Date Published: November 6, 2015
Publisher: Public Library of Science
Author(s): Adela S. Oliva Chávez, James W. Fairman, Roderick F. Felsheim, Curtis M. Nelson, Michael J. Herron, LeeAnn Higgins, Nicole Y. Burkhardt, Jonathan D. Oliver, Todd W. Markowski, Timothy J. Kurtti, Thomas E. Edwards, Ulrike G. Munderloh, Eric P. Skaar.
Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of A. phagocytophilum resulted in an insertion within the coding region of an o-methyltransferase (omt) family 3 gene. In wild-type bacteria, expression of omt was up-regulated during binding to tick cells (ISE6) at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4), but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT) were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT) was methylated in APH_0406. In vitro methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the apo (PDB_ID:4OA8), the S-adenosine homocystein-bound (PDB_ID:4OA5), the SAH-Mn2+ bound (PDB_ID:4PCA), and SAM- Mn2+ bound (PDB_ID:4PCL) X-ray crystal structures of the enzyme. Here, we characterized a mutation in A. phagocytophilum that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary mutations.
Anaplasma phagocytophilum is an obligately intracellular bacterium classified in the order Rickettsiales, and is the causative agent of Human Granulocytic Anaplasmosis (HGA) . HGA is characterized by high fevers, rigors, generalized myalgias, and severe headache. It is a potentially life-threatening disease, with 36% of patients diagnosed with HGA requiring hospitalization, 7% needing urgent care, and mortality of ~1% . The incidence of HGA has been increasing steadily, from 348 identified cases in 2000 when it first became reportable to the CDC, to 1761 cases in 2010  and 2,782 reported cases in 2013 . Similar trends are evident in other countries in Europe and Asia [reviewed in ]. In addition, A. phagocytophilum infects domestic animals such as dogs, cats, and horses, as well as wild mammals from deer and wolves to various rodents .
Genetic manipulation of A. phagocytophilum and other members of the Anaplasmataceae is difficult due to the intracellular nature of these organisms, and currently relies on random mutagenesis to study the role of specific genes during pathogenesis in the mammal and development in the tick [18,37,43]. Targeted mutagenesis in the related organism, Ehrlichia chaffeensis, proved ultimately unsuccessful as the transformants obtained were not able to persist in vitro for more than six days . The recent success of targeted mutagenesis in Rickettsia rickettsii resulting in the disruption of a major surface protein gene (ompA)  presumed to be a virulence factor without producing a detectable defect provides impetus to develop this method for other Rickettsiales, and serves as a reminder that gene function ultimately must be confirmed by mutational analysis. In this manuscript, we report the effects of the mutation of a specific gene of A. phagocytophilum that we suspect abolished its ability to infect tick cells. However, due to the lack of a complementation system in the Anaplasmataceae, we cannot completely rule out that this change in phenotype was due to secondary mutations. Nevertheless, our conclusions are supported by the effect of the methylation inhibitor AdOx, which mimicked the mutation at a concentration of 30 nM (Fig 3). Previously, Chen et al.  described an A. phagocytophilum mutant with an insertion in the dihydrolipoamide dehydrogenase 1 (lpda1) gene at the APH_0065 locus, which altered the inflammatory response during infection of mice by increasing the production of reactive oxygen species , but had no effect on in vitro growth. The mutant (ΔOMT) described here was selected in the human cell line HL-60 in which it replicated in a manner comparable to wild-type bacteria (Fig 2). The mutant had an insertion in aph_0584 encoding an o-methyltransferase (OMT) family 3 (GI: 88598384; E.C. 22.214.171.124). The inability of ΔOMT to replicate within tick cells highlighted the distinct mechanisms used by A. phagocytophilum for colonization of mammal and tick hosts.