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The LspA proteins (LspA1 and LspA2) of Haemophilus ducreyi are necessary for this pathogen to inhibit the phagocytic activity of macrophage cell lines, an event that can be correlated with a reduction in the level of active Src family protein tyrosine kinases (PTKs) in these eukaryotic cells. During studies investigating this inhibitory mechanism, it was discovered that the LspA proteins themselves were tyrosine phosphorylated after wild-type H. ducreyi cells were incubated with macrophages. LspA proteins in cell-free concentrated H. ducreyi culture supernatant fluid could also be tyrosine phosphorylated by macrophages. This ability to tyrosine phosphorylate the LspA proteins was not limited to immune cell lineages but could be accomplished by both HeLa and COS-7 cells. Kinase inhibitor studies with macrophages demonstrated that the Src family PTKs were required for this tyrosine phosphorylation activity. In silico methods and site-directed mutagenesis were used to identify EPIYG and EPVYA motifs in LspA1 that contained tyrosines that were targets for phosphorylation. A total of four tyrosines could be phosphorylated in LspA1, with LspA2 containing eight predicted tyrosine phosphorylation motifs. Purified LspA1 fusion proteins containing either the EPIYG or EPVYA motifs were shown to be phosphorylated by purified Src PTK in vitro. Macrophage lysates could also tyrosine phosphorylate the LspA proteins and an LspA1 fusion protein via a mechanism that was dependent on the presence of both divalent cations and ATP. Several motifs known to interact with or otherwise affect eukaryotic kinases were identified in the LspA proteins.
Haemophilus ducreyi is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease (34). While extremely rare in the United States, this disease is prevalent in some developing countries (7). Very little is known about the bacterial gene products necessary for development of chancroidal ulcers, although a human challenge model for experimental chancroid (4, 41) identified several genes whose function is necessary at least for the formation of pustules at an efficiency equivalent to that of the wild-type parent strain (3, 8, 12, 13, 18, 19, 40). This gram-negative organism resists phagocytosis in vitro (2, 48); this ability to resist phagocytosis is dependent on the expression of either the LspA1 or the LspA2 protein (44). These two H. ducreyi gene products are 86% identical (46) and are released by a two-partner secretion system (47). Each can be detected in cell-free culture supernatant fluid (46), with LspA1 appearing to be more abundant than LspA2 (45). The LspA1 and LspA2 proteins themselves are the largest proteins expressed by H. ducreyi, containing 4,152 and 4,919 amino acids, respectively. The documented attenuation of an lspA1 lspA2 mutant in a human challenge model (19) substantiated the involvement of these proteins in disease production in vivo.
Wild-type H. ducreyi cells expressing LspA1 and LspA2 not only resist phagocytosis but also inhibit the phagocytosis of secondary targets (e.g., opsonized animal erythrocytes) by both macrophage-like cell lines (e.g., U-937 and J774A.1) and granulocytes from peripheral blood (2, 48). Investigation of protein tyrosine phosphorylation profiles of macrophages incubated with the wild-type H. ducreyi strain or the lspA1 lspA2 mutant revealed that incubation with the former strain resulted in significant reductions in the levels of phospho-active Src family protein tyrosine kinases (PTKs) (27). These eukaryotic PTKs are among the most proximal elements in the phagocytic signaling pathway (for a review, see reference 20). Exactly how the LspA1 and LspA2 proteins facilitate this decrease in the level of active Src PTKs in the macrophage is not known but could occur by either of two different mechanisms. Internalization of these H. ducreyi proteins by endocytosis could allow the LspA proteins to affect Src family PTKs directly (e.g., by binding to PTKs) or indirectly (e.g., by activating or concentrating protein tyrosine phosphatases). Alternatively, binding of these H. ducreyi proteins to a receptor on macrophages could trigger a negative signaling cascade, similar to that which occurs when the CD47 molecule on erythrocytes binds to SIRP-α on the macrophage (43).
Our interest in how H. ducreyi could affect signaling pathways involved in phagocytosis led us to investigate protein tyrosine phosphorylation patterns in macrophages exposed to this bacterium. Unexpectedly, we found that the H. ducreyi LspA proteins are themselves tyrosine phosphorylated after incubation with macrophages. Tyrosine phosphorylation of bacterial proteins by eukaryotic kinases is not a common occurrence, although such phosphorylations can have profound effects (for a review, see reference 6). Tyrosine phosphorylation of LspA proteins also occurred with nonimmune cells. Sequence homology searches and site-directed mutagenesis led to the identification of four tyrosine residues in LspA1 that were targets for phosphorylation. The involvement of Src family PTKs in tyrosine phosphorylation of the LspA proteins was demonstrated by using both recombinant Src PTK and inhibitors of this kinase. A novel finding of this study was the fact that the LspA proteins and an LspA fusion protein could be tyrosine phosphorylated by macrophage lysates in the absence of exogenously added ATP. In silico analysis identified several predicted motifs within the LspA proteins that could be involved in facilitating tyrosine phosphorylation activity in the macrophage lysates.
