Haemophilus ducreyi Targets Src Family Protein Tyrosine Kinases To Inhibit Phagocytic Signaling
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感染与免疫杂志 2005年第12期
Department of Microbiology Center for Immunology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9048
Departments of Medicine and Pharmacology, Columbia University, New York, New York 10032
ABSTRACT
Haemophilus ducreyi, the etiologic agent of the sexually transmitted disease chancroid, has been shown to inhibit phagocytosis of both itself and secondary targets in vitro. Immunodepletion of LspA proteins from H. ducreyi culture supernatant fluid abolished this inhibitory effect, indicating that the LspA proteins are necessary for the inhibition of phagocytosis by H. ducreyi. Fluorescence microscopy revealed that macrophages incubated with wild-type H. ducreyi, but not with a lspA1 lspA2 mutant, were unable to complete development of the phagocytic cup around immunoglobulin G-opsonized targets. Examination of the phosphotyrosine protein profiles of these two sets of macrophages showed that those incubated with wild-type H. ducreyi had greatly reduced phosphorylation levels of proteins in the 50-to-60-kDa range. Subsequent experiments revealed reductions in the catalytic activities of both Lyn and Hck, two members of the Src family of protein tyrosine kinases that are known to be involved in the proximal signaling steps of Fc receptor-mediated phagocytosis. Additional experiments confirmed reductions in the levels of both active Lyn and active Hck in three different immune cell lines, but not in HeLa cells, exposed to wild-type H. ducreyi. This is the first example of a bacte-rial pathogen that suppresses Src family protein tyrosine kinase activity to subvert phagocytic signaling in hostcells.
INTRODUCTION
Haemophilus ducreyi, a gram-negative pleomorphic bacterium, is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease (42). Each year, there are an estimated 6 million cases of chancroid worldwide (44), predominantly in sub-Saharan Africa and Southeast Asia, where chancroid is a major cause of genital ulceration syndrome. Frequencies of both acquisition and transmission of human immunodeficiency virus type 1 infection are increased by the presence of chancroidal ulcers (12, 15). While chancroid is extremely uncommon in the United States, outbreaks have occurred in urban areas (19).
Despite its prevalence in some countries, chancroid remains one of the least understood sexually transmitted diseases (reviewed in reference 35). Such basic issues as the causes of tissue necrosis and the retardation of healing, both characteristic features of chancroid (35), remain to be explained. Determination of the key elements in the pathogenesis of chancroid has been a difficult task, made even more complicated by the fastidious nature of H. ducreyi. Nonetheless, it has been established that H. ducreyi cannot invade intact skin (37), and it is assumed that microabrasions sustained during sexual activity permit penetration of this bacterium into the epidermal layers. Once in this environment, H. ducreyi elaborates as yet unidentified virulence factors that result in ulceration.
In lesions generated in the human challenge model for experimental chancroid, H. ducreyi attached to phagocytes but remained extracellular at least through the pustular stage of disease (4, 5). This finding led to the hypothesis by Spinola et al. that H. ducreyi might survive in vivo by resisting phagocytosis (35). Subsequent studies by Totten and colleagues (50) as well as by Lagergard and coworkers (1) proved that not only can wild-type strains of H. ducreyi resist phagocytosis in vitro, but they can also inhibit the phagocytosis of secondary targets (e.g., opsonized erythrocytes). The fact that H. ducreyi can inhibit phagocytosis indicates that, in addition to production of the cytolethal distending toxin that can cause apoptosis in some immune cells (14, 39), this pathogen also possesses the means to escape one of the most potent effectors of both innate and acquired immunity.
We determined previously that an H. ducreyi mutant unable to express the LspA1 and LspA2 proteins lacked the ability to inhibit phagocytic activity of macrophage-like and polymorphonuclear neutrophil (PMN)-like cell lines (45). LspA1 and LspA2 are 86% identical, have calculated molecular masses of 456,211 Da and 542,660 Da, respectively, and are encoded by two of the largest prokaryotic open reading frames (ORFs) (12.5 and 14.8 kb, respectively) described to date (48). Together with the LspB outer membrane protein, LspA1 and LspA2 constitute a two-partner secretion system (20) in which LspB is the essential secretion factor (49). Soluble forms of the LspA proteins with apparent molecular masses of 160 to 270 kDa are detectable in H. ducreyi culture supernatant fluid (47, 48). Expression of either LspA1 or LspA2 is necessary to inhibit phagocytic activity; therefore, both lspA1 and lspA2 must be inactivated in order to eliminate the ability of H. ducreyi to inhibit phagocytosis (45). In addition, a lspB mutant unable to secrete LspA1 or LspA2 was also unable to inhibit phagocytosis (45). Finally, a lspA1 lspA2 mutant of H. ducreyi exhibited greatly reduced virulence in both the temperature-dependent rabbit model of experimental chancroid (47) and the human challenge model (21).
In this report, we provide additional evidence that the wild-type H. ducreyi LspA proteins are involved in the inhibition of Fc receptor (FcR)-mediated phagocytosis. More importantly, our studies demonstrate that this inhibition involves one of the most proximal signaling events in phagocytosis. Incubation of wild-type H. ducreyi with immune cells resulted in decreased phosphorylation and reduced catalytic activity of Src family protein tyrosine kinases, leading to an inability to complete phagocytic cup development. This appears to be a novel mechanism for inhibition of phagocytosis by a bacterial pathogen.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The wild-type H. ducreyi strain 35000HP and the lspA1 lspA2 mutants 35000HP.12 (47) and 35000HP12 (21) were grown as described previously on chocolate agar plates (21) at 33°C in a 95% air-5% CO2 humidified atmosphere. H. ducreyi strains were also grown in Columbia broth (Difco Laboratories, Detroit, Mich.) supplemented with 0.1% (wt/vol) Trizma base (Sigma, St. Louis, Mo.), hemin (25 μg/ml), 1% (vol/vol) IsoVitaleX (Becton Dickinson, Cockeysville, Md.), and 2.5% (vol/vol) heat-inactivated fetal bovine serum (FBS; HyClone, Logan, Utah) (48) at 33°C with agitation at 120 to 130 rpm. Concentrated culture supernatant fluids (CCS) were prepared from broth-grown H. ducreyi as described elsewhere (45). Briefly, the culture fluid was first subjected to centrifugation at 6,000 x g for 10 min at 4°C to remove the bacteria. The resultant supernatant fluid was passed through a 0.22-μm-pore-size filter and then subjected to ultracentrifugation at 125,000 x g for 1 h at 4°C to remove membrane fragments. Last, this supernatant fluid was concentrated 40-fold by using an Amicon Ultra centrifugal filter device (100,000-molecular-weight cutoff) (Millipore, Inc., Bedford, Mass.) and used immediately in phagocytosis assays.
Mammalian tissue culture growth and differentiation. The human PMN-like cell line HL-60 (ATCC CCL-240; American Type Culture Collection, Manassas, VA) and the mouse monocyte/macrophage cell lines J774A.1 (ATCC TIB-67) and RAW 264.7 (ATCC TIB-71) were cultivated as previously described (45). HeLa cells (ATCC CCL-2) and the DC2.4 dendritic cell line (kindly provided by Kenneth Rock, Dana-Farber Cancer Institute, Boston, MA) were grown in the same medium as the macrophage cell lines.
