Intracellular killing involves bactericidal molecules such as anion superoxide, proton ATPases and lysosomal enzymes delivered through fusion with endocytic compartments as described in the section II. C. In the following sections I am describing bactericidal molecules at play in mammalian phagolysosome and their mechanisms to ensure efficient bacterial killing (II. D. 1.) (Fig. 15), I am then comparing with the bactericidal molecules in Dictyostelium phagolysosome (II. D. 2.).
II. D. 1. Bacterial killing in the mammalian phagolysosome
a. The respiratory burst
• History and controversy
The discovery of the role of NADPH oxidase in bacterial elimination came from the study of the chronic granulomatous disease (CGD). CGD is a genetic disorder that is detected most frequently in patients with recurrent pneumonia and abcesses of the skin or organs. An impaired oxidative function of leukocytes in these patients is responsible for the disease (Baehner and Nathan, 1967).
Further analysis of neutrophils and macrophages from CGD patients led to the discovery of an impaired production of anion superoxide (O2.-) by the Nicotinamide Adenine Dinucleotide Phosphate oxidase (NADPH oxidase) complex in phagosomes (Mandell et al., 1970).
In normal activated macrophages, NADPH oxidase (encoded by the NOX2 gene) is present at the membrane of the phagosome and converts NADPH to NADP+, releasing one proton in the cytoplasm and 2 electrons that are translocated inside the phagosome (Ejlerskov et al., 2012). These electrons are transferred to dioxygen to produce anion superoxide (O2.-). The activity of the NADPH oxidase thus creates a voltage gradient across the phagosomal membrane and consumes lumenal protons that must be counteracted by the entry of cations, in order to prevent self-inhibition of the NADPH oxidase. The search for the compensating cation identity raised a controverse. Segal and colleagues proposed that part of the charge compensation was achieved by the entry of potassium (K+) ions through large conductance potassium channels (BK) (Ahluwalia et al., 2004;
DeCoursey, 2004; Reeves et al., 2002; Segal, 2005). These data were contradicted by later study, which failed to identify BK channels in macrophages and neutrophils, reported that inhibition of potassium channels did not affect bacterial killing in these cells and that the function of NADPH oxidase required a proton influx. Early observations of Segal and colleagues were recently retracted (2010; Femling et al., 2006).
! &#!
Later on, the identification of a proton channel at the phagosomal membrane and involved in ROS production, reopened the debate (Ramsey et al., 2006; Sasaki et al., 2006). It was named human voltage gated proton channel 1 (Hvcn1). Hvcn1 null mice have impaired ROS production in phagocytic cells and a flat proton current, associated with an acidification of the cytosol. Thus, Hvcn1 has been proposed to be the channel allowing the entry of protons inside the phagosome that counteracts the voltage gradient (Demaurex and El Chemaly, 2010; El Chemaly and Demaurex, 2012).
• ROS production
Production of ROS is a sequence of chemical reactions initiated by the production of O2.-. ROS have been shown to have oxidizing effects on bacterial component like proteins, nucleic acids and fatty acids (Haas, 2007). In the phagosomal lumen the enzyme superoxide dismutase (SOD) converts O2.- and H2O into hydrogen peroxide (H2O2), then the myeloperoxydase (MPO) converts H2O2 and chloride (Cl-) into hypochlorous acid (HOCl), the major component of household bleach.
HOCl can generate chlorine (Cl2) and chlorine derivatives, which specifically oxidize aldehyde groups (Klebanoff, 2005) (Rada and Leto, 2008).
Superoxide (O2.-) produced by NADPH oxidase can also react with nitric oxide radical (NO%) and produce peroxynitrite (ONOO-) that triggers protein oxidation and DNA fragmentation (Chakravortty and Hensel, 2003). NO% is produced by the action of the nitric oxide synthase, a membrane protein present at the phagosomal membrane, on nitric oxide (NO) produced by the oxidization of L-arginine into L-citrulline (MacMicking et al., 1997).
b. Acidification
Acidification of the newly formed phagosomal compartment containing the engulfed bacteria is rapid (2 minutes) and is due to the acquisition at the phagosomal membrane of the vacuolar H+ -ATPase due to fusion with endocytic compartments (Lukacs et al., 1990; Sun-Wada et al., 2009).
