Adhesion of professional phagocytes, like macrophages or Dictyostelium cells, to bacteria is achieved by the binding of membrane receptors at the surface of the phagocyte to specific ligand at the surface of the bacteria. In this section I am reviewing the mammalian membrane receptors allowing cells to adhere to bacteria and to phagocytose them (II. B. 1.). I am then presenting a historical perspective on the putative phagocytosis receptors identified in Dictyostelium, SadA, Phg1A and SibA (II. B. 2.).
II. B. 1. Adhesion of mammalian phagocytes to bacteria
Recognition of bacteria by phagocytes involves a large panel of molecules, on both sides.
Pathogens express at their surface molecules such as LPS or flagella described previously in this manuscript (Introduction I.), that are collectively named microbe-associated molecular patterns (MAMPs). Phagocytic cell recognizes MAMPs directly or indirectly. Direct recognition involves receptors for MAMPs at the cell surface of the phagocyte, while indirect recognition involves soluble intermediate molecules named opsonins. Opsonins bind to bacteria and are then recognized by specific receptors at the surface of phagocytes (Fig. 9). Receptors for MAMPs and opsonins are named pattern recognition receptors (PRRs) (Kumar et al., 2011). The binding of MAMPs or opsonins to PRRs triggers a pro-inflammatory response and phagocytosis.
Among MAMPs receptors, Toll-like receptors (TLRs) are mainly involved in controlling the expression of pro-inflammatory cytokines and interferon, but they can also promote phagocytosis (Manavalan et al., 2011). On the contrary scavenger receptors (SRs) have been shown to be specifically involved in phagocytosis (Areschoug and Gordon, 2009). Many receptors have been shown to bind opsonins and trigger phagocytosis, for example Fc!Rs (Indik et al., 1995), complement receptors (CR3) and other integrins (Dupuy and Caron, 2008; van Lookeren Campagne et al., 2007).
a. Toll-like receptors (TLRs)
The TLRs family is composed of 10 members in mammals. All TLRs are type I transmembrane glycoproteins with a extracellular domain containing leucine-rich repeats involved in the interaction with their ligand, a single transmembrane domain and a cytoplasmic domain containing a Toll-like Interleukine-1 receptor (TIR) motif involved in downstream signaling. Despite their common structure, TLRs bind to different ligands (Manavalan et al., 2011). Among the TLRs located at the
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cell surface there are TLRs 1, 2, 4, 5 and 6. All are involved in the recognition of a large panel of microbial or synthetic molecules. More specifically, TLR2 recognizes peptidoglycans of Gram-positive bacteria, TLR4 lipopolysaccharides (LPS) of Gram-negative bacteria (Kumar et al., 2011), and TLR5 the bacterial flagellum (Descamps et al., 2012). Besides inducing the production of pro-inflammatory cytokines, TLRs, and particularly TLR4, have been shown to mediate phagocytosis both directly and indirectly by activating Scavenger Receptor (Doyle et al., 2004; Kong and Ge, 2008).
b. Scavenger receptors (SRs)
Scavenger receptors (SRs) form a family of glycoproteins that have been first identified for their ability to bind modify lipoproteins in macrophages present in atherosclerotic plaques. In fact, their cloning revealed a wide variety of membrane proteins that have been divided in eight groups, from A to H, according to their tertiary structure (Peiser and Gordon, 2001; Peiser et al., 2000) (Murphy et al., 2005). Further analysis revealed a large panel of ligands including proteins and polysaccharides. The SR-A and SR-B families have been described in great detail and have been shown to bind bacteria. Members of the SR-A family are trimeric transmembrane glycoproteins that have been shown to recognize LPS and mediate phagocytosis of bacteria. CD36 has been the first characterized member of the B family of SRs. It is a two-transmembrane protein and has been suggested to be specifically involved in gram-positive bacteria recognition (Areschoug and Gordon, 2009).
