Discovery of New Genes Involved in Curli Production by a Uropathogenic Escherichia coli Strain from the Highly Virulent O45:K1:H7 Lineage

Abstract

Curli are bacterial surface-associated amyloid fibers that bind to the dye Congo red (CR) and facilitate uropathogenic Escherichia coli (UPEC) biofilm formation and protection against host innate defenses. Here we sequenced the genome of the curli-producing UPEC pyelonephritis strain MS7163 and showed it belongs to the highly virulent O45:K1:H7 neonatal meningitis-associated clone. MS7163 produced curli at human physiological temperature, and this correlated with biofilm growth, resistance of sessile cells to the human cationic peptide cathelicidin, and enhanced colonization of the mouse bladder. We devised a forward genetic screen using CR staining as a proxy for curli production and identified 41 genes that were required for optimal CR binding, of which 19 genes were essential for curli synthesis. Ten of these genes were novel or poorly characterized with respect to curli synthesis and included genes involved in purine de novo biosynthesis, a regulator that controls the Rcs phosphorelay system, and a novel repressor of curli production (referred to as rcpA). The involvement of these genes in curli production was confirmed by the construction of defined mutants and their complementation. The mutants did not express the curli major subunit CsgA and failed to produce curli based on CR binding. Mutation of purF (the first gene in the purine biosynthesis pathway) and rcpA also led to attenuated colonization of the mouse bladder. Overall, this work has provided new insight into the regulation of curli and the role of these amyloid fibers in UPEC biofilm formation and pathogenesis.

bolA gene involved in curli amyloids and fimbriae production in E. coli: exploring pathways to inhibit biofilm and amyloid formation

“…YESCA agar (Yeast extract and casaminoacid) plates with 30 μg mL −1 Congo red (YESCA + Congo red) were prepared and supplemented with appropriate concentrations of antibiotics and inducer aTc [7]. The plates were streaked with an overnight culture of cells and incubated at 26 °C for 48 h.…”

“…Among them, urinary tract infections (UTIs) affect millions of human beings and considered to be a community-acquired infection caused by the uropathogenic E. coli (UPEC) [4][5][6]. These UPEC strains build biofilms in the form of intracellular bacterial communities (IBCs) within the superficial facet cells of the bladder or on the surface of the bladder epithelium and catheters [7], while ExPEC strains cause a wide range of human diseases varying from elementary cystitis to bloodstream infections (BSI) with mortality up to 18% within 30-days [8]. Flagella adhesins, toxins, and siderophores are associated virulence factors found with UPEC, among which α-hemolysin is a toxin, produced by around 40-50% of UPEC isolates.…”

“…Biofilm formation in E. coli (e.g. UPEC) is controlled by several factors like curli, fimbriae, and auto transporter proteins such as adhesins, cellulose, and capsule as polysaccharides [7].…”

Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922

“…According to the analysis of transposon insertion realised in collaboration with BIOTECHVANA (Valencia, Spain), in the Tn463 mutant, the transposon was inserted in the csgA and cysB genes. The csgBAC operon encodes the curli major subunit protein CsgA, the curli nucleator protein CsgB, and the periplasmic chaperone CsgC [ 36 ]. The importance of curli fibers in biofilm formation has previously been demonstrated as being linked to the synthesis of the exopolysaccharide cellulose, a complementary factor of the biofilm matrix [ 37 ].…”

“…Thus the lack of purines tends to reduce RNA synthesis by RNA polymerase or amino acid starvation resulting in a deficient biofilm phenotype. Nhu et al found that the de novo purine biosynthesis is a critical pathway in curli production [ 36 ]. They hypothesise that a disruption in this pathway reduces the cyclic-di-GMP concentration inside the cell triggering the transcription of the master regulator csgD .…”