Abstract
The type VI secretion system (T6SS) is a widespread machine used by bacteria to control their environment and kill or disable bacterial species or eukaryotes through toxin injection. The T6SS comprises a central tube formed of stacked hexamers of hemolysin co-regulated proteins (Hcp) and terminated by a trimeric valine-glycine repeat protein G (VgrG) component, the cell puncturing device. A contractile tail sheath, formed by the TssB and TssC proteins, surrounds this tube. This syringe-like machine has been compared to an inverted phage, as both Hcp and VgrG share structural homology with tail components of Caudovirales. Here we solved the crystal structure of a tryptophan-substituted double mutant of Hcp1 from enteroaggregative Escherichia coli and compared it to the structures of other Hcps. Interestingly, we observed that the purified Hcp native protein is unable to form tubes in vitro. To better understand the rationale for observation, we measured the affinity of Hcp1 hexamers with themselves by surface plasmon resonance. The intra-hexamer interaction is weak, with a KD value of 7.2 µM. However, by engineering double cysteine mutants at defined positions, tubes of Hcp1 gathering up to 15 stacked hexamers formed in oxidative conditions. These results, together with those available in the literature regarding TssB and TssC, suggest that assembly of the T6SS tube differs significantly from that of Sipho- or Myoviridae.
Highlights
The type VI secretion system (T6SS) is a widespread versatile machine used by bacteria as a weapon to control their biotope and fight bacterial species or eukaryotes [1,2,3]
MALLS-UV experiments on Hcp1 and Hcp2 revealed that they form particles of 128 kDa and 123 kDa respectively that likely corresponds to hexamers
Electron microscopy (EM) of negative-stained Hcp1 and Hcp2 further showed that both proteins have a well-defined donut shape (Fig. 1B, D)
Summary
The type VI secretion system (T6SS) is a widespread versatile machine used by bacteria as a weapon to control their biotope and fight bacterial species or eukaryotes [1,2,3]. The long cytoplasmic structure comprised a number of subunits that share significant structural and functional similarities with bacteriophage tail proteins [8]. It is formed by a tube assembled from stacked hexamers of the hemolysin co-regulated (Hcp) protein [1,9,10]. This tube is terminated by a trimer of the valine-glycine repeat protein G (VgrG) [11] that has been hypothesized to pierce the bacterial prey cell wall [6,12] or the membrane of eukaryotes, allowing toxins delivery into the target cell [13,14,15]. The ClpV ATPase is recruited to the contracted tail sheath complex and catalyzes its disassembly, to target the TssB and TssC proteins to degradation or to allow new run of assembly [6,30]
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