The wild-type H. ducreyi strain 35000HP (4) and the H. ducreyi lspA1 lspA2 mutant 35000HPΩ12 (19) were grown on chocolate agar plates at 33°C in a humidified atmosphere containing 95% air-5% CO2, as described previously (33). For liquid culture, H. ducreyi strains were grown in a modified version of a Columbia broth-based medium (46). Escherichia coli strains used for cloning and other genetic manipulations were grown in Luria-Bertani medium supplemented with ampicillin (100 μg/ml) or chloramphenicol (30 μg/ml), when appropriate, for maintenance of plasmids.
The mouse monocyte-macrophage cell line J774A.1 (ATCC TIB-67; American Type Culture Collection, Manassas, VA) was cultivated in Dulbecco's modified Eagle's medium (MediaTech, Inc., Herndon, VA) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (FCS). The mouse monocyte-macrophage cell line RAW 264.7 (ATCC TIB-71) was cultivated as suggested by the ATCC in this same medium, as were the human epithelial cell line HeLa (ATCC CCL-2) and the monkey kidney fibroblast cell line COS-7 (ATCC CRL-1651).
To obtain LspA proteins free of bacterial cells, concentrated culture supernatant (CCS) fluid was prepared by using broth-grown H. ducreyi cells as described previously (46).
Mice were immunized with purified His-LspA1 fusion protein 8 (designated HL8) (27) which contains amino acids (aa) 2349 to 2639 from the H. ducreyi 35000HP LspA1 protein. The hybridoma fusion procedure was performed by the University of Texas Southwestern Antibody Core Facility, and culture supernatant fluid from the hybridoma fusion clones was screened by enzyme-linked immunosorbent assay with the fusion protein as antigen. Clones found positive by enzyme-linked immunosorbent assay were then screened in immunoprecipitation reactions to determine if the LspA-reactive monoclonal antibodies (MAbs) would precipitate native LspA proteins from CCS. The MAb secreted by hybridoma cell line 3H9 immunoprecipitated an LspA protein with an apparent molecular mass much greater than 250 kDa from H. ducreyi CCS. Subsequent analyses showed that MAb 3H9 binds to both LspA1 and LspA2.
Two other LspA1 fusion proteins were used to develop a polyclonal rabbit antibody reactive with the LspA1 protein. Nucleotides encoding LspA1 aa 2693 to 3053 containing the YopT-like region (46) were cloned into the pGEX-4T-2 vector to obtain a glutathione S-transferase (GST) fusion protein designated GST-Yop. Similarly, nucleotides encoding LspA1 aa 2001 to 3033 were cloned into the same plasmid vector to obtain a larger GST-LspA1 fusion protein designated GST-788.5.
Western blot analysis was performed by standard methods. In general, the phosphotyrosine (pY)-specific MAb 4G10 was used as a horseradish peroxidase (HRP) conjugate (Millipore) in Western blot analysis whereas the other antibodies used in this study were detected by using HRP-conjugated goat anti-mouse immunoglobulin G or HRP-conjugated goat anti-rabbit immunoglobulin G as secondary antibodies. When a blot needed to be probed with more than one primary antibody, the blot was stripped with Restore Western Blot Stripping Buffer (Pierce Biotechnology, Rockford, IL) and then probed with the second primary antibody.
The construction of recombinant E. coli plasmids encoding His-tagged LspA1 fusion protein fragments 7, 8, 8.5, and 9 was described previously (27). For brevity, these same fusion proteins were designated HL7, HL8, HL8.5, and HL9, respectively. These His-LspA1 fusion proteins were purified from E. coli host strain M15(pREP4) under native conditions according to the manufacturer's protocol (Qiagen, Valencia, CA).
For construction of the GSTL fusion protein containing aa 3060 to 3385 of the LspA1 protein and GST, nucleotides encoding the LspA repeat region (978 bp) (46) were amplified from H. ducreyi 35000HP genomic DNA by using Pfu polymerase (Stratagene, La Jolla, CA) together with oligonucleotide primers 1 and 2 (Table (Table1).1). The BamHI- and AvaI-digested PCR amplicon was ligated into the BamHI- and SalI-digested pGEX-4T-2 vector (GE Healthcare BioSciences Corp., Piscataway, NJ) to obtain the expression plasmid pGEX-rpt. The GSTL fusion protein was expressed in E. coli strain BL21(DE3) (pLysS) (Stratagene) and purified on a GSTrapFF 1-ml column in an AKTA fast-protein liquid chromatography system (GE Healthcare). The purified GSTL fusion protein was identified in Western blot analysis by using an anti-GST antibody (GE Healthcare) and mouse polyclonal antibody against the HL9 fusion protein (27) independently.
Site-directed mutagenesis of the DNA inserts encoding HL8 and GSTL was performed according to manufacturer's protocol with a QuikChange II site-directed mutagenesis kit (Stratagene). The relevant oligonucleotide primers are listed in Table Table11.