LspA1 polyclonal antisera. The 12.5-kb lspA1 ORF was segmented into 13 fragments of approximately 1 kb which were then amplified by PCR and cloned into the plasmid vector pQE-30 (QIAGEN, Inc., Valencia, CA) to obtain fusion proteins consisting of LspA1 protein segments with an N-terminal six-His tag. The oligonucleotide primers used to generate these PCR products are listed in Table 1. These recombinant plasmids were used to transform Escherichia coli M15 containing pREP4 (QIAGEN). To induce expression of His-LspA1 fusion proteins, isopropylthio--D-galactoside (Invitrogen, Carlsbad, CA) was added to a final concentration of 1 mM. Each His-LspA1 fusion protein was purified per the QIAGEN protocol and used to immunize 10-week-old female BALB/c mice (Charles River Laboratories Inc., Wilmington, MA).
Immunodepletion of LspA proteins. CCS from the wild-type strain 35000HP and the lspA1 lspA2 mutant 35000HP12 were incubated with heat-inactivated LspA1 antiserum or with heat-inactivated normal BALB/c mouse serum for 2 h at 4°C. Protein A/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA) was then added, and the suspension was mixed for 1 h at 4°C and then subjected to centrifugation (12,000 x g, 5 s) to remove antigen-antibody-agarose complexes. The resultant supernatant fluids were used in phagocytosis assays with HL-60 cells.
Phagocytosis assays using fluorescent microspheres as a secondary target. A 100-μl volume of CCS or immunodepleted CCS from wild-type 35000HP or the 35000HP12 mutant was added to a 96-well tissue culture plate containing differentiated HL-60 cells at a concentration of 5 x 105 cells/well. After 1 h of incubation at 33°C, streptavidin-coated green fluorescent microspheres previously opsonized with rabbit anti-streptavidin immunoglobulin G (IgG) (Rockland Immunochemicals, Gilbertsville, PA) (45) were added, and phagocytosis was measured as described elsewhere (45).
Staining of the J774A.1 actin cytoskeleton. Bacteria were grown for 15 h in liquid culture, harvested by centrifugation for 10 min at 4,000 x g, and suspended in tissue culture medium lacking FBS to an optical density at 600 nm of 0.5. These assays were performed using J774A.1 cells attached to glass coverslips in 24-well plates (1 x 105 cells/well) in tissue culture medium without FBS. The J774A.1 cells were incubated for 1 h at 33°C with H. ducreyi cells (multiplicity of infection, 25). Sheep erythrocytes opsonized with rabbit IgG (EIgG) were added to each well and incubated for 3 to 6 min at 37°C. The J774A.1 cells were washed twice with phosphate-buffered saline (PBS) and then fixed with 3.7% formaldehyde for 10 min at room temperature. The cells were then permeabilized with 0.1% (vol/vol) Triton X-100 in PBS for 3 to 5 min at room temperature. To reduce nonspecific background, 1% (wt/vol) bovine serum albumin was incubated with the coverslips for 30 min prior to addition of staining solution. The staining solution consisted of 5 μl of a methanolic stock solution of rhodamine-phalloidin (Molecular Probes, Eugene, Oreg.) and 0.5 μl of Oregon green 488-conjugated goat anti-rabbit IgG (Molecular Probes) in 200 μl of PBS for each coverslip to be stained. The staining solution was placed on the coverslip for 20min at room temperature. The coverslips were washed twice with PBS and mounted on a slide (cell side down) in Vectashield mounting medium solution (Vector Laboratories, Inc., Burlingame, Calif.). Slides were viewed using a CFI Plan Apochromat DM60X (numerical aperture, 1.4) objective and a Nikon TE-200 inverted microscope equipped with epifluorescence. Imaging was performed using MetaMorph software (v. 6.1) and Nearest Neighbors digital deconvolution (Universal Imaging Corp., Downington, PA).
Detection of phosphoproteins by Western blot analysis. Mammalian cell lines were incubated with wild-type 35000HP and the lspA1 lspA2 mutant 35000HP12 at 33°C for 4 h. After the fluid was decanted from the flasks, the monolayers were lysed with radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology Inc.). The protein content of the lysates was measured and standardized by using the Bradford method (Bio-Rad). These lysates were heated at 100°C for 5 min in digestion buffer (18), and proteins present in the lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% (wt/vol) polyacrylamide separating gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Inc., Billerica, Mass.). The membranes were blocked with PBS containing 0.05% (vol/vol) Tween 20 and 4% (wt/vol) bovine serum albumin prior to incubation with primary antibodies. Monoclonal antibody (MAb) 4G10 (Upstate Biotechnology Inc., Lake Placid, NY) was used to detect phosphotyrosine residues, whereas the rabbit polyclonal antisera -pY418 and -pY529 (BioSource International, Inc., Camarillo, CA) were used to detect specific phosphorylated forms of Src family protein tyrosine kinases. Total Src protein was detected with MAb 327 (Calbiochem, San Diego, CA), and glyceraldehyde-3-phosphate dehydrogenase was detected with MAb 6C5 (Abcam, Cambridge, MA).
In vitro protein tyrosine kinase assays. J774A.1 macrophages were incubated with medium (control), wild-type H. ducreyi 35000HP, or the lspA1 lspA2 mutant 35000HP12 for 4 h and then lysed with RIPA buffer. The protein content of the lysates was measured and standardized by using the Bradford method (Bio-Rad). Polyclonal antibodies to Lyn (sc-15; Santa Cruz) and Hck (06-833; Upstate) were added and allowed to incubate overnight at 4°C to bind these macrophage kinases in the standardized lysates. Protein A/G agarose (Santa Cruz) was then added and the suspension mixed for 1 h, followed by several washes with kinase buffer (32.5 mM Tris-HCl [pH 7.6], 6.25 mM MnCl2, and 0.0625% [vol/vol] Triton X-100). The immunoprecipitated complexes were resuspended in 40 μl of kinase buffer, and 1 μg of a purified glutathione S-transferase (GST) fusion protein containing the cytoplasmic region of the T-cell-receptor subunit (34) was added along with unlabeled ATP to a final concentration of 0.024 mM. A 1-μl portion of [-32P]ATP (specific activity, 10 μCi/μl) was added, and the reaction mixtures were incubated at 30°C for 45 min. The kinase reaction was terminated by addition of 20 μl of loading buffer followed by heating at 95°C for 5 min. Proteins in the reaction mixture were resolved by SDS-PAGE and transferred to PVDF membranes. Phosphorylated GST- protein was detected by autoradiography. Additional Western blot analyses involving different antibodies to Lyn (sc-7274; Santa Cruz), Hck (sc-1428; Santa Cruz), and (MAb 6B10.2) (46) were used to verify that equivalent amounts of the immunoprecipitated protein tyrosine kinases and purified GST- were loaded and transferred to the PVDF membranes. Relative quantitation of phosphorylated GST- was achieved by using an Amersham Biosciences Storm 820 PhosphorImager.
RESULTS
Evidence for the involvement of the LspA proteins in the inhibition of phagocytic activity. We previously reported that a lspA1 lspA2 mutant of H. ducreyi was unable to inhibit phagocytosis (45). Efforts to clone full-length H. ducreyi lspA genes into E. coli have been unsuccessful to date, precluding the use of a genetic approach to prove that the LspA proteins are directly involved in the inhibition of phagocytosis. Therefore, to obtain additional evidence that the LspA proteins are necessary for the inhibition of phagocytosis by H. ducreyi, immunodepletion experiments were performed with H. ducreyi CCS. His-tagged LspA1 fusion proteins were used to raise mouse polyclonal antibodies to different regions of the LspA1 protein for use in these immunodepletion experiments (Fig. 1). We were successful in obtaining antibodies against 10 of the 13 LspA1 segments diagramed in Fig. 1B.
