Ribonuclease 7 polymorphism rs1263872 reduces antimicrobial activity and associates with pediatric urinary tract infections
Keith R. Pierce, … , David S. Hains, John D. Spencer
Keith R. Pierce, … , David S. Hains, John D. Spencer
Published November 15, 2021
Citation Information: J Clin Invest. 2021;131(22):e149807. https://doi.org/10.1172/JCI149807.
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Ribonuclease 7 polymorphism rs1263872 reduces antimicrobial activity and associates with pediatric urinary tract infections

Abstract

Ribonuclease 7 (RNase 7) is an antimicrobial peptide that prevents urinary tract infections (UTI); however, it is yet unknown how RNASE7 genetic variations affect its antimicrobial activity and its mitigation of UTI risk. This study determined whether the RNASE7 SNP rs1263872 is more prevalent in children with UTI and defined how rs1263872 affects RNase 7’s antimicrobial activity against uropathogenic E. coli (UPEC). We performed genotyping for rs1263872 in 2 national UTI cohorts, including children enrolled in the Randomized Intervention for Children with Vesicoureteral Reflux trial or the Careful Urinary Tract Infection Evaluation study. Genotypes from these cohorts were compared with those of female controls with no UTI. To assess whether rs1263872 affects RNase 7’s antimicrobial activity, we generated RNase 7 peptides and genetically modified urothelial cultures encoding wild-type RNase 7 and its variant. Compared with controls, girls in both UTI cohorts had an increased prevalence of the RNASE7 variant. Compared with the missense variant, wild-type RNase 7 peptide showed greater bactericidal activity against UPEC. Wild-type RNase 7 overexpression in human urothelial cultures reduced UPEC invasive infection compared with mutant overexpression. These results show that children with UTI have an increased prevalence of RNASE7 rs1263872, which may increase UTI susceptibility by suppressing RNase 7’s antibacterial activity.

Authors

Keith R. Pierce, Tad Eichler, Claudia Mosquera Vasquez, Andrew L. Schwaderer, Aaron Simoni, Steven Creacy, David S. Hains, John D. Spencer

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Figure 2

Compared with wild-type RNase 7, RNASE7103Ala has reduced antimicrobial activity.

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Compared with wild-type RNase 7, RNASE7103Ala has reduced antimicrobial ...
(A) E. coli strains were incubated with 2.5 μM RNASE7103Ala (diamonds) or wild-type RNASE7103Pro (triangles). An irrelevant peptide (circles) served as control. After incubation, UPEC strains were plated on LB agar and colonies were enumerated. Results are from 4 to 5 experiments performed in duplicate (n = 4–5). Data shown represent percentages of remaining colony-forming units (mean ± SEM). (B) Displacement of LPS-bound Bodipy TR Cadaverine with 0.5 μM RNase A (circles, negative control), RNASE7103Ala (diamonds), and wild-type RNASE7103Pro (triangles). Results show the mean and SEM from 4 experiments performed in duplicate (n = 4). (C and D) UROtsa cells were infected with retroviral constructs expressing wild-type RNASE7103Pro, RNASE7103Ala, or empty vector. Mean ± SEM. (C) Representative Western blot, probed for RNase 7 and GAPDH. (D) ELISA quantified RNase 7 in culture media. Results are from 3 experiments performed in triplicate (n = 3). Mean ± SEM. (E) Extracellular bactericidal activity assays were performed using culture media from empty vector (circles), RNASE7103Ala (diamonds), and RNASE7103Pro (triangles) retrovirus-infected cells, as described in the Supplemental Methods. Graphs show the mean UPEC survival and SEM. Results are from 4 to 5 experiments performed in triplicate (n = 4–5)(mean ± SEM). (F and G) UPEC attachment (F) and invasion (G) assays were performed as outlined in the Supplemental Methods. Shown are the percentage of UPEC adhering to the cellular surface and invading empty vector– (circles), RNASE7103Ala- (diamonds), and RNASE7103Pro-transduced (triangles) UROtsa cells. Results are from 5 to 6 experiments performed in quadruplicate (n = 5–6) (mean ± SEM). ND, not detected. (H) To account for the attenuated attachment of MDR12 and UTI89ΔfimH, the ratio of UPEC attachment in RNASE7103Ala- (diamonds) and RNASE7103Pro-overexpressing (triangles) cells compared with empty vector–expressing cells is shown. (A, B, and D–G) Pairwise comparisons were made by 1-way ANOVA with Tukey’s modification or (H) Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.