Autophagy regulates Wolbachia populations across diverse symbiotic associations - PubMed (original) (raw)

Autophagy regulates Wolbachia populations across diverse symbiotic associations

Denis Voronin et al. Proc Natl Acad Sci U S A. 2012.

Abstract

Wolbachia are widespread and abundant intracellular symbionts of arthropods and filarial nematodes. Their symbiotic relationships encompass obligate mutualism, commensalism, parasitism, and pathogenicity. A consequence of these diverse associations is that Wolbachia encounter a wide range of host cells and intracellular immune defense mechanisms of invertebrates, which they must evade to maintain their populations and spread to new hosts. Here we show that autophagy, a conserved intracellular defense mechanism and regulator of cell homeostasis, is a major immune recognition and regulatory process that determines the size of Wolbachia populations. The regulation of Wolbachia populations by autophagy occurs across all distinct symbiotic relationships and can be manipulated either chemically or genetically to modulate the Wolbachia population load. The recognition and activation of host autophagy is particularly apparent in rapidly replicating strains of Wolbachia found in somatic tissues of Drosophila and filarial nematodes. In filarial nematodes, which host a mutualistic association with Wolbachia, the use of antibiotics such as doxycycline to eliminate Wolbachia has emerged as a promising approach to their treatment and control. Here we show that the activation of host nematode autophagy reduces bacterial loads to the same magnitude as antibiotic therapy; thus we identify a bactericidal mode of action targeting Wolbachia that can be exploited for the development of chemotherapeutic agents against onchocerciasis, lymphatic filariasis, and heartworm.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Association of ATG8a expression and Wolbachia in the filarial nematode B. malayi. (A_–_F) ATG8a (green in B, D, and F) colocalized with Wolbachia clusters (small red spots in A and C; large red structures are nematode nuclei) throughout the lateral chord cytoplasm of adult female B. malayi (A_–_D) and is absent from naturally _Wolbachia_-free A. viteae (E and F). (Scale bars: 50 μm. (G) Western blot (composite image) of the ATG8a protein in B. malayi (BM) and A. viteae (AV). MF-BM, B. malayi microfilaria; L3-BM, B. malayi L3; L4-BM, B. malayi L4; L4-BM-TET, B. malayi L4 treated with tetracycline in vivo for 14 d; Adult-BM, protein extract from untreated adult females; Adult-BM-TET protein extract from adult females treated with tetracycline in vivo for 6 wk. *ATG8a cytosolic form; **cleaved membrane-associated forms.

Fig. 2.

Fig. 2.

qPCR analysis of Wolbachia numbers in B. malayi after in vitro treatment with rapamycin (A_–_D), doxycycline (E), or siRNA (F). (A) Ratio of wsp/gst in microfilaria after 5 d of treatment with rapamycin (RAPA). (B) Number of wsp copies in L3 larvae treated for 5 d. (C) Number of wsp copies in L4 larvae treated for 5 d. (D) Number of wsp copies per worm in adult females treated with rapamycin or DMSO (control) for 7 d. (E) Number of wsp copies per worm in adult females treated with doxycycline (Doxy) and DMSO (control). (F) Number of wsp copies per worm in adult females treated with siRNA (bmTOR or bmATG1) or GFP as a control. *P < 0.001.

Fig. 3.

Fig. 3.

Morphological effects on B. malayi treated with rapamycin. Micrographs of hypodermal chord cells (A and B), developing embryos (C and D), and stretched microfilaria (E and F) in the uterus of adult females treated with rapamycin and control. A, B, D, and F show rapamycin-treated samples; C and E show control samples. The arrow in B indicates the fusion of the lysosome and bacteria. B, bacteria; Bi, degenerated bacteria; L, lysosomes; N, nuclei. (Scale bars: 1 μm in A and B; 15 μm in C_–_F.)

Fig. 4.

Fig. 4.

Ultrastructural localization of ATG8a protein in B. malayi and Wolbachia on the bacterial vacuole (A_–_E), bacterial cell wall (B and D), and in the bacterial cell matrix (A, B, C, E). b, bacteria; m, mitochondria. (Scale bars: 1 μm.)

Fig. 5.

Fig. 5.

Regulation of autophagy controls ATG8a expression and Wolbachia load in A. albopictus C6/36 cells. (A_–_F) Detection of ATG8a (green) in C6/36 (wAlbB) cells (A_–_C) and uninfected C6/36 cells (D_–_F) during the treatment. (A and D) Control (DMSO-treated) cells. (B and E) Rapamycin-induced cells display up-regulated ATG8a signals. (C and F) 3-MA–treated cells show suppressed expression of ATG8a. (Scale bars: 5 μm). (G and H) qPCR analysis of Wolbachia (WSP) and host actin gene copies in mosquito cells after treatment. (G) Ratio of wsp:actin in the C6/36 (wAlbB) cells treated with rapamycin, 3-MA, or DMSO (control). (H) Ratio of wsp:actin in the C6/36 (wAlbB) cells exposed to starvation (Star-Med) and treated with autophagy inhibitors Wortmannin (WM) or 3-MA. *P < 0.001.

Fig. 6.

Fig. 6.

Autophagy activation controls Wolbachia populations in Drosophila. (A) Effects of siRNA treatment on _w_MelPop populations in Drosophila cells (PC15). Ratio of wsp:RP49 in PC15 (_w_MelPop) cells. (B) Reduction of the wsp:RP49 ratio in D. melanogaster (w1118) naturally infected with _w_MelPop and treated with rapamycin (RAPA).

Fig. 7.

Fig. 7.

qPCR analysis of Wolbachia number in B. malayi after in vivo treatment with rapamycin (RAPA) or spermidine (SPN) and in controls. Reductions of Wolbachia were seen in worms treated with inducers of autophagy. White bars indicate wsp:gst ratio in L4 larvae (treated for 14 d), black bars indicate wsp:gst ratio in adult females treated for 35 d.

Fig. P1.

Fig. P1.

Autophagy regulates Wolbachia populations. (A) Autophagy membrane protein ATG8a (green) is associated with Wolbachia (small red dots) in the hypodermal chord cells of the filarial nematode B. malayi. (Large red structures are nematode nuclei,) (B) Natural populations of Wolbachia in diverse symbioses and hosts are regulated by autophagy. Chemical (3-methyladenine, 3-MA) or genetic (siRNA atg1) suppression of autophagy increases Wolbachia loads, whereas activation of autophagy by rapamycin or siRNA tor decreases bacterial load in host cells.

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