exogenous calcium was added, YPD broth was supplemented with CaCl₂ to 50mM media [67 (link)]. Minimal media (0.67% yeast nitrogen base without amino acids, 2% dextrose, 2% agar) [78 (link)] (Thermo Fisher Scientific) was used to remove the genomically integrated CRISPR/Cas9 cassette and nourseothricin selectable marker following successful gene deletion [79 (link)]. LB media (0.5% yeast extract, 1% tryptone, 1% NaCl) (Thermo Fisher Scientific) was used for the growth of DH5-α Escherichia coli strains (NEB) and cells were grown at 37°C on a rotator drum.

The synthesized SmCI-1 sequence was inserted into the pET-47b(+) bacterial expression vector (Novagen) following manufacturers specifications. BL21(DE3) (Thermo Fisher Scientific) cells were transfected with SmCI-1-containing pET-47b(+) vector, grown at 37°C in LB media (Thermo Fisher Scientific) containing 100μg/ml kanamycin (Thermo Fisher Scientific) and then induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) once the cells had grown to OD 600. Following ~4 hours of growth, the cells were concentrated by centrifugation at 10 000 rpm for 20 minutes at 4°C. The final weight of the bacteria pellet was measured and then the lysing reagent B-PER (Thermo Fisher Scientific) was added at a concentration of 4 ml/g of bacteria in combination with phenylmethylsufonyl fluoride (PMSF, final concentration of 1 mM), mixed gently and left to incubate at room temperature for 15 minutes. Before application to the 5ml 6xHIS column (GE Healthcare) for purification, the supernatant was diluted to a total protein concentration of 100 μg/ml in binding buffer (GE Healthcare). Following purification, the 6x HIS tag region of the recombinant protein was removed following the vector manufacturers specifications (Novagen). This recombinant SmCI-1 was used to generate a rabbit anti-SmCI-1 polyclonal antibody using the Custom pAb service offered by Genscript.

See Supplementary Table 1 for list of bacteria used in this study. Isolated colonies of bacteria were grown from −80°C stock aliquots frozen in 25% glycerol (Fisher) on solid or liquid LB media (Fisher) containing 200 μg/ml thymine (Arcos Pharmaceuticals) and selective antibiotics [50 μg/mL kanamycin (Sigma), 50 μg/mL ampicillin (Sigma), or 20 μg/mL chloramphenicol (Gold Biotechnology)] as required. Bacteria grown on solid media was incubated for 24–30 h at 37°C before use. Liquid media cultures were incubated in 50 mL sterile tubes for 20–24 h in a 37°C, 220 rpm dry shaking incubator. Strains grown for injection were washed with sterile phosphate buffered saline (PBS) (Rocky Mountain Biologicals) and concentration adjusted for injection (Supplementary Figure 1) and for in vitro cell viability assays.