H. ducreyi strains grown in 50-ml broth cultures overnight were harvested by centrifugation at 1,000 × g for 5 min at room temperature and then suspended in 5 ml of tissue culture medium lacking FCS. A 0.5-ml portion of this suspension was added to 9 ml of the macrophage tissue culture medium lacking FCS and adjusted to a final optical density at 600 nm of 0.5. This bacterial suspension was added to a monolayer of eukaryotic cells (90% confluent) in a 75-cm2 tissue culture flask, which was then incubated at 33°C for 4 h. The monolayer was then washed with phosphate-buffered saline (PBS) twice and lysed by the addition of 500 μl of lysis buffer consisting of 1% (vol/vol) NP-40 in Tris-buffered saline (TBS; 25 mM Tris base containing 0.8% [wt/vol] NaCl and 0.02% [wt/vol] KCl, pH 7.4), 2 mM phenylmethylsulfonyl fluoride, 1 mM protease inhibitor cocktail (Santa Cruz Biotechnology, Santa Cruz, CA) and 1 mM sodium orthovanadate. It should be noted that this lysis buffer did not contain EDTA. The protein concentration of the different lysates was measured by using a protein assay reagent (Bio-Rad Laboratories, Hercules, CA) and standardized by dilution with lysis buffer. A 20-μg portion of each lysate was incubated with 4 μg of the appropriate purified MAb at 4°C for 16 h with gentle agitation. A 50-μl portion of protein A/G-agarose (Santa Cruz Biotechnology) was added to the mixture, which was then gently agitated at 4°C for 1 h. The agarose beads were then washed with 0.8 ml of 1% (vol/vol) NP-40 in TBS five times in the cold by repeated centrifugation and resuspension and were finally suspended in 100 μl of 1× Laemmli sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. This suspension was heated at 100°C for 5 min and then loaded directly into wells for SDS-PAGE and subsequent Western blot analysis. When recombinant His-tagged or GST fusion proteins were incubated with the macrophages, a 60-μg quantity of the purified protein was used, and the immunoprecipitation procedure was performed as described above. The purified antibodies used for immunoprecipitation included mouse anti-pY MAb 4G10, mouse MAb 3H9 directed against the LspA1 fusion protein HL8 (27), mouse Penta-His MAb (Qiagen), and polyclonal goat anti-GST antibody (GE HealthCare Bio-Sciences).
The protein tyrosine kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine) (15, 21) (Calbiochem, San Diego, CA) was dissolved in dimethyl sulfoxide (DMSO) and incubated with J774A.1 macrophages for 30 min at 37°C prior to addition of native bacterial or LspA1 fusion proteins. The negative control for PP2 was PP3 (4-amino-7-phenylpyrazolo[3,4-d]pyramidine) (Calbiochem) dissolved in DMSO.
A 1-ml volume of protein tyrosine kinase working solution was freshly prepared by adding 10 μl of a 40 mM ATP solution and 10 μl of a 100 mM sodium orthovanadate solution to 980 μl of kinase buffer (25 mM HEPES [pH 7.0] containing 150 mM NaCl, 10 mM MgCl2, and 5 mM dithiothreitol). Purified recombinant chicken c-Src protein (stock concentration of 100 μg/ml) was diluted 1:1,000 in kinase buffer before use. A 40-μl reaction mixture was prepared by adding 2 μg of the purified His-LspA1 fusion protein HL7, HL8, or HL8.5 and 5 μl of diluted c-Src to 33 μl of tyrosine kinase working solution. This mixture was incubated at 30°C for 45 min before the reaction was stopped by adding 10 μl of 5× SDS-PAGE sample buffer. A 20-μl portion of the reaction mixture was subjected to SDS-PAGE and Western blot analysis, using the anti-pY antibody 4G10 coupled to HRP to detect tyrosine-phosphorylated proteins.
Tyrosine phosphorylation of His-LspA1 fusion proteins by macrophage lysates was accomplished using J774A.1 cells that had been grown in a 12-well tissue culture plate to more than 90% confluence. After two washes with PBS, the monolayers were lysed with 100 μl of 1% (vol/vol) NP-40 in TBS. Portions (6 μg) of HL8, GSTL, or a purified GST fusion protein containing the T-cell receptor ζ (zeta) subunit [GST-ζ] (39) were added to the lysates and incubated at room temperature for various time periods before 3× SDS-PAGE sample buffer was added to stop enzymatic activity. No exogenous ATP was added to these reaction mixtures. For certain experiments, apyrase (final concentration, 10 U/ml; Sigma), PP2 (final concentration, 100 μM), or EDTA (1 mM) was added to the reaction mixture.
J774A.1 macrophages grown in a 75-cm2 flask were washed and lysed with 500 μl of lysis buffer. The eukaryotic cells were further disrupted by repeated passage through a 23-gauge needle and divided into two portions. One was saved as the complete lysate. The other was subjected to centrifugation at 16,100 × g for 5 min at 4°C, and the resultant supernatant fluid was used as the soluble portion of the lysate. The complete lysate and the soluble portion were dialyzed at 4°C in a 10,000-molecular-weight cutoff dialysis cassette (Pierce Biotechnology) against 300 ml of 50 mM Tris-HCl, pH 7.5, containing 1 mM protease inhibitor cocktail and 1 mM sodium orthovanadate, with two changes of buffer over 24 h. As a control, J774A.1 cells from another flask were processed in the same manner, but the dialysis step was excluded. The complete lysate and soluble portion from this latter flask were held at 4°C while the lysates from the former flask were being dialyzed for 24 h. To measure the tyrosine phosphorylation ability of these lysates, 50-μl portions of each lysate were incubated with 1 μg of HL8 at room temperature for 1 h. To some tubes, ATP was added to a final concentration of 0.8 mM with or without the addition of a 10 μM MgCl2-10 μM MnCl2 solution. After incubation, 30 μl of 3× SDS-PAGE sample buffer was added to stop enzymatic activity.
