Global proteomic analysis of two tick-borne emerging zoonotic agents: anaplasma phagocytophilum and ehrlichia chaffeensis

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.

Keywords: Anaplasma phagocytophilum; Ehrlichia chaffeensis; human granulocytic anaplasmosis; human leukocytes; human monocytic ehrlichiosis; proteomic analysis.

Figure 1

Anaplasma phagocytophilum P44 expression maps as detected by proteomic analysis. All genes encoding P44 outer membrane proteins were plotted on the first circle. The bar heights on the second circle represented the number of P44-matching peptides detected, with higher bars indicating greater numbers of matching unique peptides. The third circle showed P44 proteins that had matched peptides but no unique peptide matches, and the fourth circle showed P44 proteins that had no peptide matches by proteomic analysis (APH_1122/P44-75, APH_1124/P44-C, and APH_1399/P44-C). The origin of the A. phagocytophilum genome was marked as (1), and the expression locus p44-18ES was highlighted by the green box. Color codes in circles 1, 2, and 4: Red, full-length p44s; Blue, truncated p44s; Green: N-terminal p44 fragments; Brown: C-terminal p44 fragments; Gray: Degenerated P44 fragments.

Figure A1

Mass tag accumulation trends. Two AMT tag databases were constructed for positively-identified peptides, with numbers of total peptides accumulated versus total numbers of MS/MS runs were plotted. More than 126,000 peptides each were identified in protein samples from A. phagocytophilum or E. chaffeensis, using both purified bacteria from infected cells or whole lysates from infected cells.

Figure A2

Expression of overlapping ORFs in A. phagocytophilum. (A) Expression of complete overlapping protein-coding ORFs in A. phagocytophilum. (B) Expression of partial overlapping protein-coding ORFs in A. phagocytophilum. Peptide sequences detected by proteomics analysis were highlighted by bold and red fonts in amino acid sequences for these overlapping ORFs.

Figure A2

Expression of overlapping ORFs in A. phagocytophilum. (A) Expression of complete overlapping protein-coding ORFs in A. phagocytophilum. (B) Expression of partial overlapping protein-coding ORFs in A. phagocytophilum. Peptide sequences detected by proteomics analysis were highlighted by bold and red fonts in amino acid sequences for these overlapping ORFs.

Figure A2

Expression of overlapping ORFs in A. phagocytophilum. (A) Expression of complete overlapping protein-coding ORFs in A. phagocytophilum. (B) Expression of partial overlapping protein-coding ORFs in A. phagocytophilum. Peptide sequences detected by proteomics analysis were highlighted by bold and red fonts in amino acid sequences for these overlapping ORFs.

Figure A2

Expression of overlapping ORFs in A. phagocytophilum. (A) Expression of complete overlapping protein-coding ORFs in A. phagocytophilum. (B) Expression of partial overlapping protein-coding ORFs in A. phagocytophilum. Peptide sequences detected by proteomics analysis were highlighted by bold and red fonts in amino acid sequences for these overlapping ORFs.

Figure A3

Expression of overlapping ORFs in E. chaffeensis. (A) Expression of complete overlapping protein-coding ORFs in E. chaffeensis. (B) Expression of overlapping RNA and protein-coding ORFs in E. chaffeensis. (C) Expression of partial overlapping proteincoding ORFs in E. chaffeensis. Peptide sequences detected by proteomics analysis were highlighted by bold and red fonts in amino acid sequences for these overlapping ORFs.

Figure A3

Expression of overlapping ORFs in E. chaffeensis. (A) Expression of complete overlapping protein-coding ORFs in E. chaffeensis. (B) Expression of overlapping RNA and protein-coding ORFs in E. chaffeensis. (C) Expression of partial overlapping proteincoding ORFs in E. chaffeensis. Peptide sequences detected by proteomics analysis were highlighted by bold and red fonts in amino acid sequences for these overlapping ORFs.

Figure A3

Expression of overlapping ORFs in E. chaffeensis. (A) Expression of complete overlapping protein-coding ORFs in E. chaffeensis. (B) Expression of overlapping RNA and protein-coding ORFs in E. chaffeensis. (C) Expression of partial overlapping proteincoding ORFs in E. chaffeensis. Peptide sequences detected by proteomics analysis were highlighted by bold and red fonts in amino acid sequences for these overlapping ORFs.