RNA interactome profiling in hypervirulent Klebsiella pneumoniae reveals small RNA ArcZ as an inhibitor of mucoviscosity and virulence

Hypervirulent Klebsiella pneumoniae (HvKP) is an emerging human pathogen causing invasive infection in immune-competent hosts. The hypervirulence is strongly linked to the overproduction of hypermucovisous capsule, but the underlining regulatory mechanism of hypermucoviscosity (HMV) has been elusive, especially at the post-transcriptional level mediated by small RNAs (sRNAs). Using a recently developed RNA interactome profiling approach, we have investigated the Hfq-associated sRNA regulatory network and established the first in vivo RNA-RNA interactome in HvKP. Our data reveal numerous interactions between sRNAs and HMV-related mRNAs, and identify a plethora of sRNA that inhibit or promote HMV. One of the strongest repressors of HMV was ArcZ, a conserved sRNA in the Enterobacteriaceae family. We found that ArcZ is activated by the master regulator of catabolite repression Crp, and down-regulates the expression of mlaA encoding an outer-membrane lipoprotein, leading to decreased HMV and virulence attenuation in mice. ArcZ significantly reduced HMV in several carbapenem-resistant and hypervirulent clinical isolates with diverse genetic background, suggesting it is an antisense RNA inhibitor of HMV with therapeutic potential. In summary, our work provides a comprehensive map of the RNA-RNA interaction network of HvKP and identifies ArcZ as a conserved repressor of HMV, providing novel insights into the mechanisms of posttranscriptional regulations of virulence. To assess the the effect of expression of ArcZ and MlaA on transcriptome, we have first applied RNA-seq to biological triplicates of K. pneumoniae WT, ΔmlaA, ΔarcZ, ΔarcZ+pZE12-ArcZ and ΔarcZ+pZE12-ArcZ-M5. WT and ΔarcZ carry the empty vector. Strains were grown in LB media containing IPTG as sRNA expression inducer. Cells at OD600 3 were harvested to construct the strand-specific cDNA library and to be sequenced.