Purine Biosynthesis Metabolically Constrains Intracellular Survival of Uropathogenic Escherichia coli

Abstract

The ability to de novo synthesize purines has been associated with the intracellular survival of multiple bacterial pathogens. Uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections, undergoes a transient intracellular lifestyle during which bacteria clonally expand into multicellular bacterial communities within the cytoplasm of bladder epithelial cells. Here, we characterized the contribution of the conserved de novo purine biosynthesis-associated locus cvpA-purF to UPEC pathogenesis. Deletion of cvpA-purF, or of purF alone, abolished de novo purine biosynthesis but did not impact bacterial adherence properties in vitro or in the bladder lumen. However, upon internalization by bladder epithelial cells, UPEC deficient in de novo purine biosynthesis was unable to expand into intracytoplasmic bacterial communities over time, unless it was extrachromosomally complemented. These findings indicate that UPEC is deprived of purine nucleotides within the intracellular niche and relies on de novo purine synthesis to meet this metabolic requirement.

Matrix stiffness regulates endosomal escape of uropathogenicE. coli

“…The specificity of the Lamp1 antibody was confirmed by staining lamp1 −/− BECs (Figure S1b). To verify that infection on soft gels resulted in cytoplasmic UPEC we used a chloroquine‐resistance assay (Fernandez‐Prada et al, ; Paetzold, Lourido, Raupach, & Zychlinsky, ; Shaffer et al, ). Chloroquine is lysosomotropic and concentrates within vesicular compartments; treating BECs with 2.5 mg/ml chloroquine leads to therapeutic levels of the drug within vesicles thereby selectively killing endosomal UPEC (Shaffer et al, ).…”

“…These morphological changes were not due merely to seeding on PA gels; when BECs were grown on gels with an elastic modulus of 30 kPa they became morphologically indistinguishable from those grown on glass coverslips ( Figure 1b The specificity of the Lamp1 antibody was confirmed by staining lamp1 −/− BECs ( Figure S1b). To verify that infection on soft gels resulted in cytoplasmic UPEC we used a chloroquine-resistance assay (Fernandez-Prada et al, 2000;Paetzold, Lourido, Raupach, & Zychlinsky, 2007;Shaffer et al, 2016). Chloroquine is lysosomotropic and concentrates within vesicular compartments;…”

“…treating BECs with 2.5 mg/ml chloroquine leads to therapeutic levels of the drug within vesicles thereby selectively killing endosomal UPEC (Shaffer et al, 2016). In infected BECs seeded on rigid substrates, chloroquine caused a 10-fold reduction in UPEC viability, whereas no significant effect was seen with cells seeded on soft gels ( Figure 1d); a similar dependence on substrate stiffness was observed with UPEC strain CFT073 (Mobley et al, 1990) demonstrating that mechanical regulation of endosomal escape was not strain specific ( Figure 1e).…”

Transposon-insertion sequencing screens unveil requirements for EHEC growth and intestinal colonization

“…6A) . cvpA lies upstream of the purine biosynthesis locus purF , and some Δ cvpA mutant phenotypes have been attributed to reduced expression on purF due to polar effects (103,106). The growth of the EHEC Δ cvpA mutant was not impaired in the absence of exogenous purines (Fig S5C), suggesting the cvpA deletion does not adversely modify purF expression.…”

Invasion of Host Cells and Tissues by Uropathogenic Bacteria

“…These include type 1 pili, the adhesin Ag43, capsule, OmpA, purine biosynthesis enzymes, and various regulators like Integration Host Factor (IHF), the QseC sensor kinase, the RNA chaperone Hfq, and the periplasmic prolyly isomerase and chaperone SurA (Fig. 5) (28, 121–129). Bacteria growing within IBCs appear to utilize non-glucose carbon sources such as galactoside and express stress resistance genes like yeaR that enable UPEC to better deal with oxidative and nitrosative stresses (97, 130).…”