Figure 1.
Fluorescence difference in gel electrophoresis (2D-DIGE) of UPEC cytoplasmic proteins during growth in urine.
Soluble proteins (50 µg) from E. coli CFT073 cultured in urine were labeled with Cy3 (green), from CFT073 grown in LB with Cy5 (red), and the pooled internal standard representing an equal amount of urine and LB soluble proteins with Cy2 (blue). The labeled proteins (150 µg) were pooled and applied to a pH 4–7 IPG strip and second dimension 10% SDS-PAGE. Green spots indicate protein features induced in urine; red spots represent proteins induced in LB medium.
Table 1.
UPEC cytoplasmic proteins differentially expressed in human urine.
Table 2.
In vivo fitness for select 2D-DIGE mutants.
Figure 2.
In vivo contribution of UPEC arginine and serine biosynthesis.
Demonstration of auxotrophic phenotypes for (A) ΔargG and (B) ΔserA in MOPS defined medium containing 0.2% glucose and 10 mM of the indicated amino acid. (C) Growth in human urine. Growth curves represent the average measurement at each time point from triplicate experiments. Individual female mice were transurethrally inoculated with 2×108 CFU of a 1∶1 mixture of wild-type and mutant bacteria. In vivo fitness at 48 h post infection (hpi) for UPEC mutants defective in (D) arginine and (E) serine biosynthesis. (F) In vivo competition between arginine and serine auxotrophy. At 48 hpi, bladders and kidneys were aseptically removed, homogenized, and plated on LB or LB containing kanamycin to determine viable counts of wild-type and mutant strains, respectively. Each dot represents the log CFU/g from an individual animal. Bars represent the median CFU/g, and the limit of detection is 200 CFU. Significant differences in colonization levels (P<0.05) were determined using a two-tailed Wilcoxon matched pairs test.
Figure 3.
In vivo contribution of UPEC peptide substrate-binding proteins.
Individual female mice were transurethrally inoculated with 2×108 CFU of a 1∶1 mixture of wild-type and mutant bacteria. In vivo fitness at 48 hpi for UPEC mutants defective in import of dipeptides (A) ΔdppA or oligopeptides (B) ΔoppA. At 48 hpi, bladders and kidneys were aseptically removed, homogenized, and plated on LB or LB containing kanamycin to determine viable counts of wild-type and mutant strains, respectively. In vivo complementation of ΔdppA was performed by inoculating mice with a mixture of wild-type CFT073 containing pGEN empty vector and ΔdppA containing pGEN empty vector or pGEN-dppA. At 48 hpi, (C) bladders and (D) kidneys were aseptically removed, homogenized, and plated on LB with ampicillin or LB containing ampicillin and kanamycin to determine viable counts of wild-type (closed symbols) and mutant strains (open symbols), respectively. Each dot represents the log CFU/g from an individual animal. Bars represent the median CFU/g, and the limit of detection is 200 CFU. Significant differences in colonization levels (P<0.05) are indicated and were determined using a two-tailed Wilcoxon matched pairs test.
Figure 4.
In vitro growth of UPEC central metabolism mutants.
Optical density of wild-type UPEC and central metabolism mutants during growth in (A) pooled and sterilized human urine from 8–10 donors and in (B) MOPS defined medium containing 0.2% glucose as the sole carbon source. Growth curves represent the average measurement at each time point from triplicate experiments.
Figure 5.
In vivo fitness of UPEC central metabolism mutants.
Individual female mice were transurethrally inoculated with 2×108 CFU of a 1∶1 mixture of wild-type and mutant bacteria. In vivo fitness at 48 hpi for UPEC mutants defective in: (A,B) glycolysis, (C) pentose phosphate pathway, (D) Entner-Doudoroff pathway, (E) TCA cycle, and (F) gluconeogenesis. At 48 hpi, bladders and kidneys were aseptically removed, homogenized, and plated on LB or LB containing kanamycin to determine viable counts of wild-type and mutant strains, respectively. Each dot represents the log CFU/g from an individual animal. Bars represent the median CFU/g, and the limit of detection is 200 CFU. Significant differences in colonization levels (P<0.05) are indicated and were determined using a two-tailed Wilcoxon matched pairs test.
Figure 6.
In vivo complementation of UPEC ΔpckA.
Individual female mice were transurethrally inoculated with 2×108 CFU of a 1∶1 mixture of wild-type CFT073 containing pGEN empty vector and ΔpckA containing pGEN empty vector or pGEN-pckA. At 48 hpi, bladders were aseptically removed, homogenized, and plated on LB with ampicillin or LB containing ampicillin and kanamycin to determine viable counts of wild-type (closed symbols) and mutant strains (open symbols), respectively. Bars represent the median CFU/g, and the limit of detection is 200 CFU. Significant differences in colonization levels (P<0.05) are indicated and were determined using a two-tailed Wilcoxon matched pairs test.
Table 3.
Growth of central metabolism mutants in vitro and in vivo.
Figure 7.
UPEC acquires amino acids and requires gluconeogenesis and the TCA cycle for fitness in vivo.
Peptide substrate-binding protein genes dppA and oppA are required to import di- and oligopeptides into the cytoplasm from the periplasm. Short peptides are degraded into amino acids in the cytoplasm and converted into pyruvate and oxaloacetate. Pyruvate is converted into acetyl-CoA and enters the TCA cycle to replenish intermediates and generate oxaloacetate. Oxaloacetate is converted to phosphoenolpyruvate by the pckA gene product during gluconeogenesis. Mutations in the indicated genes dppA, oppA, pckA, sdhB, and tpiA demonstrated fitness defects in vivo.