As reported previously (45), wild-type 35000HP CCS (Fig. 2, column A) readily inhibited phagocytosis whereas CCS from the lspA1 lspA2 mutant 35000HP12 (Fig. 2, column B) did not inhibit phagocytic activity. To confirm the involvement of the LspA proteins in this inhibition, polyclonal mouse antibodies raised against some of the His-LspA1 fusion proteins (Fig. 1B) were incubated with CCS from wild-type H. ducreyi and the lspA1 lspA2 mutant. (LspA1 is the vastly predominant form of LspA protein in wild-type H. ducreyi CCS; LspA2 is almost undetectable [47].) After removal of the immune complexes, the CCS were incubated with HL-60 cells, which were then used in phagocytosis assays with opsonized fluorescent microspheres. In initial immunodepletion experiments, we used a pool of antisera to His-LspA1 fusion proteins 4 to 10 (Fig. 1B) and successfully removed the ability of wild-type H.ducreyi CCS to inhibit phagocytosis by HL-60 cells (data not shown). Subsequent experiments were then performed using a polyclonal antibody to the His-LspA1 fusion protein 8 only (Fig. 1B). Incubation with these LspA1 antibodies caused the wild-type CCS (Fig. 2, column C) to lose its ability to inhibit phagocytic activity. Normal mouse serum (used as a negative control) (Fig. 2, column E) did not eliminate this inhibitory effect. As expected, the lack of inhibition observed with CCS from the lspA1 lspA2 mutant (Fig. 2, column B) was not affected by either of these antibody preparations (Fig. 2, columns D and F). These results provide additional evidence for the involvement of the LspA proteins in the phagocytic inhibition phenotype of wild-type H. ducreyi 35000HP.
Wild-type H. ducreyi affects phagocytic cup development. FcR-mediated phagocytosis normally involves localized actin assembly, which drives pseudopod extension, forming a "cup-like" structure. This culminates in complete target engulfment and eventual actin depolymerization (16). Cytoskeleton staining was performed and viewed by indirect immunofluorescence to detect possible differences in FcR-induced actin assembly in macrophages exposed to wild-type H. ducreyi 35000HP and the lspA1 lspA2 mutant 35000HP.12. This method allows visualization of both phagocytic cups and IgG-opsonized particles (16).
J774A.1 macrophages were incubated for 1 h with either strain prior to the addition of EIgG. After a 3-min incubation with EIgG, the cells were fixed, permeabilized, and stained with rhodamine-phalloidin to detect F-actin and with Oregon green-conjugated goat anti-rabbit IgG to detect the opsonized erythrocytes. Macrophages incubated with the lspA1 lspA2 mutant (Fig. 3, lower panels) showed normal phagocytic cup development and engulfment of opsonized erythrocytes. In contrast, exposure to wild-type H. ducreyi apparently halted progression of phagocytic cup formation around the opsonized erythrocytes, which remained extracellular (Fig. 3, upper panels). It should also be noted here that it was previously shown that exposure to wild-type H. ducreyi does not affect binding of IgG-opsonized targets to these macrophages (45).
H. ducreyi affects macrophage tyrosyl phosphoproteins. The fact that wild-type H. ducreyi had the ability to apparently halt the development of the phagocytic cup suggested a direct inhibition of phagocytic signaling and prompted us to examine the levels of phosphoproteins in these macrophages. The most proximal events in the phagocytic signaling pathway involve protein phosphorylation events (e.g., Src kinase activation, immunoreceptor tyrosine-based activation motif [ITAM] phosphorylation) catalyzed by tyrosine protein kinases (Fig. 4A) (for reviews, see references 22 and 40). To determine whether the LspA proteins were involved in perturbing these signaling events, J774A.1 macrophages were incubated with wild-type H. ducreyi 35000HP or the lspA1 lspA2 mutant 35000HP12. These phagocytes were then lysed, and their solubilized proteins were probed by Western blot analysis with the phosphotyrosine-specific MAb 4G10. The levels of several macrophage phosphotyrosine proteins were found to be drastically reduced in lysates from J774A.1 macrophages incubated with wild-type H. ducreyi (Fig. 4B, lane 1) relative to those incubated with the mutant (Fig. 4B, lane 2). These included prominent phosphotyrosine proteins with an apparent molecular weight of approximately 55,000. This effect of wild-type H. ducreyi 35000HP on phosphoproteins was not limited to J774A.1 macrophages; it was observed with RAW 264.7 macrophages as well (Fig. 4B; compare lanes 3 and 4). Decreases in the levels of these phosphoproteins were also observed when IgG-opsonized erythrocytes were added to the macrophages after incubation with wild-type H. ducreyi (data not shown).
Wild-type H. ducreyi reduces the levels of active Src family kinase members. The apparent molecular weights of the phosphoprotein(s) affected by incubation with wild-type H. ducreyi (Fig. 4B) corresponded to the approximate sizes of Src family protein tyrosine kinases which are essential for normal phagocytic activity (Fig. 4A) (11, 22). To determine whether these phosphoproteins were Src family kinases, we prepared cellular extracts from four different cell lines that had been incubated with either wild-type H. ducreyi 35000HP or the lspA1 lspA2 mutant 35000HP12 and probed these by Western blot analysis with antibodies specific for the two different tyrosine-phosphorylated forms of Src family members. To detect the active form of Src family kinases, we used a polyclonal antiserum (-pY418) reactive with the phosphorylated form of Src tyrosine residue Y418; this tyrosine is phosphorylated when Src kinases are catalytically active. To detect the inactive phosphorylated form of Src kinases, we used an antibody (-pY529) that recognizes the Src tyrosine residue which, when phosphorylated, holds Src kinases in an inactive state. Extracts from the three immune cell lineages (J774A.1 macrophages, PMN-like HL-60 cells, and DC2.4 dendritic cells) that had been incubated with wild-type H. ducreyi (Fig. 5A, lanes 2, 5, and 8, respectively) showed dramatic decreases in reactivity with the -pY418 antibody specific for active phospho-Src kinases. In contrast, extracts from cells of the same types that had been incubated with the lspA1 lspA2 mutant (Fig. 5A, lanes 3, 6, and 9) or with tissue culture medium (control) (Fig. 5A, lanes 1, 4, and 7) showed little or no reduction in reactivity with the same antibody. Interestingly, extracts from the nonimmune HeLa cell line (Fig. 5A, lanes 10, 11, and 12) showed no apparent difference in reactivity with the same antibody. In the same four cell lines incubated with wild-type H. ducreyi, we found modest and variable changes in the level of phosphorylation of the distal Src tyrosine residue Y529 (Fig. 5B). The levels of total Src tyrosine kinases were relatively unchanged within each cell type (Fig. 5C). Additional experiments indicated that a decrease in activated phospho-Src levels could be detected by Western blot analysis after just 1 h of incubation with wild-type H. ducreyi 35000HP (data not shown).
H. ducreyi reduces the catalytic activity of Src family tyrosine kinases. The Western blot data (Fig. 4B and 5A) clearly demonstrated reductions in the levels of the active forms of Src kinases. The Src family kinases Hck, Lyn, and Fgr are the predominant Src kinases present in macrophages (22); the former two tyrosine kinases are required for normal phagocytic activity, whereas Fgr plays an important negative regulatory role (17). Our Western blot data suggested that Hck and Lyn would exhibit decreases in catalytic activity. To examine this possibility, J774A.1 macrophages were incubated with H. ducreyi strains as described for Fig. 5, and then, after lysis of the macrophages, antibodies to Hck and Lyn were used to immunoprecipitate these Src family members for use in tyrosine kinase reactions. These in vitro kinase assays utilized a physiological substrate, the ITAM-containing T-cell-receptor subunit (in the form of a GST fusion protein). As can be seen from the levels of phosphorylated GST-, the catalytic activities of both Hck (Fig. 6A, lane 2) and Lyn (Fig. 6A, lane 5) were decreased in macrophages incubated with wild-type H. ducreyi but not in macrophages incubated with the lspA1 lspA2 mutant (Fig. 6A, lanes 3 and 6, respectively). PhosphorImager analysis indicated an 84% reduction in Hck activity and a 47% reduction in Lyn activity; these differences were significant (P = 0.045 for Hck and P = 0.012 for Lyn by a paired Student t test). Equivalent amounts of total Hck and Lyn kinases (Fig. 6B) and total GST- protein (Fig. 6C) were present in these samples as determined by Western blot analysis. These data indicate that the reduced levels of active Src kinases seen in Fig. 5 in the J774A.1 cells are reflected by decreases in the enzymatic activities of both Hck and Lyn.
