Interactions of Encephalitozoon cuniculi polar tube proteins

Abstract

Microsporidia are eukaryotic, obligate intracellular organisms defined by small spores that contain a single invasion organelle, the polar tube, which coils around the interior of the spore. When these parasites infect host cells, the polar tube is discharged from the anterior pole of the spore, pierces the cell, and transfers sporoplasm into the cytoplasm of the host. Three polar tube proteins (PTP1, PTP2, and PTP3) have been identified in this structure. The interactions of these proteins in the assembly and function of the polar tube are not known. This study was undertaken to examine the protein interactions of the Encephalitozoon cuniculi polar tube proteins (EcPTPs). Immunofluorescence and immunoelectron microscopy confirmed the colocalization of EcPTP1, EcPTP2, and EcPTP3 to the polar tube. Experiments using cross-linkers indicated that EcPTP1, EcPTP2, and EcPTP3 form a complex in the polar tube, which was confirmed by immunoprecipitation using EcPTP1 antiserum. Yeast two-hybrid analysis revealed that full-length EcPTP1, EcPTP2, and EcPTP3 interact with each other in vivo. Both the N and C termini of EcPTP1 were involved in these interactions, but the central region of this protein, which contains a repetitive motif, was not. Further studies of polar tube proteins and their structural interactions may help elucidate the formation of the polar tube during the invasion process.

FIG. 1.

Analysis of rPTP antisera. Top, immunoblot analysis. Protein lysates were prepared from E. cuniculi run on a 10% SDS-PAGE gel, transferred to nitrocellulose, and examined using anti-rPTP. Anti-rEcPTP1 (1:5,000 dilution), anti-rEcPTP2 (1:5,000 dilution), and anti-rEcPTP3 (1:1,000 dilution) were used. Bands corresponding to EcPTP1, EcPTP2, and EcPTP3 were seen in the corresponding immunoblots. No bands were seen in preimmune sera (data not shown). Bottom, immunoelectron microscopy. E. cuniculi spores (see Materials and Methods for fixation details). (A) Anti-rEcPTP1 (1:100 dilution); (B) anti-rEcPTP2 (1:100 dilution) (insert is an enlarged section of the image); and (C) anti-rECPTP3 (1:100 dilution). All of the antisera react with the polar tube (arrows). Bar length is 600 nm. Gold particles are 6 nm. (D) Representative negative control (preimmune serum at 1:100 dilution). No gold labeling is seen. EN, endospore; Ex, exospore; ANT, anterior portion of the polar filament; R, ribosomes; arrows, polar tubes. Bar length is 500 nm.

FIG. 2.

Immunofluorescent analysis of rPTP antibodies. Confocal images of extruded polar tubes of E. cuniculi that were initially incubated with one of the three polar tube antibodies, anti-rEcPTP1, anti-rEcPTP2, or anti-rEcPTP3. After their initial incubation with their respective antibody and tyramide signal-amplified fluorescent label, the polar tubes were dually labeled with a second polar tube antibody and a tyramide fluorescent label (see Materials and Methods). Control preimmune sera did not display any reactivity in this assay. (A, B, C) Images of an E. cuniculi polar tube incubated with anti-rEcPTP1 labeled with Alexa Fluor 488 (green) (A) and anti-rEcPTP3 labeled with Alexa Fluor 594 (red) (B). Panel C is the merged image. Note the presence of both red and green signals along the polar tube and areas of yellow signal where they overlap. Bar length for the group is 5 μm, as shown in panel A. (D, E, F) Images of an E. cuniculi polar tube incubated with anti-rEcPTP2 labeled with Alexa Fluor 488 (green) (D) and anti-rEcPTP3 labeled with Alexa Fluor 594 (red) (E). Panel F is the merged image. Note the presence of both red and green signals along the polar tube and areas of yellow signal where they overlap. Bar length for the group is 5 μm, as shown in panel D. (G, H, I) Images of an E. cuniculi polar tube incubated with anti-rEcPTP1 labeled with Alexa Fluor 488 (green) (G) and anti-rEcPTP2 labeled with Alexa Fluor 594 (red) (H). Panel I is the merged image. Note the presence of both red and green signals along the polar tube and areas of yellow signal where they overlap. Bar length for the group is 5 μm, as shown in panel G.

