Synergy between Group 2 capsules and lipopolysaccharide underpins serum resistance in Extra-intestinal Pathogenic Escherichia coli

Abstract

Escherichia coli (E. coli) is a major cause of urinary tract infections, bacteraemia, and sepsis. CFT073 is a prototypic, urosepsis isolate of sequence type (ST) 73. This laboratory, among others, has shown that strain CFT073 is resistant to serum, with capsule and other extracellular polysaccharides imparting resistance. The interplay of such polysaccharides remains under-explored. This study has shown that CFT073 mutants deficient in lipopolysaccharide (LPS) O-antigen and capsule display exquisite serum sensitivity. Additionally, O-antigen and LPS outer core mutants displayed significantly reduced surface K2 capsule, coupled with increased unbound K2 capsule being detected in the supernatant. The R1 core and O6 antigen are involved in the tethering of K2 capsule to the CFT073 cell surface, highlighting the importance of the R1 core in serum resistance. The dependence of capsule on LPS was shown to be post-transcriptional and related to changes in cell surface charge. Furthermore, immunofluorescence microscopy suggested that the surface pattern of capsule is altered in such LPS core mutants, which display a punctate capsule expression. Finally, targeting LPS biosynthesis using sub-inhibitory concentrations of a WaaG inhibitor resulted in increased serum sensitivity, antibiotic sensitivity, and reduced capsule in CFT073. Interestingly, the dependency of capsule on LPS has been observed previously in several Klebsiella pneumoniae isolates and a neonatal meningitic E. coli strain, indicating that the synergy between these polysaccharides is not just strain, serotype or species-specific but may be conserved across several pathogenic Gram-negative species. Therefore, using WaaG inhibitor derivatives or phage-derived depolymerases to target LPS is a promising avenue for co-administration with antibiotics to reduce morbidity and mortality by reducing or eliminating surface capsule. Impact statementDisseminated infections caused by E. coli place a large burden on healthcare systems globally, with incidence as well as antimicrobial resistance on the rise (1, 2). The key to designing successful therapeutic strategies is to understand how and why bacteria can cause infection, with an increased need for alternative/ preventative therapies in the age of antimicrobial resistance. ST131 is the predominant and most significant clonal group of bloodstream isolates currently (3-6). The second-most frequently isolated clonal group in E. coli bloodstream infections is that of ST73, into which the prototype used in this study, CFT073, is categorised (3, 5-7). Whilst antimicrobial resistance is not currently widespread in the ST73 clonal group, patterns of drug resistance are emerging (7). The clonal group is also associated with higher virulence scores than ST131, highlighting a need to better understand ST73 virulence to identify potential therapeutic intervention strategies (6). This study has identified a synergy between common virulence factors of sepsis-associated E. coli, capsule and LPS, which can be exploited to reduce virulence, a promising prospect in the age of antimicrobial resistance. Data summaryOligonucleotide primers for mutagenesis as well as schematic representations of gene clusters were designed based on the assembled genome sequence of strain CFT073 (GenBank accession number: AE014075.1). The authors confirm all other supporting data, code and protocols have been provided within the article or through supplementary data files.