DISCUSSION
A large number of putative virulence factors of H. ducreyi have been identified to date, including both proteins and lipooligosaccharide (see reference 35 for a review). In addition, H. ducreyi has been shown to synthesize at least two toxins (9, 25, 41) and a copper-zinc superoxide dismutase (28). However, when tested in the human challenge model for experimental chancroid (3, 37), only five of these gene products have been shown to be required for full virulence of H. ducreyi. These include the peptidoglycan-associated lipoprotein (13), the hemoglobin-binding outer membrane protein HgbA (2), the DsrA outer membrane protein (7), the flp gene cluster (36), and the LspA1 and LspA2 proteins (21). The latter two proteins have been shown, both by a previous mutant analysis (45) and by the present study, to be involved in the ability of this pathogen to inhibit phagocytosis in vitro. The findings of the present study have also revealed a novel and previously unreported capacity of H. ducreyi to affect the phosphorylation state of Src family protein tyrosine kinases.
Bacterial pathogens have devised a number of different strategies for escaping from or preventing phagocytosis (for reviews, see references 23 and 27). These range from the expression of polysaccharide capsules, which can present a simple physical impediment or even alter phagocytic signaling (e.g., Streptococcus suis [29]), to injection of effector molecules that target signaling components (e.g., Yersinia YopT [32] and Pseudomonas ExoT [38]). Phagocytosis is an extremely complex process (Fig. 4A) (for a review, see reference 40). While binding of IgG-opsonized particles or bacteria to most FcRs (except FcRIIB) will initiate the phagocytic signaling cascade, signaling through Toll-like receptors has also recently been shown to affect phagocytosis (6, 10). FcR cross-linking (by IgG-opsonized target particles) results in activation of Src family protein tyrosine kinases, with Hck, Lyn, and Fgr being predominant in murine macrophages (11). These protein tyrosine kinases catalyze the phosphorylation of ITAMs present on or associated with FcR. Once phosphorylated, FcR-associated ITAMs become docking sites for the SH2 domains of Syk and Src family protein tyrosine kinases. ITAM-bound Syk is catalytically activated by a combination of autophosphorylation and transphosphorylation by Src family protein tyrosine kinases. Activated Syk subsequently phosphorylates a number of intracellular substrates, initiating a series of downstream events, including small Rho GTPase activation, which culminate in actin filament assembly and development of the phagocytic cup (Fig. 4A). Since activation of Src family protein tyrosine kinases is a very proximal event in FcR signaling, it is likely that multiple cellular events relevant to phagocytosis (e.g., altered membrane trafficking) are also affected by the LspA proteins, in addition to actin assembly (Fig. 3).
We have established that expression of either LspA1 or LspA2 individually is necessary for H. ducreyi to inhibit phagocytic activity in vitro (45). Efforts to clone full-length H. ducreyi lspA genes into E. coli or to purify functional LspA proteins from spent H. ducreyi culture medium have been unsuccessful to date, so we used LspA-specific antibodies to provide additional proof that this inhibitory effect required these H. ducreyi proteins (Fig. 2). Other experiments showed that incubation of wild-type H. ducreyi, but not the lspA1 lspA2 mutant, with macrophages resulted in dramatic reductions in the levels of active Src protein tyrosine kinases (Fig. 5 and 6). These kinases are involved in some of the most proximal events of the phagocytic signaling pathway (Fig. 4A). This reduced enzymatic activity of the Src protein tyrosine kinases is consistent with the inability of these macrophages to complete the development of phagocytic cups (Fig. 3). We hypothesize that either (i) the LspA proteins bind to a receptor on the macrophage surface and thereby initiate a signaling cascade that abolishes phagocytic activity or (ii) the LspA proteins enter or penetrate into the phagocyte and thereby cause derangement of the normal phagocytic signaling pathway. The levels of active phospho-Src kinases in all three immune cell types tested in this study were reduced by exposure to wild-type H. ducreyi, whereas phospho-Src kinase levels in HeLa cells appeared unaffected (Fig. 5). This finding raises the possibility that a surface component present in immune cell lineages may interact with the LspA proteins, resulting in a negative signaling event. At this time, however, we cannot exclude the possibility that LspA proteins enter the macrophage via an endocytic process and exert their function in the cytoplasm, but it is not clear how these proteins would survive endosomal proteolysis and exit into the cytoplasm. An equivalently large bacterial exoprotein (i.e., RtxA) synthesized by Vibrio cholerae has been shown to directly cross-link actin intracellularly, but the method of entry or penetration of this protein into the host cell cytoplasm remains to be determined (33).
Only two regions of the LspA proteins have significant homology to known bacterial virulence factors. The N-terminal one-quarter of LspA1 (Fig. 1A) contains several features associated with secretion of other soluble virulence factors including the Bordetella pertussis filamentous hemagglutinin (20). There is also a 260-amino-acid region in the C-terminal half of the LspA proteins that has 36% identity with the YopT protein from pathogenic Yersinia species (32) (Fig. 1A). YopT is the prototype of a family of cysteine proteases involved in virulence expression and cleaves small Rho GTPases from the eukaryotic cell membrane (32), thereby altering signaling and inhibiting phagocytosis. Western blot experiments targeting the small Rho GTPases known to be involved in FcR-mediated signaling (8) showed a reduction in the level of membrane-bound Cdc42 in macrophages incubated with wild-type H. ducreyi relative to that in macrophages exposed to the lspA1 lspA2 mutant (data not shown). It is likely that this change is simply a downstream effect related to the greatly decreased levels of Src family kinase activity. Moreover, we have established that the level of membrane-bound Hck is unchanged whether the macrophages are incubated with wild-type H. ducreyi or the lspA1 lspA2 mutant (data not shown). The latter finding indicates that the YopT-like region is not cleaving Hck from the membrane. It is possible that this YopT-like region could be involved in autoprocessing of the LspA proteins, in a manner similar to that catalyzed by the Pseudomonas syringae AvrPphB protein, another member of this cysteine protease family (31).
To date, there is only one other example of a bacterial gene product that affects Src family protein tyrosine kinases. The CagA protein of Helicobacter pylori, after injection into macrophages via a type IV secretion system, is phosphorylated by Src kinase (30) and in turn causes a reduction in active phospho-Src kinase levels by increasing the activity of the protein tyrosine phosphatase SHP-2 (43). However, two independent studies indicate that CagA is not involved in the inhibition of phagocytosis caused by H. pylori (24, 26). It should be noted here that efforts to detect possible phosphorylation of the H. ducreyi LspA proteins after their interaction with macrophages have been unsuccessful to date (data not shown).
Exactly how the LspA proteins are involved in the observed reductions in the catalytic activities of the Src family protein tyrosine kinases remains to be determined. At this time, we cannot formally exclude the possibility that another, as yet unidentified H. ducreyi gene product works in concert with the LspA proteins to inhibit phagocytosis. Efforts to elucidate the precise mechanism of action of these secreted virulence factors are in progress and may provide additional insight into the pathogenesis of chancroid as well as a valuable biological tool for further dissection of Src family kinase function, regulation, and activity.
