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Infection and Immunity, November 2008, p. 4905-4912, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00851-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Anaplasma phagocytophilum Increases Cathepsin L Activity, Thereby Globally Influencing Neutrophil Function

Venetta Thomas, Swapna Samanta, and Erol Fikrig^*

Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520

Received 9 July 2008/ Returned for modification 9 August 2008/ Accepted 22 August 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, is an unusual obligate intracellular pathogen that persists in neutrophils. A. phagocytophilum increases the binding of a repressor, CCAAT displacement protein (CDP), to the gp91^phox promoter, thereby diminishing the host oxidative burst. We now show that A. phagocytophilum infection also enhances the binding of CDP to the promoters of human neutrophil peptide 1 and C/EBP—molecules important for neutrophil defense and maturation—suggesting that this is a general strategy used by this pathogen to alter polymorphonuclear leukocyte function. To explore the mechanism by which A. phagocytophilum increases CDP activity, we assessed the effects of this microbe on cathepsin L, a protease that cleaves CDP into a form with increased DNA binding ability. A. phagocytophilum infection resulted in elevated cathepsin L activity and the proteolysis of CDP. Blocking the action of cathepsin L with a chemical inhibitor or small interfering RNA targeting of this molecule caused a marked reduction in the degree of A. phagocytophilum infection. These data demonstrate that increasing cathepsin L activity is a strategy used by A. phagocytophilum to alter CDP activity and thereby globally influence neutrophil function. As therapeutic options for A. phagocytophilum and related organisms are limited, these results also identify a cellular pathway that may be targeted for the treatment of A. phagocytophilum infection.

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Anaplasma phagocytophilum, a gram-negative obligate intracellular pathogen, has a tropism for neutrophils and their precursors. The resulting disease, human granulocytic anaplasmosis, is often characterized by fever, severe headache, malaise, myalgia, leukopenia, thrombocytopenia, and elevated hepatic transaminases. The illness is rarely fatal, but death may occur as a result of opportunistic infections, often with catalase-positive organisms (7). Neutrophils infected with A. phagocytophilum demonstrate defects in several cellular functions (2, 31), suggesting that A. phagocytophilum exploits a number of pathways to survive in this hostile environment. One effect of A. phagocytophilum is the transcriptional repression of gp91^phox, a key component of the respiratory burst. We have previously shown that A. phagocytophilum alters gp91^phox expression by facilitating the binding of the repressor CCAAT displacement protein (CDP) to the gp91^phox promoter (35).

CDP is critical in the regulation of several genes associated with neutrophil differentiation and development (27). The repressive activity of CDP is generally reduced in terminally differentiated cells and correlates with the enhanced expression of target genes (23). The overexpression of CDP represses the expression of gp91^phox, neutrophil gelatinase, human neutrophil peptide (HNP), and CCAAT enhancer binding protein epsilon (C/EBP), among others (13, 15). The binding of CDP can be altered by interactions with accessory proteins such as SATB1, CBP, p300/CREB, and G9a (19, 21, 28) or by posttranslational modifications such as phosphorylation by protein kinase C, casein kinase II, and cyclin A/Cdk1, dephosphorylation by Cdc25A, and acetylation by PCAF (4, 5, 19, 24, 32).

CDP binding activity is also influenced through proteolytic processing by the cysteine protease cathepsin L. The 200-kDa form of CDP contains displacement activity (25). The removal of the N-terminal portion of this protein releases an autoinhibitory activity and facilitates stable binding to the DNA (36). A nucleus-localized isoform of cathepsin L cleaves CDP during G₁/S transition of the cell cycle, resulting in a 110-kDa fragment with enhanced DNA binding capability (26). More recently, a 90-kDa isoform of CDP with similar DNA binding ability was identified in cells of epithelial origin (11). Lysosomal cysteine proteases such as cathepsin L are known to participate in the regulation of antigen presentation by major histocompatibility complex class II and CD1d molecules (38). Cathepsin L cleavage of bacterial proteins can facilitate antigen presentation and the generation of an immune response (29), and the cleavage of viral glycoprotein by cathepsin L can facilitate viral fusion (30, 33). Here, we examined whether A. phagocytophilum infection facilitates cathepsin L activity to influence CDP binding activity and CDP-related genes.

