RfaH contributes to maximal colonization and full virulence of hypervirulent Klebsiella pneumoniae

Hypervirulent <i>K. pneumoniae</i> (hvKp) have emerged as clinically important pathogens, posing a serious threat to human health. RfaH, a transcriptional elongation factor, has been regarded as implicated in facilitating the transcription of long virulence operons in certain bacterial species. In <i>K. pneumoniae</i>, RfaH plays a vital role in promoting CPS synthesis and hypermucoviscosity, as well as mediating bacterial fitness during lung infection. In this study, we aim to conduct a systematic investigation of the roles of rfaH in the survival, dissemination, and colonization of hvKp through <i>in vitro</i> and <i>in vivo</i> assays. We found that bacterial cells and colonies displayed capsule -deficient phenotypes subsequent to the deletion of <i>rfaH</i> in <i>K. pneumoniae</i> NTUH-K2044. We confirmed that <i>rfaH</i> is required for the synthesis of capsule and lipopolysaccharide (LPS) by positively regulating the expression of CPS and LPS gene clusters. We found that the Δ<i>rfaH</i> mutant led to a significantly decreased mortality of <i>K. pneumoniae</i> in a mouse intraperitoneal infection model. We further demonstrated that the absence of <i>rfaH</i> was associated with slower bacterial growth under conditions of low nutrition or iron limitation. Δ<i>rfaH</i> displayed reduced survival rates in the presence of human serum. Besides, the engulfment of the Δ<i>rfaH</i> mutant was significantly higher than that of NTUH-K2044 by macrophages <i>in vivo</i>, indicating an indispensable role of RfaH in the phagocytosis resistance of hvKp in mice. Both mouse intranasal and intraperitoneal infection models revealed a higher bacterial clearance rate of Δ<i>rfaH</i> in lungs, livers, and spleens of mice compared to its wild type, suggesting an important role of RfaH in the bacterial survival, dissemination, and colonization of hvKp <i>in vivo</i>. Histopathological results supported that RfaH contributes to the pathogenicity of hvKp in mice. In conclusion, our study demonstrates crucial roles of RfaH in the survival, colonization and full virulence of hvKp, which provides several implications for the development of RfaH as an antibacterial target.