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 the non-hypervirulent strains which cause the majority of infections. We have performed genome-scale fitness profiling of a multidrug-resistant K. pneumoniae ST258 strain during infection of the insect Galleria mellonella, with the aim 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 that may be specific to ST258. Our analysis also identified a role for the metalloregulatory protein NfeR (also called YqjI) in virulence. Overall, this study offers new insight into the infection fitness landscape of K. pneumoniae ST258, and provides a framework for using the highly flexible, scalable G. mellonella infection model to dissect the molecular virulence mechanisms of K. pneumoniae and other bacterial pathogens.

An acquired acyltransferase promotes Klebsiella pneumoniae ST258 respiratory infection

“…Deletion of atf3 does not alter the physical properties of KP35 Neither the primary sequence nor the predicted protein structure of the KP35 atf3 gene product indicates its function. However, atf3 is colocated with multiple genes involved in capsular production (ugd, gnd; components of the cps cluster in K. pneumoniae) (Pan et al, 2015) and O-antigen synthesis (wbgU, tagGH) (Bruchmann et al, 2021;Caboni et al, 2015) (Figure 2A). To study the role of atf3 in pathogenesis, a null mutant was constructed using a recently adapted CRISPR-Cas9 system, Lambda Red Recombineering genes to improve efficiency, and Zeocin selection due to the presence of multiple antimicrobial resistance elements in the KP35 genome (McConville et al, 2020).…”

Anatomy of an Extensively Drug Resistant Klebsiella pneumoniae Outbreak in Tuscany, Italy

“…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 (ECA), a carbohydrate polymer thought to play a significant role in K. pneumoniae physiology and host interactions (26). Amino acid substitutions in the adhesin fimH and in the multifunctional regulator mgrA , were also observed in these serial isolates.…”

“…Using parsimony, we identified 8 variants found in all isolates from Italy that were missing in all ME genomes (node 1). Of particular interest were: (i) 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 (25), (ii) a NSY mutation in pabA involved in aromatic amino acid biosynthesis and previously characterized as a general infection requirement for K. pneumoniae (26), and (iii) a NSY mutation in hdfR encoding for a transcriptional regulator involved in the complex regulation of capsule production and hypermucoviscosity (27).…”

Transposon mutagenesis screen inKlebsiella pneumoniaeidentifies genetic determinants required for growth in human urine and serum

“…Transposon libraries have previously been generated in other K. pneumoniae strains, however these studies primarily focused on identifying genes that are required for a specific phenotype i.e antibiotic stress or capsular mutants, and therefore they lack a definitive list of essential genes for in vitro growth (65)(66)(67)(68)(69). To benchmark our library, we compared our data to previously published studies that did report K. pneumoniae essential gene lists: KPNIH1 (ST258), RH201207 (ST258) and ATCC 43816 (ST493) (30,70). As shown in Figure 3, the ECL8 strain is an acceptable representative for the K. pneumoniae KpI phylogroup as demonstrated by the phylogenetic and ANI (average nucleotide identity) analyses in the context of K.…”