Hypervirulent Klebsiella pneumoniae (HvKP) causes severe infections in healthy humans, leading to diseases such as pneumonia, liver abscess, meningitis, and sepsis. The major virulence factor of HvKP is the highly viscous polysaccharides on the bacterial surface, which help the bacterium escape the host immune system. Understanding how the synthesis of capsule polysaccharides is regulated and how the capsular viscosity is generated are key to understand the pathogenesis of HvKP.

A research team led by Dr. CHAO Yanji at the Shanghai Institute of Immunity and Infection of the Chinese Academy of Sciences revealed that noncoding small RNAs effectively inhibited HvKP superbug infection by targeting a novel virulence mechanism. The study was published in Nature Communications.

Small RNAs are the most abundant class of post-transcriptional regulators in bacterial pathogens, which control the expression of many other messenger RNAs via direct RNA-RNA interactions. In this study, researchers established a global RNA-RNA interaction network in HvKP using iRIL-seq, a recently developed RNA-RNA interactome profiling technology, and discovered that small RNAs inhibit the synthesis of capsule polysaccharides and a key membrane transporter required for virulence.

Based on the RNA-RNA interactome data, researchers screened more than 20 different small RNAs. They found that the small RNA ArcZ had the strongest inhibitory effect on capsular mucoviscosity, and significantly inhibited the pathogenicity of HvKP in animals. The inhibitory effect of ArcZ was highly conserved, independent of the genotype and capsule type of strains. ArcZ significantly inhibited the capsular mucoviscosity in several different hypervirulent and drug-resistant clinical strains, suggesting that ArcZ is a potent RNA inhibitor of superbugs with therapeutic potential in clinics.

Besides, researchers identified that the expression of ArcZ was activated by carbon metabolism signals and the related transcription factor CRP, and that the ArcZ downstream targets genes were mlaA and fbp, which encode the outer membrane phospholipid transporter protein MlaA and the fructose-1,6-bisphosphatase Fbp, respectively. They demonstrated that ArcZ inhibited the expression of mlaA and fbp mRNAs by a direct RNA-RNA basepairing interaction mechanism, revealing a novel CRP-ArcZ-MlaA capsular hypermucoviscosity regulatory pathway.

Since the outer membrane phospholipid transporter protein MlaA is not directly involved in the synthesis of capsule polysaccharides, researchers further elucidated the molecular mechanism by which MlaA regulates capsular mucoviscosity. Through gene knockout, single-copy gene complementation and mutation of key amino acid residuals, they found that the MlaA protein and its phospholipid transporter activity were essential for both capsular mucoviscosity and pathogenicity, establishing that MlaA is a novel virulence factor in HvKP. Inactivation of MlaA led to a significant reduction in the capsular mucoviscosity as well as a significant reduction in bacterial virulence in mice, revealing a virulence attenuation approach potentially useful for vaccine development.

This study reveals a novel virulence mechanism underlying the hypervirulent superbug infections, and identifies a sRNA molecule that neutralized this virulence mechanism. It will promote the development of novel anti-infective drugs and effective vaccines to curb superbug infections.