The prenylation inhibitor manumycin A reduces the viability of Anaplasma phagocytophilum

Abstract

Anaplasma phagocytophilum is an obligately intracellular bacterium and is the causative agent of human granulocytic anaplasmosis (HGA), an emerging and major tick-borne disease in the USA and other parts of the world. This study showed that the prenylation inhibitor manumycin A effectively blocked A. phagocytophilum infection in host cells (HL-60 or RF/6A cells). A. phagocytophilum infection activated extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase in host cells, and manumycin A treatment reduced ERK activation in A. phagocytophilum-infected host cells. As ERK activation is required for A. phagocytophilum infection, we examined whether manumycin A inhibited the bacteria directly or through host ERK signalling. Treatment of A. phagocytophilum alone with manumycin A significantly reduced the bacterial infectivity of host cells and bacterial viability in the absence of host cells, whereas pre-treatment of host cells did not inhibit bacterial infection in host cells. The inhibitory effect of manumycin A on A. phagocytophilum infection in host cells was achieved even at a concentration 100 times lower than that required for effective inhibition of mammalian cell signalling. These results suggested that manumycin A directly inactivates the bacterium, resulting in reduced infection and ERK1/2 activation. Thus, the manumycin group of drugs may have a therapeutic potential for HGA.

Fig. 1.

Manumycin A blocks A. phagocytophilum infection in host cells. Manumycin A (0, 0.01, 0.1 and 1 µM in 1 % DMSO) was added to HL-60 cells (a, b) or RF/6A cells (c) at 0 h p.i. (a, c) or 1 day p.i. (b, d). The number of bacteria was determined at 2 days p.i. (a, b) or 3 days p.i. (c). Data are expressed as means±sd (n = 3) and are representative of three independent experiments with similar results. *, P<0.05; **, P<0.01 (unpaired two-tailed t-tests). (d) At 2 days p.i., infected cells treated with 1 µM manumycin A and vehicle-control DMSO were harvested and observed by light microscopy following Diff-Quik staining. Arrows indicate A. phagocytophilum inclusions. Note the much smaller inclusions, which contained fewer bacteria, in the manumycin A-treated cell compared with the control DMSO-treated cell. Bar, 5 µm.

Fig. 2.

A. phagocytophilum infection activates ERK and manumycin A inhibits both bacterial infection and ERK activation in infected HL-60 cells. Western blot analysis was performed with antibodies specific to phosphorylated ERK (p-ERK), total ERK1/2 and P44, the A. phagocytophilum major surface protein. Uninfected and A. phagocytophilum-infected HL-60 cells (Ap) with or without manumycin A treatment were collected at 3 days p.i. α-Tubulin was used as a protein loading control to normalize each sample. The values under the bands show the ratios of band intensities of P44, p-ERK1/2 and total ERK (T-ERK) to α-tubulin, respectively. Data are representative of three independent experiments.

Fig. 3.

Pre-treatment of A. phagocytophilum alone with manumycin A inhibits bacterial infection in host cells. (a) Host-cell-free A. phagocytophilum was pre-treated with serially diluted manumycin A for 15 min at 37 °C in SPK buffer and, after washing, was inoculated onto non-treated HL-60 cells. **, P<0.01 (unpaired two-tailed t-test). (b) Host HL-60 cells were pre-treated with serially diluted manumycin A in RPMI for 15 min at 37 °C. After washing, they were inoculated with non-treated A. phagocytophilum. The number of bacteria was determined at 2 days p.i. (c) Host-cell-free A. phagocytophilum was pre-treated with 1 µM manumycin A or 1 % DMSO (control) for different time periods (2, 10 and 30 min) at 37 °C in SPK buffer. A time of 0 min indicates cells that were not exposed to manumycin A or DMSO. After washing, the cells were inoculated onto HL-60 cells. The number of bacteria was determined at 2 days p.i. Data are expressed as means±sd (n = 3) and are representative of three independent experiments with similar results. *, P<0.05; **, P<0.01 (unpaired two-tailed t-tests). P values were calculated for manumycin A-treated versus vehicle control for each time point.

Fig. 4.

Manumycin A reduces isolated A. phagocytophilum viability. Host-cell-free A. phagocytophilum was pre-treated with 1 µM manumycin A or 1 % DMSO for 15 min in SPK buffer, followed by staining for 15 min with the LIVE/DEAD BacLight Bacterial Viability kit, and was observed by fluorescence microscopy. A green signal indicates viable bacteria and a red signal indicates dead bacteria. Data are representative of three independent experiments with similar results. Bars, 5 µm.