Identifying virulence determinants of multidrug-resistant Klebsiella pneumoniae in Galleria mellonella

Abstract

Infections caused by Klebsiella pneumoniae are a major public health threat. Extensively drug-resistant and even pan-resistant strains have been reported. Understanding K. pneumoniae pathogenesis is hampered by the fact that murine models of infection offer limited resolution for non-hypervirulent strains which cause the majority of infections. The insect Galleria mellonella larva is a widely used alternative model organism for bacterial pathogens. We have performed genome-scale fitness profiling of a multidrug-resistant K. pneumoniae ST258 strain during infection of G. mellonella, to determine if this model is suitable for large-scale virulence factor discovery in this pathogen. Our results demonstrated a dominant role for surface polysaccharides in infection, with contributions from siderophores, cell envelope proteins, purine biosynthesis genes and additional genes of unknown function. Comparison with a hypervirulent strain, ATCC 43816, revealed substantial overlap in important infection-related genes, as well as additional putative virulence factors specific to ST258, reflecting strain-dependent fitness effects. Our analysis also identified a role for the metalloregulatory protein NfeR (YqjI) in virulence. Overall, this study offers new insight into the infection fitness landscape of K. pneumoniae, and provides a framework for using the highly flexible and easily scalable G. mellonella infection model to dissect molecular virulence mechanisms of bacterial pathogens.

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 ).…”

“…NSY mutations were present in genes involved in cell-wall biosynthesis/recycling ( rlpA , emtA ) and surface polysaccharide synthesis ( rffG ). The latter is part of a gene cluster that encodes the enterobacterial common antigen, a carbohydrate polymer thought to play a significant role in K. pneumoniae physiology and host interactions ( 27 ). Amino acid substitutions in the adhesin fimH and in the multifunctional regulator mgrA were also observed in these serial isolates.…”

Klebsiella pneumoniae Biofilms and Their Role in Disease Pathogenesis

“…The Galleria , however, is a controversial model. Bruchmann and colleagues, have recently published a study where they explore this model to identify genes related to MDR strains, and the model showed itself very useful and the authors were able to describe several genes related to virulence factors that are involved in biofilm formation in MDR strains ( Bruchmann et al., 2021 ). On the other hand, Russo and Macdonald have applied two models to evaluate the difference between hypervirulent and classic (less virulent) K. pneumoniae strains, and found that the mouse infection model was much more accurate than the Galleria model to differentiate the two groups which represent a disadvantage for this model ( Russo and MacDonald, 2020 ).…”

Anti-Inflammatory Metabolites in the Pathogenesis of Bacterial Infection

“…Exposure to adenosine precursors such as IMP enhances its hypermucoviscosity and production of capsular polysaccharide (CPS), both associated with infection of the alveolar epithelium ( Mike et al., 2021 ). The ability to hydrolyze and synthesize purines has been associated with the intracellular persistence of both classical and hypervirulent strains of K. pneumoniae in lung infection ( Bachman et al., 2015 ; Bruchmann et al., 2021 ). Mutations in the cytoplasmic protein adenylosuccinate synthase ( purA ) prevented K. pneumoniae from repurposing intracellular nucleosides for CPS biosynthesis and exhibited growth defects along with mutants purF , purL and purH ( Mike et al., 2021 ).…”