ACKNOWLEDGMENTS
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-AI007520-08.
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Departments of Medicine and Pharmacology, Columbia University, New York, New York 10032
ABSTRACT
Haemophilus ducreyi, the etiologic agent of the sexually transmitted disease chancroid, has been shown to inhibit phagocytosis of both itself and secondary targets in vitro. Immunodepletion of LspA proteins from H. ducreyi culture supernatant fluid abolished this inhibitory effect, indicating that the LspA proteins are necessary for the inhibition of phagocytosis by H. ducreyi. Fluorescence microscopy revealed that macrophages incubated with wild-type H. ducreyi, but not with a lspA1 lspA2 mutant, were unable to complete development of the phagocytic cup around immunoglobulin G-opsonized targets. Examination of the phosphotyrosine protein profiles of these two sets of macrophages showed that those incubated with wild-type H. ducreyi had greatly reduced phosphorylation levels of proteins in the 50-to-60-kDa range. Subsequent experiments revealed reductions in the catalytic activities of both Lyn and Hck, two members of the Src family of protein tyrosine kinases that are known to be involved in the proximal signaling steps of Fc receptor-mediated phagocytosis. Additional experiments confirmed reductions in the levels of both active Lyn and active Hck in three different immune cell lines, but not in HeLa cells, exposed to wild-type H. ducreyi. This is the first example of a bacte-rial pathogen that suppresses Src family protein tyrosine kinase activity to subvert phagocytic signaling in hostcells.
INTRODUCTION
Haemophilus ducreyi, a gram-negative pleomorphic bacterium, is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease (42). Each year, there are an estimated 6 million cases of chancroid worldwide (44), predominantly in sub-Saharan Africa and Southeast Asia, where chancroid is a major cause of genital ulceration syndrome. Frequencies of both acquisition and transmission of human immunodeficiency virus type 1 infection are increased by the presence of chancroidal ulcers (12, 15). While chancroid is extremely uncommon in the United States, outbreaks have occurred in urban areas (19).
Despite its prevalence in some countries, chancroid remains one of the least understood sexually transmitted diseases (reviewed in reference 35). Such basic issues as the causes of tissue necrosis and the retardation of healing, both characteristic features of chancroid (35), remain to be explained. Determination of the key elements in the pathogenesis of chancroid has been a difficult task, made even more complicated by the fastidious nature of H. ducreyi. Nonetheless, it has been established that H. ducreyi cannot invade intact skin (37), and it is assumed that microabrasions sustained during sexual activity permit penetration of this bacterium into the epidermal layers. Once in this environment, H. ducreyi elaborates as yet unidentified virulence factors that result in ulceration.
In lesions generated in the human challenge model for experimental chancroid, H. ducreyi attached to phagocytes but remained extracellular at least through the pustular stage of disease (4, 5). This finding led to the hypothesis by Spinola et al. that H. ducreyi might survive in vivo by resisting phagocytosis (35). Subsequent studies by Totten and colleagues (50) as well as by Lagergard and coworkers (1) proved that not only can wild-type strains of H. ducreyi resist phagocytosis in vitro, but they can also inhibit the phagocytosis of secondary targets (e.g., opsonized erythrocytes). The fact that H. ducreyi can inhibit phagocytosis indicates that, in addition to production of the cytolethal distending toxin that can cause apoptosis in some immune cells (14, 39), this pathogen also possesses the means to escape one of the most potent effectors of both innate and acquired immunity.
We determined previously that an H. ducreyi mutant unable to express the LspA1 and LspA2 proteins lacked the ability to inhibit phagocytic activity of macrophage-like and polymorphonuclear neutrophil (PMN)-like cell lines (45). LspA1 and LspA2 are 86% identical, have calculated molecular masses of 456,211 Da and 542,660 Da, respectively, and are encoded by two of the largest prokaryotic open reading frames (ORFs) (12.5 and 14.8 kb, respectively) described to date (48). Together with the LspB outer membrane protein, LspA1 and LspA2 constitute a two-partner secretion system (20) in which LspB is the essential secretion factor (49). Soluble forms of the LspA proteins with apparent molecular masses of 160 to 270 kDa are detectable in H. ducreyi culture supernatant fluid (47, 48). Expression of either LspA1 or LspA2 is necessary to inhibit phagocytic activity; therefore, both lspA1 and lspA2 must be inactivated in order to eliminate the ability of H. ducreyi to inhibit phagocytosis (45). In addition, a lspB mutant unable to secrete LspA1 or LspA2 was also unable to inhibit phagocytosis (45). Finally, a lspA1 lspA2 mutant of H. ducreyi exhibited greatly reduced virulence in both the temperature-dependent rabbit model of experimental chancroid (47) and the human challenge model (21).
In this report, we provide additional evidence that the wild-type H. ducreyi LspA proteins are involved in the inhibition of Fc receptor (FcR)-mediated phagocytosis. More importantly, our studies demonstrate that this inhibition involves one of the most proximal signaling events in phagocytosis. Incubation of wild-type H. ducreyi with immune cells resulted in decreased phosphorylation and reduced catalytic activity of Src family protein tyrosine kinases, leading to an inability to complete phagocytic cup development. This appears to be a novel mechanism for inhibition of phagocytosis by a bacterial pathogen.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The wild-type H. ducreyi strain 35000HP and the lspA1 lspA2 mutants 35000HP.12 (47) and 35000HP12 (21) were grown as described previously on chocolate agar plates (21) at 33°C in a 95% air-5% CO2 humidified atmosphere. H. ducreyi strains were also grown in Columbia broth (Difco Laboratories, Detroit, Mich.) supplemented with 0.1% (wt/vol) Trizma base (Sigma, St. Louis, Mo.), hemin (25 μg/ml), 1% (vol/vol) IsoVitaleX (Becton Dickinson, Cockeysville, Md.), and 2.5% (vol/vol) heat-inactivated fetal bovine serum (FBS; HyClone, Logan, Utah) (48) at 33°C with agitation at 120 to 130 rpm. Concentrated culture supernatant fluids (CCS) were prepared from broth-grown H. ducreyi as described elsewhere (45). Briefly, the culture fluid was first subjected to centrifugation at 6,000 x g for 10 min at 4°C to remove the bacteria. The resultant supernatant fluid was passed through a 0.22-μm-pore-size filter and then subjected to ultracentrifugation at 125,000 x g for 1 h at 4°C to remove membrane fragments. Last, this supernatant fluid was concentrated 40-fold by using an Amicon Ultra centrifugal filter device (100,000-molecular-weight cutoff) (Millipore, Inc., Bedford, Mass.) and used immediately in phagocytosis assays.
Mammalian tissue culture growth and differentiation. The human PMN-like cell line HL-60 (ATCC CCL-240; American Type Culture Collection, Manassas, VA) and the mouse monocyte/macrophage cell lines J774A.1 (ATCC TIB-67) and RAW 264.7 (ATCC TIB-71) were cultivated as previously described (45). HeLa cells (ATCC CCL-2) and the DC2.4 dendritic cell line (kindly provided by Kenneth Rock, Dana-Farber Cancer Institute, Boston, MA) were grown in the same medium as the macrophage cell lines.
LspA1 polyclonal antisera. The 12.5-kb lspA1 ORF was segmented into 13 fragments of approximately 1 kb which were then amplified by PCR and cloned into the plasmid vector pQE-30 (QIAGEN, Inc., Valencia, CA) to obtain fusion proteins consisting of LspA1 protein segments with an N-terminal six-His tag. The oligonucleotide primers used to generate these PCR products are listed in Table 1. These recombinant plasmids were used to transform Escherichia coli M15 containing pREP4 (QIAGEN). To induce expression of His-LspA1 fusion proteins, isopropylthio--D-galactoside (Invitrogen, Carlsbad, CA) was added to a final concentration of 1 mM. Each His-LspA1 fusion protein was purified per the QIAGEN protocol and used to immunize 10-week-old female BALB/c mice (Charles River Laboratories Inc., Wilmington, MA).