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Cell lines and A. phagocytophilum infection. The promyelocytic cell line (HL-60) was acquired from the American Type Culture Collection (ATCC, Manassas, VA) and maintained according to the supplier's specifications. A. phagocytophilum-infected cells were maintained as described previously; equal volumes of uninfected cells and infected cells were combined, and the mixture was diluted 1:5 with fresh medium (35). A host cell-free preparation of A. phagocytophilum was obtained as described previously (34) and used at a multiplicity of infection (MOI) of 10.

RNA and DNA preparation. mRNA from A. phagocytophilum-infected and uninfected HL-60 cells was prepared using an RNA isolation kit (Stratagene). cDNA was prepared via a reverse transcriptase PCR kit according to the recommendations of the manufacturer (Stratagene). DNA was prepared using the DNeasy blood and tissue kit from Qiagen (Valencia, CA). The gene encoding p44, the 44-kDa immunodominant protein of A. phagocytophilum, was amplified with p44 primer 1 (5'-GCCACTATGGAATTTTCTTCGGG-3') and p44 primer 2 (5'-TCAAGACCAAGGGGTATTAGAGATAG-3'). gp91^phox was detected as described previously (1). Additional genes were amplified with the following primers: the HNP gene with HNP primer 1 (5'-ATGAGGACCCTCGCCATCCTT-3') and HNP primer 2 (5'-GCAAGCTCGCAGCAGAATG-3'), the C/EBP gene with C/EBP primer 1 (5'-ATGTCCCACGGGACCTACTACGA-3') and C/EBP primer 2 (5'-ACAGTGTGCCACTTGGTACTGCAG-3'), and the actin gene with actin primer 1 (5'-AGCGGGAAATCGTGCGTG-3') and actin primer 2 (5'-CAGGGTACATGGTGGTGCC-3').

Quantitative PCR. Real-time PCR was performed using the iCycler iQ real-time detection system (Bio-Rad, Hercules, CA). The amplification of the specific genes was detected by using the iQ SYBR green supermix according to the specifications of the manufacturer (Bio-Rad) and the primers for the HNP-1, gp91^phox, C/EBP, actin, and A. phagocytophilum p44 genes described above.

ChIP. Chromatin immunoprecipitation (ChIP) was performed using the method of Li et al. (20) with some modifications. HL-60 cells or A. phagocytophilum-infected HL-60 cells (10⁷) were collected in 10 ml of medium and placed into 100-mm tissue culture plates. The cells were fixed with a final concentration of 2% formaldehyde for 30 min at 37°C. Next, they were collected and washed several times in cold phosphate-buffered saline (PBS) and then sonicated in ChIP buffer (10 mM Tris-HCl, 140 mM NaCl, 5% glycerol, 0.1% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1% Triton X-100) supplemented with protease inhibitors. Following centrifugation, the supernatant was collected and stored until immunoprecipitation. The protein concentration was determined with the Bio-Rad protein assay reagent. Supernatant samples with equal protein concentrations were incubated overnight with an antibody to CDP (Santa Cruz Biotech). The antibody/protein-DNA complexes were incubated with protein A/G agarose beads for an additional 1 h and then subjected to centrifugation, and the bound protein-DNA complexes were collected. The beads were washed several times with ChIP buffer, incubated with elution buffer (0.1 M NaHCO₃ and 1% SDS), and heated for 4 h at 65°C. Supernatants were then collected and subjected to DNA purification using the Qiagen PCR purification kit. The DNA was eluted in 30 to 50 µl and used in PCR amplification with specific primers. The C/EBP promoter was amplified with C/EBP primer 1 (5'-GCTAACCGGAATATGCTAATCAG-3') and primer 2 (5'-CCTTTCAGAGACACCTGCTC-3'), the HNP promoter was amplified with primer 1 (5'-GTCAACTGTGTTAGGAGC-3') and primer 2 (5'-ATTGTGGTGGCAAGGACA-3'), and the gp91^phox promoter was amplified with primer 1 (5'-CATGGTGGCAGAGGTTGAATGT-3') and primer 2 (5'-GTGACTGGATCATTATAGACC-3'). A portion of total lysate containing no antibody was also processed for DNA elution and purification. This sample served as a control for input DNA.

