研究セミナー Architecture of type IVa and IVb pilus machines

Type IV pili (T4P) are filamentous appendages found on many bacteria and have been classified into T4aP and T4bP. T4aP are more widespread and are involved in a variety of functions ranging from cell motility, DNA transfer, host predation to electron transfer. T4bP are less prevalent and mainly found in enteropathogenic bacteria where they are utilized in host colonization. Using electron cryotomography of intect cells and followed by subtomogram averaging, we reveal 3-D in situ structures of the T4aP machine in Myxococcus xanthus. The locations of all ten components within the structure are identified by imaging mutants with individual proteins either deleted or fused to tags. The resulting component map is tested by building hypothetical models which are seen to fit the EM maps well and satisfy all known connectivity and structural constraints. The architecture of the T4aP machine suggests new mechanistic insights into pilus extension and retraction and explains how the enigmatic switch from extension to retraction occurs. We then use the same techniques to reveal in situ structures of the T4bP machine in Vibrio cholerae. Despite the presence of several non-homologous components, the structures of the V. cholerae T4bP machine are remarkably similar to that of the M. xanthus T4aP machine. We deduce locations of the components homologous to the T4aP machine, then imaged knockout mutants to map the locations of the non-homologous proteins. The resulting architecture of the T4bP machine shows that non-homologous proteins in type IVa and IVb pilus machines form similar structures, suggesting new hypotheses for their functions.