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Journal of Bacteriology, March 2007, p. 1998-2006, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01548-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Porin Activity of Anaplasma phagocytophilum Outer Membrane Fraction and Purified P44{triangledown}

Haibin Huang, Xueqi Wang, Takane Kikuchi, Yumi Kumagai, and Yasuko Rikihisa*

Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210

Received 4 October 2006/ Accepted 8 December 2006

Anaplasma phagocytophilum, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, has significantly less coding capacity for biosynthesis and central intermediary metabolism than do free-living bacteria. Thus, A. phagocytophilum needs to usurp and acquire various compounds from its host. Here we demonstrate that the isolated outer membrane of A. phagocytophilum has porin activity, as measured by a liposome swelling assay. The activity allows the diffusion of L-glutamine, the monosaccharides arabinose and glucose, the disaccharide sucrose, and even the tetrasaccharide stachyose, and this diffusion could be inhibited with an anti-P44 monoclonal antibody. P44s are the most abundant outer membrane proteins and neutralizing targets of A. phagocytophilum. The P44 protein demonstrates characteristics consistent with porins of gram-negative bacteria, including detergent solubility, heat modifiability, a predicted structure of amphipathic and antiparallel ß-strands, an abundance of polar residues, and a C-terminal phenylalanine. We purified native P44s under two different nondenaturing conditions. When reconstituted into proteoliposomes, both purified P44s exhibited porin activity. P44s are encoded by approximately 100 p44 paralogs and go through extensive antigenic variation. The 16-transmembrane-domain ß-strands consist of conserved P44 N- and C-terminal regions. By looping out the hypervariable region, the porin structure is conserved among diverse P44 proteins yet enables antigenic variation for immunoevasion. The tricarboxylic acid (TCA) cycle of A. phagocytophilum is incomplete and requires the exogenous acquisition of L-glutamine or L-glutamate for function. Efficient diffusion of L-glutamine across the outer membrane suggests that the porin feeds the Anaplasma TCA cycle and that the relatively large pore size provides Anaplasma with the necessary metabolic intermediates from the host cytoplasm.

* Corresponding author. Mailing address: Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210. Phone: (614) 292-5661. Fax: (614) 292-6473. E-mail: rikihisa.1{at}osu.edu.

{triangledown} Published ahead of print on 15 December 2006.

Journal of Bacteriology, March 2007, p. 1998-2006, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01548-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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