Dot/Icm Effector Translocation by Legionella longbeachae Creates a Replicative Vacuole Similar to That of Legionella pneumophila despite Translocation of Distinct Effector Repertoires.

Legionella organisms are environmental bacteria and accidental human pathogens that can cause severe pneumonia, termed Legionnaires' disease. These bacteria replicate within a pathogen-derived vacuole termed the Legionella-containing vacuole (LCV). Our understanding of the development and dynamics of this vacuole is based on extensive analysis of Legionella pneumophila. Here, we have characterized the Legionella longbeachae replicative vacuole (longbeachae-LCV) and demonstrated that, despite important genomic differences, key features of the replicative LCV are comparable to those of the LCV of L. pneumophila (pneumophila-LCV). We constructed a Dot/Icm-deficient strain by deleting dotB and demonstrated the inability of this mutant to replicate inside THP-1 cells. L. longbeachae does not enter THP-1 cells as efficiently as L. pneumophila, and this is reflected in the observation that translocation of BlaM-RalFLLO (where RalFLLO is the L. longbeachae homologue of RalF) into THP-1 cells by the L. longbeachae Dot/Icm system is less efficient than that by L. pneumophila. This difference is negated in A549 cells where L. longbeachae and L. pneumophila infect with similar entry dynamics. A β-lactamase assay was employed to demonstrate the translocation of a novel family of proteins, the Rab-like effector (Rle) proteins. Immunofluorescence analysis confirmed that these proteins enter the host cell during infection and display distinct subcellular localizations, with RleA and RleC present on the longbeachae-LCV. We observed that the host Rab GTPase, Rab1, and the v-SNARE Sec22b are also recruited to the longbeachae-LCV during the early stages of infection, coinciding with the LCV avoiding endocytic maturation. These studies further our understanding of the L. longbeachae replicative vacuole, highlighting phenotypic similarities to the vacuole of L. pneumophila as well as unique aspects of LCV biology.