Abstract
ABSTRACTCurli 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.
Highlights
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
MS7163 is a curli-producing Uropathogenic Escherichia coli (UPEC) pyelonephritis strain resistant to LL-37 [24]
Because curli production and CR binding by E. coli are frequently associated with the synthesis of cellulose, we examined the MS7163 genes involved in cellulose regulation and biosynthesis
Summary
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. 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). Curli are amyloid fibers that form a key component of the UPEC extracellular biofilm matrix [18,19,20] They are highly proinflammatory [21], induce the formation of immunogenic complexes with DNA to stimulate autoimmune responses [22], and can neutralize human cathelicidin (LL-37), a soluble antimicrobial peptide that protects against UTI [23, 24]. Recent work has shown that E. coli cells produce a chemically modified phosphoethanolamine cellulose [30], and this polysaccharide can dampen curlistimulated immune induction [23, 31]
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