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

Abstract

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.

Keywords: Dictyostelium; DrkD; Kil2; Klebsiella pneumoniae; LrrkA; intracellular killing; magnesium.

Figure 1

lrrkA knockout (KO) cells are unable to feed upon Gram‐positive bacteria. (a) To determine the ability of Dictyostelium cells to feed upon Klebsiella pneumoniae (KpGe strain), Dictyostelium cells (10,000, 1,000, 100, or 10 cells) were deposited on a lawn of KpGe. After 4 days, wild‐type (WT) Dictyostelium cells created phagocytic plaques (white) in the bacterial lawn (black). LrrkA KO cells grew as well as WT cells on K. pneumoniae but were unable to grow on Bacillus subtilis. A white square indicates a growth comparable with that of WT cells, and a black square indicates a defective growth. (b) Several bacterial species were assessed as described above. LrrkA KO cells grew very poorly on both Gram‐positive bacteria tested (Micrococcus luteus and B. subtilis) but normally on other bacteria. Bs, B. subtilis; Ec B/r, Escherichia coli B/r; Kp, K. pneumoniae KpGe; Kp21, K. pneumoniae LM21; Ml, M. luteus; Pa PT531, Pseudomonas aeruginosa PT531. Scale bar: 1.5 cm

Figure 2

DrkD/LrrkA belongs to the family of leucine‐rich repeat (LRR) kinases. (a) Organisation of functional domains of DrkA–DrkD, of the Dictyostelium ROCO2 kinase, and of the human LRR kinase 1. DrkA, DrkB, and DrkC contain a putative signal peptide and a putative transmembrane domain situated N‐terminally of the kinase domain. LrrkA/DrkD, Roco 2, and human LRRK1 contain LRRs and a kinase domain. In addition, ROCO proteins (Roco 2 and human LRRK1) contain a Roc and a Cor domain. (b) Schematic phylogenetic tree representing the number of LRR kinases (with or without a Roc/COR domain) in eukaryotes (indicated in red) and the number of LRR kinases containing a full Roc/COR domain (indicated in blue)

Figure 3

General organisation of cellular compartments is similar in lrrkA knockout (KO) and in wild‐type (WT) cells. (a) Immunofluorescence labelling of p80 and H⁺‐ATPase allowed to identify lysosomes (arrowheads; p80⁺ and H⁺‐ATPase⁺) and post‐lysosomes (arrows; p80⁺ and H⁺‐ATPase⁻). (b) Immunofluorescence labelling of rhesus and H⁺‐ATPase allowed to identify contractile vacuole (circles with bar; rhesus⁺ and H⁺‐ATPase⁺). (c) Immunofluorescence labelling of recycling endosomes enriched in p25. Scale bar in (a–c): 5 μm. (d) Intracellular retention of lysosomal glycosidases is as efficient in WT as in lrrkA KO cells. After 4 days of culture in HL5 medium, Dictyostelium cells were recovered by centrifugation, and the activity of two lysosomal enzymes (NAG, N‐acetyl‐β‐glucosaminidase; NAM, α‐mannosidase) was measured in cell pellets and in supernatants using chromogenic substrates. The percentage of intracellular glycosidase activity was not different for WT and lrrkA KO cells (mean ± standard error of the mean, N = 7, paired Student's t test, NAG p = .948, NAM p = .492). (e) Intracellular levels of lysozyme activity are similar in WT and lrrkA KO cells (mean ± standard error of the mean, paired Student's t test, N = 5, p = .251)

Figure 4

Acidification of phagosomes is unaffected in lrrkA knockout (KO) cells. (a) Wild‐type (WT) and lrrkA KO cells were allowed to internalise 3‐μm‐diameter beads coated with two fluorophores (pH‐sensitive FITC and pH‐insensitive Alexa 594) and imaged every 75 s for 3 hr. The pH was deduced from the fluorescence 488/594 nm ratio (mean ± standard error of the mean, N = 4, n = 40 beads for each strain). The kinetics of acidification and the pH value in phagolysosomes were identical in lrrkA KO and in WT cells. The fluorescence ratio in acidic phagolysosomes is 0.324. (b) Calibration curve of the pH‐sensitive beads in buffer ranging from pH 1 to 9. The dynamic range allows to discriminate variations of pH from 2 to 8 (mean ± standard error of the mean, N = 3, n = 100 beads per pH). A fluorescence ratio of 0.324 corresponds to a pH value between 2.5 and 3

