Human granulocytic anaplasmosis (HGA), an emerging tick-borne zoonosis is caused by a gram-negative, obligatory intracellular bacterium, Anaplasma phagocytophilum. A. phagocytophilum has the remarkable ability to inhibit the spontaneous apoptosis of neutrophils, block the production of reactive oxygen intermediates, and replicate in membrane-bound inclusions in the cytoplasm of neutrophils. However, the A. phagocytophilum inclusions have not been fully characterized, and bacterial factors contributing to these phenomena remain unknown. In this study, we studied several molecular aspects of A. phagocytophilum pathogenesis.
(1) Characterization of A. phagocytophilum replicative inclusions. We demonstrated that A. phagocytophilum replicative inclusions had the characteristic of early autophagosomes, as shown by the presence of autophagosome markers, LC3 and double lipid bilayer membrane in the A. phagocytophilum inclusions. Furthermore our data suggested that autophagy enhanced A. phagocytophilum replication instead of inhibiting its growth.
(2) Investigation of the expression of genes encoding type IV secretion system apparatus in A. phagocytophilum. We found the expression of virB6 and virB9 was up-regulated during the bacterial growth in human neutrophils. Furthermore, differential VirB9 expression was shown to associate with the binding of A. phagocytophilum to neutrophils, and prevention of internalized bacteria from being delivered to lysosomes.
(3) Identification of a novel substrate of type IV secretion system in A. phagocytophilum. A novel substrate, Ats-1 was identified by bacterial two-hybrid screening. We demonstrated that Ats-1 targeted mitochondria of host cells, and inhibited cell apoptosis.
(4) Development of a reporter system in A. phagocytophilum to detect substrates of type IV secretion system. We made some progress to achieve the goal by constructing plasmid harboring reporter-substrate fusion gene.