IqgC is a potent regulator of macropinocytosis in the presence of NF1 and its loading to macropinosomes is dependent on RasG.

RasG is a major regulator of macropinocytosis in Dictyostelium discoideum. Its activity is under the control of an IQGAP-related protein, IqgC, which acts as a RasG-specific GAP (GTPase activating protein). IqgC colocalizes with the active Ras at the macropinosome membrane during its formation and for some time after the cup closure. However, the loss of IqgC induces only a minor enhancement of fluid uptake in axenic cells that already lack another RasGAP, NF1. Here, we show that IqgC plays an important role in the regulation of macropinocytosis in the presence of NF1 by restricting the size of macropinosomes. We further provide evidence that interaction with RasG is indispensable for the recruitment of IqgC to forming macropinocytic cups. We also demonstrate that IqgC interacts with another small GTPase from the Ras superfamily, Rab5A, but is not a GAP for Rab5A. Since mammalian Rab5 plays a key role in early endosome maturation, we hypothesized that IqgC could be involved in macropinosome maturation via its interaction with Rab5A. Although an excessive amount of Rab5A reduces the RasGAP activity of IqgC in vitro and correlates with IqgC dissociation from endosomes in vivo, the physiological significance of the Rab5A-IqgC interaction remains elusive.

Extracellular and intracellular destruction of Pseudomonas aeruginosa by Dictyostelium discoideum phagocytes mobilize different antibacterial mechanisms.

Ingestion and killing of bacteria by phagocytic cells are critical processes to protect the human body from bacterial infections. In addition, some immune cells (neutrophils, NK cells) can release microbicidal molecules in the extracellular medium to eliminate non-ingested microorganism. Molecular mechanisms involved in the resulting intracellular and extracellular killing are still poorly understood. In this study, we used the amoeba Dictyostelium discoideum as a model phagocyte to investigate the mechanisms allowing intracellular and extracellular killing of Pseudomonas aeruginosa. When a D. discoideum cell establishes a close contact with a P. aeruginosa bacterium, it can either ingest it and kill it in phagosomes, or kill it extracellularly, allowing a direct side-by-side comparison of these two killing modalities. Efficient intracellular destruction of P. aeruginosa requires the presence of the Kil2 pump in the phagosomal membrane. On the contrary, extracellular lysis is independent on Kil2 but requires the expression of the superoxide-producing protein NoxA, and the extracellular release of the AplA bacteriolytic protein. These results shed new light on the molecular mechanisms allowing elimination of P. aeruginosa bacteria by phagocytic cells.

A quantitative comparison of antibodies against epitope tags for immunofluorescence detection.

Epitope tags recognized by specific antibodies have been widely used over the last few decades, notably to localize tagged proteins within cells by immunofluorescence. The diversity of tags and antibodies usually prevents a side-by-side comparison of the efficiency with which each antibody recognizes its cognate tag. We expressed chimeric proteins, each composed of an invariant domain (IL2Ra) associated with a specific epitope tag. Double immunofluorescence allowed us to quantify in parallel the reference signal generated by the anti-IL2Ra antibody and the signal generated by the anti-epitope tag antibody. Since all antibodies used in this study were recombinant antibodies fused to the same mouse Fc domain, the generated signals were directly comparable. Three groups of tags/antibodies were revealed: 'good' antibodies generated high signals even when used at a low concentration (50 ng·mL-1 ), 'fair' antibodies generated a high signal only at high concentrations (5000 ng·mL-1 ), and 'mediocre' antibodies generated positive but weak signals. Except for an anti-myc antibody, similar results were obtained when cells were fixed in paraformaldehyde or methanol. These results provide a side-by-side quantitative evaluation of different tag/antibody pairs. This information will be useful to optimize the choice of epitope tags and to choose optimal antibodies.

Sequential action of antibacterial effectors in Dictyostelium discoideum phagosomes.

Mammalian professional phagocytic cells ingest and kill invading microorganisms and prevent the development of bacterial infections. Our understanding of the sequence of events that results in bacterial killing and permeabilization in phagosomes is still largely incomplete. In this study, we used the Dictyostelium discoideum amoeba as a model phagocyte to study the fate of the bacteria Klebsiella pneumoniae inside phagosomes. Our analysis distinguishes three consecutive phases: bacteria first lose their ability to divide (killing), then their cytosolic content is altered (permeabilization), and finally their DNA is degraded (digestion). Phagosomal acidification and production of free radicals are necessary for rapid killing, membrane-permeabilizing proteins BpiC and AlyL are required for efficient permeabilization. These results illustrate how a combination of genetic and microscopical tools can be used to finely dissect the molecular events leading to bacterial killing and permeabilization in a maturing phagosome.

