Identification of NAD-RNAs in the multidrug-resistant pathogen Klebsiella pneumoniae
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Abstract
Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme that is involved in many cellular processes. Pathogens often target the NAD metabolism during infection, and defects in this pathway have been linked to various human disorders. As a result, NAD metabolism has become a potential target for therapy. Recently, it was discovered that NAD can be covalently attached to the 5’ end of various RNAs, linking an important metabolite with gene expression. NAD-RNAs are present across all domains of life. In this study, we identified NAD-RNAs in the multidrug-resistant human pathogen Klebsiella pneumoniae using LC-MS/MS and NAD captureSeq. The synthesis and removal of cap structures are essential for RNA lifecycle regulation and gene expression control. NudC is a well-characterized Nudix hydrolase that specifically removes NAD caps from RNA. Our research showed that NudC from K. pneumoniae is active on NAD-RNAs. We examined the effects of knocking out nudC on bacterial phenotype, growth, proteome, metabolome, transcriptome, and NAD epitranscriptome. Our findings provided the first evidence that altered NAD-RNA metabolism impacts the virulence of K. pneumoniae. The thick extracellular polysaccharide capsule is one of the key virulence factors of K. pneumoniae. Using shotgun proteomics and liquid chromatography-mass spectrometry, we detected downregulation of two CPS-related proteins, Wzc and WbaP, which are conserved within the cps gene cluster and involved in capsule initiation and export. The Wzi protein catalyses the final step of CPS biosynthesis and plays an important role in capsule surface organization. NAD captureSeq data showed that the mRNA of wzi is NAD-capped, along with bfr, bfd, and fur, which encode proteins critical for iron homeostasis in bacterial cells, especially during infection. No significant differences in phenotype or growth were observed between the NudC mutant and the wild type. However, a significant reduction in phagocytosis occurred when human macrophages were infected with the NudC mutant. Additionally, we observed a decreased infection rate of ∆NudC strains in Galleria mellonella compared to wild-type K. pneumoniae.
We propose that deleting the decapping enzyme NudC changes the NAD-RNA metabolism in K. pneumoniae. We hypothesize that this, in turn, impacts the virulence of K. pneumoniae by disrupting CPS biosynthesis through altered gene expression of key components such as wzc, wbaP, and wzi, as well as affecting iron homeostasis by influencing gene expression of NAD-capped transcripts like bfr, bfd, and fur. However, the exact mechanism linking NAD-capped RNA metabolism to these virulence-related pathways is still unclear and requires further investigation.
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Issued: 2025-11-19
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FB17:Biologie
Language
en
Keywords
RNA modificationNAD-RNAK. pneumoniaeMacrophage infectionG. mellonella infectionTranscriptomicProteomicEpitranscriptomic
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Funding Organisations:
LOEWE Research Cluster Diffusible Signals
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Shanmuganathan, Hamrithaa: Identification of NAD-RNAs in the multidrug-resistant pathogen Klebsiella pneumoniae. : 2025-11-19.
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