c. Opsono-receptors
Fc!Rs and integrins such as complement receptor 3 (CR3) recognize opsonins. More specifically, Fc!Rs recognize immunoglobulins and CR3 the complement’s molecule iC3b. Polysaccharides such as fibronectin and vitronectin are recognized by other integrins (Aderem and Underhill, 1999;
Ulanova et al., 2009; Underhill and Ozinsky, 2002). Immunoglobulins, iC3b, fibronectin, vitronectin are typical opsonins, present in the serum. There are four families of Fc! receptors identified to date: Fc!RI, Fc!RII, Fc!RIII and Fc!RIV (Nimmerjahn and Ravetch, 2006). They all possess immunoglobulin (Ig)-like domains in their extracellular domain, which confer affinity for the Fc constant region of IgG. The binding of Fc!Rs to IgG triggers the phosphorylation of a immune-tyrosine-based activation motifs (ITAMs). In the case of the Fc! receptor IIa, ITAMs are located in the cytoplasmic domain of the receptor.
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Figure 9: Interaction of PRRs with MAMPs.
Bacteria express molecules at their surface collectively designated microbe associated molecular pattern (MAMPs). MAMPs are recognized by receptors named pattern recognition receptors (PRRs) located at the surface of host cell. Well-characterized PRRs include Toll-like receptors (TLRs), Fc! receptors (Fc!Rs), scavenger receptors (SRs) and integrins such as complement receptor (CR3). TLRs recognize specifically LPS and flagella, SRs recognize multiple ligands including polysaccharides, lipids and LPS, and the opsonins including immunoglobulins (Ig), inactivated complement fragment 3b (iC3b) or polysaccharides such as vitronectin, are recognized by Fc!Rs, CR3 and other integrins respectively.
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In the case of the Fc!RI, III and IV, ITAMs are located in the associated ! chain. These phosphorylation events activate downstream signalization and lead to efficient particle uptake (Indik et al., 1995). Integrins are a wide family of heterodimeric proteins formed by two single chain transmembrane proteins named # and $. In humans, # and $ 18 sub-units can form 24 heterodimers (Dupuy and Caron, 2008). The main function of integrins is to participate in cellular adhesion. Some integrins bind bacteria and trigger bacterial engulfment. The first phagocytic integrin described was CR3 (also named #M$2). Activation of integrins requires stimuli that proceed in two steps: first an “inside-out” mechanism and second an “outside-in” mechanism. The
“inside-out” mechanism includes binding of talin to the conserved NPXY motif in the cytoplasmic tail of $2, leading to a conformational change of the integrin and allowing binding of the integrin to its extracellular ligand (e. g. iC3b in the case of the CR3). The binding of the extracellular ligand activates the “outside-in mechanism”, which leads to an actin cytoskeleton reorganization and to the subsequent engulfment of the opsonized particle. Some integrins can bind specific bacterial adhesins such as the invasin of Yersinia species but most integrins bind to opsonins such as fibronectin or vitronectin that bind bacteria like Staphylococcus aureus or Pseudomonas aeruginosa (Ulanova et al., 2009).
II. B. 2. Receptors for phagocytosis in Dictyostelium
Similar to mammalian phagocytes, amoebae must first adhere to bacteria by using adhesion molecules at their plasma membrane. Vogel and coworkers have first suggested the existence of three types of receptors: a non-specific “hydrophobic” receptor, a lectin-type receptor and an unknown receptor involved in the binding to hydrophilic substrates. For this, they randomly mutagenesized Dictyostelium cells and selected mutants unable to phagocytose tungsten beads.
They then analyzed the specificity of the phagocytosis defect by allowing mutant cells to engulf various particles. From their observations they concluded that Dictyostelium possess at least three receptor. A first non-specific receptor binds to hydrophobic substrates such as latex beads. A second lectin-type receptor binds to glucose, such as the glucose residues at the surface of E.coli B/R. And a third receptor that recognizes hydrophilic substrate such as protein-coated latex beads (Vogel et al., 1980).
Since three membrane proteins essential for phagocytosis have been shown: Phg1, SadA and SibA proteins. We recently showed that only SibA is directly involved in adhesion and phagocytosis of hydrophilic particles, and that Phg1A and SadA have regulatory effects on SibA expression. I am describing these three proteins.