We previously reported that the level of active Src family PTKs was reduced in J774A.1 murine macrophages when they were incubated with wild-type H. ducreyi 35000HP whereas incubation with the lspA1 lspA2 mutant 35000HPΩ12 did not appear to alter the level of active Src family PTKs (27). To determine whether the tyrosine phosphorylation state of additional proteins was regulated by H. ducreyi, macrophages were incubated with wild-type H. ducreyi 35000HP or the lspA1 lspA2 mutant and lysed, and tyrosine-phosphorylated proteins were immunoprecipitated with the pY MAb 4G10. The tyrosine phosphorylation pattern was assessed by using the HRP-conjugated pY MAb 4G10 in Western blot analysis (Fig. (Fig.1A).1A). A tyrosine-phosphorylated protein with an apparent molecular weight much greater than 250,000 was present in the lysate of the macrophages that had been incubated with H. ducreyi 35000HP (Fig. (Fig.1A,1A, lane 1) but was absent from the lysate of macrophages that had been incubated with the lspA1 lspA2 mutant (Fig. (Fig.1A,1A, lane 2).
Based on its apparent molecular weight, we suspected that this very large protein containing pY might be LspA1 and/or LspA2, which have calculated masses of 456,141 and 542,559, respectively (46). To confirm the identity of this phospho-antigen(s), we used the LspA1- and LspA2-specific MAb 3H9 to immunoprecipitate the LspA proteins present in these lysates. The immunoprecipitates were then probed with the pY MAb 4G10 in Western blot analysis. A very large antigen reactive with MAb 4G10 was present in the immunoprecipitate derived from macrophages incubated with the wild-type H. ducreyi strain (Fig. (Fig.1B,1B, lane 1) but lacking in the immunoprecipitate from the macrophages incubated with the lspA1 lspA2 mutant (Fig. (Fig.1B,1B, lane 2). When the MAb 3H9-derived immunoprecipitates were probed with polyclonal rabbit antibody raised against a different region of the LspA1 protein (contained in the GST-Yop fusion protein), this same very large immunoreactive antigen was detected only in the lysate from the macrophages incubated with wild-type H. ducreyi (Fig. (Fig.1C,1C, lane 1). These results provide the first evidence that the LspA proteins are tyrosine phosphorylated upon incubation with macrophages.
To determine whether the LspA proteins could be tyrosine phosphorylated by other cell types, the H. ducreyi wild-type strain 35000HP and the lspA1 lspA2 mutant were incubated with four different cell lines. These included RAW 264.7 murine macrophages (Fig. (Fig.1D,1D, lanes 1 and 2), J774A.1 murine macrophages (Fig. (Fig.1D,1D, lanes 3 and 4), human HeLa cells (Fig. (Fig.1D,1D, lanes 5 and 6), and COS-7 monkey kidney fibroblasts (Fig. (Fig.1D,1D, lanes 7 and 8). Tyrosine-phosphorylated LspA proteins could be detected in all of the immunoprecipitates from the cell lines incubated with the wild-type H. ducreyi strain (Fig. (Fig.1D,1D, lanes 1, 3, 5, and 7) but not in those from the cell lines incubated with the mutant (Fig. (Fig.1D,1D, lanes 2, 4, 6, and 8).
To determine whether the LspA proteins could be tyrosine-phosphorylated in the absence of H. ducreyi cells, we incubated H. ducreyi CCS with J774A.1 macrophages and then used MAb 3H9 to immunoprecipitate the LspA proteins after lysis of the macrophages. The LspA proteins present in H. ducreyi 35000HP CCS (Fig. (Fig.1E,1E, lane 1) were tyrosine phosphorylated after incubation with macrophages. No such immunoreactive band was observed in the immunoprecipitate derived from the use of the CCS from the lspA1 lspA2 mutant (Fig. (Fig.1E,1E, lane 2). This phosphorylated antigen was confirmed to be the LspA proteins in a control experiment using a rabbit polyclonal antiserum against the LspA1 protein (data not shown).
To determine whether Src family PTKs in the macrophages were responsible for the tyrosine phosphorylation of the LspA proteins, we used PP2, a selective inhibitor of Src family PTKs. This inhibitor was incubated with J774A.1 macrophages prior to the addition of bacterial cells. Treatment of the macrophages with PP2 decreased tyrosine phosphorylation of the LspA proteins in a dose-dependent manner (Fig. (Fig.2A,2A, lanes 2 to 4). Reduction of LspA phosphorylation was detected with as little as 10 μM PP2 (Fig. (Fig.2A,2A, lane 3). PP3, a nonactive analogue of PP2, was used as a negative control and did not detectably inhibit LspA phosphorylation at a concentration of 100 μM (Fig. (Fig.2A,2A, lane 5). Probing of this same blot with polyclonal GST-Yop antibody indicated that similar amounts of LspA proteins had been immunoprecipitated by MAb 3H9 in all five reactions (Fig. (Fig.2B,2B, lane 1 to 5). These results strongly suggest that Src family PTK activity is required for the tyrosine phosphorylation of the LspA proteins.