Immunodepletion of LspA proteins. CCS from the wild-type strain 35000HP and the lspA1 lspA2 mutant 35000HP12 were incubated with heat-inactivated LspA1 antiserum or with heat-inactivated normal BALB/c mouse serum for 2 h at 4°C. Protein A/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA) was then added, and the suspension was mixed for 1 h at 4°C and then subjected to centrifugation (12,000 x g, 5 s) to remove antigen-antibody-agarose complexes. The resultant supernatant fluids were used in phagocytosis assays with HL-60 cells.
Phagocytosis assays using fluorescent microspheres as a secondary target. A 100-μl volume of CCS or immunodepleted CCS from wild-type 35000HP or the 35000HP12 mutant was added to a 96-well tissue culture plate containing differentiated HL-60 cells at a concentration of 5 x 105 cells/well. After 1 h of incubation at 33°C, streptavidin-coated green fluorescent microspheres previously opsonized with rabbit anti-streptavidin immunoglobulin G (IgG) (Rockland Immunochemicals, Gilbertsville, PA) (45) were added, and phagocytosis was measured as described elsewhere (45).
Staining of the J774A.1 actin cytoskeleton. Bacteria were grown for 15 h in liquid culture, harvested by centrifugation for 10 min at 4,000 x g, and suspended in tissue culture medium lacking FBS to an optical density at 600 nm of 0.5. These assays were performed using J774A.1 cells attached to glass coverslips in 24-well plates (1 x 105 cells/well) in tissue culture medium without FBS. The J774A.1 cells were incubated for 1 h at 33°C with H. ducreyi cells (multiplicity of infection, 25). Sheep erythrocytes opsonized with rabbit IgG (EIgG) were added to each well and incubated for 3 to 6 min at 37°C. The J774A.1 cells were washed twice with phosphate-buffered saline (PBS) and then fixed with 3.7% formaldehyde for 10 min at room temperature. The cells were then permeabilized with 0.1% (vol/vol) Triton X-100 in PBS for 3 to 5 min at room temperature. To reduce nonspecific background, 1% (wt/vol) bovine serum albumin was incubated with the coverslips for 30 min prior to addition of staining solution. The staining solution consisted of 5 μl of a methanolic stock solution of rhodamine-phalloidin (Molecular Probes, Eugene, Oreg.) and 0.5 μl of Oregon green 488-conjugated goat anti-rabbit IgG (Molecular Probes) in 200 μl of PBS for each coverslip to be stained. The staining solution was placed on the coverslip for 20min at room temperature. The coverslips were washed twice with PBS and mounted on a slide (cell side down) in Vectashield mounting medium solution (Vector Laboratories, Inc., Burlingame, Calif.). Slides were viewed using a CFI Plan Apochromat DM60X (numerical aperture, 1.4) objective and a Nikon TE-200 inverted microscope equipped with epifluorescence. Imaging was performed using MetaMorph software (v. 6.1) and Nearest Neighbors digital deconvolution (Universal Imaging Corp., Downington, PA).
Detection of phosphoproteins by Western blot analysis. Mammalian cell lines were incubated with wild-type 35000HP and the lspA1 lspA2 mutant 35000HP12 at 33°C for 4 h. After the fluid was decanted from the flasks, the monolayers were lysed with radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology Inc.). The protein content of the lysates was measured and standardized by using the Bradford method (Bio-Rad). These lysates were heated at 100°C for 5 min in digestion buffer (18), and proteins present in the lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% (wt/vol) polyacrylamide separating gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Inc., Billerica, Mass.). The membranes were blocked with PBS containing 0.05% (vol/vol) Tween 20 and 4% (wt/vol) bovine serum albumin prior to incubation with primary antibodies. Monoclonal antibody (MAb) 4G10 (Upstate Biotechnology Inc., Lake Placid, NY) was used to detect phosphotyrosine residues, whereas the rabbit polyclonal antisera -pY418 and -pY529 (BioSource International, Inc., Camarillo, CA) were used to detect specific phosphorylated forms of Src family protein tyrosine kinases. Total Src protein was detected with MAb 327 (Calbiochem, San Diego, CA), and glyceraldehyde-3-phosphate dehydrogenase was detected with MAb 6C5 (Abcam, Cambridge, MA).
In vitro protein tyrosine kinase assays. J774A.1 macrophages were incubated with medium (control), wild-type H. ducreyi 35000HP, or the lspA1 lspA2 mutant 35000HP12 for 4 h and then lysed with RIPA buffer. The protein content of the lysates was measured and standardized by using the Bradford method (Bio-Rad). Polyclonal antibodies to Lyn (sc-15; Santa Cruz) and Hck (06-833; Upstate) were added and allowed to incubate overnight at 4°C to bind these macrophage kinases in the standardized lysates. Protein A/G agarose (Santa Cruz) was then added and the suspension mixed for 1 h, followed by several washes with kinase buffer (32.5 mM Tris-HCl [pH 7.6], 6.25 mM MnCl2, and 0.0625% [vol/vol] Triton X-100). The immunoprecipitated complexes were resuspended in 40 μl of kinase buffer, and 1 μg of a purified glutathione S-transferase (GST) fusion protein containing the cytoplasmic region of the T-cell-receptor subunit (34) was added along with unlabeled ATP to a final concentration of 0.024 mM. A 1-μl portion of [-32P]ATP (specific activity, 10 μCi/μl) was added, and the reaction mixtures were incubated at 30°C for 45 min. The kinase reaction was terminated by addition of 20 μl of loading buffer followed by heating at 95°C for 5 min. Proteins in the reaction mixture were resolved by SDS-PAGE and transferred to PVDF membranes. Phosphorylated GST- protein was detected by autoradiography. Additional Western blot analyses involving different antibodies to Lyn (sc-7274; Santa Cruz), Hck (sc-1428; Santa Cruz), and (MAb 6B10.2) (46) were used to verify that equivalent amounts of the immunoprecipitated protein tyrosine kinases and purified GST- were loaded and transferred to the PVDF membranes. Relative quantitation of phosphorylated GST- was achieved by using an Amersham Biosciences Storm 820 PhosphorImager.
RESULTS
Evidence for the involvement of the LspA proteins in the inhibition of phagocytic activity. We previously reported that a lspA1 lspA2 mutant of H. ducreyi was unable to inhibit phagocytosis (45). Efforts to clone full-length H. ducreyi lspA genes into E. coli have been unsuccessful to date, precluding the use of a genetic approach to prove that the LspA proteins are directly involved in the inhibition of phagocytosis. Therefore, to obtain additional evidence that the LspA proteins are necessary for the inhibition of phagocytosis by H. ducreyi, immunodepletion experiments were performed with H. ducreyi CCS. His-tagged LspA1 fusion proteins were used to raise mouse polyclonal antibodies to different regions of the LspA1 protein for use in these immunodepletion experiments (Fig. 1). We were successful in obtaining antibodies against 10 of the 13 LspA1 segments diagramed in Fig. 1B.
