Epigenetic silencing of host cell defense genes enhances intracellular survival of the rickettsial pathogen Anaplasma phagocytophilum

Abstract

Intracellular bacteria have evolved mechanisms that promote survival within hostile host environments, often resulting in functional dysregulation and disease. Using the Anaplasma phagocytophilum-infected granulocyte model, we establish a link between host chromatin modifications, defense gene transcription and intracellular bacterial infection. Infection of THP-1 cells with A. phagocytophilum led to silencing of host defense gene expression. Histone deacetylase 1 (HDAC1) expression, activity and binding to the defense gene promoters significantly increased during infection, which resulted in decreased histone H3 acetylation in infected cells. HDAC1 overexpression enhanced infection, whereas pharmacologic and siRNA HDAC1 inhibition significantly decreased bacterial load. HDAC2 does not seem to be involved, since HDAC2 silencing by siRNA had no effect on A. phagocytophilum intracellular propagation. These data indicate that HDAC up-regulation and epigenetic silencing of host cell defense genes is required for A. phagocytophilum infection. Bacterial epigenetic regulation of host cell gene transcription could be a general mechanism that enhances intracellular pathogen survival while altering cell function and promoting disease.

Figure 1. Down-regulation of host defense genes with A. phagocytophilum infection of THP-1 cells.

RNA was extracted from infected and uninfected THP-1 cells 48 hours post-infection and expression of defense genes was quantitated by qRT-PCR. IL8 and FTH expression were used as up-regulation controls. Gene expression changes were expressed as transcription fold-change in infected cells with respect to uninfected cells. Numbers <1 denote down-regulation and >1 indicate up-regulation.

Figure 2. The pattern of histone post-translational modifications and HDAC1 binding in the defense gene promoters is affected by A. phagocytophilum infection of THP-1 cells.

Chromatin from infected and uninfected THP-1 cells was prepared 48 hours post-infection. The histone modification pattern and HDAC1 binding at the defense gene promoters was analyzed by ChIP using antibodies specific for (A) Ac-H3, (B) Me-H3 and (C) HDAC1. Immunoprecipitated DNA fragments were quantitated by qPCR using primers specific for each promoter region. Changes were expressed as the ratio of immunoprecipitated chromatin target from infected to uninfected cells enriched with respect to input chromatin.

Figure 3. HDAC expression and activity is increased by A. phagocytophilum infection.

(A) RNA from infected and uninfected cells was extracted and HDAC1 and HDAC2 expression was quantitated by qRT-PCR. Transcription fold-change with respect to uninfected cells was calculated. (B) HDAC1 and HDAC2 in infected and uninfected THP-1 cells were detected by immunoblotting. Band intensity was determined by densitometric analysis and protein expression level changes with respect to the initial expression level in uninfected cells were calculated. Samples were normalized for β-actin content. The example shown is representative of 3 separate experiments with similar results. (C) HDAC activity in nuclear extracts of infected and uninfected cells at 48 hpi was determined using a fluorescent assay kit. Nuclear extract from HeLa cells was used as control.

Figure 4. HDAC inhibition impairs the ability of A. phagocytophilum to propagate intracellularly.

THP-1 cells were infected with cell-free A. phagocytophilum and incubated with or without the HDAC inhibitors TSA and sodium butyrate. Twenty-four hours post infection, cells were collected and A. phagocytophilum infection level was determined by qPCR. The first point represents infection level in the absence of HDAC inhibitor. Cells were incubated with (A) TSA or (B) sodium butyrate. Infection levels were normalized for β-actin gene content to account for differences in the number of viable cells. (C) Cell-free A. phagocytophilum preincubated with 100 nM TSA for 2 hours prior to infection was used to infect THP-1 cells as described. A. phagocytophilum load was determined by qPCR 24 hours later.

Figure 5. HDAC1 silences defense gene expression and facilitates A. phagocytophilum infection of THP-1 cells.

(A) THP-1 cells were transfected with HDAC1 or HDAC2 siRNA and were infected 24 hours after transfection with cell-free A. phagocytophilum. The A. phagocytophilum load was determined 24 h after infection by qPCR and normalized to infection levels in non-transfected cells. (B) THP-1 cells were transfected with HDAC1 siRNA for silencing or with pHDAC1-FLAG plasmid to overexpress HDAC1 and were infected 24 hours after transfection with cell-free GFP-expressing HGE-1 strain of A. phagocytophilum. The percent of infected cells was determined 24 h after infection by flow cytometry and normalized to infection levels in untreated cells. (C) Expression of defense genes by THP-1 cells infected with A. phagocytophilum–infected or transfected with pHDAC1-FLAG plasmid or HDAC1 siRNA was determined by qRT-PCR. Gene expression was normalized to housekeeping genes and to the level of expression of untreated, uninfected THP-1 cells.