Antibodies and inhibitors. Antibodies to cathepsin L were obtained from Cell Signaling Technology (Beverly, MA), BD Bioscience (San Diego, CA), and Calbiochem of EMD Bioscience Inc. (Darmstadt, Germany). Phosphorylated tyrosine (phosphorylated at Tyr-100 and Tyr-102) was obtained from Cell Signaling Technology. Actin and CDP (C-20) antibodies were obtained from Santa Cruz Biotech (Santa Cruz, CA). Secondary antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies to PSGL-1 (PL1) and a mouse immunoglobulin G1 (IgG1) isotype control were acquired from BD Bioscience. Cathepsin L inhibitors (I and III) and a cathepsin B inhibitor (CA-074) were purchased from Calbiochem.

EMSA. The gel shift assay was performed as described previously (35) with the Lightshift chemiluminescent electrophoretic mobility shift assay (EMSA) kit (Pierce, Rockford, IL). Complementary biotinylated oligonucleotides consisting of the CDP binding site CCAAT-CGAT (25) were annealed and then loaded onto a 2% agarose gel. The annealed CCAAT-CGAT oligonucleotide was extracted with the QIAquick gel extraction kit (Qiagen). The isolated double-stranded oligonucleotide was added to a 20-µl reaction mix consisting of 5 µg of nuclear extract from either uninfected HL-60 cells or A. phagocytophilum-infected HL-60 cells, along with DNA binding buffer, glycerol, MgCl₂, NP-40, and poly(dI-dC) at concentrations based on the recommendations of the EMSA kit manufacturer. For supershifting, 5 to 10 µg of anti-CDP was incubated with the DNA-extract complexes for 20 min on ice.

Immunoblotting. Immunoblotting was performed as described previously (35). Briefly, whole-cell extracts from uninfected HL-60 cells and (90%) A. phagocytophilum-infected cells were produced by lysing with modified radioimmunoprecipitation assay buffer prepared based on the protocol from Upstate (Temecula, CA); 1x radioimmunoprecipitation assay buffer contained 50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% deoxycholate, 150 mM NaCl, and 1 mM EDTA. Protease and phosphatase inhibitors (1 mM NaF and Na₃VO₄) plus 1x protease inhibitor cocktail (Roche, Indianapolis, IN) were added on the day of extraction. Extracts were stored at –80°C until use. Samples (20 µg) of the cell extracts were resolved on an SDS-10% polyacrylamide gel. The proteins were then transferred onto a nitrocellulose membrane. Depending on the primary antibody, the membrane was incubated with either 5% bovine serum albumin or milk in Tween 20-Tris-buffered saline to bind nonspecific sites. For antibody blocking, cells were incubated with 10 µg of mouse IgG1 or PL1 (anti-PSGL-1)/ml for 1 h prior to infection with A. phagocytophilum. For inhibitor treatment, cells were preincubated with dimethyl sulfoxide (DMSO) or 10 µM cathepsin B or cathepsin L inhibitor I for 1 h prior to infection. A host cell-free preparation of A. phagocytophilum was added at a MOI of 10 for the times indicated below, and the infected samples were processed as described previously.

RNA interference and transfection. Small interfering RNA (siRNA) for cathepsin L, designed based on the sequence (5'-GGCGATGCACAACAGATTATT-3') described by Pager and Dutch (30), and negative control 1 siRNA were obtained from Ambion (Austin, TX). HL-60 cells (2 x 10⁶) were nucleofected with 1 µg of siRNA by using the nucleofector from Amaxa Biosystems (Gaithersburg, MD) with kit V and program T-19 according to the manufacturer's specifications. Nucleofected cells were collected after 48 h and incubated with a host cell-free preparation of A. phagocytophilum for 2 h. Following centrifugation (Hettich Rotina 46R; Hettich, Tuttlingen, Germany) at 1,200 rpm to pellet cells and bound bacteria, the cell pellet was collected and washed once with culture medium. The supernatant was removed, and the cells were cultured in fresh medium for 48 to 72 h.

Detection of cathepsin L activity. A rhodamine-labeled cathepsin L substrate [(benzyloxycarbonyl-Phe-Arg)₂-R110] was obtained from Molecular Probes (Eugene, OR). An intracellular proteinase assay was performed according to the recommendations of the substrate manufacturer. HL-60 cells were infected with a host cell-free preparation of A. phagocytophilum (MOI of 10) for approximately 24 h. Cells were pelleted and resuspended in HEPES-buffered saline at a concentration of 10⁶ cells/ml and stored on ice. A 10 µM concentration of the substrate was added, and cells were incubated for 20 min on ice. Cells were sedimented, resuspended in PBS containing 4% formaldehyde, and analyzed with the FACSCaliber machine using the CellQuest software (Becton Dickinson, San Jose, CA). When a cathepsin L inhibitor was tested, cells were preincubated with 10 µM inhibitor or diluent (DMSO) for 1 h prior to infection and processed as described above.

