Dataset Information

Enhancement of Cell Adhesion by Anaplasma phagocytophilum Nucleolin-Interacting Protein AFAP.

ABSTRACT: Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis (HGA), is an obligate intracellular Gram-negative bacterium. During infection, A. phagocytophilum enhances the adhesion of neutrophils to the infected endothelial cells. However, the bacterial factors contributing to this phenomenon remain unknown. In this study, we characterized a type IV secretion system substrate of A. phagocytophilum, AFAP (an actin filament-associated Anaplasma phagocytophilum protein) and found that it dynamically changed its pattern and subcellular location in cells and enhanced cell adhesion. Tandem affinity purification combined with mass spectrometry identified host nucleolin as an AFAP-interacting protein. Further study showed the disruption of nucleolin by RNA interference, and the treatment of a nucleolin-binding DNA aptamer AS1411 attenuated AFAP-mediated cell adhesion, indicating that AFAP enhanced cell adhesion in a nucleolin-dependent manner. The characterization of cell adhesion-enhancing AFAP and the identification of host nucleolin as its interaction partner may help understand the mechanism underlying A. phagocytophilum-promoting cell adhesion, facilitating the elucidation of HGA pathogenesis.

SUBMITTER: Tang H

PROVIDER: S-EPMC9965380 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Enhancement of Cell Adhesion by Anaplasma phagocytophilum Nucleolin-Interacting Protein AFAP.

Tang Hongcheng H Zhang Daxiu D Jiang Fenfen F Yu Lifeng L Tang Hui H Zhu Jiafeng J Wu Shuyan S Niu Hua H

Journal of personalized medicine 20230208 2

Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis (HGA), is an obligate intracellular Gram-negative bacterium. During infection, A. phagocytophilum enhances the adhesion of neutrophils to the infected endothelial cells. However, the bacterial factors contributing to this phenomenon remain unknown. In this study, we characterized a type IV secretion system substrate of A. phagocytophilum, AFAP (an actin filament-associated Anaplasma phagocy ...[more]

PMID: 36836536

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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.

Dataset Information

Enhancement of Cell Adhesion by Anaplasma phagocytophilum Nucleolin-Interacting Protein AFAP.

Publications

Enhancement of Cell Adhesion by <i>Anaplasma phagocytophilum</i> Nucleolin-Interacting Protein AFAP.

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