Anaplasma phagocytophilum increases cathepsin L activity, thereby globally influencing neutrophil function

Abstract

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/EBPepsilon--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.

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 (αCDP). 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 gp91^phox, HNP, and C/EBPɛ 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 gp91^phox, HNP, and C/EBPɛ 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 gp91^phox (C and F), HNP (D and G), and C/EBPɛ (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/EBPɛ (E and H) normalized to the expression (copies) of actin is reported. Due to the variability in results among experiments, the level of gp91^phox expression (C and F) was determined by normalization to the level of actin expression followed by comparison to the level of gp91^phox expression in the untreated control cells, and the results are reported as the increase in gp91^phox expression (n-fold) over that in the untreated controls. Asterisks denote statistical significance, with P values of <0.05.

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 (αCDP). 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.

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.

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.

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.

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.