Tool development for Anaplasma phagocytophilum to understand determinants of infection

PROJECT SUMMARY
Rickettsiales are a diverse order of obligate, intracellular bacteria often transmitted by arthropods and pose a
substantial threat to public health. Rickettsial organisms manipulate a wide array of host cell processes to gain
entry and establish a replicative niche, while simultaneously avoiding host cell defenses. How rickettsial
pathogens mediate this complex network of interactions is not well understood. A central feature of all rickettsial
pathogens is the Type 4 Secretion System (T4SS), which secretes effector proteins into the cytosol and
manipulates biological processes in the host cell. However, few T4SS translocated effectors have been identified
or characterized from rickettsial organisms owing, in part, to the lack of genetic tools available among these
bacteria. Their obligate intracellular nature has historically hindered genetic manipulation of Rickettsiales.
Recently, we achieved targeted gene deletion in the rickettsial pathogen A. phagocytophilum using allelic
exchange by homologous recombination. In the R61 portion of this proposal we will leverage this technique to
add to the A. phagocytophilum genetic toolbox by developing 1) a T4SS effector translocation assay for use in
the organism of study, 2) conditional expression systems, and 3) luminescent reporters. Using these tools in the
R33 phase we will: 4) characterize the A. phagocytophilum T4SS effector repertoire, 5) evaluate the contributions
of essential A. phagocytophilum effectors to infection, and 6) monitor the expression kinetics of effectors in vivo.
The objectives of this proposal will expand the tractability of A. phagocytophilum and our understanding of the
host-pathogen interactions it navigates during infection through elucidation to the T4 effector repertoire, and an
understanding of how, when and where these genes are regulated. The impact of this proposal will extend to
other rickettsial organisms, as these approaches can be adapted for use in related intracellular pathogens.

Grant Number: 5R61AI179933-03
NIH Institute/Center: NIH
Principal Investigator: Kelly Brayton