Abstract
Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis (HGA) is an obligate intracellular Gram-negative bacterium with the genome size of 1.47 megabases. The intracellular life style and small size of genome suggest that A. phagocytophilum has to modulate a multitude of host cell physiological processes to facilitate its replication. One strategy employed by A. phagocytophilum is through its type IV secretion system (T4SS), which translocates bacterial effectors into target cells to disrupt normal cellular activities. In this study we developed a TEM-1 β-lactamase based protein translocation assay and applied this assay for identification of A. phagocytophilum T4SS effectors. An A. phagocytophilum hypothetical protein, APH0215 is identified as a T4SS effector protein and found interacting with trans-Golgi network in transfected cells. Hereby, this protein translocation assay developed in this study will facilitate the identification of A. phagocytophilum T4SS effectors and elucidation of HGA pathogenesis.
Highlights
Bacterial type IV secretion system (T4SS) is a translocation apparatus which transports macromolecules, such as DNA and protein substrates from bacteria to prokaryotic and eukaryotic cells, facilitating dissemination of mobile genetic elements, or dysregulating physiological processes of host cells, resulting in disease development[1]
Chinese hamster ovary K1 (CHO K1) cells incubated with SM10 λpir expressing TraG-VirD4 and TEM-1-Ats-1 have higher percent of blue cells than those incubated with SM10 λpir expressing TraG-VirD4 and TEM-1 (0.367 ± 0.012% vs. 0.040 ± 0.010%) (Fig. 2), suggesting that TEM-1-Ats-1 is transported into CHO K1 cells
A myriad of effector proteins was identified in intracellular bacteria, such as L. pneumophila, C. burnetii, and Brucella[31,36,37], a very limited number of T4SS substrates was revealed in A. phagocytophilum
Summary
Bacterial type IV secretion system (T4SS) is a translocation apparatus which transports macromolecules, such as DNA and protein substrates from bacteria to prokaryotic and eukaryotic cells, facilitating dissemination of mobile genetic elements, or dysregulating physiological processes of host cells, resulting in disease development[1]. Shared among bacterial P- and F-type conjugation systems, as well as Agrobacterium tumefaciens VirB/ VirD4 system, T4ASS transporter is composed of functionally distinct modules including pilus, outer membrane core complex (OMCC), inner membrane complex (IMC) and type IV coupling protein (T4CP)[1,2]. These modules are assembled with a conserved set of approximately 11 VirB proteins from VirB1 to VirB11, and VirD4 subunit, among of which VirD4 is a T4CP2. By application of this assay and immunofluorescence labeling, an A. phagocytophilum hypothetical protein, APH0215 is identified as a T4SS effector protein and found interacting with trans-Golgi network in transfected cells
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