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a. Phg1
In 2000, in a screen for mutants presenting a phagocytosis defect of latex beads, random mutagenesis was performed on D. discoideum (Cornillon et al., 2000). The method used was the Restriction Enzyme-Mediated Integration (REMI), which results the random insertion of a blasticidin resistant plasmid in the genome of D. discoideum (Kuspa, 2006; Kuspa and Loomis, 1992). Phagocytosis defective mutants were cloned and the insertion region sequenced. Cornillon and colleagues obtained three clones and identified the genes disrupted in each of them. In one clone the disrupted gene encoded Myosin VII, a protein known to be involved in phagocytosis in D.
discoideum (Titus, 1999). The two other clones had an insertion in the same gene, and were named by the authors PHG1. The PHG1 family is composed of 3 members: PHG1A, PHG1B and PHG1C.
Phg1 proteins have a conserved general structure made of a N-terminal signal peptide, followed by a large variable extracellular domain and containing nine putative transmembrane domains. Based on their structure and sequence, Phg1 proteins belong to the transmembrane 9 superfamily (TM9SF) that exists in other organisms such as human or Drosophila. Based on sequences alignment, this family has been divided in two subgroups: I and II (Fig. 10). The subgroup I includes proteins with a short extracellular domain (220 amino acids) and a characteristic amino-acid motif at the position 50: VGPYxNxQETY. Proteins of the subgroup II are characterized by a longer extracellular domain (280 amino acids) with a conserved motif right after the signal peptide:
FY(V/L)PG(VL)AP (Benghezal et al., 2003). Phg1A belongs to the subgroup II and Phg1B to the subgroup I. Phg1C exhibit mixed features and cannot be include in the one or the other of these subgroups. The precise function of these proteins remains largely unknown but their conservation among evolution suggests an important physiological function.
Further analysis of phg1 knock-out cells revealed that the phagocytosis defect was due to an adhesion defect rather than to an actin-cytoskeleton reorganization defect. Accordingly, macropinocytosis, which involves membrane extension mechanisms similar to phagocytosis, was not affected in these cells. Furthermore, analysis of cell surface proteins revealed strong differences compared to WT cells, suggesting that Phg1 acts as a regulator of the cell surface protein composition rather than as a receptor for phagocytosis (Benghezal et al., 2003).
Immunoblotting experiments revealed the presence of Phg1A at the cell surface, unfortunately we could not develop antibodies that recognize the native protein, and could not perform
immunofluorescence experiments. Orthologs of Phg1 have been identified in other organisms such as Drosophila, humans and zebrafish, named transmembrane nine superfamily (TM9SF), as well as in yeast and named transmembrane nine (Tmn) proteins (Bergeret et al., 2008; Froquet et al., 2008;
Lozupone et al., 2009; Pruvot et al., 2010).
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Figure 10: Phylogenetic tree of TM9SF proteins
This phylogenetic tree has been obtained by comparing amino acids sequences of TM9SF proteins from human (Hs), mouse (Mm), drosophila (Dm), yeast (Sc), zebrafish (Dr) and D. discoideum Phg1A, Phg1B and Phg1C proteins (MEGA software). TM9SF proteins can be divided in two main group based on structural features. While Phg1A belongs to the group II, like the TM9SF4 proteins, Phg1B belongs to the group I, like the TM9SF1 proteins. Phg1C cannot be classified in any of these groups.
