A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impact Klebsiella pneumoniae fitness

Abstract

Hypervirulent K. pneumoniae (hvKp) is a distinct pathotype that causes invasive community-acquired infections in healthy individuals. Hypermucoviscosity (hmv) is a major phenotype associated with hvKp characterized by copious capsule production and poor sedimentation. Dissecting the individual functions of CPS production and hmv in hvKp has been hindered by the conflation of these two properties. Although hmv requires capsular polysaccharide (CPS) biosynthesis, other cellular factors may also be required and some fitness phenotypes ascribed to CPS may be distinctly attributed to hmv. To address this challenge, we systematically identified genes that impact capsule and hmv. We generated a condensed, ordered transposon library in hypervirulent strain KPPR1, then evaluated the CPS production and hmv phenotypes of the 3,733 transposon mutants, representing 72% of all open reading frames in the genome. We employed forward and reverse genetic screens to evaluate effects of novel and known genes on CPS biosynthesis and hmv. These screens expand our understanding of core genes that coordinate CPS biosynthesis and hmv, as well as identify central metabolism genes that distinctly impact CPS biosynthesis or hmv, specifically those related to purine metabolism, pyruvate metabolism and the TCA cycle. Six representative mutants, with varying effect on CPS biosynthesis and hmv, were evaluated for their impact on CPS thickness, serum resistance, host cell association, and fitness in a murine model of disseminating pneumonia. Altogether, these data demonstrate that hmv requires both CPS biosynthesis and other cellular factors, and that hmv and CPS may serve distinct functions during pathogenesis. The integration of hmv and CPS to the metabolic status of the cell suggests that hvKp may require certain nutrients to specifically cause deep tissue infections.

Anatomy of an extensively drug-resistant Klebsiella pneumoniae outbreak in Tuscany, Italy

“…A total of eight variants were shared in all isolates from Italy but missing in all Middle East genomes (node 1). Of particular interest were 1) a predicted LOF mutation in cycA which encodes a serine/alanine transporter that, when functional, allows the antibiotic d -cycloserine to cross the cell wall and reach its cytoplasmic target ( 26 ), 2) an NSY mutation in pabA involved in aromatic amino acid biosynthesis and previously characterized as a general infection requirement for K. pneumoniae ( 27 ), and 3) an NSY mutation in hdfR encoding for a transcriptional regulator involved in the complex regulation of capsule production and hypermucoviscosity ( 28 ).…”

Mechanistic Insights into the Capsule-Targeting Depolymerase from a Klebsiella pneumoniae Bacteriophage

“…2c ). Note that while many genes are required for full capsule production or hypermucoidy in K. pneumoniae ATCC 43816 and its derivative K. pneumoniae KPPR1 ( 30 , 36 ), only those genes where mutation is predicted to result in a complete loss of capsule were required for RAD2 infection. To validate the role of the capsule for RAD2 infection, an acapsular mutant of B5055 which lacks wzb and wzc was used as an infection host ( 37 ).…”

Genomic insights into virulence factors affecting tissue-invasive Klebsiella pneumoniae infection

“…Nevertheless, our results indicate that the K1 genotype, rmpA , and aerobactin are substantial predictors of hvKp infection. Each of these virulence factors may affect pathogenesis independently but in a coordinated manner; a recent study suggested that capsule biosynthesis, hypermucoviscosity, and metabolism coordinately affect K. pneumoniae fitness [ 23 ]. Conversely, a positive string test was inferior to these predictors and was less accurate for identifying hvKp (sensitivity of 84.2%, specificity of 43.3%, and positive LR of 1.49 [95% CI: 0.99–1.79]) than reported previously (sensitivity of 89% and specificity of 91%) [ 2 ].…”