Table 1.
Essential genes for ExPEC growth in serum.
Fig 1.
Overnight cultures of each indicated strain were inoculated 1:100 into LB medium (left panel) or fresh mouse serum (right panel). OD600 nm was measured every 0.5 hours. The ExPEC PCN033 strain served as a positive control with robust growth in serum, while the E. coli MC4100 strain was used as a negative control that was unable to grow in serum.
Fig 2.
De novo nucleotide biosynthesis genes are critical for ExPEC virulence.
A. Animal infection assays. Mice were intraperitoneally injected with 2 × 10⁵ CFU of mid-log phase cultures of each indicated strain. Bacterial burdens in tissues were quantified at each indicated time point. B. In vitro stress response assays. Mid-log phase cultures of each strain were diluted to 1 × 106 CFU/ml, and 5 μL of each cell suspension was spotted onto the indicated agar plates, followed by incubation at 37°C. C. Cell adhesion assay. PK-15 cells were infected with mid-log phase cultures of ExPEC PCN033 and its mutants at a multiplicity of infection (MOI) of 10:1 and incubated at 37°C for one hour. The cell culture was then washed twice with sterile PBS, followed by the addition of sterile water and incubation at 4°C for one hour to lyse cells. The lysate was serially diluted and plated on LB agar for bacterial enumeration. D. Intracellular survival assay. Bacterial cultures were mixed with RAW264.7 cells at an MOI of 10:1 and incubated at 37°C for one hour. After washing twice with sterile PBS, unphagocytized bacteria were killed by incubation with PBS containing 50 μg/ml chloramphenicol. Cells were then cultured in fresh DMEM. After two hours, macrophages were lysed as described above for bacterial enumeration. Statistical significance was assessed using one-way ANOVA. Significance levels were defined as ns (not significant), P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****).
Fig 3.
De novo nucleotide biosynthesis genes show significant upregulation during serum incubation and infection.
A. Statistics of up- and down-regulated genes in ExPEC after serum incubation. B. KEGG pathway enrichment analysis of differentially expressed genes (DEGs). The DEGs identified in transcriptomic analysis were subjected to KEGG pathway enrichment, with the top 10 enriched pathways shown. C. Schematic diagram of upregulated genes in the de novo nucleotide biosynthesis pathway. Upregulated genes are marked with brown boxes. D. Expression of the purE gene during mouse infection. ExPEC PCN033 carrying either pCDF-J23110-Lux or pCDF-purR-Lux was used to infect mice. Mice were humanely euthanized at indicated time points, followed by tissue collection and homogenization for luminescence measurement and bacterial load quantification. Statistical significance was assessed using one-way ANOVA. Significance levels were defined as ns (not significant) and P < 0.0001 (****).
Fig 4.
Expression analysis of de novo purine biosynthesis genes in ExPEC under different conditions and strains.
Gene expression was determined using RT-qPCR with WT, ΔpurR, and purR-OE strains in M9 medium (A) or serum (B), with or without supplementation of 0.1 mM guanine (Gua) or hypoxanthine (Hyp). Statistical significance was assessed using one-way ANOVA. Significance levels were defined as ns (not significant), P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).
Fig 5.
PurR contributes to in vivo fitness of ExPEC.
A. Competitive infection assays. Cells of ΔpurR or purR-OE strains were mixed with ExPEC PCN033 at a 1:1 ratio, and the mixture containing a total of 2 × 105 CFU was intraperitoneally injected into mice. At twelve hours post-infection, mice were humanely euthanized, and bacterial burden in tissue samples was quantified for each strain. The competitive index (CI) was calculated by dividing the ratio of the two strains’ loads in the tissues by their corresponding ratio in the inoculum. Statistical analysis was performed using Student’s t-test by comparing the CI of the indicated strain (ΔpurR or purR-OE) with that of the WT strain. Significance levels were defined as ns (not significant), P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***). B. Mouse survival assays. 6 × 105 CFU cells of the mid-log phase WT, ΔpurR, or purR-OE strains were used to intraperitoneally inject mice, respectively. Survival of each group was recorded every day. n = 8. Statistical differences were examined using the Gehan-Breslow-Wilcoxon test, and * indicates P < 0.05. C. In vivo colonization assays. 2 × 105 CFU cells of the mid-log phase WT, ΔpurR, or purR-OE strains were used to infect mice via intraperitoneal injection, respectively. Mice from each group were euthanized at 12, 24, and 36 hpi, and the blood, brain, liver, and spleen samples were collected. The tissues were homogenized, diluted with sterile saline, and plated on LA plates. Colonies were counted after overnight incubation. Statistical significance was assessed using one-way ANOVA. Significance levels were defined as ns (not significant), P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).
Fig 6.
Purine precursors influence the binding affinity of PurR to its target.
A. Quantification of intracellular purine precursors. ExPEC PCN033 was cultured to mid-log phase in either mouse serum or LB medium. Intracellular metabolites were extracted, and the concentrations of guanine and hypoxanthine were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). B. Molecular docking of PurR with guanine and hypoxanthine. The three-dimensional structures of PurR-guanine and PurR-hypoxanthine complexes, along with their ipTM and pTM scores, were predicted using a locally implemented AlphaFold3 server. The PurR protein is depicted in blue, while the ligands (guanine or hypoxanthine) are shown in red. The residues in the binding pocket are shown in green. C & E. Electrophoretic mobility shift assay (EMSA) analysis of PurR binding to the purE promoter region. Purified His-tagged PurR (C) or PurRmut (E) at various concentrations was incubated with the purE promoter DNA fragment in binding buffer at room temperature for 30 minutes. The reaction mixtures were separated on 6% native polyacrylamide gels, stained with GelRed, and visualized using a gel imaging system. D & F. Effect of guanine and hypoxanthine on PurR-DNA binding. PurR (D) or PurRmut (F) at a non-binding concentration (0.1 ng/μL) was incubated with the purE promoter region in the presence or absence of varying concentrations of guanine (Gua) or hypoxanthine (Hyp). The binding reactions were electrophoresed on 6% native polyacrylamide gels, stained with GelRed, and analyzed using a gel imaging system.