5-ethyl-2'-deoxyuridine fragilizes Klebsiella pneumoniae outer wall and facilitates intracellular killing by phagocytic cells.

Klebsiella pneumoniae is the causative agent of a variety of severe infections. Many K. pneumoniae strains are resistant to multiple antibiotics, and this situation creates a need for new antibacterial molecules. K. pneumoniae pathogenicity relies largely on its ability to escape phagocytosis and intracellular killing by phagocytic cells. Interfering with these escape mechanisms may allow to decrease bacterial virulence and to combat infections. In this study, we used Dictyostelium discoideum as a model phagocyte to screen a collection of 1,099 chemical compounds. Phg1A KO D. discoideum cells cannot feed upon K. pneumoniae bacteria, unless bacteria bear mutations decreasing their virulence. We identified 3 non-antibiotic compounds that restored growth of phg1A KO cells on K. pneumoniae, and we characterized the mode of action of one of them, 5-ethyl-2'-deoxyuridine (K2). K2-treated bacteria were more rapidly killed in D. discoideum phagosomes than non-treated bacteria. They were more sensitive to polymyxin and their outer membrane was more accessible to a hydrophobic fluorescent probe. These results suggest that K2 acts by rendering the membrane of K. pneumoniae accessible to antibacterial effectors. K2 was effective on three different K. pneumoniae strains, and acted at concentrations as low as 3 µM. K2 has previously been used to treat viral infections but its precise molecular mechanism of action in K. pneumoniae remains to be determined.

The Fate of Bacteria of the Bacillus cereus Group in the Amoeba Environment.

The Bacillus cereus sensu lato group consists of several closely related species, including B. anthracis, B. cereus sensu stricto, and B. thuringiensis. Spores of these pathogenic bacteria are commonly found in the soil but evidence suggests that they are unable to grow in such a natural environment in the absence of nutrient input. Amoebas have been reported to be an amplifier for several species of pathogenic bacteria and their potential involvement to explain the large amount of B. thuringiensis and B. cereus spores in soil has been frequently proposed. Here, we studied the fate of Bacillus and amoebas when cultured together. We show that the virulence factors produced by B. thuringiensis and B. cereus do not affect the amoeba Acanthamoeba castellanii, which, on the contrary, can phagocytose and effectively digest vegetative Bacillus cells to grow and prevent the formation of cysts. Bacterial spores can germinate in the amoeba environment and the vegetative cells can then form chains or aggregates that appear to be less efficiently phagocyted by the amoeba. The use of transcriptional fusions between fluorescent reporter genes and stationary phase- and sporulation-specific promoters showed that the sporulation process occurs more efficiently in the presence of amoebas than in their absence. Moreover, our results showed the amoeba environment to promote spore germination and allow the bacteria to complete their developmental cycle. Overall, this study suggests that the amoeba-Bacillus interaction creates a virtuous circle in which each protagonist helps the other to develop.

Learning objectives: an epiphany.

A new Bachelor-Master curriculum in Biomedical Sciences was created at the University of Geneva in 2017. As we organized the new curriculum, we discovered the usefulness of learning objectives. This goal-oriented approach of teaching proved essential to determine the overall structure of the teaching program, as well as the content of specific courses, and the nature of the examinations. It led us to include innovative elements in the program, preparing students for real-life situations. Finally, it convinced us to change our role as teachers, in order to engage students in a more active learning relationship.

Intracellular targeting of Cisd2/Miner1 to the endoplasmic reticulum.

BACKGROUND: Cisd1 and Cisd2 proteins share very similar structures with an N-terminal membrane-anchoring domain and a C-terminal cytosolic domain containing an iron-cluster binding domain and ending with a C-terminal KKxx sequence. Despite sharing a similar structure, Cisd1 and Cisd2 are anchored to different compartments: mitochondria for Cisd1 and endoplasmic reticulum for Cisd2. The aim of this study was to identify the protein motifs targeting Cisd2 to the ER and ensuring its retention in this compartment. RESULTS: We used new recombinant antibodies to localize Cisd1 and Cisd2 proteins, as well as various protein chimeras. Cisd2 is targeted to the ER by its N-terminal sequence. It is then retained in the ER by the combined action of a C-terminal COPI-binding KKxx ER retrieval motif, and of an ER-targeting transmembrane domain. As previously reported for Cisd1, Cisd2 can alter the morphology of the compartment in which it accumulates. CONCLUSION: Although they share a very similar structure, Cisd1 and Cisd2 use largely different intracellular targeting motifs to reach their target compartment (mitochondria and endoplasmic reticulum, respectively).

Role of LrrkA in the Control of Phagocytosis and Cell Motility in Dictyostelium discoideum.