As reported previously (45), wild-type 35000HP CCS (Fig. 2, column A) readily inhibited phagocytosis whereas CCS from the lspA1 lspA2 mutant 35000HP12 (Fig. 2, column B) did not inhibit phagocytic activity. To confirm the involvement of the LspA proteins in this inhibition, polyclonal mouse antibodies raised against some of the His-LspA1 fusion proteins (Fig. 1B) were incubated with CCS from wild-type H. ducreyi and the lspA1 lspA2 mutant. (LspA1 is the vastly predominant form of LspA protein in wild-type H. ducreyi CCS; LspA2 is almost undetectable [47].) After removal of the immune complexes, the CCS were incubated with HL-60 cells, which were then used in phagocytosis assays with opsonized fluorescent microspheres. In initial immunodepletion experiments, we used a pool of antisera to His-LspA1 fusion proteins 4 to 10 (Fig. 1B) and successfully removed the ability of wild-type H.ducreyi CCS to inhibit phagocytosis by HL-60 cells (data not shown). Subsequent experiments were then performed using a polyclonal antibody to the His-LspA1 fusion protein 8 only (Fig. 1B). Incubation with these LspA1 antibodies caused the wild-type CCS (Fig. 2, column C) to lose its ability to inhibit phagocytic activity. Normal mouse serum (used as a negative control) (Fig. 2, column E) did not eliminate this inhibitory effect. As expected, the lack of inhibition observed with CCS from the lspA1 lspA2 mutant (Fig. 2, column B) was not affected by either of these antibody preparations (Fig. 2, columns D and F). These results provide additional evidence for the involvement of the LspA proteins in the phagocytic inhibition phenotype of wild-type H. ducreyi 35000HP.
Wild-type H. ducreyi affects phagocytic cup development. FcR-mediated phagocytosis normally involves localized actin assembly, which drives pseudopod extension, forming a "cup-like" structure. This culminates in complete target engulfment and eventual actin depolymerization (16). Cytoskeleton staining was performed and viewed by indirect immunofluorescence to detect possible differences in FcR-induced actin assembly in macrophages exposed to wild-type H. ducreyi 35000HP and the lspA1 lspA2 mutant 35000HP.12. This method allows visualization of both phagocytic cups and IgG-opsonized particles (16).
J774A.1 macrophages were incubated for 1 h with either strain prior to the addition of EIgG. After a 3-min incubation with EIgG, the cells were fixed, permeabilized, and stained with rhodamine-phalloidin to detect F-actin and with Oregon green-conjugated goat anti-rabbit IgG to detect the opsonized erythrocytes. Macrophages incubated with the lspA1 lspA2 mutant (Fig. 3, lower panels) showed normal phagocytic cup development and engulfment of opsonized erythrocytes. In contrast, exposure to wild-type H. ducreyi apparently halted progression of phagocytic cup formation around the opsonized erythrocytes, which remained extracellular (Fig. 3, upper panels). It should also be noted here that it was previously shown that exposure to wild-type H. ducreyi does not affect binding of IgG-opsonized targets to these macrophages (45).
H. ducreyi affects macrophage tyrosyl phosphoproteins. The fact that wild-type H. ducreyi had the ability to apparently halt the development of the phagocytic cup suggested a direct inhibition of phagocytic signaling and prompted us to examine the levels of phosphoproteins in these macrophages. The most proximal events in the phagocytic signaling pathway involve protein phosphorylation events (e.g., Src kinase activation, immunoreceptor tyrosine-based activation motif [ITAM] phosphorylation) catalyzed by tyrosine protein kinases (Fig. 4A) (for reviews, see references 22 and 40). To determine whether the LspA proteins were involved in perturbing these signaling events, J774A.1 macrophages were incubated with wild-type H. ducreyi 35000HP or the lspA1 lspA2 mutant 35000HP12. These phagocytes were then lysed, and their solubilized proteins were probed by Western blot analysis with the phosphotyrosine-specific MAb 4G10. The levels of several macrophage phosphotyrosine proteins were found to be drastically reduced in lysates from J774A.1 macrophages incubated with wild-type H. ducreyi (Fig. 4B, lane 1) relative to those incubated with the mutant (Fig. 4B, lane 2). These included prominent phosphotyrosine proteins with an apparent molecular weight of approximately 55,000. This effect of wild-type H. ducreyi 35000HP on phosphoproteins was not limited to J774A.1 macrophages; it was observed with RAW 264.7 macrophages as well (Fig. 4B; compare lanes 3 and 4). Decreases in the levels of these phosphoproteins were also observed when IgG-opsonized erythrocytes were added to the macrophages after incubation with wild-type H. ducreyi (data not shown).
Wild-type H. ducreyi reduces the levels of active Src family kinase members. The apparent molecular weights of the phosphoprotein(s) affected by incubation with wild-type H. ducreyi (Fig. 4B) corresponded to the approximate sizes of Src family protein tyrosine kinases which are essential for normal phagocytic activity (Fig. 4A) (11, 22). To determine whether these phosphoproteins were Src family kinases, we prepared cellular extracts from four different cell lines that had been incubated with either wild-type H. ducreyi 35000HP or the lspA1 lspA2 mutant 35000HP12 and probed these by Western blot analysis with antibodies specific for the two different tyrosine-phosphorylated forms of Src family members. To detect the active form of Src family kinases, we used a polyclonal antiserum (-pY418) reactive with the phosphorylated form of Src tyrosine residue Y418; this tyrosine is phosphorylated when Src kinases are catalytically active. To detect the inactive phosphorylated form of Src kinases, we used an antibody (-pY529) that recognizes the Src tyrosine residue which, when phosphorylated, holds Src kinases in an inactive state. Extracts from the three immune cell lineages (J774A.1 macrophages, PMN-like HL-60 cells, and DC2.4 dendritic cells) that had been incubated with wild-type H. ducreyi (Fig. 5A, lanes 2, 5, and 8, respectively) showed dramatic decreases in reactivity with the -pY418 antibody specific for active phospho-Src kinases. In contrast, extracts from cells of the same types that had been incubated with the lspA1 lspA2 mutant (Fig. 5A, lanes 3, 6, and 9) or with tissue culture medium (control) (Fig. 5A, lanes 1, 4, and 7) showed little or no reduction in reactivity with the same antibody. Interestingly, extracts from the nonimmune HeLa cell line (Fig. 5A, lanes 10, 11, and 12) showed no apparent difference in reactivity with the same antibody. In the same four cell lines incubated with wild-type H. ducreyi, we found modest and variable changes in the level of phosphorylation of the distal Src tyrosine residue Y529 (Fig. 5B). The levels of total Src tyrosine kinases were relatively unchanged within each cell type (Fig. 5C). Additional experiments indicated that a decrease in activated phospho-Src levels could be detected by Western blot analysis after just 1 h of incubation with wild-type H. ducreyi 35000HP (data not shown).
H. ducreyi reduces the catalytic activity of Src family tyrosine kinases. The Western blot data (Fig. 4B and 5A) clearly demonstrated reductions in the levels of the active forms of Src kinases. The Src family kinases Hck, Lyn, and Fgr are the predominant Src kinases present in macrophages (22); the former two tyrosine kinases are required for normal phagocytic activity, whereas Fgr plays an important negative regulatory role (17). Our Western blot data suggested that Hck and Lyn would exhibit decreases in catalytic activity. To examine this possibility, J774A.1 macrophages were incubated with H. ducreyi strains as described for Fig. 5, and then, after lysis of the macrophages, antibodies to Hck and Lyn were used to immunoprecipitate these Src family members for use in tyrosine kinase reactions. These in vitro kinase assays utilized a physiological substrate, the ITAM-containing T-cell-receptor subunit (in the form of a GST fusion protein). As can be seen from the levels of phosphorylated GST-, the catalytic activities of both Hck (Fig. 6A, lane 2) and Lyn (Fig. 6A, lane 5) were decreased in macrophages incubated with wild-type H. ducreyi but not in macrophages incubated with the lspA1 lspA2 mutant (Fig. 6A, lanes 3 and 6, respectively). PhosphorImager analysis indicated an 84% reduction in Hck activity and a 47% reduction in Lyn activity; these differences were significant (P = 0.045 for Hck and P = 0.012 for Lyn by a paired Student t test). Equivalent amounts of total Hck and Lyn kinases (Fig. 6B) and total GST- protein (Fig. 6C) were present in these samples as determined by Western blot analysis. These data indicate that the reduced levels of active Src kinases seen in Fig. 5 in the J774A.1 cells are reflected by decreases in the enzymatic activities of both Hck and Lyn.
