SummaryBacterial ingestion and killing by phagocytic cells are essential processes to protect the human body from infectious microorganisms. However, only few proteins implicated in intracellular bacterial killing have been identified to date. We used Dictyostelium discoideum, a phagocytic bacterial predator, to study intracellular killing. In a random genetic screen we identified Kil2, a type V P-ATPase as an essential element for efficient intracellular killing of Klebsiella pneumoniae bacteria. Interestingly, kil2 knockout cells still killed efficiently several other species of bacteria, and did not show enhanced susceptibility to Mycobacterium marinum intracellular replication. Kil2 is present in the phagosomal membrane, and its structure suggests that it pumps cations into the phagosomal lumen. The killing defect of kil2 knockout cells was rescued by the addition of magnesium ions, suggesting that Kil2 may function as a magnesium pump. In agreement with this, kil2 mutant cells exhibited a specific defect for growth at high concentrations of magnesium. Phagosomal protease activity was lower in kil2 mutant cells than in wild-type cells, a phenotype reversed by the addition of magnesium to the medium. Kil2 may act as a magnesium pump maintaining magnesium concentration in phagosomes, thus ensuring optimal activity of phagosomal proteases and efficient killing of bacteria.

Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) is an important regulator of vesicle trafficking. The authors show that, in Dictyostelium, WASH is required for lysosomal hydrolase recycling and function. WASH is therefore crucial for both autophagic and phagocytic degradation, and cells lacking WASH are unable to either survive starvation or grow on certain bacteria.

By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum . PIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5) 2 ) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation in Dictyostelium . Disruption of PIKfyve inhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently, PIKfyve - cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lacking PIKfyve are more susceptible to infection by the intracellular pathogen Legionella pneumophila . We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes.

Aurora-A kinase has recently been shown to be deregulated in thyroid cancer cells and tissues. Among the Aurora-A substrates identified, transforming acidic coiled-coil (TACC3), a member of the TACC family, plays an important role in cell cycle progression and alterations of its expression occur in different cancer tissues. In this study, we demonstrated the expression of the TACC3 gene in normal human thyroid cells (HTU5), and its modulation at both mRNA and protein levels during cell cycle. Its expression was found, with respect to HTU5 cells, unchanged in cells derived from a benign thyroid follicular tumor (HTU42), and significantly reduced in cell lines derived from follicular (FTC-133), papillary (B-CPAP), and anaplastic thyroid carcinomas (CAL-62 and 8305C). Moreover, in 16 differentiated thyroid cancer tissues, TACC3 mRNA levels were found, with respect to normal matched tissues, reduced by twofold in 56% of cases and increased by twofold in 44% of cases. In the same tissues, a correlation between the expression of the TACC3 and Aurora-A mRNAs was observed. TACC3 and Aurora-A interact in vivo in thyroid cells and both proteins localized onto the mitotic structure of thyroid cells. Finally, TACC3 localization on spindle microtubule was no more observed following the inhibition of Aurora kinase activity by VX-680. We propose that Aurora-A and TACC3 interaction is important to control the mitotic spindle organization required for proper chromosome segregation.

Highlights d We identify a new regulator that shapes macropinocytic and phagocytic cups d Shaping protrusions into cups requires differential regulation of Ras and Rac d Cups are organized by integrating interactions with phospholipids and multiple GTPases d Defective cup formation causes a target shape-specific defect in phagocytosis

