Contribution of Structural Domains to the Activity of Ribonuclease 7 against Uropathogenic Bacteria

Abstract

Ribonuclease 7 (RNase 7) is a 14.5-kDa peptide that possesses potent antimicrobial properties against Gram-negative and Grampositive bacteria and is expressed in a variety of epithelial tissues. Little is known about its mechanisms of action and the determinants of its antimicrobial properties. The objective of this study was to identify the intrinsic functional domains of RNase 7 that influence its activity against uropathogenic bacteria. A series of RNase 7 fragments were generated that contained different components of its secondary motifs starting from both the N terminus and the C terminus of RNase 7. We determined the antimicrobial properties of each fragment against both Gram-positive Staphylococcus saprophyticus and Gram-negative Escherichia coli and Proteus mirabilis. RNase 7 fragments displayed significant differences in their antimicrobial activity profiles. Compared to N-terminal fragments, C-terminal fragments showed uniformly decreased activity against Gram-negative E. coli and P. mirabilis and Gram-positive S. saprophyticus. Fragments that lack ␤-sheets 1, 3, and 4 also demonstrated significantly decreased activities. We have also identified one fragment with at least 4-fold increased potency against both E. coli and Staphylococcus compared to full-length peptide. We identified distinct regions of the peptide that are independently responsible for Gram-negative and Gram-positive activity. Our results suggest that distinct mechanisms are responsible for RNase 7's antimicrobial activity against various uropathogens.

Two Human Host Defense Ribonucleases against Mycobacteria, the Eosinophil Cationic Protein (RNase 3) and RNase 7

“…1C). Previous works have outlined that the main determinants for the human RNases antimicrobial action are clustered at the N terminus region, and derived peptides were designed as potential lead pharmacophores (39,40,64,65). Accordingly, synthetic peptides corresponding to the first 45 residues of both RNases, encompassing the first ␣1-␣3 helices (Fig.…”

Amplifying renal immunity: the role of antimicrobial peptides in pyelonephritis

“…98,104 The addition of recombinant RNase 7 peptide to cultured UPEC results in bactericidal and bacteriostatic activity at low micromolar concentrations, matching levels observed in human urine. 99,103 These findings suggest that RNase 7 constitutes a front-line epithelial-derived shield against invading uropathogens. 107 Unlike the epithelial-derived RNase 7, RNase 6 is a myeloid-derived AMP that is highly conserved in humans and rodents.…”

“…Indeed, members of AMP families such as defensins, ribonucleases, and cathelicidins exhibit salt and pH sensitivity. [52][53][54] Moreover, the reduction of disulphide bonds activates human β-defensin 1, a finding that has potential implications for tubules of the renal medulla, where lower oxygen tension could favour the predominance of reduced isoforms. 55 Thus, changes in renal physiology will have key implications for local AMP function in the kidney.…”

“…101 In addition to these lysine residues, distinct regions of RNase 7 seem to facilitate its antimicrobial activity against certain pathogens, including UPEC. 101,103 Both the ribonuclease and bactericidal activity of RNase 7 are regulated through its tight association with the ribonuclease inhibitor, which blocks binding of RNase 7 to bacterial cell wall components such as lipopolysaccharide. Expression of ribonuclease inhibitor decreases in pyelonephritis, attributed at least in part to its proteolytic degradation.…”

Carbonic anhydrase 2 deficiency leads to increased pyelonephritis susceptibility

“…Our group has demonstrated that certain urinary antimicrobial peptides can have a wide variety of activity at different urinary pHs. Specifically, activity is reduced at extremes of pH and optimum around physiologic 7.4 (48). The host must balance altering growth conditions for bacteria while not detrimentally affecting its innate defenses.…”