<i>B. recurrentis</i> spirochetemia in SCID BEIGE and SCID mice.

<p><i>B. recurrentis</i> established a relatively low-grade (compared to <i>B. duttonii</i>, see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000522#pntd-0000522-g001" target="_blank">Figure 1C, D</a>), stable infection in SCID BEIGE and SCID mice, but not in BALB/c or NUDE animals. Strain A17 (panel A) caused a higher (p<0.01) level of spirochetemia than did strain A11 (panel B). No difference in <i>B. recurrentis</i> spirochetemia was observed between SCID BEIGE and SCID mice. Data are presented as medians with 25^th and 75^th percentiles.</p

Splenomegaly in infected mice.

<p>Although expansion of B- and T-cells is the major cause of splenomegaly in wild-type animals, SCID BEIGE mice infected with <i>B. recurrentis</i> A17 displayed significant (p<0.01) splenomegaly,which indicates a T-, B-, and NK-cell-independent immune response.</p

<i>B. duttonii</i> spirochetemia in mice.

<p>BALB/c (A), NUDE (B), SCID (C), and SCID BEIGE (D) mice. <i>B. duttonii</i> established infection in all four mouse strains. Spirochetemia was significantly (p = 0.02) higher in B-cell-deficient mice (C, D). No differences were observed between wild-type and T-cell-deficient mice (A vs. B) or between SCID mice and NK-cell-deficient SCID BEIGE mice (C vs. D). Data are presented as medians with 25^th and 75^th percentiles. Notice that the scales of the y-axes in this figure differ from those in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000522#pntd-0000522-g002" target="_blank">Figures 2</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000522#pntd-0000522-g004" target="_blank">4</a>.</p

Effect of clodronate-induced macrophage depletion on spirochetemia.

<p>Mice were injected with clodronate liposomes in PBS to deplete macrophages (and other phagocytic cells) in the blood and in organs in contact with the blood (e.g., the spleen and liver). PBS was used as a negative control. The injections (indicated by black triangles) were given every fifth day, starting one day before infection. A. BALB/c mice infected with <i>B. duttonii</i> developed very high spirochetemia, and all except one individual (dotted line) died at day 6 p.i. B. <i>B. duttonii</i>-infected SCID BEIGE mice treated with clodronate developed uncontrolled spirochetemia, and they all died before day 8 p.i. Control SCID BEIGE and BALB/c mice injected with PBS had much lower spirochetemia compared to their macrophage-depleted counterparts. C. SCID BEIGE mice treated with clodronate and infected with <i>B. recurrentis</i> A17 developed high but not lethal spirochetemia. The maximum spirochetemia in any of the control SCID BEIGE and BALB/c mice injected with PBS was 7.3×10⁶/ml. Even between the two peaks shown for the <i>B. recurrentis</i>-infected, clodronate-treated mice, the median spirochetemia was never below 8×10⁶/ml. <i>B. recurrentis</i> was unable to establish detectable spirochetemia in macrophage-depleted BALB/c mice.</p

Dependence of the single-channel conductance of P66 on the molecular mass (A) and the hydrodynamic radius (B) of the nonelectrolytes.

<p>G_(+NE)/G_(−NE) is the ratio of the mean single-channel channel conductance in the presence of NEs (taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078272#pone-0078272-t001" target="_blank">table 1</a>) to that in the absence of NEs (11.0 nS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078272#pone.0078272-BarcenaUribarri1" target="_blank">[1]</a>). Molecular masses and hydrodynamic radii of the nonelectrolytes were taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078272#pone-0078272-t001" target="_blank">table 1</a>. The bars indicate absolute errors.</p

PEG 400-induced blockage of P66 on the single-channel level.

<p>Small amounts of highly diluted P66 (1∶1,000 in 1% Genapol) was added to both sides of a diphytanoyl PC membrane. After reconstitution of one single 11 nS P66 unit, 90 mM PEG 400 was added to both sides of the membrane. The P66 conductance was blocked stepwise exhibiting subconductance steps of approximately 1.5 nS; temperature = 20°C; applied voltage = 20 mV.</p

Power density spectrum of PEG 600-induced current noise of 61 P66 channels.

<p>Trace 1 shows the control, the aqueous phase contained 1- and maltohexaose-induced current noises resulted in similar power density spectra (data not shown).</p

Titration of the P66-induced membrane conductance with PEG 600 (A) and maltohexaose (B).

<p>The membrane was formed with PC/<i>n</i>-decane. The aqueous phase contained ∼100 ng ml⁻¹ P66, 1 M KCl and respective nonelectrolytes in the concentration as indicated; temperature = 20°C; applied voltage = 20 mV.</p

Blue native (BN) PAGE, SDS PAGE and WB analysis of the P66 complex.

<p>The complete outer membrane fraction of <i>B. burgdorferi</i> B31 was applied to a 4–16% BN PAGE. A 460 kDa band that reacted to the P66 antibody was extracted from the gel and loaded again in a 4–16% BN PAGE (BN/BN PAGE). This gel was stained with silver nitrate (SN) and subjected to a western blot (WB) against P66 (A). The 460 kDa band was extracted from the gel using detergents and resolved again under denaturing conditions in glycine and tricine SDS PAGE (B). A whole lane from the BN/BN PAGE containing the P66 complex was resolved in a second dimension SDS PAGE (C). The elution of the 460 kDa band from the BN/BN PAGE was tested in planar bilayers for pore-forming activity. Step-like increases in the conductance of the membrane were observed after adding the sample to the salt solution (D, left panel) and at least 100 insertional events were summarized in a histogram (D, right panel).</p

Transmission electron micrograph showing immunogold labeled DipA in the outer membrane of the <i>B. burgdorferi</i> Osp-less mutant B313.

<p>Ultrathin cryosections were prepared from the <i>B. burgdorferi</i> Osp-less mutant B313 at −110°C, embedded in gelatin. The immungold particles were visualized by sequential incubation of the fixed cells by the polyclonal rabbit antibodies detecting (A) DipA or (B) pre-serum and the anti-rabbit antibodies GAR10.</p