Hcp-dependent delivery of type VI secretion system effectors by Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, utilises the type VI secretion system (T6SS) for virulence and bacterial competition by firing toxic effectors into eukaryotes and prokaryotes. The T6SS is a macromolecular complex that propels an effector-loaded spear into target cells. The effectors are attached to the VgrG/PAAR tip or the Hcp tube of the spear. There are three P. aeruginosa T6SSs, H1-H3-T6SS, delivering seventeen characterised effectors - most are delivered by the VgrG tip and usually encoded next to the vgrG gene. We further contributed to understanding the delivery of VgrG effectors, determining the importance of particular PAAR proteins and the preference for VgrG-PAAR-effector combinations at the spear tip.

The few known Hcp tube-delivered effectors, Tse1-3, were previously identified through secretome screening. We performed a protein pull-down screen and identified several putative effectors that interact with the P. aeruginosa Hcp tubes (Hcp1, Hcp2 or Hcp3). Three of these putative effectors cause toxicity in bacteria. Previous studies suggest the Tse1-3 effectors travel inside the Hcp1 tube, since the Hcp1 inner tube residues were essential for Tse1-3 secretion. We revealed that Hcp2 and Hcp3 inner tube residues were also important for interaction with two newly identified effectors, suggesting a common delivery mode for multiple effectors via the inside of the Hcp tube. Whilst the known diameter of the Hcp inner tube is 40 Å, whether effectors have to conform to this size limit was until now unknown. We identified a size limit where, using a large Tse1 chimera, secretion and H1-T6SS-dependent killing was inhibited, since the Hcp1-Tse1 chimeric complex blocked tube formation and subsequent firing.

These findings advance our understanding of Hcp-delivered effectors. The approach used to identify and characterise new effectors is directly applicable to other organisms, wherein antibacterial effectors discovered could be used to design future antimicrobials.