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. 2014 May 20;5(3):e01178-14.
doi: 10.1128/mBio.01178-14.

The Haemophilus Ducreyi LspA1 Protein Inhibits Phagocytosis by Using a New Mechanism Involving Activation of C-terminal Src Kinase

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The Haemophilus Ducreyi LspA1 Protein Inhibits Phagocytosis by Using a New Mechanism Involving Activation of C-terminal Src Kinase

Dana A Dodd et al. mBio. .
Free PMC article

Abstract

Haemophilus ducreyi causes chancroid, a sexually transmitted infection. A primary means by which this pathogen causes disease involves eluding phagocytosis; however, the molecular basis for this escape mechanism has been poorly understood. Here, we report that the LspA virulence factors of H. ducreyi inhibit phagocytosis by stimulating the catalytic activity of C-terminal Src kinase (Csk), which itself inhibits Src family protein tyrosine kinases (SFKs) that promote phagocytosis. Inhibitory activity could be localized to a 37-kDa domain (designated YL2) of the 456-kDa LspA1 protein. The YL2 domain impaired ingestion of IgG-opsonized targets and decreased levels of active SFKs when expressed in mammalian cells. YL2 contains tyrosine residues in two EPIYG motifs that are phosphorylated in mammalian cells. These tyrosine residues were essential for YL2-based inhibition of phagocytosis. Csk was identified as the predominant mammalian protein interacting with YL2, and a dominant-negative Csk rescued phagocytosis in the presence of YL2. Purified Csk phosphorylated the tyrosines in the YL2 EPIYG motifs. Phosphorylated YL2 increased Csk catalytic activity, resulting in positive feedback, such that YL2 can be phosphorylated by the same kinase that it activates. Finally, we found that the Helicobacter pylori CagA protein also inhibited phagocytosis in a Csk-dependent manner, raising the possibility that this may be a general mechanism among diverse bacteria. Harnessing Csk to subvert the Fcγ receptor (FcγR)-mediated phagocytic pathway represents a new bacterial mechanism for circumventing a crucial component of the innate immune response and may potentially affect other SFK-involved cellular pathways.

Importance: Phagocytosis is a critical component of the immune system that enables pathogens to be contained and cleared. A number of bacterial pathogens have developed specific strategies to either physically evade phagocytosis or block the intracellular signaling required for phagocytic activity. Haemophilus ducreyi, a sexually transmitted pathogen, secretes a 4,153-amino-acid (aa) protein (LspA1) that effectively inhibits FcγR-mediated phagocytic activity. In this study, we show that a 294-aa domain within this bacterial protein binds to C-terminal Src kinase (Csk) and stimulates its catalytic activity, resulting in a significant attenuation of Src kinase activity and consequent inhibition of phagocytosis. The ability to inhibit phagocytosis via Csk is not unique to H. ducreyi, because we found that the Helicobacter pylori CagA protein also inhibits phagocytosis in a Csk-dependent manner. Harnessing Csk to subvert the FcγR-mediated phagocytic pathway represents a new bacterial effector mechanism for circumventing the innate immune response.