The structure and function of this highly conserved transporter were mainly characterized in yeast (Nelson et al., 2000). Vacuolar H+-ATPase is a multiprotein complex, which involves a membrane part, the V0 complex, and a cytosolic part, the V1 complex. In its activated form, the cytosolic part associates with the transmembrane part to form a pump. The cytosolic part hydrolyses ATP into ADP, providing the energy necessary to transport protons inside the phagosome via a channel formed by the membrane part. During phagosome maturation, V H+-ATPase assembles at the plasma membrane of the nascent phagosome and accumulates in the maturating phagosome, creating a pH reported to be as low as 4.5 (Steinberg et al., 2007). V H+-ATPase activity is
! &$!
electrogenic and requires counter-ions transfer to support acidification, either exit of cations or entry of anions. A transporter of potassium (K+) and Cl-/H+ antiporter like the chloride channels (CLCs) have been proposed to ensure this function but their role in maintaining acidic pH is still under investigation (Mindell, 2012). An acidic pH is required for hydrolytic enzymes activity as well as for production of hydrogen peroxide in macrophages (Vieira et al., 2002). The importance of acidification is illustrated by the studies on intracellular replication of Mycobacterium tuberculosis that requires exclusion of V H+-ATPase from phagosomal membrane, thus preventing acidification of the phagosomal lumen (Rohde et al., 2007; Wong et al., 2011).
c. Ions
Besides protons, ions are important regulators of physiological functions including bacterial killing.
Indeed pathogenic bacteria manipulate phagosomal ionic concentrations in order to survive and replicate (Nusse, 2011). Iron (Fe3+), chloride (Cl-), potassium (K+) and magnesium (Mg2+) have been shown to be involved in bacterial killing.
Like many organisms, bacterial redox-dependent enzymes require iron. Proteins that control iron homeostasis in the cell, and more particularly in the phagosome are essential for immunity against pathogens (Johnson and Wessling-Resnick, 2012). The natural resistance associated macrophage protein 1 (Nramp1) is a divalent metal transporter that is associated to late endosome/phagolysosome, and has been shown to deplete iron and manganese from the lumen of the phagosome, depriving bacteria of these ions and leading to a bacteriostatic effect (Forbes and Gros, 2003; Gruenheid et al., 1997).
The two transporters of Cl-, the cystic fibrosis transmembrane conductance receptor (CFTR) and the chloride channel 3 (CLC-3) have been shown to promote entry of Cl- inside the phagosome. The Cl -ions are necessary for the production of hypochlorous acid (HOCl) by the action of myeloperoxidase on H2O2, a reaction essential for bacterial killing (Moreland et al., 2006; Painter et al., 2010). Studies of bacterial survival in phagosomes revealed that while P.
aeruginosa is sensitive to Cl-, L. monocytogenes exploit CFTR to escape from phagosomes. Indeed, Radtke and colleagues showed that L. monocytogenes requires phagosomal Cl- to escape from phagosome. These results highlight the role of ions in bacterial elimination as well as the diversity of bacterial virulence mechanisms (Painter et al., 2008; Radtke et al., 2011).The hypothesis of a prominent role for K+ in NADPH oxidase activity has been refuted as discussed previously in this chapter. Investigations on another putative role of K+ in bacterial killing suffer from the lack of efficient probes to measure intraphagosomal potassium fluctuations (Nusse, 2011). One possible starting point, as suggested by Nüse et al, for further studies on the role of potassium would be to
! &%!
study the Kef potassium channel expressed by M. tuberculosis, that triggers entry of K+ inside the phagosome, and paradoxically appears to impede bacterial survival (Butler et al., 2010).
Finally, a few studies analyzed the role of magnesium (Mg2+) in bacterial killing. One in vitro study revealed that Mg2+ activates a human elastase HLE by detaching it from the glycoaminoglycans of the matrix of the granules in neutrophils (Kostoulas et al., 1997). Consistent with this report, Martin-Orozco et al showed that depletion of Mg2+ in phagosome favored the growth of Salmonella enterica (Martin-Orozco et al., 2006).
d. Lysosomal enzymes
Lysosomal enzymes are soluble proteins often referred to as acid hydrolases. They include a wide variety of enzymes such as nucleases involved in DNA or RNA degradation, proteases, such as cathepsins and collagenases, glycosidases such as #-glucosidase, "-mannosidase and lysozyme, and lipases like sphingomyelinases, esterases (Flannagan et al., 2009). One of the most studied families of acid hydrolases is the Cathepsin family. It is composed of eleven members in mammals. Most of them are cysteine proteases. These enzymes are produced in an inactive form and require protease activation, low pH and glycosaminoglycans to be activated. After synthesis and postranlational modification in the Golgi they are transported to lysosomes (Conus and Simon, 2010).
Lysozyme is glycosidase, which specifically cleaves glycosidic bonds of peptidoglycan (PGN), a component of bacterial cell wall (Nakimbugwe et al., 2006a). Degradation of PGN by lysozyme has been shown to trigger bacterial killing as well as inflammation response.