LrrkA is a Dictyostelium discoideum kinase with leucine-rich repeats. LrrkA stimulates Kil2 and intra-phagosomal killing of ingested bacteria in response to folate. In this study, we show that genetic inactivation of lrrkA also causes a previously unnoticed phenotype: lrrkA KO cells exhibit enhanced phagocytosis and cell motility compared to parental cells. This phenotype is cell autonomous, is reversible upon re-expression of LrrkA, and is not due to an abnormal response to inhibitory quorum-sensing factors secreted by D. discoideum in its medium. In addition, folate increases motility in parental D. discoideum cells, but not in lrrkA KO cells, suggesting that LrrkA plays a pivotal role in the cellular response to folate. On the contrary, lrrkA KO cells regulate gene transcription in response to folate in a manner indistinguishable from parental cells. Overall, based on analysis of mutant phenotypes, we identify gene products that participate in the control of intracellular killing, cell motility, and gene transcription in response to folate. These observations reveal a mechanism by which D. discoideum encountering bacterially-secreted folate can migrate, engulf, and kill bacteria more efficiently.

How Phagocytic Cells Kill Different Bacteria: a Quantitative Analysis Using Dictyostelium discoideum.

Ingestion and killing of bacteria by phagocytic cells protect the human body against infections. While many mechanisms have been proposed to account for bacterial killing in phagosomes, their relative importance, redundancy, and specificity remain unclear. In this study, we used the Dictyostelium discoideum amoeba as a model phagocyte and quantified the requirement of 11 individual gene products, including nine putative effectors, for the killing of bacteria. This analysis revealed that radically different mechanisms are required to kill Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis AlyL, a lysozyme-like protein equipped with a distinct bacteriolytic region, plays a specific role in the intracellular killing of K. pneumoniae, with assistance from BpiC and Aoah, two lipopolysaccharide (LPS)-binding proteins. Rapid killing of E. coli and P. aeruginosa requires the presence of BpiC and of the NoxA NADPH oxidase. No single effector tested is essential for rapid killing of S. aureus or B. subtilis Overall, our observations reveal an unsuspected degree of specificity in the elimination of bacteria in phagosomes.IMPORTANCE Phagocytic cells ingest and kill bacteria, a process essential for the defense of the human body against infections. Many potential killing mechanisms have been identified in phagocytic cells, including free radicals, toxic ions, enzymes, and permeabilizing peptides. Yet fundamental questions remain unanswered: what is the relative importance of these mechanisms, how redundant are they, and are different mechanisms used to kill different species of bacteria? We addressed these questions using Dictyostelium discoideum, a model phagocytic cell amenable to genetic manipulations and quantitative analysis. Our results reveal that vastly different mechanisms are required to kill different species of bacteria. This very high degree of specificity was unexpected and indicates that a lot remains to be discovered about how phagocytic cells eliminate bacteria.

A recombinant antibody toolbox for Dictyostelium discoideum.

OBJECTIVE: The amoeba Dictyostelium discoideum has been a valuable model organism to study numerous facets of eukaryotic cell biology, such as cell motility, cell adhesion, macropinocytosis and phagocytosis, host-pathogen interactions and multicellular development. However, the relative small size of the Dictyostelium community hampers the production and distribution of reagents and tools, such as antibodies, by commercial vendors. RESULTS: For the past 5 years, our laboratory has worked to promote an increased use of recombinant antibodies (rAbs) by academic laboratories. Here we report our efforts to ensure that Dictyostelium researchers have access to rAbs. Using hybridoma sequencing and phage display techniques, we generated a panel of recombinant antibodies against D. discoideum antigens, providing a useful and reliable set of reagents for labelling and characterization of proteins and subcellular compartments in D. discoideum, accessible to the entire Dictyostelium community.

Transcriptional Responses of Dictyostelium discoideum Exposed to Different Classes of Bacteria.

Dictyostelium discoideum amoebae feed by ingesting bacteria, then killing them in phagosomes. Ingestion and killing of different bacteria have been shown to rely on largely different molecular mechanisms. One would thus expect that D. discoideum adapts its ingestion and killing machinery when encountering different bacteria. In this study, we investigated by RNA sequencing if and how D. discoideum amoebae respond to the presence of different bacteria by modifying their gene expression patterns. Each bacterial species analyzed induced a specific modification of the transcriptome. Bacteria such as Bacillus subtilis, Klebsiella pneumoniae, or Mycobacterium marinum induced a specific and different transcriptional response, while Micrococcus luteus did not trigger a significant gene regulation. Although folate has been proposed to be one of the key molecules secreted by bacteria and recognized by hunting amoebae, it elicited a very specific and restricted transcriptional signature, distinct from that triggered by any bacteria analyzed here. Our results indicate that D. discoideum amoebae respond in a highly specific, almost non-overlapping manner to different species of bacteria. We additionally identify specific sets of genes that can be used as reporters of the response of D. discoideum to different bacteria.

