Project description:Polymyxins are increasingly used as the critical last-resort therapeutic options for multidrug-resistant gram-negative bacteria. Unfortunately, polymyxin resistance has increased gradually for the last few years. Although studies on mechanisms of polymyxin are expanding, system-wide analyses of the underlying mechanism for polymyxin resistance and stress response are still lacking. To understand how Klebsiella pneumoniae adapt to colistin (polymyxin E) pressure, we carried out proteomic analysis of Klebsiella pneumoniae strain cultured with different concentrations of colistin. Our results showed that the proteomic responses to colistin treatment in Klebsiella pneumoniae involving several pathways, including (i) gluconeogenesis and TCA cycle; (ii) arginine biosynthesis; (iii) porphyrin and chlorophyll metabolism; and (iv) enterobactin biosynthesis. Interestingly, decreased abundance of class A β-lactamases including TEM, SHV-11, SHV-4 were observed in cells treated with colistin. Moreover, we also present comprehensive proteome atlases of paired polymyxin-susceptible and -resistant Klebsiella pneumoniae strains. The polymyxin-resistant strain Ci, a mutant of Klebsiella pneumoniae ATCC BAA 2146, showed missense mutation in crrB. The crrB mutant Ci, which displayed lipid A modification with 4-amino-4-deoxy-L-arabinose (L-Ara4N) and palmitoylation, showed striking increases of CrrAB, PmrAB, PhoPQ, ArnBCADT and PagP. We hypothesize that crrB mutations induce elevated expression of the arnBCADTEF operon and pagP via PmrAB and PhoPQ. Moreover, multidrug efflux pump KexD, which was induced by crrB mutation, also contributed to colistin resistance. Overall, our results demonstrated proteomic responses to colistin treatment and the mechanism of CrrB-mediate colistin resistance, which may further offer valuable information to manage polymyxin resistance.
Project description:To investigate the role of outer membrane vesicles (OMVs) and related proteins in iron acquisition mechanism of hypervirulent Klebsiella pneumoniae (HVKP) and classic Klebsiella pneumoniae (cKP).
Project description:The emergence of colistin resistance in carbapenem-resistant and extended-spectrum ß-lactamase (ESBL)-producing bacteria is a significant threat to human health, and new treatment strategies are urgently required. Here we investigated the ability of the safe-for-human use ionophore PBT2 to restore antibiotic sensitivity in several polymyxin-resistant, ESBL-producing, carbapenem resistant Gram-negative human pathogens. PBT2 was observed to resensitize Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa to last-resort polymyxin class antibiotics, including a ‘next generation’ polymyxin derivative, FADDI-287. To gain additional insight into the potential mechanism of action of PBT2, we analyzed the transcriptome of K. pneumoniae and E. coli in the presence of sub-inhibitory concentrations of PBT2. Treatment with PBT2 was associated with multiple stress responses in both K. pneumoniae and E. coli. Significant changes in the transcription of transition metal ion homeostasis genes were observed in both strains.
Project description:Klebsiella pneumoniae is an important human pathogen, causing various infections. Apart from traditional virulence factors, there remains a significant gap in the discovery and research of new chromosomal virulence factors. CpxR is a two-component system (TCS) response regulator, but its impact on the virulence of Klebsiella pneumoniae have not been conclusively determined. For the effect of CpxR on K. pneumoniae virulence, the cpxR deletion(ΔcpxR) strain exhibited reduced serum resistance and attenuated pathogenicity in both Galleria mellonella larvae and mouse infection models compared to the wild-type strain. To identify CpxR-regulated virulence genes, RNA-seq analysis was conducted, followed by deletion of transcription downregulated genes in the ΔcpxR strain. Through serum resistance assays and Galleria mellonella infection experiments, a novel potential virulence factor, KPHS_28080, was identified. Deletion of KPHS_28080 impaired serum survival and proliferation in carbapenem-resistant strains HS11286 and hypervirulent strain ATCC 43816. Furthermore, the ATCC 43816 ΔKPHS_28080 strain showed significantly reduced colonization, proliferation, and multi-organ dissemination capacity in mice, accompanied by diminished pathogenicity. The KPHS_28080 promoter contains a conserved CpxR binding motif, where CpxR binding enhances promoter activity and elevates gene transcription. Sequence alignment revealed that KPHS_28080 is widely conserved across Klebsiella pneumoniae strains, establishing it as a novel chromosome-encoded virulence factor. These results provide a new insight into the CpxR regulation of K. pneumoniae virulence and chromosomal virulence factors.