Figure 1.
bbe31 expression profile throughout representative stages of the natural spirochete life cycle.
bbe31 is preferentially expressed in fed tick. It is induced from the 2nd day, expressed in all the tested tissues including gut, hemolymph and salivary glands, and showed the highest expression level in the fed gut. Both mean and SD were calculated from 2 independent experiments, with 3 mRNA samples each experiment. In vitro, B.burgdorferi N40 grown in BSK medium with a concentration of 1×107/ml; Larva, I.scapularis larva fed on N40-infected mouse till engorged; day4, larva 4-day after feeding; day 21; larva 21-day after feeding; day1 Gut, day2 Gut and day3 Gut, guts of N40-nymphs feeding on C3H mice for 24, 48 and 72 hours; SG, salivary glands; HL, hemolymph; Injection, mouse inoculated with 1*106 B.burgdorferi N40; day1, day2 and day3, mouse localized skin 24, 48 or 72 hours after B.burgdorferi N40 infection through needle inoculation; skin, heart, bladder and joint, mouse tissues collected 21 days after N40-infected nymphs feeding on C3H mice.
Figure 2.
Subcellular localization of BBE31.
(A) Protease digestion assay. Intact B. burgdorferi N40 cells were incubated with Protease K in the absence (-) or presence (+) of 0.05% Triton X-100. After digestion, cells were lysed and proteins were fractionated by SDS-PAGE. Immunoblots were developed with anti-BBE31 or anti-BB0365 (an inner membrane protein). 0, 20, 200: Protease K concentration is 0, 20 or 200 µg/ml. (B) Indirect immunofluorescence staining. Intact unfixed or methanol-fixed B. burgdorferi N40 were incubated with primary rabbit anti-BBE31 (right panels) or rabbit anti-BB0365 (left panels) antibodies, then incubated with the secondary antibodies Alexa 488-labelled goat anti-rabbit. Magnification, x63. Scale bar represents 20 µm.
Figure 3.
BBE31 antibodies block B.burgdorferi migration from I.scapularis gut to the salivary glands.
(A) B.burgdorferi burden in I.scapularis gut and salivary glands assayed by q-PCR. N40-infected nymphs fed on C3H mice, which were injected with normal rabbit serum (NRS), BBE31 antiserum (BBE31 Ab) or BBI16 antiserum (BBI16 Ab), for 66 hours. DNA was extracted from both gut and salivary glands for q-PCR analysis. Each dot represents 5 guts or pairs of salivary glands. Data were collected from 3 studies of tick-feedings. *: P<0.05. (B) B.burgdorferi burden in the I.scapularis gut and salivary glands assayed by confocal microscopy. Murine immunization and tick feeding are the same as that in part (A). The spirochetes were probed with FITC-labeled goat anti-borrelia antibodies (shown in green), the nucleolus were stained with TO-PRO-3 (shown in blue). Orthogonal views (xz and yz axes) revealing the distribution of spirochetes through the full thickness of the infected salivary glands are shown. The gut and salivary gland samples were examined with the magnification of 63x and 25x, respectively. Scale bar represents 20 µm.
Figure 4.
Influence of BBE31 antibodies on B.burgdorferi infectivity in mice.
(A) BBE31 antibodies protect mouse from infection by tick-transmitted B. burgdorferi. Six of N40-infected nymphs were placed on 1 mouse, which has been injected with normal rabbit serum (NRS), BBE31 antiserum (E31) or BBI16 antiserum (I16), allowing to feed till exhausted. Twenty-one days later, the B. burgdorferi burden in mouse skin, bladder, heart and joints tissues were measured by q-PCR. Each dot represents 1 mouse. *: p<0.05, **: p<0.01. (B) BBE31 antibodies can't protect mice from infection by needle-inoculated B. burgdorferi. Mice were injected with rabbit normal serum (NRS) or BBE31 antiserum (E31). Twenty-four hours later, 102 of cultured B.burgdorferi N40 were intraperitoneally inoculated into the mice. B.burgdorferi burden in murine tissues were measured 21 days after Borrelia inoculation. Each dot represents 1 mouse. (C) Mice protection by BBE31 antiserum determined by in vitro culture of skin, bladder, heart and joints. Mice tissues used for culture were the same as those described above (4A and 4B). 102 Bb inoculation and 103 Bb inoculation: 102 or 103 of B.burgdorferi N40 in BSK medium were needle-inoculated into each C3H mouse. NA: not assayed.
Figure 5.
F(ab)2 fragments of anti-BBE31 IgG interfere with B.burgdorferi migration from I.scapularis gut into hemolymph.