We next attempted to identify the tyrosine residues that were being phosphorylated in the LspA proteins. Five bacterial effector proteins have been shown, to date, to be tyrosine phosphorylated by eukaryotic cells. These include the enteropathogenic E. coli Tir protein (22), Helicobacter pylori CagA (36), Chlamydia trachomatis Tarp (9), Bartonella henselae BepD (35), and Anaplasma phagocytophilum AnkA (17). Each of these proteins contains multiple predicted or proven tyrosine phosphorylation sites. Notably, the sequences immediately adjacent to the relevant tyrosine residue are conserved in all of these proteins. The −3 position is always glutamic acid, the −1 position is isoleucine or leucine, and the +1 position is a small or acidic residue (6). There are 110 tyrosine residues present in LspA1. Homology comparisons revealed two EPIYG motifs and two EPVYA motifs in LspA1 that resembled the known phosphorylation motifs of other bacterial effectors. The EPIYG motifs (Fig. (Fig.3)3) are embedded in a 52-aa repeat sequence located 2,463 aa from the N terminus of LspA1 and 2,592 aa from the N terminus of LspA2 whereas the EPVYA motifs (Fig. (Fig.3)3) are located in the 319-aa repeat region that appears once in LspA1 and three times in LspA2 (46).
To obtain evidence that these motifs could be tyrosine phosphorylation targets, we took advantage of the fact that we had previously cloned 13 approximately 1-kb fragments spanning the entire H. ducreyi 35000HP lspA1 open reading frame (Fig. (Fig.3B)3B) into the pQE30 vector to generate His-tagged LspA1 fusion proteins (27). HL8 contained the 52-aa repeat sequences with the EPIYG motifs (Fig. (Fig.3C).3C). To test if recombinant LspA1 fusion proteins could be tyrosine phosphorylated by macrophages, we incubated HL7, HL8, and HL8.5 with J774A.1 macrophages and then used a His-tag-specific MAb to immunoprecipitate these three fusion proteins. It should be noted that neither HL7 nor HL8.5 contained a predicted tyrosine phosphorylation motif. Probing with the pY MAb 4G10 revealed that only HL8 (Fig. (Fig.4A,4A, lane 2) was phosphorylated whereas neither HL7 (Fig. (Fig.4A,4A, lane 1) nor HL8.5 (Fig. (Fig.4A,4A, lane 3) was phosphorylated. This same blot was stripped and probed with polyclonal antibody raised against GST-788.5 to confirm that similar amounts of fusion protein were present in the immunoprecipitates (Fig. (Fig.4B,4B, lanes 1 to 3). Phosphorylation of HL8 by the macrophages was effectively inhibited by 100 μM PP2 (Fig. (Fig.4C,4C, lane 1). To confirm that equivalent quantities of HL8 were present in the immunoprecipitates shown in Fig. Fig.4C,4C, the blot was stripped and then probed with MAb 3H9, which was originally raised against HL8 (Fig. (Fig.4D,4D, lanes 1 to 3).
The two EPVYA motifs are located at the end of HL9 and at the beginning of HL10 (Fig. 3B and D). Because both HL9 and HL10 were poorly expressed in E. coli (data not shown), we constructed the 63-kDa GSTL fusion protein containing the 319-aa repeat region that included the two EPVYA motifs (Fig. 3B and D). When purified GSTL was incubated with macrophages, it was readily tyrosine phosphorylated (Fig. (Fig.4E,4E, lanes 1 and 3), and the phosphorylation was inhibited by PP2 (Fig. (Fig.4E,4E, lane 2). Taken together, the results suggested that the EPIYG motif (in HL8) and the EPVYA motif (in GSTL) are tyrosine phosphorylated by macrophages.
