Yersinia enterocolitica type III secretion injectisomes form regularly spaced clusters, which incorporate new machines upon activation

© 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd. Summary: Bacterial type III secretion systems or injectisomes are multiprotein complexes directly transporting bacterial effector proteins into eukaryotic host cells. To investigate the distribution of injectisomes in the bacterium and the influence of activation of the system on that distribution, we combined in vivo fluorescent imaging and high-resolution in situ visualization of Yersinia enterocolitica injectisomes by cryo-electron tomography. Fluorescence microscopy showed the injectisomes as regularly distributed spots around the bacterial cell. Under secreting conditions (absence of Ca2+), the intensity of single spots significantly increased compared with non-secreting conditions (presence of Ca2+), in line with an overall up-regulation of expression levels of all components. Single injectisomes observed by cryo-electron tomography tended to cluster at distances less than 100nm, suggesting that the observed fluorescent spots correspond to evenly distributed clusters of injectisomes, rather than single injectisomes. The up-regulation of injectisome components led to an increase in the number of injectisomes per cluster rather than the formation of new clusters. We suggest that injectisome clustering may allow more effective secretion into the host cells. We show that bacterial type III secretion systems, molecular syringes that bacteria use to inject proteins into eukaryotic host cells, are arranged in small clusters on the membrane of Yersinia enterocolitica. When we activated the system by Calcium chelation, new injectisomes were formed, most of which associated next to the existing clusters. Clustering of type III secretion systems may ensure fast and efficient protein injection and thereby reduce the risk for bacteria to be detected by the host immune system.