Anaplasma phagocytophilum is an obligate intracellular bacterium that invades neutrophils to cause the emerging and potentially fatal infection, human granulocytic anaplasmosis. The host cell-derived vacuole in which A. phagocytophilum resides hijacks recycling endosomes, a process that is essential for the bacterium's survival. How this unique pathogen-occupied organelle commandeers endocytic sorting is poorly understood. Monoubiquitination has recently emerged as a posttranslational modification that regulates endocytic sorting, particularly the recycling endosome pathway. Monoubiquitin is itself a sorting signal, and proteins decorated with the moiety make extensive contacts with endocytic machinery. We discovered that the A. phagocytophilum vacuolar membrane accumulates monoubiquitin and that P100, an effector that localizes to the vacuolar membrane, carries three F-boxes. The F-box motif was first identified in eukaryotes and interacts with the SCF ubiquitin ligase complex, which catalyzes ubiquitination of proteins. Monoubiquitin colocalizes with GFP-P100 when it is ectopically expressed in eukaryotic cells and with endogenous P100 on the A. phagocytophilum vacuolar membrane. The P100 region that contains the F-box motif is exposed on the cytosolic face of the A. phagocytophilum vacuolar membrane and competitively inhibits bacterial growth when it is ectopically expressed in infected cells. P100 is a novel bacterial effector because, while many microbial F-box proteins exploit host cell polyubiquitination, P100 is the first example of an F-box effector that co-opts monoubiquitination. Our investigations will test the hypothesis that the P100 F-boxes recruit the SCF ubiquitin ligase complex to direct monoubiquitination of host proteins on the A. phagocytophilum vacuolar membrane and that doing so is important for bacterial growth. In doing so, we will decipher a newly discovered bacterial pathogenic mechanism and will advance knowledge of the strategies by which microbes co-opt ubiquitin pathways to thrive in diverse, even microbiocidal, host cells.

Public Health Relevance

Human granulocytic anaplasmosis (HGA) is an infection caused by a bacterium that hijacks host cell processes to reside in a specialized compartment. The proposed research will define how a bacterial protein intercepts a host cell process to form the protective niche that aids pathogen survival. Doing so will advance knowledge of how bacteria survive in host cells and may bolster strategies for treating or preventing HGA.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI105364-01A1
Application #
8637532
Study Section
Special Emphasis Panel (ZRG1-IDM-B (80))
Program Officer
Perdue, Samuel S
Project Start
2013-12-10
Project End
2015-11-30
Budget Start
2013-12-10
Budget End
2014-11-30
Support Year
1
Fiscal Year
2014
Total Cost
$205,875
Indirect Cost
$70,875
Name
Virginia Commonwealth University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298