The detection of cathepsins L and B by fluorescence microscopy was performed with detection kits from Biomol International (Plymouth Meeting, PA). Following infection with A. phagocytophilum at an MOI of 10, cells were pelleted and resuspended at 10⁶ cells/ml in fresh culture medium. A substrate for cathepsin L, CV-(FR)₂, or cathepsin B, CV-(RR)₂, was added, and the suspension was incubated for 45 min at 37°C. Cells were mixed periodically to limit sedimentation. Cells were counterstained with Hoechst stain for 10 min. Following counterstaining, cells were centrifuged, resuspended in PBS, and visualized using a fluorescence microscope.

Statistical analysis. Statistical analysis was performed using the Student t test. A P value of less than 0.05 was considered significant.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
CDP binding and repression activities are augmented by A. phagocytophilum infection. In our previous work, we demonstrated that reductions in the transcription factors PU.1, ELF-1, and interferon regulatory factor 1 during A. phagocytophilum infection are associated with the enhanced binding of CDP to the proximal promoter of the gp91^phox gene (35). The enhanced binding of CDP to all the binding sites within the gp91^phox proximal promoter suggested that the binding activity of CDP itself was likely to be influenced by this infection. To determine if the general binding ability of CDP was altered during A. phagocytophilum infection, we assessed the binding of CDP to an oligonucleotide containing a consensus CDP binding site (25). Protein lysates from A. phagocytophilum-infected cells showed elevated binding compared to that of lysates from uninfected cells (Fig. 1A). The incubation of lysates with antibody to CDP results in a supershift of the slower-migrating complex, indicating the presence of CDP.

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FIG. 1. Enhancement of CDP binding and repression activities due to A. phagocytophilum. (A) EMSA of CDP interaction with the CDP binding site (CCAAT-CGAT). Nuclear extracts from uninfected (–) or A. phagocytophilum (Ap)-infected (+) cells show binding to the CDP binding site. The CDP band shift is denoted by an arrow. A supershift of the CDP band is seen with the addition of the CDP antibody (CDP). This result is representative of results from at least three experiments showing similar trends. (B) ChIP analysis of CDP binding to the promoters of the gp91phox, HNP, and C/EBP genes. Chromatin-cross-linked extracts from uninfected and A. phagocytophilum-infected cells were immunoprecipitated with the CDP antibody. Purified DNA was amplified with primers specific for the promoters of the gp91phox, HNP, and C/EBP genes. Amplification from extracts not subjected to CDP immunoprecipitation was used as the input control. This result is representative of results from at least three experiments with similar findings. (C to H) Expression of gp91phox (C and F), HNP (D and G), and C/EBP (E and H) genes in uninfected [(–) Ap] and A. phagocytophilum-infected [(+) Ap] cells. Following infection for 48 h, the cells were either left untreated (C through E) or stimulated with ATRA (F through H) for an additional 48 h before RNA processing. The relative expression of HNP (D and G) and C/EBP (E and H) normalized to the expression (copies) of actin is reported. Due to the variability in results among experiments, the level of gp91phox expression (C and F) was determined by normalization to the level of actin expression followed by comparison to the level of gp91phox expression in the untreated control cells, and the results are reported as the increase in gp91phox expression (n-fold) over that in the untreated controls. Asterisks denote statistical significance, with P values of <0.05.

Since the binding ability of CDP is altered by A. phagocytophilum infection, we determined whether the interaction of CDP with the promoters of other myeloid genes was also elevated. We assessed binding to the promoters of the genes for C/EBP, which is critical for secondary granule protein expression (9, 15, 18), and the antimicrobial peptide HNP-1. The promoter of the gp91^phox gene served as a control. ChIP showed detectable binding of CDP to all the promoters tested (Fig. 1B). In the presence of A. phagocytophilum, there was increased binding of CDP to the promoters of the C/EBP and HNP genes (Fig. 1B). As expected, A. phagocytophilum infection enhanced the binding of CDP to the gp91^phox promoter as well (Fig. 1B).

