Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2024

Study on effects and mechanisms of type II secretion system secreted protein PulA on the virulence of Klebsiella pneumoniae (#117)

Xiangkuo Zheng 1 2 , Weiliang Zeng 1 , Yan Liu 1 , Luozhu Feng 1 , Lei Chen 1 , Xiaodong Zhang 1 , Lijiang Chen 1 , Tao Chen 1 , Yao Sun 1 , Tieli Zhou 1
  1. Department of Clinical Laboratory, Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
  2. Department of Clinical Medicine, Medical School, Huzhou University, Huzhou, Zhejiang, China

Objectives: Klebsiella pneumoniae can cause sever clinical infection due to its hypervirulence and multiple durg-resistance. Type Ⅱ secretion system (T2SS) delivers a variety of toxins and enzymes, which confer a survival advantage to pathogenic and non-pathogenic species. Here, we investigate clinical characteristics, role, and underlying mechanisms of T2SS-secreted protein PulA on the virulence of K. pneumoniae.

Methods: 407 K. pneumoniae isolates causing bloodstream infections (BSIs) were collected, and further relevant clinical data were retrospectively analyzed. In order to determine the virulence factors and pathogenicity, we performed, virulence genes determination, biofilm formation assay, serum resistance assay, cytotoxicity assay, Galleria mellonella larvae infection assay, macrophage infection model, and mouse lung infection model. K. pneumoniae MGH78578ΔpulA, MGH78578ΔpulD, MGH78578ΔpulA+pulA mutants, and the engineered bacteria pET28a–pulA/BL21(DE3) which expressing T2SS secreted protein PulA was constructed. LPS-stimulated macrophage RAW264.7 analysis, RT-qPCR, ELISA, and western blotting was conducted to investigate the regulatory effects of PulA on the expression of NF-κB signaling pathway in macrophages.

Results: 85.01% (346/407) K. pneumoniae were found to harbor the genes encoding T2SS. Patients with T2SS+ K. pneumoniae-induced BSIs had higher white blood cell (WBC) count and worse clinical outcomes. T2SS+ K. pneumoniae carried more virulence genes, including aerobactin (iucA), rmpA, and peg-344. Mouse lung tissue exhibited weakened inflammatory infiltration, reduced bacterial burden, and decreased expression levels of proinflammatory cytokines IL-1β, IL-6, and TNF-α in the MGH78578ΔpulA and MGH78578ΔpulD infection groups. PulA promoted RAW264.7 to secrete more proinflammatory cytokines. The phosphorylation level of P65, IKK, and IκBα was upregulated in PulA-treated groups.

Discussion: T2SS is widely distributed among K. pneumoniae isolates. T2SS+ K. pneumoniae carry more virulence genes, and patients infected with these isolates showed poorer clinical outcomes.T2SS appears to enhance the virulence and pathogenicity of K. pneumoniae. PulA significantly upregulates the phosphorylation levels of P65, IKK, and IκBα of NF-κB inflammatory signaling pathway of LPS-induced macrophages, thereby promoting the secretion of inflammatory cytokines TNF-α, IL-6, and IL-1β.