Effect of increasing concentrations of sodium acetate on levels of borrelial proteins under laboratory growth conditions.

<p>Equivalent numbers of spirochetes from <i>B. burgdorferi</i> B31-A3 propagated in BSK-II medium with 6% NRS at laboratory growth conditions (pH 7.6/32°C) with increasing concentrations of supplemental NaOAc (from 0 mM –90 mM) were resolved by SDS-12.5%PAGE. The gels were stained with Coomassie blue (A) or the separated proteins were electrotransfered onto PVDF membranes. Immunoblots (B-G) were probed with anti-serum against the antigens listed to the right of the blots. Blots were developed using the Enhanced Chemiluminescence system. Numbers to the left of the panels indicate the molecular mass standards in kilodaltons proximate to each of the antigens. Increased levels of protein expression were seen with increasing concentrations of NaOAc for all proteins of the mevalonate pathway (B); OppA5 (C); gene regulators RpoS, CsrA_Bb, and BosR (D); AckA (E); pathogenesis-related proteins DbpA, BBK32, and OspC (F); NapA (G). Decreased levels of protein expression were seen with Pta (E). Acetate levels had no effect on the other proteins tested.</p

Kinetic values for HMG-CoA reductases of different species^a.

a<p><i>K</i>_m and <i>V</i>_max for <i>B. burgdorferi</i> were derived from the experiments described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038171#s2" target="_blank">Materials and Methods</a> and are the average of three independent replicates. <i>K</i>_m is in units of µM and <i>V</i>_max is in units of µmol NADPH oxidized minute⁻¹ (mg protein)⁻¹_.<i>L. monocytogenes</i> (<i>Listeria monocytogenes</i>), <i>S. aureus</i> (<i>Staphylococcus aureus</i>), <i>P.</i><i>mevalonii</i> (<i>Pseudomonas mevalonii</i>), <i>H. volcanii</i> (<i>Haloferax volcanii</i>).</p

Statins inhibit the enzyme activity of <i>B. burgdorferi</i> HMGR.

<p>(A) Lysates purified HMGR as indicated above the respective lanes were resolved by SDS-12.5%PAGE. Gels were stained with Coomassie blue. Recombinant HMGR of <i>B. burgdorferi</i> purified by FPLC. Lane 1, non-induced <i>E. coli</i> containing pMAL-p2X/<i>hmgr</i>. Lane 2, <i>E. coli</i> containing pMAL-p2X/<i>hmgr</i> induced with 1 mM IPTG. Lane 3, Purified MBP-HMGR fusion protein. (B) Recombinant <i>B. burgdorferi</i> HMGR activity was measures as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038171#s2" target="_blank">Materials and Methods</a>. Statins were added to the enzyme reaction at a concentration of 250 µM. The velocity of the control reaction was calculated as 1.41±0.10 µmol NADPH oxidized minute⁻¹ (mg protein)⁻¹, set as 100% maximal activity in the presence of the statin diluent, DMSO, alone_. Simvastatin lowered HMGR activity to 48.2% of maximal (velocity of 0.87±0.08 µmol NADPH oxidized minute⁻¹ (mg protein)⁻¹), while lovastatin reduced HMGR activity to 61.4% of maximal activity (velocity of 0.68±0.07 µmol NADPH oxidized minute⁻¹ (mg protein)⁻¹). Data shown are the average of three independent assays. Asterisks indicate samples whose values are statistically significantly different between control and treated conditions (**, <i>P</i><0.01).</p

Protective immunity conferred by mucosal vaccination.