Given that infection with A. phagocytophilum can augment the binding of CDP to the promoters of the C/EBP and HNP-1 genes, we next assessed the levels of transcription of these genes following infection. Basal levels of C/EBP and HNP-1 expression can be detected by reverse transcriptase PCR (Fig. 1D and E). Infection with A. phagocytophilum suppressed the basal transcription. All-trans retinoic acid (ATRA) is known to stimulate the expression of gp91^phox, C/EBP, and HNP-1 (14). HL-60 cells were infected for 48 h and then stimulated with ATRA to determine if transcriptional repression could be detected after stimulation. In uninfected cells, stimulation with ATRA elevated the expression of C/EBP (compare Fig. 1E and H). Infection with A. phagocytophilum inhibited the ATRA-induced expression by more than threefold. HNP gene expression was also augmented upon stimulation with ATRA (compare Fig. 1D and G). Infection with A. phagocytophilum reduced the transcription of the HNP gene as well. As expected, the transcription of the gp91^phox gene was reduced upon infection (Fig. 1C and 1F).

Elevated cathepsin L activity correlates with the proteolytic processing of CDP. In subsequent analyses, we observed the proteolytic processing of CDP in cells infected with A. phagocytophilum (Fig. 2A). While uninfected cells contained the 200-kDa full-length protein, infected cells contained several products detected by the CDP antibody. This result correlated with the generation of 110- and 75-kDa products which have been described previously for CDP (10, 12).

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FIG. 2. The proteolytic processing of CDP correlates with elevated cathepsin L activity. (A) Western blot of nuclear extracts from uninfected cells (–) and A. phagocytophilum (Ap)-infected cells (+) probed with an antibody to CDP (CDP). The arrows indicate the bands affected by infection. This result is representative of data from at least three experiments with similar results. Numbers to the left are molecular size markers, in kilodaltons. (B) FACS analysis of cathepsin L (CTSL) activity in uninfected [(–) Ap] and A. phagocytophilum-infected [(+) Ap] cells. The filled histogram represents the background staining. This experiment was done in duplicate with quadruplicate samples of A. phagocytophilum-infected cells. (C) Graphic representation of the proportions of uninfected and A. phagocytophilum-infected cells positive for cathepsin L activity. The asterisk denotes statistical significance, with a P value of <0.05.

The proteolytic processing of CDP has been attributed previously to cathepsin L activity (10, 11). We therefore investigated the level of cathepsin L activity in cells infected with A. phagocytophilum. Utilizing fluorescence-activated cell sorter (FACS) analysis, we observed that uninfected cells contained detectable cathepsin L activity above the background level in unstained cells (Fig. 2B). Infection with A. phagocytophilum increased the level of cathepsin L activity approximately fourfold (Fig. 2B and C).

A. phagocytophilum elevates cathepsin L but not cathepsin B. Cathepsin B is also a lysosomal cysteine protease and may have effects similar to those of cathepsin L (8, 22). To determine if the effect of A. phagocytophilum was unique to cathepsin L, we assessed the levels of cathepsin B and cathepsin L in cells infected with A. phagocytophilum by using immunofluorescence. Our data show that in the presence of A. phagocytophilum, the accumulation of cathepsin B was not altered compared to that in uninfected cells (Fig. 3A to C). However, in the presence of A. phagocytophilum, the accumulation of cathepsin L was enhanced (Fig. 3A to C).

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FIG. 3. Upregulation of cathepsin L and not cathepsin B by A. phagocytophilum. (A and B) Microscopic detection of cathepsin B (CTSB) and cathepsin L (CTSL) activities in the absence (–) and presence (+) of A. phagocytophilum (Ap). Cathepsin staining is shown in red, and the nuclei are stained blue. (C) Graphic representation of the proportions of uninfected [(–) Ap] and A. phagocytophilum-infected [(+) Ap] cells positive for cathepsin B and L activities. The asterisk denotes statistical significance, with a P value of <0.05, in the comparison indicated by the dashed line.

