Porin activity of Anaplasma phagocytophilum outer membrane fraction and purified P44

Abstract

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 beta-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 beta-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.

FIG. 1.

Optical density changes in liposomes reconstituted with A. phagocytophilum outer membrane proteins diluted in an isosmotic concentration of solutes of various sizes. The proteoliposomes reconstituted with 3 μg of A. phagocytophilum outer membrane proteins were diluted in isosmotic solutions of 33 mM stachyose (□), 33 mM arabinose (▪), 33 mM l-glutamine (▴), 33 mM glucose (•), and 33 mM sucrose (⧫). The y axis represents a range of A₄₀₀ values of 0.2. The traces were made after some vertical displacement so that they all started from the same initial point for easy comparison. Results shown are representative of three independent experiments.

FIG. 2.

Effects of different concentrations of stachyose on the diffusion rate of proteoliposomes reconstituted with A. phagocytophilum outer membrane proteins. A total of 10 μg (A) or 0.63 μg (B) of A. phagocytophilum outer membrane proteins were reconstituted into proteoliposomes. Proteoliposomes were diluted in 25 mM (□), 33 mM (▴), and 40 mM (•) stachyose solutions. The y axis represents a range of A₄₀₀ values of 0.25. The traces in each panel were made after some vertical displacement so that they all started from the same initial point for easy comparison. Results shown are representative of three independent experiments.

FIG. 3.

Diffusion rates of proteoliposomes reconstituted with different amounts of A. phagocytophilum outer membrane proteins. Different amounts of A. phagocytophilum outer membrane proteins (0 to 12 μg) were reconstituted into proteoliposomes. Proteoliposome suspensions were diluted in a 33 mM isosmotic arabinose or stachyose solution. The change in optical density at 400 nm (OD₄₀₀) was recorded, and the initial rates of OD₄₀₀ decrease in arabinose (•) and stachyose (○) were calculated using readings from between 10 and 20 s after the start of the reading. Results shown are representative of three independent experiments.

FIG. 4.

Effect of MAb 5C11 on diffusion rate of proteoliposomes reconstituted with outer membrane proteins. (A) Optical density changes in MAb 5C11-treated (▵) and IgG2b isotype control-treated (▪) outer membrane proteins (0.88 μg) reconstituted into proteoliposomes. The proteoliposome suspensions were diluted in an isosmotic 33 mM arabinose solution. The traces were made after some vertical displacement so that they all start from the same initial point for easy comparison. The y axis represents a range of A₄₀₀ values of 0.1. (B) Swelling rate of proteoliposomes reconstituted with MAb 5C11- or IgG2b isotype control-treated A. phagocytophilum outer membrane proteins (0.80 μg). The swelling rate was calculated using readings of the OD₄₀₀ decrease between 0 and 60 s after the start of reading. Data are presented as means and standard deviations for triplicate samples. *, significant difference from isotype control (P < 0.05). Results shown are representative of two independent experiments.

FIG. 5.

Heat modifiability of P44s. A. phagocytophilum outer membrane proteins were subjected to 12% SDS-PAGE, followed by either GelCode blue staining (A) or Western blotting using MAb 5C11 as the primary antibody (B). A. phagocytophilum outer membrane proteins in the SDS-PAGE sample buffer were treated at room temperature (lanes 1) or boiled for 5 min (lanes 2). Numbers to the left of each panel show molecular mass standards in kDa. Results shown are representative of three independent experiments.

FIG. 6.

Pore-forming activity of gel-purified P44. (A) Gel-eluted P44 proteins (0.62 μg/lane) and molecular size standards (M) were subjected to SDS-PAGE with 0.02% SDS (final concentration) in running buffer, followed by GelCode blue staining. Gel-purified P44 proteins were treated in SDS-PAGE sample buffer with 0.4% SDS (final concentration) at room temperature (lane 1) or treated in standard SDS sample buffer and boiled for 5 min (lane 2). Numbers to the left of the panel show the molecular mass standards in kDa. (B) Optical density changes in proteoliposomes reconstituted with gel-purified P44. A total of 2.5 μg of gel-purified P44s were reconstituted into proteoliposomes. The proteoliposome suspensions were diluted in isosmotic 33 mM arabinose (▴) and 33 mM l-glutamine (▪) solutions. The traces were made after some vertical displacement so that they all start from the same initial point for easy comparison. The y axis represents a range of A₄₀₀ values of 0.1. (C) Swelling rates of proteoliposomes reconstituted with purified P44s in the presence of arabinose and l-glutamine. A total of 2.5 μg of gel-purified P44s were reconstituted into proteoliposomes. The swelling rate was calculated using readings of the OD₄₀₀ decrease between 0 and 60 s after the start of reading. Data are presented as means and standard deviations of triplicate samples. *, significant difference from proteoliposomes without proteins in the presence of arabinose or l-glutamine (P < 0.05). Results shown are representative of three independent experiments.

FIG. 7.

Pore-forming activity of HPLC-purified P44s. (A) HPLC-purified P44s. The fractions containing P44s and the least contaminants were subjected to 12% SDS-PAGE followed by GelCode blue staining. The HPLC fraction showed a protein with a 36-kDa calculated molecular mass. The molecular mass of denatured P44 proteins was approximately 44 kDa. Numbers to the left of the panel show the molecular mass standards in kDa. (B) Optical density changes in proteoliposomes reconstituted with HPLC-purified P44s. A total of 1.2 μg of HPLC-purified P44s were reconstituted into proteoliposomes. The proteoliposome suspensions were diluted in isosmotic 33 mM stachyose (▪) and 33 mM arabinose (▴) solutions. The y axis represents a range of A₄₀₀ values of 0.2.

FIG. 8.

Secondary structure prediction for P44-18. (A) Membrane criterion profile for P44-18. The solid blue line shows normal β-strands. The dotted red line shows twisted β-strands. The x axis shows the amino acid number, starting from the N terminus of mature P44-18. Numbers in red at the top of each peak are predicted β-strands, from the N to the C terminus. (B) Secondary structure drawing based on results from panel A, showing individual amino acids. Amino acids in boxes are predicted to be in β-strands, and amino acids in circles are predicted to be in the extracellular loop or periplasmic turn. Amino acids with a yellow background are in the N-terminal conserved region, amino acids with a purple background are semiconserved amino acids at the border of the N-terminal conserved region and the central hypervariable region, amino acids with a green background are in the C-terminal conserved region, and amino acids without any background are in the central hypervariable region. Amino acids in bold red form the epitope recognized by MAb 5C11, amino acids in blue show the C-C region (35), and those in bold form the epitope recognized by MAb 3E65 within the hypervariable region.