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Abstract: FR-PO696

Ribonuclease Inhibitor Regulates Host Antimicrobial Activity toward Uropathogenic Escherichia coli

Session Information

  • Pediatric Nephrology - 1
    October 25, 2024 | Location: Exhibit Hall, Convention Center
    Abstract Time: 10:00 AM - 12:00 PM

Category: Pediatric Nephrology

  • 1900 Pediatric Nephrology

Authors

  • Ali, Yusuf, Kidney and Urinary Tract Center, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States
  • Cortado, Hanna H., Kidney and Urinary Tract Center, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States
  • Li, Birong, Kidney and Urinary Tract Center, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States
  • Becknell, Brian, Kidney and Urinary Tract Center, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States
Background

Members of the Ribonuclease A Superfamily exert bactericidal activity toward uropathogenic Escherichia coli (UPEC) and their activity is regulated by an endogenously expressed high affinity binding partner, Ribonuclease inhibitor (RI). Here, we tested the hypothesis that RI serves a critical role in regulating the antimicrobial activity of RNase 3.

Methods

UPEC colony forming units (CFU) were enumerated to assess the antimicrobial activity of recombinant RNase 3 protein in the presence and absence of RI. We tested the interaction, colocalization and UPEC mediated regulation of RNase 3 by RI in human MOLM-13 cells, monocytes, and neutrophils by co-immunoprecipitation (Co-IP), Western blotting and immunofluorescence (IF). Binding of RNase 3 with RI was studied by native gel electrophoresis and visualized by silver staining. Using Cre/LoxP recombination, we deleted RI in murine phagocytes. We transurethrally inoculated control and RI conditional knockout mice with UPEC and enumerated bacterial burden in urinary tract tissues.

Results

RNase 3 exhibited potent antimicrobial activity towards UPEC, which was suppressed by the addition of RI. RI and RNase 3 colocalize and form a complex in monocytes and neutrophils that dissociates with UPEC treatment. Recombinant RNase 3 and RI proteins associate directly in cell-free conditions, and this is disrupted by pre-incubation with H2O2, suggesting a redox dependent interaction. Phagocyte-specific RI conditional knockout mice exhibited augmented UPEC clearance during experimental UTI, consistent with a model in which RI limits antimicrobial RNase activity.

Conclusion

RI forms a reversible complex with RNase 3 to limit its antimicrobial activity toward UPEC. UPEC infection dissociates this complex – possibly as a consequence of oxidation - resulting in augmented RNase 3 activity and UPEC clearance. Studies in RI knockout mice substantiate its role as a negative regulator of phagocyte antimicrobial activity in vivo.