<p>Animals (n = 6 per group) were vaccinated either i.t. (panel A) or orally (panel B) with either 10⁵ CFU U112, 10⁷ CFU U112Δ<i>iglB</i>, or mock-vaccinated with PBS and rested for 30 days. Rats were challenged i.t. with 1.25×10⁴ CFU SCHU S4 (approx. 25 LD₅₀) and monitored daily for morbidity and mortality. Kaplan-Meier survival analysis revealed protection following immunization with U112 by both i.t. and oral routes were significant (<i>p</i><0.001 and <i>p</i><0.005, respectively) over mock immunization. Protection conferred by U112Δ<i>iglB</i> vaccination was significant over mock control (<i>p</i><0.01 (i.t.) and <i>p</i><0.005 (oral)). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047639#s3" target="_blank">Results</a> are representative of two independent experiments.</p

<i>Borrelia burgdorferi</i> strains used in this study.

<p><i>Borrelia burgdorferi</i> strains used in this study.</p

Sequence comparison of <i>B. burgdorferi</i> mevalonate pathway proteins to characterized homologs in other bacterial species.

<p>Sequence comparison of <i>B. burgdorferi</i> mevalonate pathway proteins to characterized homologs in other bacterial species.</p

Respiratory antibody response following mucosal vaccination.

<p>Animals (n = 3 per group) were vaccinated i.t. (top panel) or orally (bottom panel) with either 10⁵ CFU U112, 10⁷ CFU U112Δ<i>iglB</i>, or mock vaccinated with PBS and rested for 28 days. Animals were euthanized to obtain bronchioalveolar lavage fluid (BALF) which was assayed by ELISA. Significant differences were observed between U112 and U112Δ<i>iglB</i> i.t. vaccinated and respective mock groups for total Ig and IgG2a (*<i>p</i><0.05, **<i>p</i><0.01). Animals primed with U112Δ<i>iglB</i> exhibited responses which were comparable to or higher than that observed for U112-primed rats. Horizontal lines represent mean of each group. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047639#s3" target="_blank">Results</a> are representative of two separate experiments.</p

Sensitivity of the HMGR overexpression strain, TR1, to statins.

<p>Equivalent numbers of spirochetes from <i>B. burgdorferi</i> ML23 or TR1 propagated in BSK-II medium with 6% NRS at laboratory growth conditions (pH 7.6/32°C) treated with 5 mM IPTG for 16 hrs to induce HMGR expression as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038171#s2" target="_blank">Materials and Methods</a> and were resolved by SDS-12.5%PAGE. The gels were stained with Coomassie blue (A) or the separated proteins were electrotransfered onto PVDF membranes. Immunoblot (B) was probed with anti-serum against HMGR. Blots were developed using the Enhanced Chemiluminescence system. Numbers to the left of the panels indicate the molecular mass standards in kilodaltons. (C) Spirochetes were analyzed for sensitivity to increasing concentrations of stains. Data shown are the average of three independent assays; bars indicate the standard deviation. Data were subjected to the unpaired Student's <i>t</i> test implemented in Excel software. Asterisks indicate samples whose values are statistically significantly different between control and overexpression strains (**, <i>P</i><0.01).</p

Levels of proteins of the MP in <i>B. burgdorferi</i>

<p>. Equivalent number of spirochetes from <i>B. burgdorferi</i> strain B31-A3 propagated in BSK-II medium with 6% NRS under conditions that either mimicked the unfed-tick (pH 7.6/23°C; Lane 1) or fed-tick (pH 6.8/37°C; Lane 2) to a density of 5 × 10⁷ spirochetes/ml were resolved by SDS-12.5%PAGE. Gels were stained with Coomassie blue (A) or separated proteins were electrotransfered onto PVDF membranes. (B) Immunoblots were incubated with mouse serum against purified HMGR, MvaD, Pmk, Mvk and P66 respectively. Blots were developed using the Enhanced Chemiluminescence system. Numbers to the left of the panels indicate the molecular mass standards in kilodaltons proximate to each of the antigens. Higher levels of HMGR expression were seen under fed-tick conditions when compared to unfed-tick conditions while the other three proteins showed higher levels of expression under unfed-tick conditions when compared to fed-tick conditions.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p