Anaplasma phagocytophilum Ats-1 Is Imported into Host Cell Mitochondria and Interferes with Apoptosis Induction

Abstract

Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, infects human neutrophils and inhibits mitochondria-mediated apoptosis. Bacterial factors involved in this process are unknown. In the present study, we screened a genomic DNA library of A. phagocytophilum for effectors of the type IV secretion system by a bacterial two-hybrid system, using A. phagocytophilum VirD4 as bait. A hypothetical protein was identified as a putative effector, hereby named Anaplasma translocated substrate 1 (Ats-1). Using triple immunofluorescence labeling and Western blot analysis of infected cells, including human neutrophils, we determined that Ats-1 is abundantly expressed by A. phagocytophilum, translocated across the inclusion membrane, localized in the host cell mitochondria, and cleaved. Ectopically expressed Ats-1 targeted mitochondria in an N-terminal 17 residue-dependent manner, localized in matrix or at the inner membrane, and was cleaved as native protein, which required residues 55–57. In vitro-translated Ats-1 was imported in a receptor-dependent manner into isolated mitochondria. Ats-1 inhibited etoposide-induced cytochrome c release from mitochondria, PARP cleavage, and apoptosis in mammalian cells, as well as Bax-induced yeast apoptosis. Ats-1(55–57) had significantly reduced anti-apoptotic activity. Bax redistribution was inhibited in both etoposide-induced and Bax-induced apoptosis by Ats-1. Taken together, Ats-1 is the first example of a bacterial protein that traverses five membranes and prevents apoptosis at the mitochondria. Anaplasma phagocytophilum is the pathogen that causes human granulocytic anaplasmosis, an emerging infectious disease. As an obligate intracellular organism, this bacterium cannot reproduce outside of eukaryotic cells due to the loss of many genes that are present in free-living bacteria. Paradoxically, it specifically infects short-lived white blood cells that play critical roles in anti-microbial defense, by subverting a number of host innate immune responses including programmed cell death (apoptosis). A. phagocytophilum factors that are involved in this process are largely unknown. In this study, we first searched A. phagocytophilum proteins that are secreted by its specialized secretion system into eukaryotic cells. We found a protein of unknown function, here named Ats-1, which is abundantly produced by A. phagocytophilum and traverses five membranes to enter the mitochondria of human cells. Our further study showed that Ats-1 reduces the sensitivity of mitochondria to respond to apoptosis-inducing factors, leading to the inhibition of host cell apoptosis. Thus, present findings identified a bacterial protein that allows infected white blood cells to live longer to support bacterial growth. The absence of similarity of the sequence or the mode of action to any other known cell death suppressor suggests that Ats-1 defines a previously undescribed class of anti-apoptotic protein. This protein and the mechanism thereof may provide insight regarding a new therapeutic target for treatment of human granulocytic anaplasmosis.