2020MWINDPhD.pdf (10.79 MB)
The role of Type VI Secretion System in the virulence of Klebsiella pneumoniae
thesisposted on 2020-07-21, 10:05 authored by David Mwin
The enterobacteria, especially the multidrug-resistant strains, pose a serious health threat with evolving virulence mechanisms against their host. The opportunistic pathogen, K. pneumoniae, is a leading cause of urinary tract infection, blood and pneumonia in patients at the hospital and remains a common isolate in community-acquired disease. Many bacteria, including K. pneumoniae, explore a range of factors /mechanisms such as secreted toxin and antibiotic resistance that may enhance their survival, virulence, and evasion of the host immune system. Type VI Secretion Systems (T6SSs) is a transmembrane “spring-loaded” toxin-translocating nanomolecular machinery recently characterized in over 25% of Proteobacteria. T6SS shares both structural and protein homology with phage tail and is considered to be reminiscent of the bacteriophage puncturing device. While available data suggest a diverse role of the toxin puncturing T6SS device in many Gram-negative bacteria, no experimental data have demonstrated the putative T6SS gene clusters’ role in the virulence of K. pneumoniae as at the time that this research work was conducted. In silico analysis, using a full range of bioinformatic tools were used to identify and map the T6SS gene cluster (T6SS1 and T6SS3) in K. pneumoniae. A library of molecular genetic tools was constructed via a novel strategy and used to disarm the resistance in MDR K. pneumoniae, which enhanced the safety and genetic manipulation of the strain for T6SS functional studies. Mainly, three T6SS mutants, ΔT1 (T6SS1 mutant), ΔT3 (T6SS3 mutant), and ΔT1ΔT3 (T6SS1/T6SS3 mutant) generated via lambda red recombination allelic exchange and were examined for the role of the T6SS gene clusters in K. pneumoniae. The data obtained from the various assays and analysis suggest that K. pneumoniae via T6SS mediated antibacterial virulence against other competing bacteria and played a significant role T4SS-mediated conjugal transfer of mobile genetic elements. K. pneumoniae used T6SS to resist amoeba phagocytosis and enhance subsequent survival within the cell. Also, T6SS increased in vivo virulence in Galleria larvae, host cell invasion, survival, actin filament polymerisation and activation of host pro-inflammatory innate immunity. Thus, the putative T6SS gene clusters may be mediating a multipurpose virulence against host organism and other bacteria using pre-emptive contact-dependent strikes and toxin secretion.
Supervisor(s)Yassine Amrani; Shaun Heaphy; Kumar Rajakumar
Date of award2020-06-04
Author affiliationDepartment of Infection, Immunity, and Inflammation
Awarding institutionUniversity of Leicester