Figure 5

Intracellular killing of Klebsiella pneumoniae is impaired in lrrkA knockout (KO) cells. Dictyostelium cells were incubated with GFP‐expressing K. pneumoniae (Kp‐GFP) in phosphate buffer–sorbitol for 2 hr. Cells were observed by phase‐contrast and fluorescence microscopy, and the ingestion and intracellular killing of Kp‐GFP were monitored. (a) The probability of bacterial survival following ingestion is represented as a Kaplan–Meyer estimator for one experiment in wild‐type (WT) cells (white squares) and lrrkA KO cells (black squares). (b) For each experiment, the survival of bacteria was determined by measuring the area under the survival curve from 0 to 75 min. Each dot is the result of a separate experiment. Intracellular killing was significantly slower in lrrkA KO cells and in kil2 KO cells than in WT cells (p = .013; paired Student's t test, N = 8 independent experiments). Intracellular killing was not slower in lrrkA–kil2 KO cells than in kil2 KO cells (p = .510; paired Student's t test, N = 7 independent experiments), but it was significantly slower in lrrkA–kil1 KO cells than in kil1 KO cells (p = 4.10⁻⁴; paired Student's t test, N = 6 independent experiments). Total number of events observed: WT = 351, lrrkA = 496, kil2 = 274, lrrkA–kil2 = 252, kil1 = 120, and lrrkA–kil1 = 120

Figure 6

Proteolytic activity in phagosomes is reduced in lrrkA knockout (KO) cells. (a) Cells were allowed to internalise beads coated with a quenched fluorophore (DQ™ Green BSA) and a proteolysis‐insensitive dye (Alexa 594) and were imaged every 75 s. Representative, successive pictures of a wild‐type (WT) cell ingesting a bead and processing it over 80 min are shown. Upon degradation of BSA by proteases, the DQ™ Green is released in phagosomes, and the corresponding fluorescence increases. (b) To quantify proteolysis, we plotted the 488/594‐nm fluorescence ratio as a function of time following phagocytosis. In all cells, DQ green fluorescence reached a plateau approximately 40 min after phagocytosis. The fluorescence ratio was lower in lrrkA KO cells than in WT cells and even lower in kil2 KO cells or in kil2–lrrkA KO cells (mean ± standard error of the mean, N = 4, n = 40 beads for each strain)

Figure 7

Exogenous addition of magnesium restores intracellular killing and proteolytic activity in lrrkA knockout (KO), kil2 KO, and lrrkA–kil2 KO cells. (a) Intracellular killing of Klebsiella pneumoniae was determined in wild‐type (WT), lrrkA KO, kil2 KO, and lrrkA–kil2 KO cells in the presence or absence of MgCl₂ (1 mM), as described in Figure 5. In these experiments, MgCl₂ was directly added in the medium containing cells and bacteria. Exogenous MgCl₂ accelerated significantly intracellular killing in all mutant cells (p < 10⁻⁴; paired Student's t test, N = 5 independent experiments). (b) Proteolytic activity was measured in phagosomes in the presence of 1 mM of MgCl₂ as detailed in Figure 6. Normal levels of phagosomal proteolytic activity were restored in all mutant cells by addition of exogeneous magnesium

Figure 8

Far1, LrrkA, and Kil2 participate in a folate‐sensitive pathway stimulating intracellular killing. Intracellular (IC) killing of Klebsiella pneumoniae (Kp) was determined in far1 knockout (KO), lrrkA KO, kil2 KO, and kil1 KO cells in the presence or absence of folate (1 mM), as described in Figure 5 (N = 5). Each dot represents the difference of area under the curve for each strain with or without folate. All values above zero indicate that the addition of folate slowed intracellular killing. Conversely a value below zero indicates that addition of folate accelerated intracellular killing. Folate stimulated intracellular killing in kil1 KO cells but not in far1 KO, lrrkA KO, and kil2 KO cells, revealing the role of Far1, LrrkA, and Kil2 in increasing killing upon folate sensing

Figure 9

Intracellular killing of Klebsiella pneumoniae. This simple scheme describes our working model incorporating the results described in this study. Folate activates Far1, which activates LrrkA, resulting in the stimulation of the activity of Kil2 and transfer of magnesium ions (Mg²⁺) from the cytosol to the phagosomal lumen. In the presence of increased levels of magnesium ions, lysosomal enzymes kill more efficiently ingested K. pneumoniae bacteria (Kp). Kil1 and Vps13F play a distinct role in the delivery of properly modified enzymes (notably proteases) to phagosomes