Role of GuaB, the inosine-5'-monophosphate dehydrogenase of uropathogenic Escherichia coli pathogenicity: a key factor for bladder infection

Abstract

Uropathogenic Escherichia coli (UPEC) is the primary causative agent of urinary tract infections (UTIs). This bacterium infects the bladder through the urinary tract, and some bacteria ascend to the kidneys, leading to more severe conditions such as pyelonephritis and sepsis. The infection of the bladder by UPEC is a critical initial step in the development of UTIs. Once inside the bladder, UPEC forms microcolonies both within and outside bladder epithelial cells, allowing it to persist in the bladder by resisting urine flow, innate immunity, and antimicrobials. In this study, to look for novel factors of UPEC that contribute to bladder infection and persistence, we analyzed proteins expressed at significant levels by UPEC in the bladder using a UTI mouse model. Mass spectrometry detected over 30 candidate proteins, including those already reported to play important roles in bladder infection, such as OmpA, components of the iron acquisition system, and the Tol/Pal system. Among them, GuaB (inosine-5'-monophosphate dehydrogenase) emerged as a key factor for bladder infection. The GuaB mutant showed impaired growth in urine-mimicking conditions and reduced infection efficiency, attributed to decreased GMP production. GuaB expression was upregulated in urine-like conditions, influenced by urea. This highlights GuaB's role in UPEC pathogenicity. These findings suggest GuaB as a potential target for new therapeutic strategies against UPEC-related UTIs, especially amid growing antimicrobial resistance.IMPORTANCEUropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infections (UTIs). UTI caused by UPEC is often recurrent, and repeated use of antimicrobial agents is feared to lead to the spread of drug-resistant strains. In fact, quinolone-resistant and extended spectrum β-lactamase-producing strains have been rapidly increasing since 2000. Therefore, improvement of current treatment methods, including new therapeutic agents against UPEC, is desired. In this study, we analyzed proteins significantly expressed in the bladder of UTI mice by proteomic analysis in order to identify new factors contributing to UPEC infection of the bladder. Among them, we found GuaB (inosine-5'-monophosphate dehydrogenase), which is important for bladder infection. Furthermore, we characterized the role of GuaB in bladder infection and the mechanism by which GuaB induces its expression in urine. Our findings will contribute not only to further understanding of UPEC pathogenesis but also to the development of new antimicrobial strategies.

Keywords: antimicrobial resistance (AMR); bacterial pathogenesis; biofilm; cystitis; inosine monophosphate dehydrogenase (IMPDH); proteome; pyelonephritis; urinary tract infection (UTI); uropathogenic Escherichia coli (UPEC); virulence.

Fig 1

Proteins from the bladders of infected and uninfected mice. Proteins were separated by SDS-PAGE using a 5%–20% acrylamide gel and stained with Coomassie brilliant blue. Lanes labeled NB are proteins isolated from the bladders of uninfected mice, and lanes labeled 4B and 6B are proteins isolated from the bladders of infected mice. Numbers 1–11, pointed to the right in red, indicate protein bands observed in the infected samples but not in the uninfected sample. Locations of molecular mass standards (in kDa) are indicated on the left (lane marked “M”).

Fig 2

Colonization by the UPEC GU2018_CL13 parent strain and its derivative mutants in the bladders of UTI mice. At 48 hours post-infection, cell numbers of bacteria isolated from the bladder were determined as CFU. Numbers of mice are five except for the parent strain, the guaB mutant, and the selD mutant. The data for the selD mutant are for four mice because one infected mouse died. The parent strain and the guaB mutant were also initially tested with five mice, but since the CFU of the guaB mutant infected group was very low at this point, therefore another five mice were added to both the parent and guaB mutant strains for confirmation. Each data point represents a sample from an individual mouse. Horizontal bars show geometric mean values. *P < 0.05. Asterisks denote significance for values relative to the parent strain. The P value was determined by the Mann-Whitney test.

Fig 3

Growth in the parent strain, the guaB mutant (guaB mut.) or the parent and the guaB mutant carrying the pTH18kr empty vector (parent/pTH18kr and guaB mut./pTH18kr) or pTH18krguaB, the guaB expression plasmid (guaB mut./pTH18krguaB). Bacteria were cultured in LB medium (A) and mAUM containing glycine with and without 2 mM xanthine or 10 mM guanosine monophosphate (GMP) (B through D). Bacterial growth was monitored by measuring OD₆₀₀. Data plotted are the means for two biological replicates; error bars indicate the ranges. We performed this assay twice, then similar results were obtained.

Fig 4

Internalization in uroepithelial cells, swarming motilities, and hemolytic activities of the parent strain, the guaB mutant (guaB mut.) or the parent and the guaB mutant carrying the pTH18kr empty vector (parent/pTH18kr and guaB mut./pTH18kr) or pTH18krguaB, the guaB expression plasmid (guaB mut./pTh18krguaB). (A) Numbers of internalized bacteria are represented. Data plotted are the means of three biological replicates; error bars indicate standard deviations, *P < 0.05. Asterisks denote significance for values relative to parent or parent/pTH18kr. The P value was determined by the unpaired t test. (B) Swarming motilities were evaluated as bacterial migrations on LB medium containing 0.25% agar. (C) Diameters reflecting bacterial migration on the agar. Data are means from three independent experiments; error bars indicate standard deviations. (D) Hemolysis is indicated by areas of transparency around the bacteria on LB agar plates containing 5% sheep erythrocytes and 10 mM CaCl₂. We performed this experiment twice, then similar results were obtained.

Fig 5

Transcript levels of guaA, guaB, fimH, and papG genes and guaB/A promoter activities. The parent strain was cultured in LB medium and mAUM containing glycine. (A) Transcript levels of target genes were described as relative values to that of rpoD (housekeeping gene). Data plotted are the means from three independent experiments; error bars indicate the standard deviations. (B) β-Galactosidase activities corresponding to guaB/A promoter activities in the parent strain containing pNNguaB-P, the lacZ reporter plasmid. Data plotted are the means for three biological replicates; error bars indicate the standard deviations. We performed this experiment twice, then similar results were obtained. *P < 0.05 relative to the value for LB medium. The P value was determined by the unpaired t test.

Fig 6

Regulatory factors of the guaB/A gene. (A) Transcript levels of the guaB/A-regulatory genes in the parent strain cultured in LB medium and mAUM containing glycine. Transcript levels of target genes were described as relative values to that of rpoD (housekeeping gene). Data plotted are the means from three independent experiments; error bars indicate the standard deviations. (B) Intracellular concentration of cAMP in the parent strain cultured in LB medium and mAUM containing glycine. The intracellular concentration of cAMP was the amount of cAMP (represented as pmol) per cells producing 1 mg of protein. Data plotted are the means for three biological replicates; error bars indicate the standard deviations. We performed this experiment twice, then similar results were obtained. (C) β-Galactosidase activities corresponding to guaB/A promoter activities in the parent strain containing pNNguaB-P, the lacZ reporter plasmid. Bacteria were cultured in LB medium with and without urea (170 mM), uric acid (0.4 mM), or creatinine (7 mM). Data plotted are the means for three biological replicates; error bars indicate the standard deviations. We performed this experiment twice, then similar results were obtained. *P < 0.05 relative to the value for plain LB medium. The P value was determined by the unpaired t test. (D) Growth in the parent strain cultured in LB medium with and without 170 mM urea. Bacterial growth was monitored by measuring OD₆₀₀. Data plotted are the means for two biological replicates; error bars indicate the ranges. We performed this assay twice, then similar results were obtained.