Identification of Anti-Mycobacterium and Anti-Legionella Compounds With Potential Distinctive Structural Scaffolds From an HD-PBL Using Phenotypic Screens in Amoebae Host Models.

Tubercular Mycobacteria and Legionella pneumophila are the causative agents of potentially fatal respiratory diseases due to their intrinsic pathogenesis but also due to the emergence of antibiotic resistance that limits treatment options. The aim of our study was to explore the antimicrobial activity of a small ligand-based chemical library of 1255 structurally diverse compounds. These compounds were screened in a combination of three assays, two monitoring the intracellular growth of the pathogenic bacteria, Mycobacterium marinum and L. pneumophila, and one assessing virulence of M. marinum. We set up these assays using two amoeba strains, the genetically tractable social amoeba Dictyostelium discoideum and the free-living amoeba Acanthamoeba castellanii. In summary, 64 (5.1%) compounds showed anti-infective/anti-virulence activity in at least one of the three assays. The intracellular assays hit rate varied between 1.7% (n = 22) for M. marinum and 2.8% (n = 35) for L. pneumophila with seven compounds in common for both pathogens. In parallel, 1.2% (n = 15) of the tested compounds were able to restore D. discoideum growth in the presence of M. marinum spiked in a lawn of food bacteria. We also validated the generality of the hits identified in the A. castellanii-M. marinum anti-infective screen using the D. discoideum-M. marinum host-pathogen model. The characterization of anti-infective and antibacterial hits in the latter infection model revealed compounds able to reduce intracellular growth more than 50% at 30 µM. Moreover, the chemical space and physico-chemical properties of the anti-M. marinum hits were compared to standard and candidate Mycobacterium tuberculosis (Mtb) drugs using ChemGPS-NP. A principle component analysis identified separate clusters for anti-M. marinum and anti-L. pneumophila hits unveiling the potentially new physico-chemical properties of these hits compared to standard and candidate M. tuberculosis drugs. Our studies underscore the relevance of using a combination of low-cost and low-complexity assays with full 3R compliance in concert with a rationalized focused library of compounds to identify new chemical scaffolds and to dissect some of their properties prior to taking further steps toward compound development.

The multifarious lysozyme arsenal of Dictyostelium discoideum.

Dictyostelium discoideum is a free-living soil amoeba which feeds upon bacteria. To bind, ingest, and kill bacteria, D. discoideum uses molecular mechanisms analogous to those found in professional phagocytic cells of multicellular organisms. D. discoideum is equipped with a large arsenal of antimicrobial peptides and proteins including amoebapore-like peptides and lysozymes. This review describes the family of lysozymes in D. discoideum. We identified 22 genes potentially encoding four different types of lysozymes in the D. discoideum genome. Although most of these genes are also present in the genomes of other amoebal species, no other organism is as well-equipped with lysozyme genes as D. discoideum.

LrrkA, a kinase with leucine-rich repeats, links folate sensing with Kil2 activity and intracellular killing.

Phagocytic cells ingest bacteria by phagocytosis and kill them efficiently inside phagolysosomes. The molecular mechanisms involved in intracellular killing and their regulation are complex and still incompletely understood. Dictyostelium discoideum has been used as a model to discover and to study new gene products involved in intracellular killing of ingested bacteria. In this study, we performed random mutagenesis of Dictyostelium cells and isolated a mutant defective for growth on bacteria. This mutant is characterized by the genetic inactivation of the lrrkA gene, which encodes a protein with a kinase domain and leucine-rich repeats. LrrkA knockout (KO) cells kill ingested Klebsiella pneumoniae bacteria inefficiently. This defect is not additive to the killing defect observed in kil2 KO cells, suggesting that the function of Kil2 is partially controlled by LrrkA. Indeed, lrrkA KO cells exhibit a phenotype similar to that of kil2 KO cells: Intraphagosomal proteolysis is inefficient, and both intraphagosomal killing and proteolysis are restored upon exogenous supplementation with magnesium ions. Bacterially secreted folate stimulates intracellular killing in Dictyostelium cells, but this stimulation is lost in cells with genetic inactivation of kil2, lrrkA, or far1. Together, these results indicate that the stimulation of intracellular killing by folate involves Far1 (the cell surface receptor for folate), LrrkA, and Kil2. This study is the first identification of a signalling pathway regulating intraphagosomal bacterial killing in Dictyostelium cells.