The Legionella effector LtpM is a new type of phosphoinositide-activated glucosyltransferase.

Legionella pneumophila causes Legionnaires' disease, a severe form of pneumonia. L. pneumophila translocates more than 300 effectors into host cells via its Dot/Icm (Defective in organelle trafficking/Intracellular multiplication) type IV secretion system to enable its replication in target cells. Here, we studied the effector LtpM, which is encoded in a recombination hot spot in L. pneumophila Paris. We show that a C-terminal phosphoinositol 3-phosphate (PI3P)-binding domain, also found in otherwise unrelated effectors, targets LtpM to the Legionella-containing vacuole and to early and late endosomes. LtpM expression in yeast caused cytotoxicity. Sequence comparison and structural homology modeling of the N-terminal domain of LtpM uncovered a remote similarity to the glycosyltransferase (GT) toxin PaTox from the bacterium Photorhabdus asymbiotica; however, instead of the canonical DxD motif of GT-A type glycosyltransferases, essential for enzyme activity and divalent cation coordination, we found that a DxN motif is present in LtpM. Using UDP-glucose as sugar donor, we show that purified LtpM nevertheless exhibits glucohydrolase and autoglucosylation activity in vitro and demonstrate that PI3P binding activates LtpM's glucosyltransferase activity toward protein substrates. Substitution of the aspartate or the asparagine in the DxN motif abolished the activity of LtpM. Moreover, whereas all glycosyltransferase toxins and effectors identified so far depend on the presence of divalent cations, LtpM is active in their absence. Proteins containing LtpM-like GT domains are encoded in the genomes of other L. pneumophila isolates and species, suggesting that LtpM is the first member of a novel family of glycosyltransferase effectors employed to subvert hosts.

The Rab-binding Profiles of Bacterial Virulence Factors during Infection.

Legionella pneumophila, the causative agent of Legionnaire's disease, uses its type IV secretion system to translocate over 300 effector proteins into host cells. These effectors subvert host cell signaling pathways to ensure bacterial proliferation. Despite their importance for pathogenesis, the roles of most of the effectors are yet to be characterized. Key to understanding the function of effectors is the identification of host proteins they bind during infection. We previously developed a novel tandem-affinity purification (TAP) approach using hexahistidine and BirA-specific biotinylation tags for isolating translocated effector complexes from infected cells whose composition were subsequently deciphered by mass spectrometry. Here we further advanced the workflow for the TAP approach and determined the infection-dependent interactomes of the effectors SidM and LidA, which were previously reported to promiscuously bind multiple Rab GTPases in vitro. In this study we defined a stringent subset of Rab GTPases targeted by SidM and LidA during infection, comprising of Rab1A, 1B, 6, and 10; in addition, LidA targets Rab14 and 18. Taken together, this study illustrates the power of this approach to profile the intracellular interactomes of bacterial effectors during infection.

Creating a customized intracellular niche: subversion of host cell signaling by Legionella type IV secretion system effectors.

The Gram-negative facultative intracellular pathogen Legionella pneumophila infects a wide range of different protozoa in the environment and also human alveolar macrophages upon inhalation of contaminated aerosols. Inside its hosts, it creates a defined and unique compartment, termed the Legionella-containing vacuole (LCV), for survival and replication. To establish the LCV, L. pneumophila uses its Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effector proteins into the host cell. Although it has become apparent in the past years that these effectors subvert a multitude of cellular processes and allow Legionella to take control of host cell vesicle trafficking, transcription, and translation, the exact function of the vast majority of effectors still remains unknown. This is partly due to high functional redundancy among the effectors, which renders conventional genetic approaches to elucidate their role ineffective. Here, we review the current knowledge about Legionella T4SS effectors, highlight open questions, and discuss new methods that promise to facilitate the characterization of T4SS effector functions in the future.

UV-triggered affinity capture identifies interactions between the Plasmodium falciparum multidrug resistance protein 1 (PfMDR1) and antimalarial agents in live parasitized cells.