Figures

FIG 1
FIG 1
Inhibition of FcγR-mediated phagocytosis by fragments of LspA1. (A) Schematic showing the C-terminal half of the 4,153-aa LspA1 protein. The L1 (aa 1998 to 2348), YL2 (aa 2349 to 2643), and YL3 (aa 3149 to 3448) segments are shaded. The tyrosines that were phosphorylated when recombinant LspA1 proteins were incubated with macrophages (28) are labeled with asterisks. (B) Lysates from HEK293T cells transfected with the indicated plasmids were subjected to immunoprecipitation with anti-Myc beads. Samples were run on two gels. One gel was probed with the phosphotyrosine monoclonal antibody 4G10 (pY), and the other with Myc antibody (Myc). (C and D) COS-7 cells cotransfected with FcγRIIA-GFP and the indicated plasmids were allowed to ingest fluorescently labeled opsonized latex beads. The number of phagocytosed beads per cell (phagocytosis index) was quantitated for each sample. Both results are representative of three independent experiments. (E) COS-7 cells were cotransfected with FcγRIIA-GFP and the indicated plasmids. Red opsonized beads were incubated with transfected cells for 45 min. Samples were then placed on ice, and external beads were labeled blue. The plasma membrane of 1 cell for each sample is shown as a white line. Beads that bound and remained outside the cell are purple in the merge images, whereas beads that were internalized are red in the merge images. It can be seen that the COS-7 cells transfected with YL2 ingested the fewest opsonized beads. The scale bar is 20 µm. (F) RAW264.7 cells transfected with the indicated plasmids were allowed to ingest fluorescent opsonized beads, and phagocytosis ability was quantitated. The result is representative of two independent experiments. Error bars are ± standard deviation (SD) (n = 3 samples) (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
FIG 2
FIG 2
YL2 reduces SFK activity. (A) HEK293T cells cotransfected with either pcDNA3.1 vector (V), Myc-tagged L1, YL2, or YL2-DM constructs, and FcγRIIA-GFP were allowed to phagocytose fluorescently labeled IgG-opsonized beads. The phagocytosis index was determined for each sample. Error bars are ±SD (n = 3 samples) (***, P < 0.001). The result is representative of three independent experiments. (B) Total cell lysates (TCL) from HEK293T cells were probed in Western blot analysis with antibodies recognizing active Src (pY418), inactive Src (pY529), pan Src, Myc, or GAPDH. It should be noted that the amount of cell lysate loaded on the gel for the pY418 blot was 10-fold greater than that used for the pY529 blot.
FIG 3
FIG 3
Pulldown-based detection of YL2-binding proteins in mammalian cells. HEK293T cells cotransfected with L1, YL2, or YL2-DM (DM) containing Strep tags and either Csk-FLAG (A), Nck2-FLAG (B), or Grb2-FLAG (C) were pulled down with either Strep-Tactin beads or FLAG antibody-coated beads. Samples of the pulldowns (IP) and total cell lysate (TCL) were run in SDS-PAGE, and then Western blotting was performed with either FLAG or Strep primary antibodies. (D) Schematic of Csk-derived constructs. (E) HEK293T cells were cotransfected with YL2-Strep and each of the Csk-derived constructs with a FLAG tag. Proteins were either pulled down with anti-FLAG or Strep-Tactin beads. Precipitates (IP) and TCL were probed in Western blot analysis with antibodies against Strep or FLAG.
FIG 4
FIG 4
A dominant-negative Csk (Csk-DN) rescues phagocytosis in the presence of YL2 and is unable to phosphorylate YL2. (A) COS-7 cells cotransfected with three plasmids encoding FcγRIIA, either EGFP-L1 or EGFP-YL2, and either EGFP (control), Csk-DN, or Csk-WT had opsonized fluorescently labeled latex beads added. The phagocytosis index for each sample was quantitated. Error bars are ±SD (n = 3 samples) (***, P < 0.001). The result is representative of two independent experiments. (B) HEK293T cells were cotransfected with YL2-Strep and either Lyn, Csk-DN, or Csk-WT with a FLAG tag. YL2 was precipitated with Strep-Tactin beads. Samples were split into two portions, and one was treated with Antarctic phosphatase (AP). The paired samples were then probed in Western blot analysis with either pY or Strep antibodies.
FIG 5
FIG 5
YL2 enhances Csk activity and is phosphorylated by Csk. (A) Recombinant Csk (65 nM) mixed with recombinant YL2 (at 6.25, 12.5, 25, or 50 nM), YL2-DM (DM) (at 50 nM), or L1 (at 50 nM) was added to wells of a universal tyrosine kinase assay containing bound poly (Glu-Tyr) peptides. Tyrosine phosphorylation of the bound peptide was detected with a horseradish peroxidase (HRP)-conjugated pY antibody, and absorbance at OD450 was compared to a standard curve of a PTK control provided in the kit. One unit of enzyme is the amount needed to incorporate 1 pmol of phosphate into the substrate (KVEKIGEGTYGVVYK; residues 6 to 20 of p34cdc2) in 1 min. Error bars are ±SD (n = 3) (**, P < 0.01). The result is representative of three independent experiments. (B) Recombinant Csk (65 nM) and recombinant YL2 (50 nM) were incubated together with various concentrations of ATP. Western blots of the reactions were probed with either the phosphotyrosine monoclonal antibody 4G10 (pY), a monoclonal antibody directed against YL2, or an antibody to Csk.
FIG 6
FIG 6
CagA from H. pylori inhibits phagocytosis in a Csk-dependent manner. (A) Schematic of the CagA proteins from three strains of H. pylori. The darker portions from approximately aa 400 to the C-terminal end were the sections of the corresponding cagA genes that were cloned into the mammalian expression vector. Dark vertical lines indicate where EPIYA motifs are located. (B) Western blot analysis showing expression of the CagA fusion proteins as detected by probing with GFP antibody. (C) Phagocytosis indices obtained for COS-7 cells cotransfected with FcγRIIA and L1, YL2, or the three different CagA constructs. The result is representative of three independent experiments. (D) COS-7 cells were cotransfected with FcγRIIA-GFP and either vector, L1, YL2, or the G27 CagA construct. For three samples, Csk-DN was also cotransfected. Both YL2 and G27 CagA inhibited phagocytosis, and this inhibition was alleviated by the expression of Csk-DN. Error bars are ±SD (n = 3 samples) (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). These results are all representative of three independent experiments.
FIG 7
FIG 7
A model for YL2 inhibition of FcγRIIA-mediated phagocytosis. (A) In a typical phagocytic cell, IgG-opsonized targets bind to FcγRIIA receptors, resulting in clustering of these receptors and activation of SFKs. These active SFKs phosphorylate the ITAM domains of the receptors. When the ITAMs are phosphorylated, Syk kinases will be activated and a multistep signal transduction pathway ensues, ultimately resulting in actin polymerization and phagocytosis. (B) When the YL2 portion of the H. ducreyi LspA1 protein is present in the phagocytic cell, it binds Csk and thereby stimulates the kinase activity of Csk. The increased Csk kinase activity results in the inactivation of SFKs. Without active SFKs, the ITAMs are not effectively phosphorylated, Syk kinase is not activated, and phagocytosis cannot proceed.

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