Abstract

The bacterial Type VI secretion system (T6SS) delivers proteins into target cells using fast contraction of a long sheath anchored to the cell envelope and wrapped around an inner Hcp tube associated with the secreted proteins. Mechanisms of sheath assembly and length regulation are unclear. Here we study these processes using spheroplasts formed from ampicillin-treated Vibrio cholerae. We show that spheroplasts secrete Hcp and deliver T6SS substrates into neighbouring cells. Imaging of sheath dynamics shows that the sheath length correlates with the diameter of spheroplasts and may reach up to several micrometres. Analysis of sheath assembly after partial photobleaching shows that subunits are exclusively added to the sheath at the end that is distal from the baseplate and cell envelope attachment. We suggest that this mode of assembly is likely common for all phage-like contractile nanomachines, because of the conservation of the structures and connectivity of sheath subunits.

Highlights

  • The bacterial Type VI secretion system (T6SS) delivers proteins into target cells using fast contraction of a long sheath anchored to the cell envelope and wrapped around an inner Hcp tube associated with the secreted proteins

  • To test if T6SS remains active in such cells, we exposed exponentially growing V. cholerae cells expressing

  • Quantification of spheroplast induction and T6SS dynamics revealed that exposure to ampicillin for 40 min at 37 °C generated the highest proportion (89.75%) of spheroplasts displaying dynamic T6SS sheaths (1,007 out of 1,122 cells; Fig. 1d, Supplementary Movie 1)

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Summary

Introduction

The bacterial Type VI secretion system (T6SS) delivers proteins into target cells using fast contraction of a long sheath anchored to the cell envelope and wrapped around an inner Hcp tube associated with the secreted proteins. 8) to form a baseplate together with VgrG/PAAR spike complex associated with T6SS effectors[9,10,11,12] This likely triggers polymerization of Hcp tube and VipA/VipB (TssB/TssC) sheath wrapped around the tube[13,14,15]. T6SS sheaths were observed to assemble across the whole cell suggesting that sheath length is limited only by cell diameter This was shown by direct observations of TssB in Vibrio cholerae, Serratia marcescens, and E. coli[18,19,20] as well as indirectly by imaging of ClpV localization in P. aeruginosa, Burkholderia thailandensis and Bacteroidetes[21,22,23,24]. Increased cell size during spheroplast formation correlates with increased sheath length, which allowed us to partially photobleach assembling sheaths and determine at which end of the sheath the soluble subunits are incorporated

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Results
Conclusion

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