VgrG Spike Dictates PAAR Requirement for the Assembly of the Type VI Secretion System

Abstract

Widely employed by Gram-negative pathogens for competition and pathogenesis, the type six protein secretion system (T6SS) can inject toxic effectors into neighboring cells through the penetration of a spear-like structure comprising a long Hcp tube and a VgrG-PAAR spike complex. The cone-shaped PAAR is believed to sharpen the T6SS spear for penetration but it remains unclear why PAAR is required for T6SS functions in some bacteria but dispensable in others. Here, we report the conditional requirement of PAAR for T6SS functions in Aeromonas dhakensis, an emerging human pathogen that may cause severe bacteremia. By deleting the two PAAR paralogs, we show that PAAR is not required for T6SS secretion, bacterial killing, or specific effector delivery in A. dhakensis. By constructing combinatorial PAAR and vgrG deletions, we demonstrate that deletion of individual PAAR moderately reduced T6SS functions but double or triple deletions of PAAR in the vgrG deletion mutants severely impaired T6SS functions. Notably, the auxiliary-cluster-encoded PAAR2 and VgrG3 are less critical than the main-cluster-encoded PAAR1 and VgrG1&2 proteins to T6SS functions. In addition, PAAR1 but not PAAR2 contributes to antieukaryotic virulence in amoeba. Our data suggest that, for a multi-PAAR T6SS, the variable role of PAAR paralogs correlates with the VgrG-spike composition that collectively dictates T6SS assembly.