Site-directed mutagenesis was used to confirm the identity of the tyrosine phosphorylation targets in these two LspA1 fusion proteins. The tyrosine residues Y133 and Y209 in the EPIYG motifs of the HL8 fusion protein (Fig. (Fig.3C)3C) were individually and jointly replaced with phenylalanine residues by site-directed mutagenesis. Similarly, the tyrosine residues Y210 and Y290 in the EPVYA motifs of the GSTL fusion protein (Fig. (Fig.3D)3D) were replaced with phenylalanine residues. One additional tyrosine residue in each fusion protein (Y89 in HL8 and Y82 in GSTL) (Fig. 3C and D) was also converted to a phenylalanine as a negative control. When the HL8-derived mutant proteins were incubated with macrophages, the Y133F/Y209F double mutant (Fig. (Fig.5A,5A, lane 6) was not tyrosine phosphorylated. In contrast, the other three single mutants (Fig. (Fig.5A,5A, lanes 3 to 5) were as readily tyrosine phosphorylated by the macrophages as was HL8 (Fig. (Fig.5A,5A, lane 2). The same membrane was stripped and then probed with the HL8-reactive MAb 3H9 to ensure that equivalent amounts of wild-type and mutant fusion proteins were immunoprecipitated by the His tag antibody (Fig. (Fig.5B).5B). Similarly, only when both Y210 and Y290 in GSTL were mutated to phenylalanine residues in the Y210F/Y290F double mutant (Fig. (Fig.5C,5C, lane 5) was tyrosine phosphorylation abolished, whereas all three of the single mutants (Fig. (Fig.5C,5C, lanes 2 to 4) and the wild-type GSTL (Fig. (Fig.5C,5C, lane 1) were tyrosine phosphorylated by the macrophages. A mouse polyclonal antiserum against the HL9 fusion protein was used to reprobe the same blot to confirm that similar amounts of the GST-tagged fusion proteins were immunoprecipitated by the anti-GST antibody (Fig. (Fig.5D).5D). These results indicated that both the EPIYG and the EPVYA motifs in the LspA1 sequence can serve as tyrosine phosphorylation sites.
To confirm that the HL8 and GSTL fusion proteins can function as substrates for Src PTK, we performed in vitro kinase reactions with a purified recombinant chicken c-Src (28). Both HL8 (Fig. (Fig.6A,6A, lanes 2 and 3) and GSTL (Fig. (Fig.6B,6B, lane 1) were tyrosine phosphorylated by this purified recombinant enzyme. The tyrosine phosphorylation of these two different LspA fusion proteins was inhibited by the Src PTK-specific inhibitor PP2 (Fig. (Fig.6A,6A, lanes 4 and 5, and B, lane 3).
In preliminary experiments involving the use of different detergent strengths to lyse macrophages after incubation with H. ducreyi, we noted that a higher level of LspA phosphorylation was detected in the presence of the stronger detergent. This finding raised the possibility that perhaps the LspA proteins remained in the extracellular state (with respect to the macrophage) and were being phosphorylated by macrophage PTKs after integrity of the macrophages had been lost (i.e., postlysis). In Fig. Fig.7,7, HL8 was shown to be phosphorylated even immediately after it was mixed with J774A.1 lysates without extended incubation (i.e., the nominal 0 time point) (Fig. (Fig.7A,7A, lane 2). It must be noted that no exogenous ATP was added to these reactions mixtures. As the incubation time increased, more phosphorylated HL8 was detected (Fig. (Fig.7A,7A, lanes 3 and 4). In contrast, another protein (i.e., a GST fusion involving the T-cell-receptor ζ subunit that is a physiological substrate of Src PTKs both in vivo and in vitro [26, 27]) was not detectably tyrosine phosphorylated after lysis of the macrophages (Fig. (Fig.7A,7A, lanes 5 to 7). To confirm that this GST-ζ fusion protein had the potential to be phosphorylated, it was incubated with recombinant c-Src PTK and ATP in a standard kinase reaction (Fig. (Fig.7A,7A, lane 8). The same blot was stripped and reprobed with an anti-GST antibody to confirm that GST-ζ was present (Fig. (Fig.7B,7B, lanes 5 to 8). Additional experiments using the GSTL fusion protein showed that, in contrast to HL8, this LspA1-derived fusion construct was not tyrosine phosphorylated by macrophage lysates. Control experiments showed that the lack of tyrosine phosphorylation of GSTL was not caused by the presence of the GST tag (data not shown).
To further explore the mechanism by which macrophage lysates tyrosine phosphorylate HL8, we incubated HL8 (or PBS) with these lysates in the presence or absence of the Src PTK inhibitor PP2 (Fig. (Fig.8).8). Tyrosine-phosphorylated HL8 was readily detectable in the absence of PP2 (Fig. (Fig.8A,8A, lanes 5 and 6) but was not detected when PP2 was included in the reaction mixture (Fig. (Fig.8A,8A, lanes 7 and 8). The same blot was stripped and probed with the HL8-reactive MAb 3H9 to confirm that equivalent amounts of HL8 were present in all four reaction products (Fig. (Fig.8B,8B, lanes 5 to 8). To confirm that PP2 effectively reduced the level of active Src PTKs in these macrophage lysates, this blot was stripped again and then probed with polyclonal antibody to active Src (i.e., anti-pY418). When PP2 was added to the lysates (Fig. (Fig.8C,8C, lanes 3 and 4 and lanes 7 and 8), there was very little or no detectable active Src PTK. These results indicated that PP2 inhibited the kinase activity in these macrophage lysates, similar to the inhibition exerted by PP2 on purified Src kinase (Fig. (Fig.66).
Because divalent metal cations are required for kinase activity (42), we tested the effect of EDTA on tyrosine phosphorylation of HL8 by these macrophage lysates. EDTA (1 mM) completely inhibited the tyrosine phosphorylation of HL8 (Fig. (Fig.9A,9A, lanes 1 to 5), whereas increasing amounts of HL8 were phosphorylated over time in the absence of this chelator (Fig. (Fig.9A,9A, lanes 7 to 11).
