Background

Human-pathogenic bacteria of the Burkholderia pseudomallei group in part cause severe infectious diseases. Multi-drug resistance and virulence factors complicate an efficient therapy. The intracellular lifestyle of the pathogen relies on phagosomal escape, actin-based intracellular motility, and fusion of host cell membranes. The type-III secretion system T3SSBsa is one important virulence factor promoting the intracellular persistence with Burkholderia invasion proteins (Bip) being an essential part of it. The translocator/effector protein BipC is homologous to Salmonella SipC and Shigella IpaC proteins, thus BipC might share the ability of interact with host cell F-actin.

Question

The work presented here focuses on the molecular characterisation of BipC to further elucidate the impact of BipC on host cell invasion and phagosomal escape with emphasises on actin binding properties.

Methods

Sequence alignment and structure prediction were used to identify structural similarities between BipC and the homologs SipC and IpaC. The interaction with actin was performed in in vitro studies with recombinant BipC. Ectopic expression of fluorescently labeled BipC was performed in primary human macrophages. CRISPRi-mediated bipC knockdown in the model organism B. thailandensis E264 was used in cellular infection assays.

Results

The structural similarity between the homologous SipC and IpaC to BipC was used to select possible actin binding interfaces and design protein fragments of BipC. Also, ectopically expressed BipC-SiriusGFP in primary human macrophages binds to actin-rich podosomes and localizes at the subcortical F-actin network. The knockdown of bipC in B. thailandensis results in increased number of actin-coated intracellular bacterial cells and shows a clear reduction in the percentage of invasion and phagosomal escape.

Conclusions

The results indicate an essential contribution of the B. thailandensis virulence factor BipC to the pathogenicity of Burkholderia infections.