sp|Q92544|TM9S4_HUMAN_Transmembrane_9_superfamily_member_4_OS_Homo_sapiens_GN_TM9SF4_PE_1_SV_2 sp|Q8BH24|TM9S4_MOUSE_Transmembrane_9_superfamily_member_4_OS_Mus_musculus_GN_Tm9sf4_PE_2_SV_1 tr|E2D669|E2D669_DANRE_TM9SF4_OS_Danio_rerio_PE_2_SV_1
tr|Q9V3N6|Q9V3N6 DROME GH02822p OS Drosophila melanogaster GN TM9SF4 PE 2 SV 1
sp|Q55FP0|PHG1A_DICDI_Putative_phagocytic_receptor_1a_OS_Dictyostelium_discoideum_GN_phg1a_PE_2_SV_1 tr|Q9VIK1|Q9VIK1 DROME CG9318 OS Drosophila melanogaster GN CG9318 PE 2 SV 1
tr|Q7ZUF5|Q7ZUF5_DANRE_Transmembrane_9_superfamily_member_2_OS_Danio_rerio_GN_tm9sf2_PE_2_SV_1 tr|Q7ZV33|Q7ZV33 DANRE Transmembrane 9 superfamily member 3 OS Danio rerio GN tm9sf3 PE 2 SV 1
tr|Q9VRN1|Q9VRN1 DROME CG10590 OS Drosophila melanogaster GN CG10590 PE 2 SV 2
sp|Q54ZW0|PHG1B_DICDI_Putative_phagocytic_receptor_1b_OS_Dictyostelium_discoideum_GN_phg1b_PE_2_SV_1
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In Drosophila, mutation in TM9SF4 gene, the ortholog of Phg1A protein, leads to impaired adhesion and phagocytosis. In human metastatic cells, Lozupone and colleagues reported a role for TM9SF4 in phagocytic/cannibalistic activity of these cells.
b. SadA
In 2002 another putative phagocytosis receptor was identified by Fey and colleagues and named SadA, for substrate adhesion deficient (Fey et al., 2002). Fey et al performed REMI mutagenesis and selected clones that lost the ability to adhere to plastic petri-dishes. Analysis of the disrupted gene in a selected clone lead to the identification of a gene encoding a protein with 9 putative transmembrane domain with no clear orthologs in other organisms. Analysis of the amino acids sequence revealed the presence of three EGF-like repeats that are conserved motifs of about 40 amino acids containing six cysteines that have the ability to form disulfide bonds. These motifs have been shown in many instances to play a role in protein-protein interactions (Takagi et al., 2001). Based on the observation that SadA null mutants have a strong adhesion defect, that SadA-GFP mainly localizes to the cell surface and that the extracellular domain of SadA contains EGF-like repeats, the authors proposed that SadA could be a phagocytosis receptor in Dicytostelium.
Another study by the same team showed that the cytoplasmic tail of SadA, when phosphorylated, recruits Cortexilin I, a protein involved in F-actin recruitment (Kowal and Chisholm, 2011).
c. SibA
In 2006 Cornillon and colleagues identified a cell surface adhesion molecule, presenting features also found in human beta-integrins: the N-terminal part contains a Von Willebrand factor type A domain (VWA), the transmembrane domain contains a glycine motif (GxxxG) and the cytoplasmic part contains two NPxY motifs. They named it SibA, for similar to integrin beta (Cornillon et al., 2006). SibA null mutants have an adhesion defect to latex beads, that are hydrophilic substrate, but neither to Klebsiella bacteria nor to establish cell-cell adhesion, thus SibA could be the “hydrophilic”
receptor hypothesized by Vogel and coworkers. Moreover, there are four homologues of SibA: B to E. Only SibC has been shown, as SibA, to be involved in adhesion process in vegetative cells.
Analysis of sibC KO revealed redundant functions with SibA, since it is involved in adhesion to the same types of substrates but the expression of SibC depends on a quorum-sensing regulated mechanism and decrease when cell density increase, while expression of SibA is not affected. Thus
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SibA was shown to be the major adhesion protein expressed at the cell surface of vegetative cells (Cornillon et al., 2008).
During my PhD I participated in a study, which showed the regulatory roles of Phg1A and SadA in the surface expression of SibA, rather than a direct role for these two proteins in adhesion mechanisms (see Results II). This study led us to investigate more precisely the role of Phg1A in the sorting of SibA. The results I obtained (see Results III) show that Phg1A recognizes directly the transmembrane domain of SibA, allowing the localization of SibA at the cell surface.
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