Environmental and cellular factors affecting the localization of T6SS proteins in Burkholderia thailandensis.

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.

Polar localization of the ATPase ClpV-5 occurs independent of type VI secretion system apparatus proteins in Burkholderia thailandensis.

OBJECTIVE: ClpV, the ATPase of the type VI secretion system (T6SS) recycles cytoplasmic T6SS proteins following effector translocation. Fluorescent protein fusions to ClpV showed that it localizes to discrete and dynamic foci. ClpV-1-sfGFP of the bacterial cell targeting T6SS-1 of Burkholderia thailandensis exhibits a virtually random localization, whereas ClpV-5-sfGFP of the T6SS-5 targeting host cells is located at one or both poles. The mechanisms underlying the differential localization pattern are not known. Previous analysis of T6SSs, which target bacterial cells revealed that ClpV foci formation is dependent on components of the T6SS. Here, we investigated if the T6SS-5 apparatus confers polar localization of ClpV-5. RESULTS: ClpV-5-sfGFP foci formation and localization was examined in a B. thailandensis mutant harboring a deletion of the entire T6SS-5 gene cluster. We found that ClpV-5-sfGFP localization to discrete foci was not abolished in the absence of the T6SS-5 apparatus. Furthermore, the number of ClpV-5-sfGFP foci displaying a polar localization was not significantly different from that of ClpV-5-sfGFP expressed in the wild type genetic background. These findings suggest the presence of a T6SS-independent localization mechanism for ClpV-5 of the T6SS-5 targeting host cells.