Interestingly, Shimada and colleagues showed that bacteria Staphylococcus aureus escape killing by modifying their PGN making it indigestible for lysozyme (Shimada et al., 2010).
In this introduction I aimed to give a general overview of the internalization and killing processes in mammals.
Many questions remain such as does actin drives membrane extension during engulfment? Do cations such as K+ or Mg2+ have a role in hydrolytic enzyme activity? What is the relative importance of the different bactericidal mechanisms?
! &&!
Figure 15: Bacterial killing in phagolysosome. Most known effectors of bacterial elimination are depicted on this scheme. The synergistic activities of the NADPH oxidase, the V H+-ATPase, hydrolytic enzymes and ions pumps/channels is required for efficient bacterial killing. The production of superoxide by the NADPH oxidase promotes the formation of highly toxic oxidative compounds. The V H+-ATPase provides the acidification of the phagosomal lumen and participates in hydrolytic enzymes activities. Pumps or channels allow the entry or the exit of ions. The entry of Cl- and the exit of Fe2+ have deleterious effects on a variety of bacteria like
! &'!
II. D. 2. Intracellular killing in Dictyostelium
As in mammals, phagolysosomes in Dictyostelium contain lysosomal enzymes, specific ions concentrations and exhibit an acidic pH, all of these parameters participate in bacterial elimination.
Several tools have been used to decipher killing mechanisms in Dictyostelium. Mutagenesis targeting genes known to be involved in phagocytosis in mammals has allowed the comparison of bacterial killing mechanisms in both organisms. Among the similarities, several components of the Dictyostelium phagosome have orthologs in mammals such as the NADPH oxidase, the V H+ -ATPase, cysteine proteinases and the Nramp1 ion transporter. Random mutagenesis allowed the discovery of new genes involved in bacterial killing such as PHG1, KIL1 and KIL2.
In the following sections I will briefly discuss the similarities between mammals and Dictyostelium.
Then I will describe more in detail the new genes identified in Dictyostelium and present the results I obtained that aimed to clarify their roles in bacterial killing.
a. The role of mammalian orthologs
As in mammals, it is suggested that acidification of the lysosome is required for some acid hydrolases to function (Temesvari et al., 1996). By using a specific inhibitor of V H+-ATPase, Temesvari and colleagues showed that an impaired acidification of endolysosomal compartments delays the processing and targeting of the lysosomal enzyme "-mannosidase to the lysosome. In addition to the role of pH in regulating lysosomal enzyme activity, Peracino and colleagues showed that acidification of the phagosomal lumen by V H+-ATPases is required for iron eflux from the phagosome thereby preventing the growth of Legionella (Peracino et al., 2010) (see below, ions flux paragraph). The precise role of acidification in bacterial killing is still under investigation.
Orthologs of the mammalian NADPH oxidase have been identified in Dictyostelium (Lardy et al., 2005). Among them, three flavocytochromes large subunits (NoxA, NoxB and NoxC), and the small subunit p22phox have been identified. The authors also knock-out cells of the four proteins mentioned above and observed developmental defects but the growth on Klebsiella aerogenes was not affected, suggesting that NADPH oxidase is not required for the ingestion and killing of these bacteria. Benghezal and colleagues analyzed the growth of the noxA KO, which has been shown to be the major isoform expressed at the vegetative stage, on K. aerogenes and on Bacillus subtilis as well as the killing of these two bacteria and reported no defect compared to WT cells (Benghezal et al., 2006). Hence, contrary to mammalian phagocytes, superoxides generated by the NADPH oxidase are unlikely to participate in bacterial elimination in Dictyostelium.
! &(!
Several lysosomal enzymes have been identified in Dictyostelium. The "-mannosidase and #-glucosidase have been the most extensively studied, as well as cysteine proteinases similar to mammalian Cathepsins (Cardelli et al., 1986; Mehta et al., 1995). Finally, Muller and colleagues identified the first lysozyme-like protein in Dictyostelium (Braun et al., 1972; Muller et al., 2005).
This enzyme, like in mammals, degrades specifically the bacterial cell wall.
