Binding of Host Cell Surface Protein Disulfide Isomerase by Anaplasma phagocytophilum Asp14 Enables Pathogen Infection

Abstract

Diverse intracellular pathogens rely on eukaryotic cell surface disulfide reductases to invade host cells. Pharmacologic inhibition of these enzymes is cytotoxic, making it impractical for treatment. Identifying and mechanistically dissecting microbial proteins that co-opt surface reductases could reveal novel targets for disrupting this common infection strategy. Anaplasma phagocytophilum invades neutrophils by an incompletely defined mechanism to cause the potentially fatal disease granulocytic anaplasmosis. The bacterium’s adhesin, Asp14, contributes to invasion by virtue of its C terminus engaging an unknown receptor. Yeast-two hybrid analysis identified protein disulfide isomerase (PDI) as an Asp14 binding partner. Coimmunoprecipitation confirmed the interaction and validated it to be Asp14 C terminus dependent. PDI knockdown and antibody-mediated inhibition of PDI reductase activity impaired A. phagocytophilum infection of but not binding to host cells. Infection during PDI inhibition was rescued when the bacterial but not host cell surface disulfide bonds were chemically reduced with tris(2-carboxyethyl)phosphine-HCl (TCEP). TCEP also restored bacterial infectivity in the presence of an Asp14 C terminus blocking antibody that otherwise inhibits infection. A. phagocytophilum failed to productively infect myeloid-specific-PDI conditional-knockout mice, marking the first demonstration of in vivo microbial dependency on PDI for infection. Mutational analyses identified the Asp14 C-terminal residues that are critical for binding PDI. Thus, Asp14 binds and brings PDI proximal to A. phagocytophilum surface disulfide bonds that it reduces, which enables cellular and in vivo infection. IMPORTANCE Anaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis, an emerging potentially fatal disease and the second-most common tick-borne illness in the United States. Treatment options are limited, and no vaccine exists. Due to the bacterium’s obligatory intracellular lifestyle, A. phagocytophilum survival and pathogenesis are predicated on its ability to enter host cells. Understanding its invasion mechanism will yield new targets for preventing bacterial entry and, hence, disease. We report a novel entry pathway in which the A. phagocytophilum outer membrane protein Asp14 binds host cell surface protein disulfide isomerase via specific C-terminal residues to promote reduction of bacterial surface disulfide bonds, which is critical for cellular invasion and productive infection in vivo. Targeting the Asp14 C terminus could be used to prevent/treat granulocytic anaplasmosis. Our findings have broad implications, as a thematically similar approach could be applied to block infection by other intracellular microbes that exploit cell surface reductases.

An Ixodes scapularis Protein Disulfide Isomerase Contributes to Borrelia burgdorferi Colonization of the Vector

“…phagocytophilum infection of neutrophils. The study also showed that the reductase activity of PDI was critical to facilitate this infection (27)…”

“…Consistent with this hypothesis, in parasites such as Schistosoma mansoni and Leishmania major, secreted PDIs have been suggested to facilitate parasite-host interactions conducive to successful infection of the host (26). A recent study by Green et al (27) has shown that a membrane bound PDI on mammalian neutrophils binds an Anaplasma phagocytophilum surface protein Asp14 and facilitates A.…”

Ehrlichia chaffeensis EplA Interaction With Host Cell Protein Disulfide Isomerase Promotes Infection

“…EplA exhibits 28.2% identity and 66.9% similarity to Asp14 and 76.7% identity and 92.3% similarity to Ecaj_0636. EplA residues 95-104 and Ecaj_0636 amino acids 89-98 align with Asp14 residues 113-124 (Asp14 113−124 ) that constitute the adhesin's PDI binding domain (Green et al, 2020 ). Because of the importance of Asp14 to A. phagocytophilum infectivity (Kahlon et al, 2013 ; Green et al, 2020 ), the relevance of EplA to E. chaffeensis pathogenesis was examined.…”

“…inhibits Trx1 activity-dependent HIV cellular entry (Stantchev et al, 2012). Both antibodies also antagonize A. phagocytophilum infection (Green et al, 2020). The THP-1 cells were incubated with E. chaffeensis DC bacteria and examined for EcVs 24 h later.…”

“…It is enriched in the endoplasmic reticulum, but is also found in the nucleus, cytoplasm, and at the cell surface (Ali Khan and Mutus, 2014 ). PDI at the cell surface functions exclusively as a thiol reductase (Jiang et al, 1999 ; Zai et al, 1999 ; Gallina et al, 2002 ), and this activity is important for internalization into host cells by HIV, Dengue virus, Leishmania chagasi, Chlamydia trachomatis , and another Anaplasmataceae member, Anaplasma phagocytophilum (Barbouche et al, 2003 ; Ou and Silver, 2006 ; Abromaitis and Stephens, 2009 ; Santos et al, 2009 ; Reiser et al, 2012 ; Stantchev et al, 2012 ; Wan et al, 2012 ; Diwaker et al, 2015 ; Green et al, 2020 ). The A. phagocytophilum adhesin, Asp14 (14-kDa A. phagocytophilum surface protein) engages PDI on myeloid cell surfaces to bring the pathogen in sufficient proximity to the enzyme such that it reduces bacterial surface disulfide bridges as a critical step in infection (Green et al, 2020 ).…”