Anaplasma phagocytophilum Dihydrolipoamide Dehydrogenase 1 Affects Host-Derived Immunopathology during Microbial Colonization

Abstract

f Anaplasma phagocytophilum is a tick-borne rickettsial pathogen that provokes an acute inflammatory response during mammalian infection. The illness caused by A. phagocytophilum, human granulocytic anaplasmosis, occurs irrespective of pathogen load and results instead from host-derived immunopathology. Thus, characterizing A. phagocytophilum genes that affect the inflammatory process is critical for understanding disease etiology. By using an A. phagocytophilum Himar1 transposon mutant library, we showed that a single transposon insertion into the A. phagocytophilum dihydrolipoamide dehydrogenase 1 gene (lpda1 [APH_0065]) affects inflammation during infection. A. phagocytophilum lacking lpda1 revealed enlargement of the spleen, increased splenic extramedullary hematopoiesis, and altered clinicopathological abnormalities during mammalian colonization. Furthermore, LPDA1-derived immunopathology was independent of neutrophil infection and correlated with enhanced reactive oxygen species from NADPH oxidase and nuclear factor (NF)-B signaling in macrophages. Taken together, these findings suggest the presence of different signaling pathways in neutrophils and macrophages during A. phagocytophilum invasion and highlight the importance of LPDA1 as an immunopathological molecule.

The Tick Salivary Protein Sialostatin L2 Inhibits Caspase-1-Mediated Inflammation during Anaplasma phagocytophilum Infection

“…A. phagocytophilum infection was monitored under gentamicin protection assay conditions, as previously described (25,39). By using confocal microscopy, we verified that A. phagocytophilum formed small microcolonies (morulae) inside cells (Fig.…”

“…We detected reactive oxygen species (ROS) by using the fluorescence probe 2=,7=-dichlorofluorescin diacetate (H2DCFDA) (Invitrogen), as described previously (25). Fluorescence in 96-well plates was recorded over time by use of a Spectra MAX Gemini EM microplate reader (Molecular Devices) using a fluorescein isothiocyanate (FITC) filter (excitation, 485 nm; and emission, 538 nm).…”

“…A. phagocytophilum strain HZ was grown in HL-60 cells (ATCC CCL-240). Cells were maintained in Iscove's modified Dulbecco's medium (IMDM) with L-glutamine, HEPES (Thermo Scientific), and 20% heat-inactivated fetal bovine serum (FBS) in 5% CO 2 and humidified air at 37°C (25). A. phagocytophilum organisms multiply as microcolonies (morulae) inside cells.…”

“…3A to C). Previously, we showed that A. phagocytophilum did not affect mitochondrial ROS in macrophages (25). Thus, we investigated whether NADPH ROS were involved in caspase-1 function in the A. phagocytophilum model.…”

“…Therefore, we used nox2 Ϫ/Ϫ mice, which do not produce ROS from NADPH oxidase (25). We measured A. phagocytophiluminduced ROS production in macrophages (25).…”