Inhibition of cathepsin L activity. To determine the importance of cathepsin L activity in A. phagocytophilum infection, we first analyzed the effect of cathepsin L inhibitor I on the A. phagocytophilum-induced cathepsin L activity. Using FACS analysis, we showed that, as before, uninfected cells contained basal cathepsin L activity (Fig. 4B). Infection with A. phagocytophilum augmented that activity (Fig. 4C). The preincubation of cells with the inhibitor to cathepsin L significantly (P < 0.01) diminished the ability of A. phagocytophilum to induce cathepsin L activity (Fig. 4D).

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FIG. 4. The inhibition of cathepsin L activity alters A. phagocytophilum infection. Cells were treated with either diluent (DMSO) or 10 µM cathepsin L inhibitor I for 1 h prior to infection. (A to D) FACS analysis of cathepsin L (CTSL) activity in uninfected [(–) Ap], A. phagocytophilum-infected [(+) Ap], and cathepsin L inhibitor-treated and A. phagocytophilum-infected [inhibitor/(+) Ap] cells. Unstained cells served as a control. The percentages of cells with cathepsin L (CTSL) activity are shown in the upper right quadrants. These data are representative of data from at least five experiments with similar results. (E) Western blot to determine the effect of cathepsin L inhibition on A. phagocytophilum infection. HL-60 cells were treated with either cathepsin L inhibitor I, cathepsin L inhibitor III, or a combination of both inhibitors prior to infection (+) with A. phagocytophilum (Ap). The 44-kDa protein of A. phagocytophilum (p44) was detected. Actin served as a control for loading. –, uninfected, untreated control cells. (F) Quantitative PCR analysis of A. phagocytophilum p44 DNA from the cells represented in panel E. –, no inhibitor. (G) Quantitative PCR analysis of A. phagocytophilum p44 DNA from uninfected cells and A. phagocytophilum-infected cells. Cells were treated with either cathepsin L inhibitor I (CTSL) or cathepsin B inhibitor (CTSB) and then infected with A. phagocytophilum. –, no inhibitor. This experiment was done in duplicate with similar results. Asterisks denote statistical significance.

We assessed the effect of inhibition on infection with A. phagocytophilum. Using Western blotting and PCR analysis of the immunodominant 44-kDa protein of A. phagocytophilum, we observed that the pretreatment of cells with cathepsin L inhibitor I and inhibitor III altered the levels of A. phagocytophilum bacteria (Fig. 4E and F). Inhibitor I was more effective toward both the protein and DNA concentrations and was used throughout the study. The inhibitory effect upon infection was unique to cathepsin L. The infection of cells preincubated with the inhibitor of cathepsin B was not significantly different from the infection of untreated cells (Fig. 4G). In contrast, the inhibition of cathepsin L resulted in a severe decrease (P < 0.05) of A. phagocytophilum DNA.

Cathepsin L inhibition does not affect receptor binding and signaling. The binding of A. phagocytophilum to the PSGL-1 receptor results in tyrosine phosphorylation (34). Due to the effect of cathepsin L inhibition on the levels of A. phagocytophilum bacteria, we investigated whether cathepsin L was altering the binding of A. phagocytophilum to the receptor and subsequent phosphorylation induced via receptor binding. Cells were pretreated with cathepsin L inhibitor I, incubated with the receptor-blocking antibody or an isotype control antibody, and then infected with A. phagocytophilum. The blocking of the receptor (PSGL-1) abolished phosphorylation and reduced the level of bound A. phagocytophilum bacteria (Fig. 5A). The inhibition of cathepsin L did not alter the binding of or the phosphorylation induced by A. phagocytophilum. After 4 h of infection, there was no detectable effect on A. phagocytophilum. At 24 and 72 h after infection, a reduction in A. phagocytophilum protein in the presence of the cathepsin L inhibitor was detected (Fig. 5B).

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FIG. 5. The inhibition of cathepsin L does not affect the initial binding of and infection with A. phagocytophilum. (A) Western blot of cells left untreated (–) or treated (+) with 10 µM cathepsin L (CTSL) inhibitor I and then infected with A. phagocytophilum (Ap +) in the presence (+) of the receptor-blocking antibody (PL1) or isotype control antibody (Ig). Cell extracts were prepared after 4 h of infection. The blot was probed with the antibodies to phosphorylated tyrosine (pY), A. phagocytophilum (p44), and actin as a control. –, absence of infection or blocking antibody. (B) Extracts from untreated cells (–) and cathepsin L inhibitor-treated (+) uninfected (Ap –) and A. phagocytophilum-infected (Ap +) cells were prepared either 24 or 72 h after infection. Blots were probed with antibodies to A. phagocytophilum (p44) and actin. (C) Quantitative PCR analysis of A. phagocytophilum p44 from uninfected [(–) Ap] and A. phagocytophilum-infected [(+) Ap] cells in the presence (+) or absence (–) of a cathepsin L inhibitor. (D) Quantitative PCR analysis of A. phagocytophilum p44 from uninfected and A. phagocytophilum-infected cells. Twenty-four hours after infection, cells were incubated with (+) or without (–) cathepsin L inhibitor I. The experiment was done in duplicate with similar results. Asterisks denote statistical significance, with P values of <0.05.