We next used the enzyme apyrase to deplete ATP from the reaction mixture. Apyrase hydrolyzes ATP to yield AMP and inorganic phosphate (23). In the presence of apyrase, tyrosine phosphorylation of HL8 did not occur (Fig. (Fig.9B,9B, lanes 5 to 8). This result suggested that ATP (derived from the macrophages) was required in this postlysis tyrosine phosphorylation of HL8. To confirm that the endogenous ATP from the macrophages facilitated the tyrosine phosphorylation of HL8, macrophage lysates were dialyzed (10,000-molecular-weight cutoff membrane) prior to being incubated with HL8. When macrophage lysates, whether dialyzed or not, were held at 4°C for 24 h, they lost their ability to tyrosine phosphorylate HL8 (Fig. (Fig.9C,9C, lanes 1 to 4), likely as the result of ATP hydrolysis. Significantly, the addition of ATP to the undialyzed lysates resulted in phosphorylation of HL8 (Fig. (Fig.9C,9C, lanes 5 and 6). In contrast, the dialyzed lysates regained their phosphorylation ability only when both ATP and the divalent cations Mg2+ and Mn2+ were added (Fig. (Fig.9C,9C, lanes 11 and 12). This requirement for divalent cations with the dialyzed lysates is consistent with the EDTA-based inhibition of tyrosine phosphorylation activity in freshly prepared lysates (Fig. (Fig.9A9A).
This unexpected ability of the HL8 protein to be tyrosine phosphorylated by macrophage lysates prompted us to examine both LspA1 and LspA2 for additional motifs that might be involved in kinase binding or regulation. ScanSite-based analysis (scansite.mit.edu) revealed that both LspA1 (Fig. (Fig.10)10) and LspA2 (data not shown) contained multiple predicted SH2 and SH3 domain binding motifs. More specifically, HL8 was shown to contain three SH2 domain binding motifs and two SH3 domain binding motifs in addition to the two tyrosine phosphorylation sites (Fig. (Fig.10).10). The GSTL fusion protein, which could not be tyrosine phosphorylated by macrophage lysates, contained one SH2 domain binding motif and two SH3 domain binding motifs in addition to the tyrosine phosphorylation sites. The presence of SH2 and SH3 domain binding motifs in both HL8 and GSTL raised the possibility that a sequence unique to HL8 was required for tyrosine phosphorylation of HL8 by macrophage lysates. Consistent with this, analysis by CLUSTAL W alignment revealed that a region of HL8 (Fig. (Fig.10)10) has homology (29% identity) with the Pho81 minimum domain, a sequence previously shown to be involved in regulation of certain eukaryotic kinases (16, 49).
The LspA proteins of H. ducreyi remain among the largest prokaryotic polypeptides described to date. Despite their very large size (>4,000 aa), only two regions within the LspA proteins were previously shown to have any significant homology with functional domains characterized in other bacterial proteins (46). A region near the N terminus of the LspA proteins contains a predicted secretion signal which resembles that present in the FhaB protein of Bordetella pertussis (11) (Fig. (Fig.3A).3A). In the C-terminal half of the LspA proteins, there is a domain that has homology with the YopT proteins of pathogenic Yersinia species (1) (Fig. (Fig.3A).3A). In the present study, we show that the LspA proteins also contain motifs that are targets for tyrosine phosphorylation by macrophages.
When either whole H. ducreyi bacteria or H. ducreyi CCS was incubated with J774A.1 macrophages, the LspA proteins were tyrosine phosphorylated. Tyrosine phosphorylation of the LspA proteins could also be accomplished by nonimmune cells. In macrophages, tyrosine phosphorylation of the LspA proteins was found to be dependent on Src family PTKs. Site-directed mutagenesis of LspA1 fusion proteins allowed identification of motifs that contained tyrosines that were targets for phosphorylation by macrophage-encoded kinases. It should be noted that the LspA1 fusion proteins used in these site-directed mutagenesis experiments may fold differently from the much larger, full-length LspA1 protein. Use of a full-length LspA1 protein in site-directed mutagenesis studies will be necessary to confirm the identity of these tyrosine phosphorylation sites. Interestingly, tyrosine phosphorylation of one of these LspA1 fusion proteins could also be accomplished by macrophage lysates in an ATP- and divalent cation-dependent manner.
There are only a few reports of bacterial effector molecules that are tyrosine phosphorylated by eukaryotic kinases, and the function of the individual phosphorylated bacterial proteins varies widely. The Tir protein of enteropathogenic E. coli (22) and the CagA protein of H. pylori (36) are the best-studied examples of these types of effector molecules (for a review see reference 6). Tir and CagA are injected directly into the eukaryotic cell by type III (10) and type IV secretion systems (5), respectively. Similarly, with the other three bacterial effector molecules that have been reported to be tyrosine phosphorylated (9, 17, 35), injection-type secretion systems (i.e., type III or type IV) have been demonstrated or inferred to be necessary for the subsequent phosphorylation event (9, 25, 35). The lack of a type III secretion system in H. ducreyi (Robert S. Munson, Jr., personal communication) precludes direct injection of LspA proteins into macrophages by this particular method. In addition, although H. ducreyi contains the flp gene cluster that encodes a secretion system which shares conserved sequence features with some type IV secretion systems (31, 32), inactivation of the tadA gene in the flp operon did not eliminate the ability of H. ducreyi to reduce the level of active Src PTKs in macrophages (data not shown). In addition, the absence of the tadA gene product did not prevent tyrosine phosphorylation of the LspA proteins by macrophages (data not shown).