A representative of a new class of potent antimalarials with an unknown mode of action was recently described. To identify the molecular target of this class of antimalarials, we employed a photo-reactive affinity capture method to find parasite proteins specifically interacting with the capture compound in living parasitized cells. The capture reagent retained the antimalarial properties of the parent molecule (ACT-213615) and accumulated within parasites. We identified several proteins interacting with the capture compound and established a functional interaction between ACT-213615 and PfMDR1. We surmise that PfMDR1 may play a role in the antimalarial activity of the piperazine-containing compound ACT-213615.

Innate recognition by neutrophil granulocytes differs between Neisseria gonorrhoeae strains causing local or disseminating infections.

Members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family serve as cellular receptors for Neisseria gonorrhoeae. More specifically, neisserial colony opacity (OpaCEA)) proteins bind to epithelial CEACAMs (CEACAM1, CEA, CEACAM6) to promote bacterial colonization of the mucosa. In contrast, recognition by CEACAM3, expressed by human granulocytes, results in uptake and destruction of Opa(CEA)-expressing bacteria. Therefore, CEACAM3-mediated uptake might limit the spread of gonococci. However, some strains can cause disseminating gonococcal infections (DGIs), and it is currently unknown how these strains escape detection by granulocyte CEACAM3. Therefore, the opa gene loci from N. gonorrhoeae strain VP1, which was derived from a patient with disseminated gonococcal disease, were cloned and constitutively expressed in Escherichia coli. Similar to Opa proteins of the nondisseminating strain MS11, the majority of Opa proteins from strain VP1 bound epithelial CEACAMs and promoted CEACAM-initiated responses by epithelial cells. In sharp contrast to the Opa proteins of strain MS11, the Opa proteins of strain VP1 failed to interact with the human granulocyte receptor CEACAM3. Accordingly, bacteria expressing VP1 Opa proteins were not taken up by primary human granulocytes and did not trigger a strong oxidative burst. Analysis of Opa variants from four additional clinical DGI isolates again demonstrated a lack of CEACAM3 binding. In summary, our results reveal that particular N. gonorrhoeae strains express an Opa protein repertoire allowing engagement of epithelial CEACAMs for successful mucosal colonization, while avoiding recognition and elimination via CEACAM3-mediated phagocytosis. A failure of CEACAM3-mediated innate immune detection might be linked to the ability of gonococci to cause disseminated infections.

LtpD is a novel Legionella pneumophila effector that binds phosphatidylinositol 3-phosphate and inositol monophosphatase IMPA1.

The Dot/Icm type IV secretion system (T4SS) of Legionella pneumophila is crucial for the pathogen to survive in protozoa and cause human disease. Although more than 275 effector proteins are delivered into the host cell by the T4SS, the function of the majority is unknown. Here we have characterized the Dot/Icm effector LtpD. During infection, LtpD localized to the cytoplasmic face of the membrane of the Legionella-containing vacuole (LCV). In A549 lung epithelial cells, ectopically expressed LtpD localized to large vesicular structures that contained markers of endosomal compartments. Systematic analysis of LtpD fragments identified an internal 17-kDa fragment, LtpD471-626, which was essential for targeting ectopically expressed LtpD to vesicular structures and for the association of translocated LtpD with the LCV. LtpD471-626 bound directly to phosphatidylinositol 3-phosphate [PtdIns(3)P] in vitro and colocalized with the PtdIns(3)P markers FYVE and SetA in cotransfected cells. LtpD was also found to bind the host cell enzyme inositol (myo)-1 (or 4)-monophosphatase 1, an important phosphatase involved in phosphoinositide production. Analysis of the role of LtpD in infection showed that LtpD is involved in bacterial replication in THP-1 macrophages, the larvae of Galleria mellonella, and mouse lungs. Together, these data suggest that LtpD is a novel phosphoinositide-binding L. pneumophila effector that has a role in intracellular bacterial replication.