Functional Genomics of Type VI Secretion System in Hypervirulent Klebsiella pneumoniae Isolates from Bacteraemia Cases in Southeast Nigeria

Hypervirulent Klebsiella pneumoniae (hvKp) strains harbor a distinct virulence arsenal including the type VI secretion system (T6SS), which enables bacterial competition and host cell manipulation. The co-emergence of hypervirulence and carbapenem resistance in hvKp isolates from Nigerian clinical settings constitutes a critical unmet research and clinical challenge. This dissertation presents original functional genomic characterization of the T6SS in hvKp isolates from bacteraemia patients at University of Nigeria Teaching Hospital, Enugu, and Nnamdi Azikiwe University Teaching Hospital, Nnewi, Anambra State, Southeast Nigeria. Clinical hvKp isolates were identified by hypermucoviscosity string test and PCR detection of virulence markers rmpA, iucA, iroB, and peg-344. Whole-genome sequencing of 32 confirmed hvKp isolates was performed on Illumina NovaSeq with hybrid assembly using Nanopore long reads for plasmid resolution. T6SS gene cluster identification employed MacSyFinder with the TXSScan database. Conditional CRISPR-Cas12a knockdown of T6SS structural components (hcp, vgrG, tssB) was performed in selected isolates. Interbacterial competition assays and Galleria mellonella infection models assessed T6SS-dependent virulence contribution. All 32 hvKp isolates harbored at least one complete T6SS gene cluster. T6SS-mediated competitive killing of E. coli prey cells was demonstrated in 27 isolates, with knockdown of hcp abolishing killing activity. Galleria mellonella survival assays confirmed significantly reduced virulence upon T6SS inactivation (p less than 0.001). Genomic analysis identified a novel T6SS auxiliary cluster encoding a unique VgrG spike protein with predicted cytotoxic effector domain absent from reference databases. Seventeen isolates co-harbored the virulence megaplasmid and carbapenem resistance genes on the same plasmid backbone, confirming convergent evolution of resistance and hypervirulence. These findings characterize the T6SS as a critical virulence determinant in Southeast Nigerian hvKp clinical strains and identify novel effector proteins as potential intervention targets. Keywords: Klebsiella pneumoniae, type VI secretion system, hypervirulence, functional genomics, Southeast Nigeria.