To finish, an orthologue of the mammalian Nramp1 has been detected in Dictyostelium (Peracino et al., 2006). It is a divalent metal transporter found in the phagosomal membrane. Peracino and colleagues generated Dictyostelium nramp1 KO mutant and analyzed its ability to kill bacteria (Peracino et al., 2006). They showed that Legionnela prevent assembly of the V H+-ATPase but not Nramp1at the phagosomal membrane. Absence of acidic pH triggers influx activity of the Nramp1 transporter rather than efflux, loading the phagosomal vacuole with divalent cation such as Fe2+ and thereby promoting bacterial growth (Peracino et al., 2010).
b. New gene products implicated in killing
• Phg1 and Kil1 proteins
The role of Phg1A in adhesion has been clarified and it is now assumed that Phg1A act as a regulator of adhesion by modifying cell surface expression of adhesion molecules like SibA (Froquet et al., 2012) (see also the part D of this manuscript). Interestingly phg1A KO cells also exhibit a defective growth on and killing of Klebsiella, which cannot account for an adhesion defect since internalization of Klebsiella is not affected in this mutant. These defects were restored by the overexpression in phg1A KO of the protein Kil1 (Benghezal et al., 2006) (Fig. 18). Analysis of the killing of Klebsiella by kil1 KO revealed a defect intermediate to those observed in WT and in phg1A KO cells.
Prediction structure suggested that Kil1 is a single pass type II transmembrane domain, therefore locating the N-terminus of this protein on the lumenal face. Sequence alignment revealed homology to the human heparate sulfate sulfotransferase 4 (NDST4). This enzyme replace acetyl group from N-acetyl-D-glucosamine (GlcNac) residues with sulfate (Aikawa et al., 2001). This enzyme has thus a role in the general organization of heparin sulfate proteoglycans that constitute membrane and extracellular proteoglycans matrix. Four homologues exist in mammals: NDST1 to 4.
Interestingly Kil1 is the only sulfotransferase detected in Dictyostelium (Benghezal et al., 2006) (see general discussion).
Lelong et al performed REMI mutagenesis and selected clones, which fail to grow on a lawn of Klebsiella as well as to kill these bacteria. They finally obtained one clone that they named Kil2 for
! &)!
its killing defect. By performing sequence alignment they found that Kil2 share many similarities with the members of the P-type ATPase family, proteins that transport ions across membranes.
Analysis of the kil2 KO revealed that the Kil2 is involved in phagosome physiology. More precisely, they found out that Kil2 transports magnesium from the cytoplasm to the lumen of the phagosome, and that the presence of this specific ion in the phagosome is required for the killing of Klebsiella (Lelong et al., 2011). The exact mechanism of Mg2+ in bacterial killing is unknown but it has been suggested that cations such as Mg2+ could participate in acid hydrolases releasing in the phagosomal lumen (Kostoulas et al., 1997).
• Kil2
The protein Kil2 has been identified in our laboratory as a P-type ATPase that translocates magnesium inside the phagosome. kil2 knock-out cells fail in killing Klebsiella bacteria but not other species such as Bacillus subtilis (Fig. 16), suggesting that magnesium is specifically required for the killing of Klebsiella (Lelong et al., 2011).
Addition of magnesium in the medium allows the growth of kil2 KO on a lawn of Klebsiella as well as the killing of these bacteria. It has been suggested that magnesium allows the activation of hydrolases by detaching them from the glycosaminoglycans matrix (Kostoulas et al., 1997). The study of Lelong and colleagues is the first report demonstrating a role for magnesium in bacterial elimination.
The specific defects of phg1A KO, kil2 KO and kil1 KO in the killing of Klebsiella (Fig. 16) led us to address the question of the putative interrelation of these genes in Klebsiella elimination. In addition, we analyzed the role of Phg1B in this process, which has been seen to have synergistic effects with Phg1A in adhesion process, but whose role in Klebsiella elimination has not been investigated further.
During my PhD I showed that the killing defect of phg1A KO is mainly due to the loss of Kil1 in this mutant. I showed that Phg1B act in a synergistic manner to Phg1A. Finally I showed that Kil2 participate in bacterial killing in a distinct mechanism. (see Results IV).
! '+!
Figure 16: Specific growth defect of phg1A KO, kil2 KO and kil1 KO cells on K. aerogenes. As previously detailed, plaque assay is the common test to assessed virulence of a bacterial strain toward different Dictyostelium strains. The method is described in the figure 5. The gradient of virulence extends from bacteria permissives (green squares) to bacteria non-permissives (red) for the growth of Dictyostelium, including intermediate phenotype (orange). Here is presented the virulence of Escherichia coli DH5", Klebsiella aerogenes (Ka), Micrococcus lutueus (Ml), Bacillus subtilis (Bs) and Pseudomonas aeruginosa (PT5) toward Dictyostelium WT and mutant strains. M.
luteus and B. subtilis are two Gram-positive bacteria.
WT (DH1) phg1a KO
kil1 KO kil2 KO
phg1b KO phg1a KO+KIL1
DH5! Ka Bs PT5
Ml
! '*!
!
!
!
!
!
!
!
!
!
!
!
!
RESULTS
! '"!
RESULTS