Role of outer membrane vesicles of hypervirulent Klebsiella pneumoniae isolated from bovine uterine discharge in pro-inflammatory dysregulation

Abstract

Numerous research studies have established a connection between hypervirulent Klebsiella pneumo-niae (hvKP) and bovine abortion, as well as other complications and clinical illnesses. These bacteria, which belong to the Gram-negative group, release outer membrane vesicles (OMVs), believed to play a crucial role in transporting effector molecules to their target cells. To investigate this further, OMVs of hvKP were collected from 47 clinical isolates from bovine uterine discharge samples using ultra-centrifugation. Confirmation of the 47 isolates was carried out through a standard PCR test targeting the 16S rRNA gene. Subsequently, a multiplex PCR was used to verify positive 16S rRNA gene re-sults by detecting specific virulence genes (fimH, acrAB, traT, and rmpA). The standard PCR test showed that 27 out of the 47 isolates (57.45%) tested positive for the 16S rRNA gene, with all 27 of these isolates also testing positive for the rmpA and traT genes. Additionally, 30% of the isolates (8.1 out of 27) tested positive for AcrAB, while 70% of the isolates (18.9 out of 27) tested positive for FimH. During SDS-Western blotting, ten protein bands with molecular weights ranging from 18 to >60 kDa were identified in K. pneumoniae-derived OMV preparations. These bands included OmpX, flagellin, MipA, OmpA, OmpC, and OmpF (4244 kDa), a strong OmpA band at 45 KDa, and a faint band at 60 kDa of GroEL as an oligomer of OmpA. Furthermore, hvKP OMVs displayed varying cytotoxic effects on the L929 and THP-1 cell lines. Notably, incubating THP-1 cells with high con-centrations of hvKP OMVs inhibited IL-1β and TNF responses, indicating that these OMVs may not only facilitate the internalisation of the bacterium by THP-1 cells but also suppress the cells’ innate immune response to hvKP infection. This ability of hvKP OMVs to evade the immune system and modulate host responses contributes significantly to the bacterium’s pathogenicity.

Keywords

• bovine uterine discharge
• hvkp
• omvs
• pro-inflammatory response
• sds-western blotting
• virulence genes