In Dictyostelium, the cytoskeletal proteins Actin binding protein 1 (Abp1) and the class I myosin MyoK directly interact and couple actin dynamics to membrane deformation during phagocytosis. Together with the kinase PakB, they build a regulatory switch that controls the efficiency of uptake of large particles. As a basis for further functional dissection, exhaustive phagosome proteomics was performed and established that about 1300 proteins participate in phagosome biogenesis. Then, quantitative and comparative proteomic analysis of phagosome maturation was performed to investigate the impact of the absence of MyoK or Abp1. Immunoblots and two-dimensional differential gel electrophoresis of phagosomes isolated from myoK-null and abp1-null cells were used to determine the relative abundance of proteins during the course of maturation. Immunoblot profiling showed that absence of Abp1 alters the maturation profile of its direct binding partners such as actin and the Arp2/3 complex, suggesting that Abp1 directly regulates actin dynamics at the phagosome. Comparative twodimensional differential gel electrophoresis analysis resulted in the quantification of mutant-to-wild type abundance ratios at all stages of maturation for over one hundred identified proteins. Coordinated temporal changes in these ratio profiles determined the classification of identified proteins into functional groups. Ratio profiling revealed that the early delivery of ER proteins to the phagosome was affected by the absence of MyoK and was coupled to a reciprocal imbalance in the delivery of the vacuolar proton pump and Rab11 GTPases. As direct functional consequences, a delayed acidification and a reduced intraphagosomal proteolysis were demonstrated in vivo in myoK-null cells. In conclusion, the absence of MyoK alters the balance of the contributions of the ER and an endo-lysosomal compartment, and slows down phagosome acidification as well as the speed and efficiency of particle degradation inside the phagosome. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.017608, 886 -900, 2012.Professional phagocytes, ranging from phagotrophic protozoa to specialized cells of the innate immune system such as macrophages, neutrophils, or dendritic cells, ingest and digest large particles (Ͼ 250 nm). Indeed, the core machineries acting in phagocytosis have been conserved as its basic purpose evolved from predation and feeding to antigen presentation (1). Particle recognition by the phagocytes initiate the internalization process and triggers remodeling of the plasma membrane and its underlying cytoskeleton, to project a circular cup-shaped lamella around the particle. The phagocytic cup finally encloses the particle into a de novo membranebound vacuole, the phagosome. Endomembrane compartments, such as early and late endosomes (2-4) and the endoplasmic reticulum (5), are recruited to provide membrane to form the phagosome. Modifications of the environment within the closed phagosome generates a microbicidal milieu and leads to particle degradation. M...

Proteins that contain Eps15 homology domains (EHDs) in their C-terminus are newly identified key regulators of endosomal membrane trafficking. Here, we show that D. discoideum contains a single EHD protein (referred to as EHD) that localizes to endosomal compartments and newly formed phagosomes. We provide the first evidence that EHD regulates phagosome maturation. Deletion of EHD results in defects in intraphagosomal proteolysis and acidification. These defects are linked to early delivery of lysosomal enzymes and fast retrieval of the vacuolar H + -ATPase in maturing phagosomes. We also demonstrate that EHD physically interacts with DymA. Our results indicate that EHD and DymA can associate independently with endomembranes, and yet they share identical kinetics in recruitment to phagosomes and release during phagosome maturation. Functional analysis of ehd − , dymA − and double dymA − ehd − knockout strains indicate that DymA and EHD play non-redundant and independent functions in phagosome maturation. Finally, we show that the absence of EHD leads to increased tubulation of endosomes, indicating that EHD participates in the scission of endosomal tubules, as reported for DymA.

Phagocytic cells take up, kill and digest microbes by a process called phagocytosis. To this end, these cells bind the particle, rearrange their actin cytoskeleton, and orchestrate transport of digestive factors to the particle-containing phagosome. The mammalian lysosomal membrane protein LIMP-2 (also known as SCARB2) and CD36, members of the class B of scavenger receptors, play a crucial role in lysosomal enzyme trafficking and uptake of mycobacteria, respectively, and generally in host cell defences against intracellular pathogens. Here, we show that the LIMP-2 homologue LmpA regulates phagocytosis and phagolysosome biogenesis. The knockdown mutant is highly affected in actin-dependent processes, such as particle uptake, cellular spreading and motility. Additionally, the cells are severely impaired in phagosomal acidification and proteolysis, likely explaining the higher susceptibility to infection with the pathogenic bacterium , a close cousin of the human pathogen Furthermore, we bring evidence that LmpB is a functional homologue of CD36 and specifically mediates uptake of mycobacteria. Altogether, these data indicate a role for LmpA and LmpB, ancestors of the family of which LIMP-2 and CD36 are members, in lysosome biogenesis and host cell defence.