Human Ribonuclease 6 Has a Protective Role during Experimental Urinary Tract Infection

Ruiz-Rosado, Juan de Dios; Cortado, Hanna; Kercsmar, Macie; Li, Birong; Ballash, Gregory; Cotzomi-Ortega, Israel; Sanchez-Zamora, Yuriko I.; Gupta, Sudipti; Ching, Christina; Boix, Ester; Jackson, Ashley R.; Spencer, John David; Becknell, Brian

doi:10.1159/000534736

Cited by 5 publications

(5 citation statements)

References 43 publications

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“…Human and mouse RNase 6 kill UPEC at low micromolar concentrations without exerting cytotoxicity toward host cells [ 23 , 24 ]. Recently, we determined that human RNASE6 transgenic mice are protected from experimental UTI, consistent with the concept that future interventions to augment the level and/or activity of RNase 6 may reduce the incidence and severity of UTI [ 25 , 26 ]. In this study, we generated mice with a Rnase6 EGFP knock-in allele to study the cellular sources of Rnase6 along with the consequences of its deletion on UPEC killing and susceptibility to experimental UTI.…”

Section: Introductionsupporting

confidence: 66%

“…Isolation of total RNA, reverse transcription, and quantitative PCR detection were performed as described [ 25 ]. Results were analyzed by the 2 −ΔΔCT method, normalizing to Gapdh .…”

Section: Methodsmentioning

confidence: 99%

“…Bone marrow-derived macrophages (BMDMs) were derived as described and seeded into 24-well plates at 200,000 cells/well in antibiotic-free medium [ 25 ]. The next day, UPEC was added at the indicated multiplicity of infection (MOI) and the plate was centrifuged 750 g for 5 min to allow for efficient UPEC attachment.…”

Section: Methodsmentioning

confidence: 99%

“…Rnase6 +/+ and Rnase6 EGFP/EGFP BMDMs were incubated with UPEC (MOI 10) for 30 min and 3 h to investigate early apoptosis and late apoptosis/necrosis, respectively. Cells undergoing apoptosis and/or necrosis were identified by flow cytometry based on Annexin V and 7-AAD positivity as described [ 25 ].…”

Section: Methodsmentioning

confidence: 99%

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Murine Ribonuclease 6 Limits Bacterial Dissemination During Experimental Urinary Tract Infection

Cortado,

Kercsmar,

et al. 2024

J Innate Immun

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Introduction: The Ribonuclease (RNase) A superfamily encodes cationic antimicrobial proteins with potent microbicidal activity toward uropathogenic bacteria. Ribonuclease 6 (RNase6) is an evolutionarily conserved, leukocyte-derived antimicrobial peptide with potent microbicidal activity toward uropathogenic Escherichia coli (UPEC), the most common cause of bacterial urinary tract infections (UTI). In this study, we generated Rnase6 deficient mice to investigate the hypothesis that endogenous RNase 6 limits host susceptibility to UTI. Methods: We generated a Rnase6EGFP knock-in allele to identify cellular sources of Rnase6 and determine the consequences of homozygous Rnase6 deletion on antimicrobial activity and UTI susceptibility. Results: We identified monocytes and macrophages as the primary cellular sources of Rnase6 in bladders and kidneys of Rnase6EGFP/+ mice. Rnase6 deficiency (i.e., Rnase6EGFP/EGFP) resulted in increased upper urinary tract UPEC burden during experimental UTI, compared to Rnase6+/+ controls. UPEC displayed increased intracellular survival in Rnase6 deficient macrophages. Conclusion: Our findings establish that RNase6 prevents pyelonephritis by promoting intracellular UPEC killing in monocytes and macrophages and reinforce the overarching contributions of endogenous antimicrobial RNase A proteins to host UTI defense.

show abstract

Section: Introductionsupporting

confidence: 66%

“…Isolation of total RNA, reverse transcription, and quantitative PCR detection were performed as described [ 25 ]. Results were analyzed by the 2 −ΔΔCT method, normalizing to Gapdh .…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Murine Ribonuclease 6 Limits Bacterial Dissemination During Experimental Urinary Tract Infection

