The type VI secretion system (T6SS) injects effector proteins into neighboring bacteria and host cells. Effector translocation is driven by contraction of a tubular sheath in the cytoplasm that expels an inner needle across the cell envelope. The AAA + ATPase ClpV disassembles and recycles the contracted sheath. While ClpV-1-GFP of the Burkholderia T6SS-1, which targets prokaryotic cells, assembles into randomly localized foci, ClpV-5-GFP of the virulence-associated T6SS-5 displays a polar distribution. The mechanisms underlying the localization of T6SSs to a particular site in the bacterial cell are currently unknown. We recently showed that ClpV-5-GFP retains its polar localization in the absence of all T6SS-5 components during infection of host cells. Herein, we set out to identify factors involved in the distribution of ClpV-5 and ClpV-1 in Burkholderia thailandensis. We show that focal assembly and polar localization of ClpV-5-GFP is not dependent on the intracellular host cell environment, known to contain the signal to induce T6SS-5 gene expression. In contrast to ClpV-5-GFP, localization of ClpV-1-GFP was dependent on the cognate T6SS. Foci formation of both ClpV5-GFP and ClpV-1-GFP was decreased by D cycloserine-mediated inhibition of peptidoglycan synthesis while treatment of B. thailandensis with A22 blocking the cytoskeletal protein MreB did not affect assembly of ClpV-5 and ClpV-1 into single discrete foci. Furthermore, we found that surface contact promotes but is not essential for localization of ClpV-5-GFP to the pole whereas expression of clpV-1-gfp appears to be induced by surface contact. In summary, the study provides novel insights into the localization of ClpV ATPases of T6SSs targeting prokaryotic and eukaryotic cells.

VI型分泌系统（T6SS）将效应蛋白注射到附近的细菌和宿主细胞中。效应器易位是由细胞质中管状鞘的收缩驱动的，该鞘将内针穿过细胞膜。AAA + ATPase ClpV分解并回收收缩的鞘。而伯克霍尔德氏菌的ClpV-1-GFP靶向原核细胞的T6SS-1组装成随机定位的病灶，与毒力相关的T6SS-5的ClpV-5-GFP显示出极性分布。目前尚不了解将T6SS定位到细菌细胞中特定位点的机制。我们最近显示，在宿主细胞感染期间，在没有所有T6SS-5成分的情况下，ClpV-5-GFP保留了其极性定位。在这里，我们着手确定涉及泰国伯克霍尔德氏菌ClpV-5和ClpV-1分布的因素。我们表明ClpV-5-GFP的焦点大会和极性定位不依赖于细胞内宿主细胞环境，已知包含诱导T6SS-5基因表达的信号。与ClpV-5-GFP不同，ClpV-1-GFP的定位取决于相关的T6SS。D环丝氨酸介导的肽聚糖合成的抑制作用减少了ClpV5-GFP和ClpV-1-GFP的形成，而泰国A. B22用B22阻断细胞骨架蛋白MreB的处理不影响ClpV-5和ClpV-1的组装。单个离散病灶。此外，我们发现表面接触促进但不是ClpV-5-GFP定位于极点的必需条件，而clpV-1-gfp的表达似乎是由表面接触引起的。总而言之，该研究为靶向原核和真核细胞的T6SS的ClpV ATPase的定位提供了新颖的见解。