Abstract
Anaplasma phagocytophilum and Ehrlichia chaffeensis are obligatory intracellular α-proteobacteria that infect human leukocytes and cause potentially fatal emerging zoonoses. In the present study, we determined global protein expression profiles of these bacteria cultured in the human promyelocytic leukemia cell line, HL-60. Mass spectrometric (MS) analyses identified a total of 1,212 A. phagocytophilum and 1,021 E. chaffeensis proteins, representing 89.3 and 92.3% of the predicted bacterial proteomes, respectively. Nearly all bacterial proteins (≥99%) with known functions were expressed, whereas only approximately 80% of “hypothetical” proteins were detected in infected human cells. Quantitative MS/MS analyses indicated that highly expressed proteins in both bacteria included chaperones, enzymes involved in biosynthesis and metabolism, and outer membrane proteins, such as A. phagocytophilum P44 and E. chaffeensis P28/OMP-1. Among 113 A. phagocytophilum p44 paralogous genes, 110 of them were expressed and 88 of them were encoded by pseudogenes. In addition, bacterial infection of HL-60 cells up-regulated the expression of human proteins involved mostly in cytoskeleton components, vesicular trafficking, cell signaling, and energy metabolism, but down-regulated some pattern recognition receptors involved in innate immunity. Our proteomics data represent a comprehensive analysis of A. phagocytophilum and E. chaffeensis proteomes, and provide a quantitative view of human host protein expression profiles regulated by bacterial infection. The availability of these proteomic data will provide new insights into biology and pathogenesis of these obligatory intracellular pathogens.
Highlights
Anaplasma phagocytophilum and Ehrlichia chaffeensis are small, pleomorphic gram-negative bacteria that belong to the family Anaplasmataceae in the order Rickettsiales, the class α-proteobacteria (Dumler et al, 2001; Rikihisa, 2010b)
Overview of A. phagocytophilum and E. chaffeensis proteins identified by proteomics In order to identify and quantitate the comprehensive protein expression profiles of A. phagocytophilum and E. chaffeensis, 18 A. phagocytophilum protein samples and 14 E. chaffeensis samples were prepared from purified host cell-free bacteria and infected HL-60 cells using three different protein extraction protocols as described; each contained approximately 1 mg of peptides after tryptic digestion and column clean-up
The database for E. chaffeensis contained 49 datasets from purified bacteria and 192 datasets associated with E. chaffeensis-infected host cells
Summary
Anaplasma phagocytophilum and Ehrlichia chaffeensis are small (ca. 0.4 by 1.5 μm), pleomorphic gram-negative bacteria that belong to the family Anaplasmataceae in the order Rickettsiales, the class α-proteobacteria (Dumler et al, 2001; Rikihisa, 2010b). Anaplasma phagocytophilum and Ehrlichia chaffeensis are small The infection of humans by A. phagocytophilum and E. chaffeensis causes human granulocytic anaplasmosis [HGA, first reported in 1994, formerly known as human granulocytic ehrlichiosis (HGE)] and human monocytic ehrlichiosis (HME, first reported in 1987), respectively (Maeda et al, 1987; Chen et al, 1994). HGA and HME are similar systemic febrile diseases characterized by fever, headache, myalgia, anorexia, and chills, and are frequently accompanied by leukopenia, thrombocytopenia, anemia, and elevations in serum hepatic aminotransferases (Paddock and Childs, 2003; Bakken and Dumler, 2008; Thomas et al, 2009). Neurological signs are more frequently reported with HME than HGA (Paddock and Childs, 2003). As important life-threatening tick-borne emerging zoonoses, HGA and HME were designated as nationally notifiable diseases by US Centers for Disease Control and Prevention in 1998
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