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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.

SUBMITTER: Green RS

PROVIDER: S-EPMC6989111 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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

Green Ryan S RS Naimi Waheeda A WA Oliver Lee D LD O'Bier Nathaniel N Cho Jaehyung J Conrad Daniel H DH Martin Rebecca K RK Marconi Richard T RT Carlyon Jason A JA

mBio 20200128 1

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. <i>Anaplasma phagocytophilum</i> invades neutrophils by an incompletely defined mechanism to cause the potentially fatal disease gran ...[more]

PMID: 31992623

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Project description:BackgroundThe zoonotic intracellular alpha-proteobacterium Anaplasma phagocytophilum is a tick-transmitted pathogen. The associations between vertebrate reservoirs and vectors are described as wide-ranging, and it was previously shown that the pathogenicity of A. phagocytophilum differs depending on the combination of pathogen variant and infected host species. This leads to the question of whether there are variations in particular gene loci associated with different virulence. Therefore, this study aims at clarifying existing host-variant combinations and detecting possible reservoir hosts. To understand these interactions, a complex toolset for molecular epidemiology, phylogeny and network theory was applied.MethodsSequences of up to four gene loci (msp4, msp2, groEL and 16S rRNA) were evaluated for different isolates from variable host species, including, for example, dogs, cattle and deer. Variant typing was conducted for each gene locus individually, and combinations of different gene loci were analysed to gain more detailed information about the genetic plasticity of A. phagocytophilum. Results were displayed as minimum spanning nets and correlation nets.ResultsThe highest diversity of variants for all gene loci was observed in roe deer. In cattle, a reduced number of variants for 16S rRNA [only 16S-20(W) and 16S-22(Y)] but multiple variants of msp4 and groEL were found. For dogs, two msp4 variants [m4-20 and m4-2(B/C)] were found to be linked to different variants of the other three gene loci, creating two main combinations of gene loci variants. Cattle are placed centrally in the minimum spanning net analyses, indicating a crucial role in the transmission cycles by possibly bridging the vector-wildlife cycle to infections of humans and domestic animals. The minimum spanning nets confirmed previously described epidemiological cycles of the bacterium in Europe, showing separation of variants originating from wildlife animals only and a set of variants shared by wild and domestic animals.ConclusionsIn this comprehensive study of 1280 sequences, we found a high number of gene variants only occurring in specific hosts. Additionally, different hosts show unique but also shared variant combinations. The use of our four gene loci expand the knowledge of host-pathogen interactions and may be a starting point to predict future spread and infection risks of A. phagocytophilum in Europe.

Project description:SUMOylation, the covalent attachment of a member of the small ubiquitin-like modifier (SUMO) family of proteins to lysines in target substrates, is an essential post-translational modification in eukaryotes. Microbial manipulation of SUMOylation recently emerged as a key virulence strategy for viruses and facultative intracellular bacteria, the latter of which have only been shown to deploy effectors that negatively regulate SUMOylation. Here, we demonstrate that the obligate intracellular bacterium, Anaplasma phagocytophilum, utilizes an effector, AmpA (A. phagocytophilum post-translationally modified protein A) that becomes SUMOylated in host cells and this is important for the pathogen's survival. We previously discovered that AmpA (formerly APH1387) localizes to the A. phagocytophilum-occupied vacuolar membrane (AVM). Algorithmic prediction analyses denoted AmpA as a candidate for SUMOylation. We verified this phenomenon using a SUMO affinity matrix to precipitate both native AmpA and ectopically expressed green fluorescent protein (GFP)-tagged AmpA. SUMOylation of AmpA was lysine dependent, as SUMO affinity beads failed to precipitate a GFP-AmpA protein when its lysine residues were substituted with arginine. Ectopically expressed and endogenous AmpA were poly-SUMOylated, which was consistent with the observation that AmpA colocalizes with SUMO2/3 at the AVM. Only late during the infection cycle did AmpA colocalize with SUMO1, which terminally caps poly-SUMO2/3 chains. AmpA was also detected in the cytosol of infected host cells, further supporting its secretion and likely participation in interactions that aid pathogen survival. Indeed, whereas siRNA-mediated knockdown of Ubc9 - a necessary enzyme for SUMOylation - slightly bolstered A. phagocytophilum infection, pharmacologically inhibiting SUMOylation in infected cells significantly reduced the bacterial load. Ectopically expressed GFP-AmpA served as a competitive agonist against native AmpA in infected cells, while lysine-deficient GFP-AmpA was less effective, implying that modification of AmpA lysines is important for infection. Collectively, these data show that AmpA becomes directly SUMOylated during infection, representing a novel tactic for A. phagocytophilum survival.

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

Publications

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

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