The machinery at endoplasmic reticulum-plasma membrane contact sites contributes to spatial regulation of multiple Legionella effector proteins.

The Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P)-binding domain first described in the effector DrrA (SidM). This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV), and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM) and the endoplasmic reticulum (ER) modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase IIIα (PI4KIIIα) was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIIIα was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis.

The role of Rab GTPases in the transport of vacuoles containing Legionella pneumophila and Coxiella burnetii.

Intracellular pathogens survive in eukaryotic cells by evading a variety of host defences. To avoid degradation through the endocytic pathway, intracellular bacteria must adapt their phagosomes into protective compartments that promote bacterial replication. Legionella pneumophila and Coxiella burnetii are Gram-negative intracellular pathogens that remodel their phagosomes by co-opting components of the host cell, including Rab GTPases. L. pneumophila and C. burnetii are related phylogenetically and share an analogous type IV secretion system for delivering bacterial effectors into the host cell. Some of these effectors mimic eukaryotic biochemical activities to recruit and modify Rabs at the vacuole. In the present review, we cover how these bacterial species, which utilize divergent strategies to establish replicative vacuoles, use translocated proteins to manipulate host Rabs, as well as exploring which Rabs are implicated in vacuolar biogenesis in these two organisms.

From structure to solutions: the role of basic research in developing anthrax countermeasures: Microbiology Graduate Program Seminar: Anthrax toxin.

Dr. John Collier traced the discoveries that elucidated the structure and function of the anthrax toxin in his talk "Anthrax Toxin," which was part of the Microbiology Graduate Program Seminar Series at Yale School of Medicine on February 23, 2012. Dr. Collier, Professor of Microbiology and Immunobiology at Harvard University, began by noting the advantages to studying anthrax pathogenesis in a biosafety level-1 lab. This designation does not merely facilitate his research, but also reflects a larger trend of basic research being leveraged to develop translational applications. Basic research on toxin structure has led to the development of a vaccine by Dr. Collier's group. Next-generation prophylactics also may stem from recent discoveries uncovering a role for cellular cofactors that mediate toxin function. Finally, basic research into the toxin substructure has facilitated efforts to change the receptor tropism to target dysregulated cells for therapeutic purposes. The urgency around biodefense agents makes the choice of research priorities a salient issue. As such, this author submits that basic research occupies a unique and lucrative niche driving clinical applications.

Hepatocyte nuclear factor-1 as marker of epithelial phenotype reveals marrow-derived hepatocytes, but not duct cells, after liver injury in mice.

The potential bone marrow origin of hepatocytes, cholangiocytes, and ductal progenitor cells in the liver was examined in female mice after transplantation of bone marrow cells from male green fluorescent protein (GFP) transgenic donors. Following stable hematopoietic engraftment, the livers of the recipients were injured with carbon tetrachloride (CCl(4), with or without local irradiation of the liver) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC, with or without local irradiation of the liver). The presence of numerous marrow-derived, GFP-positive inflammatory cells had the potential to lead to erroneous interpretation of marrow-derived hepatocytes, cholangiocytes, and ductal progenitor cells. Identification of marrow-derived ductal progenitor or cholangiocyte phenotype using colocalization of GFP or Y chromosome with pancytokeratin staining also failed to distinguish epithelial cells from closely apposed inflammatory cells. To address this inadequacy, we developed a rigorous new immunofluorescence protocol to identify marrow-derived epithelial cells in the liver using Y chromosome (donor marker) and hepatocyte nuclear factor-1 (HNF1, a nuclear marker of liver epithelial, nonhematopoietic phenotype). Using the Y/HNF1 method, rare (approximately one in 20,000) hepatocytes in female mice transplanted with male bone marrow contained a donor-derived Y chromosome. On the other hand, no Y chromosomes were found in cholangiocytes or ductal progenitor cells in mice with liver injury due to DDC or CCl(4). The use of a nuclear marker of mature hepatocytes or cholangiocytes, such as HNF1, improves discrimination of marrow-derived epithelial cells in tissue sections.

AMPylation is critical for Rab1 localization to vacuoles containing Legionella pneumophila.

UNLABELLED: Legionella pneumophila is an intracellular pathogen that resides within a membrane-bound compartment that is derived from vesicles exiting the endoplasmic reticulum (ER). To create this compartment, these bacteria use a type IV secretion system to deliver effector proteins that subvert host cell functions. Several Legionella effector proteins modulate the function of the host protein Rab1, which is a GTPase that is recruited to the Legionella-containing vacuole (LCV). Here, we examined which of the Rab1-directed enzymatic activities displayed by Legionella effectors are important for localizing the Rab1 protein to the LCV membrane. The guanine nucleotide exchange factor (GEF) domain in the effector protein DrrA (SidM) was essential for Rab1 recruitment to the LCV and Rab1 AMPylation by the nucleotidyltransferase domain in DrrA was important for Rab1 retention. Legionella organisms producing mutant DrrA proteins that were severely attenuated for GEF activity in vitro retained the ability to localize Rab1 to the LCV. Rab1 localization to the LCV mediated by these GEF-defective mutants required AMPylation. Importantly, we found that efficient localization of Rab1 to the LCV occurred when Rab1 GEF activity and Rab1 AMPylation activity were provided by separate proteins. Rab1 phosphocholination (PCylation) by the effector protein AnkX, however, was unable to substitute for Rab1 AMPylation. Lastly, the defect in Rab1 localization to the LCV in AMPylation-deficient strains of Legionella was partially suppressed if the GTPase-activating protein (GAP) LepB was eliminated. Thus, our data indicate that AMPylation of Rab1 is an effective strategy to maintain this GTPase on the LCV membrane. IMPORTANCE: Activities that enable the intracellular pathogen Legionella pneumophila to subvert the function of the host protein Rab1 were investigated. Our data show that a posttranslational modification called AMPylation is critical for maintaining a pool of Rab1 on the LCV membrane. AMPylation of Rab1 led to the accumulation of GTP-bound Rab1 on the LCV membrane by protecting the protein from inactivation by GAPs. Importantly, PCylation of Rab1 by the Legionella effector protein AnkX was neither necessary nor sufficient to maintain Rab1 on the LCV, indicating that AMPylation and PCylation represent functionally distinct activities. We conclude that modification of Rab1 by AMPylation is an effective strategy to spatially and temporally regulate the function of this GTPase on a membrane-bound organelle.