Since the effect of inhibition was observed after 24 h, we assessed whether cells previously infected with A. phagocytophilum were also susceptible to cathepsin L inhibition. Cells were first infected and then treated with the cathepsin L inhibitor. As described previously, a quantitative PCR analysis of A. phagocytophilum DNA showed that cells treated with the inhibitor prior to infection had a severe loss (P < 0.05) of A. phagocytophilum DNA (Fig. 5C). When cells were infected for 24 h and then treated with the inhibitor, a loss of A. phagocytophilum DNA was also detected (P < 0.05) (Fig. 5D).

siRNA targeting of cathepsin L alters A. phagocytophilum infection. We next used siRNA knockdown of cathepsin L to verify the effect on A. phagocytophilum infection. siRNA targeting of cathepsin L reduced the level of cathepsin L as detected by Western blotting (Fig. 6A). At 48 h postinfection, the level of A. phagocytophilum p44 protein in cells targeted for cathepsin L knockdown was reduced compared to, but not statistically different from, that in cells treated with control siRNA (Fig. 6A and B). Quantitative PCR showed that the pattern for p44 DNA was similar (Fig. 6D). At 72 h postinfection, increases in p44 protein in both the cathepsin L-targeted and control cells were detected (Fig. 6A and C). However, the level of protein in the cathepsin L-targeted cells was reduced compared to that in the control cells. Quantitative PCR analysis of the p44 DNA showed a threefold reduction (P < 0.05) in A. phagocytophilum DNA in cathepsin L-targeted cells compared to that in the control (Fig. 6D). This result suggests that although the cells may be initially infected to similar degrees, the propagation of A. phagocytophilum is severely altered in the absence of cathepsin L.

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FIG. 6. siRNA targeting of cathepsin L reduces A. phagocytophilum propagation. (A) Extracts from cells treated with nonspecific siRNA (NS) and siRNA specific for cathepsin L (CTSL) and then infected (+) with A. phagocytophilum (Ap) were probed for A. phagocytophilum p44 and cathepsin L. Actin was also detected as a control for loading. –, uninfected control cells. (B and C) Graphic presentation of p44 levels from the analysis presented in panel A. The relative p44 densities in control cells (NS) and siRNA-targeted cells (CTSL) at 48 h (B) and 72 h (C) postinfection with A. phagocytophilum are expressed as percentages of the actin densities. (D) Quantitative PCR analysis of A. phagocytophilum DNA after 48 and 72 h of infection. Infected cells targeted for cathepsin L knockdown (CTSL) or treated with nonspecific siRNA (NS) were assessed. These data are representative of data from at least three experiments with similar results. The asterisk denotes statistical significance between samples indicated with the dashed line.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
During A. phagocytophilum infection, the elevated binding of CDP to the promoter of the gp91^phox gene contributes to gene repression. We have presented data showing that this enhanced DNA binding ability is not unique to the gp91^phox gene. The general binding capacity of CDP was altered during infection, resulting in enhanced binding to the promoters of other CDP-regulated genes, such as the HNP and C/EBP genes. The enhanced binding of CDP correlated with the repression of basal and ATRA-induced gene expression. In the presence of A. phagocytophilum, CDP undergoes proteolytic processing, resulting in fragments of CDP. The 200-kDa CDP protein containing the cut repeats CR1, CR2, and CR3 and the homeodomain (HD) binding regions has displacement activity. Fragments of CDP containing CR2-CR3-HD and CR3-HD have stable binding activity (3, 25). The generation of these isoforms can occur in cases of active cell proliferation (11). This result implies that the cleavage products of CDP observed during infection with A. phagocytophilum may contribute to the A. phagocytophilum-induced gene repression.