Cortado,

Kercsmar,

et al. 2024

J Innate Immun

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“…Interestingly, recombinant human and mouse RNase 6 proteins exhibited bactericidal activity toward uropathogenic Escherichia coli (UPEC) at low-micromolar concentrations [ 16 ]. Accordingly, RNASE 6 transgenic mice are less susceptible to UPEC induced experimental UTI than non-transgenic controls [ 29 ]. In this study, we have characterized the most common non-synonymous RNASE6 SNP in the human population and assessed its impact on RNase 6 antimicrobial activity toward UPEC.…”

Section: Introductionmentioning

confidence: 99%

A Common Polymorphism in RNASE6 Impacts Its Antimicrobial Activity toward Uropathogenic Escherichia coli

Anguita,

Prats-Ejarque,

Moussaoui

et al. 2024

IJMS

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Human Ribonuclease (RNase) 6 is a monocyte and macrophage-derived protein with potent antimicrobial activity toward uropathogenic bacteria. The RNASE6 gene is heterogeneous in humans due to the presence of single nucleotide polymorphisms (SNPs). RNASE6 rs1045922 is the most common non-synonymous SNP, resulting in a G to A substitution that determines an arginine (R) to glutamine (Q) transversion at position 66 in the protein sequence. By structural analysis we observed that R66Q substitution significantly reduces the positive electrostatic charge at the protein surface. Here, we generated both recombinant RNase 6-R66 and -Q66 protein variants and determined their antimicrobial activity toward uropathogenic Escherichia coli (UPEC), the most common cause of UTI. We found that the R66 variant, encoded by the major SNP rs1045922 allele, exhibited superior bactericidal activity in comparison to the Q66 variant. The higher bactericidal activity of R66 variant correlated with an increase in the protein lipopolysaccharide binding and bacterial agglutination abilities, while retaining the same enzymatic efficiency. These findings encourage further work to evaluate RNASE6 SNP distribution and its impact in UTI susceptibility.

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Bacterial RNA sensing by TLR8 requires RNase 6 processing and is inhibited by RNA 2’O-methylation

Nunes,

Breitenbach,

Pawusch

et al. 2024

EMBO Rep

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TLR8 senses single-stranded RNA (ssRNA) fragments, processed via cleavage by ribonuclease (RNase) T2 and RNase A family members. Processing by these RNases releases uridines and purine-terminated residues resulting in TLR8 activation. Monocytes show high expression of RNase 6, yet this RNase has not been analyzed for its physiological contribution to the recognition of bacterial RNA by TLR8. Here, we show a role for RNase 6 in TLR8 activation. BLaER1 cells, transdifferentiated into monocyte-like cells, as well as primary monocytes deficient for RNASE6 show a dampened TLR8-dependent response upon stimulation with isolated bacterial RNA (bRNA) and also upon infection with live bacteria. Pretreatment of bacterial RNA with recombinant RNase 6 generates fragments that induce TLR8 stimulation in RNase 6 knockout cells. 2’O-RNA methyl modification, when introduced at the first uridine in the UA dinucleotide, impairs processing by RNase 6 and dampens TLR8 stimulation. In summary, our data show that RNase 6 processes bacterial RNA and generates uridine-terminated breakdown products that activate TLR8.

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Human Ribonuclease 6 Has a Protective Role during Experimental Urinary Tract Infection

Cited by 5 publications

References 43 publications

Murine Ribonuclease 6 Limits Bacterial Dissemination During Experimental Urinary Tract Infection

Murine Ribonuclease 6 Limits Bacterial Dissemination During Experimental Urinary Tract Infection

A Common Polymorphism in RNASE6 Impacts Its Antimicrobial Activity toward Uropathogenic Escherichia coli

Bacterial RNA sensing by TLR8 requires RNase 6 processing and is inhibited by RNA 2’O-methylation

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