(A) Borreliacidal activity of BBE31 F(ab)2 fragments against B. burgdorferi. B.burgdorferi N40 were incubated in BSK medium in the absence (Control) or presence of F(ab)2 fragments prepared from normal rabbit serum [NRS F(ab)2] or BBE31 antiserum [BBE31 F(ab)2]. A borreliacidal serum from a patient with diagnosed Lyme Disease (B.burgdorferi antisera) was used as a positive control. The number of spirochetes was assessed by dark-field microscopy after 24 and 48 hours of incubation. Data are presented relative to controls without treatment. Data represent the number of spirochetes remaining viable after treatment (mean ± SD, n = 4). Difference between control and NRS F(ab)2- as well as BBE31 F(ab)2-treated samples were not statistically significant. (B) B.burgdorferi burden in I.scapularis hemolymph assayed by q-PCR. N40-infected nymphs fed on C3H mice, which had been injected with F(ab)2 fragments of either normal rabbit IgG (NRS-F(ab)2) or anti-BBE31 IgG (BBE31-F(ab)2), for 66 hours. DNA was extracted from the hemolymph for q-PCR analysis. Each dot represents hemolymph from 5 ticks. Data were collected from 2 times of tick-feedings. *: p<0.05. (C) B.burgdorferi burden in I.scapularis hemolymph assayed by confocal microscope. Tick feeding is the same as that described in part (A). The spirochetes in tick hemolymph were probed with FITC-labeled goat anti-borrelia antibodies (shown in green), the hemocyte nucleolus were stained with TO-PRO-3 (shown in blue). The FITC and TO-PRO-3 images were examined at ×25 magnifications and are presented as merged images. Scale bar represents 20 µm. (D) Spirochetes lacking bbe31 can survive in tick hemolymph and invade the salivary glands. B.burgdorferi wild-type strain MSK5 and the lp25-lacking isolate ML23 were grown in BSK medium, resuspended in PBS and microinjected into partially-fed clean nymphal hemocoel (clean nymphs fed on C3H mice for 48 hours before microinjection). Six hours later, both hemolymph (HL) and salivary glands (SG) were collected for B.burgdorferi quantification by q-PCR. Mean and SD were calculated from 5 mRNA samples and 5 ticks were grouped for 1 mRNA sample.
Figure 6.
Confirmation of the interaction between BBE31 and TRE31 in vitro.
(A) Nucleotide and predicted amino acid sequences of TRE31. The tre31 nucleotide sequence was deduced from the alignment of sequenced tick gut genes, which were expressed by yeast cells enriched through biotinylated BBE31, with several databases (BLAST search, GenBank and EMBL nonredundant databases). Two I.scapularis contigs IscW_ISCW017272 and IscW_ISCW017271 (arrows) were identified from the databases, which showed 100% and 98% nucleotide identity with tre31 N-terminal and C-terminal sequences, respectively. The predicted peptide sequences of both contigs are in-frame with each other and constitute a protein of 302 amino acids. Among the 16 sequenced tre31 copies expressed by enriched yeast cells, 4 different N-terminals were found (shading). The longest TRE31 copy is from amino acid 31 to 302. Protein sequence analysis through SignalP 3.0 revealed a signal peptide in the N-terminal (underlining) with the cleavage site between amino acid 23 (A) and 24 (K). (B) BBE31specifically interacts with yeast cells expressing TRE31. Plasmid containing tre31 or the empty vector pYD1 was introduced into yeast competent cell EYB100, respectively. Both kinds of yeast cells were grown in SDCAA, induced in SGCAA, enriched through biotinylated BBE31 or OspC, and enriched cells eluted from the MACS column for quantification. Protein interaction was assessed by the elution percentage. (C) tre31 expression in tick. tre31 expression in unfed clean nymphal gut (Unfed Clean), N40-infected unfed nymphal gut (Unfed Gut), guts and salivary glands of N40-nymphs fed on C3H mice for 66 hours (Fed Gut and Fed SG), was measure by q-RT-PCR. Mean and SD were calculated from 6 mRNA samples.
Figure 7.
tre31 dsRNA interfered with the migration of B.burgdorferi within I.scapularis during spirochete transmission from tick to the host.
(A) tre31 mRNA was dramatically reduced within tre31 dsRNA-treated tick guts. Equal volumes of tre31 dsRNA (tre31 ds) or Elute Buffer (Buffer) were delivered directly into the gut of N40-infected Ixodes nymphs via microinjection. Ticks were allowed to rest for 3 hours and feed on C3H mice for 66 hr. tre31 expression in tick gut was measured by q-RT-PCR. (**: p<0.01) (B) B.burgdoferi burden in the tick gut was not influenced by tre31 deficiency. (C) B.burgdoferi burden in tick salivary glands was decreased by tre31 deficiency (*: p<0.05). (D) B.burgdoferi burden in tick hemolymph was decreased by tre31 deficiency (*: p<0.05). Each dot represents 2 tick gut or salivary glands, or 4 hemolymph. Experiments were repeated two times.