The truly novel finding from the present study is the ability of the LspA1 fusion proteins to be tyrosine phosphorylated by macrophage lysates produced by detergent solubilization. In addition, incubation of wild-type H. ducreyi bacteria with macrophage lysates also resulted in tyrosine phosphorylation of the LspA proteins (data not shown). These results were unexpected because after lysis of the macrophages the concentration of ATP (necessary for kinase activity) would be very minimal, and it is generally accepted that little or no tyrosine phosphorylation of proteins can be accomplished by eukaryotic cell lysates in the absence of exogenously added ATP. These data raised the possibility that either the LspA proteins have a strong interaction with macrophage-encoded PTKs or that the LspA proteins can bind ATP, thereby concentrating it and making it available for PTK activity. In this regard, it should be noted that the LspA proteins each contain a putative ATP/GTP binding domain (Walker motif A; GINTKGKT, aa 2206 to 2213 in LspA1 and aa 2344 to 2351 in LspA2). However, efforts to directly demonstrate ATP binding by the LspA fusion protein HL8 have been unsuccessful to date.
We also looked for other potential domains that might explain why the HL8 and GSTL fusion proteins differed in their ability to be tyrosine phosphorylated by macrophage lysates even though both of these fusion proteins were readily tyrosine phosphorylated by cells and purified c-Src PTK. The presence of more SH2 domain binding motifs in HL8 (three) than in GSTL (one) (Fig. (Fig.10)10) raises the possibility that HL8 may bind a macrophage PTK more effectively than does GSTL. Perhaps more significantly, the Pho81 minimum domain homology in HL8 (Fig. (Fig.10)10) distinguishes HL8 again from GSTL. Pho81 is a 130-kDa eukaryotic protein that inhibits the cyclin-dependent kinase Pho80-Pho85 under low-phosphate conditions (16). An 80-aa motif in Pho81 (the Pho81 minimum domain; aa 645 to 724) is sufficient to inhibit Pho80-Pho85 (16), and a 22-aa region within this motif has been shown to interact with the signaling molecule diphosphoinositol heptakisphosphate (24). How the Pho81 minimum domain-like region in LspA1 might affect tyrosine phosphorylation of this H. ducreyi molecule is not immediately apparent. However, it must be noted that CLUSTAL W-based analysis of the other bacterial proteins (Tir, CagA, Tarp, BepD, and AnkA) known to be tyrosine phosphorylated by eukaryotic cells revealed that they all had less homology with the Pho81 minimum domain than LspA1. In addition, within the primary amino acid sequence of each protein, the proven or putative tyrosine phosphorylation sites were generally distant from the region with any homology to the Pho81 minimum domain (data not shown).
Taken together, these data suggest that a previously undescribed interaction between the LspA proteins and both eukaryotic PTKs and related regulatory factors may provide an explanation for the unusual ability of these H. ducreyi proteins to be tyrosine phosphorylated by macrophage lysates. At the very least, the ability of the LspA proteins to undergo tyrosine phosphorylation by cell lysates raises the interesting possibility that extracellular LspA proteins in chancroidal lesions could be tyrosine phosphorylated and then contribute at some level to the disease process in chancroid. Whether the LspA proteins have to be tyrosine phosphorylated to allow H. ducreyi to inhibit phagocytosis also remains to be determined.
The basis for the physical interaction between the LspA proteins and the macrophage at the molecular level remains to be elucidated. Even in the absence of specific injection systems, other large bacterial exoproteins including the Vibrio cholerae RTX toxin (>450 kDa) (14) and the enteroaggregative E. coli plasmid-encoded toxin, Pet (104 kDa) (30), can gain access to the cytoplasm of the eukaryotic cell, where they exert their effects. Pet has been shown to be taken up in apparently intact form by clathrin-mediated endocytosis (29, 30). With the V. cholerae RTX toxin, recent evidence indicates that after translocation of part of the toxin across the eukaryotic membrane, a cysteine protease domain within the RTX holotoxin cleaves the toxin at several different sites, thereby releasing one or more other activity domains into the cell cytoplasm (38). It is therefore interesting that a region in the LspA proteins (Fig. (Fig.3)3) has similarity to the YopT cysteine protease (37). Whether this YopT-like region in the LspA proteins plays any role in the ability of H. ducreyi to inhibit phagocytosis is currently under investigation.
This study was supported by U.S. Public Health Service grant AI32011 to E.J.H. J.R.M. was supported by U.S. Public Health Service training grant 5-T32-AI007520.
We thank Maria Labandeira-Rey for very helpful suggestions.
Editor: S. R. Blanke
Published ahead of print on 4 August 2008.