Understanding the contribution of CRISPR CAS adaptive immune system in pathogenicity and virulence of Klebsiella pneumoniae

Abstract

This study explored the contribution of CRISPR-Cas systems in the pathogenicity and virulence of Klebsiella pneumoniae, using the standard ATCC 43816 strain and clinical isolates. The study conducted in-depth in silico analysis of CRISPR-Cas systems in Klebsiella species, identifying three distinct subtypes (type IE, type IE*, and type IF) and their respective genomic locations. Spacer analysis revealed limited targeting of plasmids and phages. In clinical isolates, CRISPR-Cas system was found in 12% of the strains, type IE* being more prevalent (80%) than type IE (20%) system. Sequencing of type IE* and IE CRISPR systems revealed differences in spacer targeting, protospacer adjacent motifs, and direct repeats. Notably, spacers from type IE* strains exhibited enhanced plasmid and phage targeting. Furthermore, an association was observed between CRISPR-Cas presence and antibiotic susceptibility. Type IE* strains exhibited higher susceptibility to antibiotics. Similarly, CRISPR-Cas systems also impacted phage susceptibility of K. pneumoniae isolates, with type IE strains showing higher protection towards phages. The analysis of quorum sensing profile of K. pneumoniae strains revealed variability in quorum sensing production. Isolates carrying type IE systems were devoid of both the quorum sensing systems. Furthermore, assessment of virulence attributes revealed that type IE CRISPR-Cas strains were robust biofilm formers and this system also influenced polysaccharide production, and mucoviscosity. Virulence gene expression analysis demonstrated up-regulation and down-regulation of specific genes in different strains, correlating with the presence or absence of CRISPR-Cas. The establishment of a burn wound infection model in mice revealed that type IE strains exhibited higher pathogenicity, supported by histopathological changes, heightened inflammatory markers, and cytokine levels.