DISCUSSION
A large number of putative virulence factors of H. ducreyi have been identified to date, including both proteins and lipooligosaccharide (see reference 35 for a review). In addition, H. ducreyi has been shown to synthesize at least two toxins (9, 25, 41) and a copper-zinc superoxide dismutase (28). However, when tested in the human challenge model for experimental chancroid (3, 37), only five of these gene products have been shown to be required for full virulence of H. ducreyi. These include the peptidoglycan-associated lipoprotein (13), the hemoglobin-binding outer membrane protein HgbA (2), the DsrA outer membrane protein (7), the flp gene cluster (36), and the LspA1 and LspA2 proteins (21). The latter two proteins have been shown, both by a previous mutant analysis (45) and by the present study, to be involved in the ability of this pathogen to inhibit phagocytosis in vitro. The findings of the present study have also revealed a novel and previously unreported capacity of H. ducreyi to affect the phosphorylation state of Src family protein tyrosine kinases.
Bacterial pathogens have devised a number of different strategies for escaping from or preventing phagocytosis (for reviews, see references 23 and 27). These range from the expression of polysaccharide capsules, which can present a simple physical impediment or even alter phagocytic signaling (e.g., Streptococcus suis [29]), to injection of effector molecules that target signaling components (e.g., Yersinia YopT [32] and Pseudomonas ExoT [38]). Phagocytosis is an extremely complex process (Fig. 4A) (for a review, see reference 40). While binding of IgG-opsonized particles or bacteria to most FcRs (except FcRIIB) will initiate the phagocytic signaling cascade, signaling through Toll-like receptors has also recently been shown to affect phagocytosis (6, 10). FcR cross-linking (by IgG-opsonized target particles) results in activation of Src family protein tyrosine kinases, with Hck, Lyn, and Fgr being predominant in murine macrophages (11). These protein tyrosine kinases catalyze the phosphorylation of ITAMs present on or associated with FcR. Once phosphorylated, FcR-associated ITAMs become docking sites for the SH2 domains of Syk and Src family protein tyrosine kinases. ITAM-bound Syk is catalytically activated by a combination of autophosphorylation and transphosphorylation by Src family protein tyrosine kinases. Activated Syk subsequently phosphorylates a number of intracellular substrates, initiating a series of downstream events, including small Rho GTPase activation, which culminate in actin filament assembly and development of the phagocytic cup (Fig. 4A). Since activation of Src family protein tyrosine kinases is a very proximal event in FcR signaling, it is likely that multiple cellular events relevant to phagocytosis (e.g., altered membrane trafficking) are also affected by the LspA proteins, in addition to actin assembly (Fig. 3).
We have established that expression of either LspA1 or LspA2 individually is necessary for H. ducreyi to inhibit phagocytic activity in vitro (45). Efforts to clone full-length H. ducreyi lspA genes into E. coli or to purify functional LspA proteins from spent H. ducreyi culture medium have been unsuccessful to date, so we used LspA-specific antibodies to provide additional proof that this inhibitory effect required these H. ducreyi proteins (Fig. 2). Other experiments showed that incubation of wild-type H. ducreyi, but not the lspA1 lspA2 mutant, with macrophages resulted in dramatic reductions in the levels of active Src protein tyrosine kinases (Fig. 5 and 6). These kinases are involved in some of the most proximal events of the phagocytic signaling pathway (Fig. 4A). This reduced enzymatic activity of the Src protein tyrosine kinases is consistent with the inability of these macrophages to complete the development of phagocytic cups (Fig. 3). We hypothesize that either (i) the LspA proteins bind to a receptor on the macrophage surface and thereby initiate a signaling cascade that abolishes phagocytic activity or (ii) the LspA proteins enter or penetrate into the phagocyte and thereby cause derangement of the normal phagocytic signaling pathway. The levels of active phospho-Src kinases in all three immune cell types tested in this study were reduced by exposure to wild-type H. ducreyi, whereas phospho-Src kinase levels in HeLa cells appeared unaffected (Fig. 5). This finding raises the possibility that a surface component present in immune cell lineages may interact with the LspA proteins, resulting in a negative signaling event. At this time, however, we cannot exclude the possibility that LspA proteins enter the macrophage via an endocytic process and exert their function in the cytoplasm, but it is not clear how these proteins would survive endosomal proteolysis and exit into the cytoplasm. An equivalently large bacterial exoprotein (i.e., RtxA) synthesized by Vibrio cholerae has been shown to directly cross-link actin intracellularly, but the method of entry or penetration of this protein into the host cell cytoplasm remains to be determined (33).
Only two regions of the LspA proteins have significant homology to known bacterial virulence factors. The N-terminal one-quarter of LspA1 (Fig. 1A) contains several features associated with secretion of other soluble virulence factors including the Bordetella pertussis filamentous hemagglutinin (20). There is also a 260-amino-acid region in the C-terminal half of the LspA proteins that has 36% identity with the YopT protein from pathogenic Yersinia species (32) (Fig. 1A). YopT is the prototype of a family of cysteine proteases involved in virulence expression and cleaves small Rho GTPases from the eukaryotic cell membrane (32), thereby altering signaling and inhibiting phagocytosis. Western blot experiments targeting the small Rho GTPases known to be involved in FcR-mediated signaling (8) showed a reduction in the level of membrane-bound Cdc42 in macrophages incubated with wild-type H. ducreyi relative to that in macrophages exposed to the lspA1 lspA2 mutant (data not shown). It is likely that this change is simply a downstream effect related to the greatly decreased levels of Src family kinase activity. Moreover, we have established that the level of membrane-bound Hck is unchanged whether the macrophages are incubated with wild-type H. ducreyi or the lspA1 lspA2 mutant (data not shown). The latter finding indicates that the YopT-like region is not cleaving Hck from the membrane. It is possible that this YopT-like region could be involved in autoprocessing of the LspA proteins, in a manner similar to that catalyzed by the Pseudomonas syringae AvrPphB protein, another member of this cysteine protease family (31).
To date, there is only one other example of a bacterial gene product that affects Src family protein tyrosine kinases. The CagA protein of Helicobacter pylori, after injection into macrophages via a type IV secretion system, is phosphorylated by Src kinase (30) and in turn causes a reduction in active phospho-Src kinase levels by increasing the activity of the protein tyrosine phosphatase SHP-2 (43). However, two independent studies indicate that CagA is not involved in the inhibition of phagocytosis caused by H. pylori (24, 26). It should be noted here that efforts to detect possible phosphorylation of the H. ducreyi LspA proteins after their interaction with macrophages have been unsuccessful to date (data not shown).
Exactly how the LspA proteins are involved in the observed reductions in the catalytic activities of the Src family protein tyrosine kinases remains to be determined. At this time, we cannot formally exclude the possibility that another, as yet unidentified H. ducreyi gene product works in concert with the LspA proteins to inhibit phagocytosis. Efforts to elucidate the precise mechanism of action of these secreted virulence factors are in progress and may provide additional insight into the pathogenesis of chancroid as well as a valuable biological tool for further dissection of Src family kinase function, regulation, and activity.
ACKNOWLEDGMENTS
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-AI007520-08.
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