FIG. 3.

EcPTP1, EcPTP2, and EcPTP3 form a protein complex. (A) SDS-PAGE analysis. Coomassie blue-stained gel of protein samples made from E. cuniculi spores, cross-linked with 4 mM DTSSP, without (−) or with (+) treatment with 2-mercaptoethanol (25% vol/vol). Protein markers are in kDa. A large complex (arrow) at the top of the gel disappeared with mercaptoethanol treatment. (B) Immunoprecipitation analysis. Protein samples were extracted from E. cuniculi, cross-linked with DTSSP, and then immunoprecipitated with a control anti-mouse IgG (lane 2) or with anti-rEcPTP1 (lane 3). Lane 1 shows the protein markers (kDa standards). Immunoprecipitates were treated with gel sample buffer (containing reducing agents) and run on 12% SDS-PAGE, transferred to nitrocellulose, and probed with anti-rEcPTP1 (1:2,000), anti-rEcPTP2 (1:2,000), or anti-rEcPTP3 (1:2,000). Reactions were detected with an alkaline phosphatase-conjugated anti-mouse IgG (1:5,000) and detected with BCIP-NBT. Anti-rEcPTP1 was able to precipitate a complex which contained EcPTP1, EcPTP2, and EcPTP3. The control antiserum did not precipitate this complex.

FIG. 4.

Yeast two-hybrid analysis of PTP interactions. This illustrates the interactions of the pAD and pBD (bait and prey) constructs containing full-length EcPTP1, full-length EcPTP2, and full-length EcPTP3. The negative-control (pBDwt/pADplasminC) and positive-control (pBDwt/pADwt) reactions are provided. This demonstrates that all three EcPTPs can interact with themselves and each other, although the domains responsible for this interaction remain to be determined.

FIG. 5.

Yeast two-hybrid analysis of the interaction of EcPTP1 N-terminal and C-terminal domains. This illustrates the interactions of the pAD and pBD (bait and prey) constructs containing full-length EcPTP1 (EcPTP1-FL), N-terminal Ec-PTP1 (EcPTP1-NT), and C-terminal EcPTP1 (EcPTP1-CT). The negative-control (pBDwt/pADplasminC) and positive-control (pBDwt/pADwt) reactions are provided. This demonstrates that both the N- and C-terminal regions of EcPTP1 can interact with each other and themselves.

FIG. 6.

Yeast two-hybrid analysis of the interactions of the EcPTP1 N-terminal, C-terminal, and central-repeat domains of EcPTP2 and EcPTP3. (A) Interactions between EcPTP1-FL as the bait and EcPTP1-FL (1), EcPTP1-NT (2), the central region of EcPTP1 (EcPTP1-Cent) (3), or EcPTP1-CT (4) as the prey. (B) Interactions between EcPTP2-FL as the bait and EcPTP1-FL (1), EcPTP1-NT (2), EcPTP1-Cent (3), or EcPTP1-CT (4) as the prey. (C) Interactions between EcPTP3-FL as the bait and EcPTP1-FL (1), EcPTP1-NT (2), EcPTP1-Cent (3), or EcPTP1-CT (4) as the prey. A positive control (pBDwt/pADwt) (P) and a negative control (pBDwt/pADplasminC) (N) are shown in each group. This demonstrates that the central region of EcPTP1 does not contain any interacting domains. In addition, while EcPTP1-FL can interact with either EcPTP3-FL or EcPTP2-FL, neither the N-terminal nor the C-terminal domains are sufficient for this interaction to occur.