The Capsule Regulatory Network of Klebsiella pneumoniae Defined by density-TraDISort

Klebsiella pneumoniae infections affect infants and the immunocompromised, and the recent emergence of hypervirulent and multidrug-resistant K. pneumoniae lineages is a critical health care concern. Hypervirulence in K. pneumoniae is mediated by several factors, including the overproduction of extracellular capsule. However, the full details of how K. pneumoniae capsule biosynthesis is achieved or regulated are not known. We have developed a robust and sensitive procedure to identify genes influencing capsule production, density-TraDISort, which combines density gradient centrifugation with transposon insertion sequencing. We have used this method to explore capsule regulation in two clinically relevant Klebsiella strains, K. pneumoniae NTUH-K2044 (capsule type K1) and K. pneumoniae ATCC 43816 (capsule type K2). We identified multiple genes required for full capsule production in K. pneumoniae, as well as putative suppressors of capsule in NTUH-K2044, and have validated the results of our screen with targeted knockout mutants. Further investigation of several of the K. pneumoniae capsule regulators identified—ArgR, MprA/KvrB, SlyA/KvrA, and the Sap ABC transporter—revealed effects on capsule amount and architecture, serum resistance, and virulence. We show that capsule production in K. pneumoniae is at the center of a complex regulatory network involving multiple global regulators and environmental cues and that the majority of capsule regulatory genes are located in the core genome. Overall, our findings expand our understanding of how capsule is regulated in this medically important pathogen and provide a technology that can be easily implemented to study capsule regulation in other bacterial species.