Cathepsin L is the protease responsible for the cleavage of CDP into the 110- and 90-kDa isoforms (10, 11). We found that A. phagocytophilum elevated the activity of this enzyme, indicating that increased cathepsin L activity during A. phagocytophilum infection was responsible for the proteolytic cleavage of CDP and the subsequent gene repression. Basal cathepsin L activity was detected by flow cytometry and immunofluorescence microscopy; infection with A. phagocytophilum stimulated this activity. We also looked at the effect of A. phagocytophilum on cathepsin B, which has activity similar to that of cathepsin L. The activity of cathepsin B was not altered by A. phagocytophilum infection, demonstrating the specific influence of A. phagocytophilum.

We blocked the activity of cathepsin L to assess the effect on A. phagocytophilum infection. The chemical inhibition of cathepsin L was able to inhibit the A. phagocytophilum-induced activity of cathepsin L and reduce the degree of A. phagocytophilum infection. A. phagocytophilum infection was affected post-receptor binding in cells treated with the cathepsin L inhibitor. Cells were initially bound and infected with comparable levels of A. phagocytophilum. After 24 h, however, the level of A. phagocytophilum protein in inhibitor-treated cells showed a reduction. At this point, A. phagocytophilum DNA was significantly affected by the inhibition of cathepsin L. We verified this effect by using siRNA targeting cathepsin L and observed similar results. Upon cathepsin L targeting, the level of p44, while not dramatically altered in the early stage of infection, showed a reduction at the later stage of infection. This delayed effect may be explained by the incomplete knockdown of cathepsin L. In the cathepsin L-targeted cells, the gene expression was not completely knocked down, which may have allowed for some level of cathepsin L activity to facilitate infection. Compared to the chemical inhibition of cathepsin L, inhibition by siRNA targeting appeared to be delayed, with greater repression observed after prolonged infection. This finding suggests that the cells were initially infected to similar degrees. With continued infection, the proliferation of A. phagocytophilum was affected by the loss of cathepsin L.

A. phagocytophilum alters the expression of antimicrobial peptide HNP-1 and C/EBP, a transcription factor critical for myeloid differentiation and secondary granule gene expression (17). Defects in C/EBP result in a depressed respiratory burst and a lack of the primary granule defensin and secondary granule proteins such as lactoferrin, gelatinase, and collagenase (9, 18). Mice lacking C/EBP have impaired host defense and succumb to secondary infections (16, 37). Neutrophils from these mice show defects in bacterial uptake and phagocyte killing (16). Humans with specific granule disorder suffer from recurrent bacterial infections (6, 17). In vitro, the overexpression of CDP can inhibit the expression of HNP, C/EBP, lactoferrin, neutrophil collagenase, and neutrophil gelatinase and contribute to defects in neutrophil maturation (13, 15). In our work, we demonstrated that the DNA binding activity of CDP was augmented by infection with A. phagocytophilum. The elevated binding of CDP to the promoters of the HNP and C/EBP genes resulted in transcriptional repression. This finding implies that a similar phenomenon of global gene repression with possible secondary granule deficiency may also occur with A. phagocytophilum infection.

In summary, A. phagocytophilum induces cathepsin L activity, resulting in the proteolytic processing of CDP, the elevated binding of CDP, and the repression of several CDP-regulated genes. The inhibition of cathepsin L, either by a chemical inhibitor or by siRNA, altered A. phagocytophilum propagation, demonstrating the importance of this pathway in A. phagocytophilum infection. These data show that A. phagocytophilum has developed methods to globally alter neutrophil function in order to facilitate survival. As the therapeutic options for A. phagocytophilum infection are limited, particularly for individuals that cannot take tetracycline derivatives, the findings of these studies suggest potential cellular targets that may be effective for new therapeutics against this pathogen.

 
We thank Juan Anguita, Deborah Beck, Peter Gaines, and Nancy Berliner for their technical assistance and helpful discussion.

This work is supported by grants from the National Institutes of Health.

 
* Corresponding author. Mailing address: Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, S525A, 300 Cedar St., P.O. Box 208031, New Haven, CT 06520-8031. Phone: (203) 785-2453. Fax: (203) 785-7053. E-mail: erol.fikrig{at}yale.edu

Published ahead of print on 2 September 2008.

Editor: A. J. Bäumler

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Infection and Immunity, November 2008, p. 4905-4912, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00851-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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