Absence of \u3ci\u3esodA\u3c/i\u3e Increases the Levels of Oxidation of Key Metabolic Determinants of \u3ci\u3eBorrelia burgdorferi\u3c/i\u3e

Borrelia burgdorferi, the causative agent of Lyme disease, alters its gene expression in response to environmental signals unique to its tick vector or vertebrate hosts. B. burgdorferi carries one superoxide dismutase gene (sodA) capable of controlling intracellular superoxide levels. Previously, sodA was shown to be essential for infection of B. burgdorferi in the C3H/HeN model of Lyme disease. We employed two-dimensional electrophoresis (2-DE) and immunoblot analysis with antibodies specific to carbonylated proteins to identify targets that were differentially oxidized in the soluble fractions of the sodA mutant compared to its isogenic parental control strain following treatment with an endogenous superoxide generator, methyl viologen (MV, paraquat). HPLC-ESI-MS/MS analysis of oxidized proteins revealed that several proteins of the glycolytic pathway (BB0057, BB0020, BB0348) exhibited increased carbonylation in the sodA mutant treated with MV. Levels of ATP and NAD/NADH were reduced in the sodA mutant compared with the parental strain following treatment with MV and could be attributed to increased levels of oxidation of proteins of the glycolytic pathway. In addition, a chaperone, HtpG (BB0560), and outer surface protein A (OspA, BBA15) were also observed to be oxidized in the sodA mutant. Immunoblot analysis revealed reduced levels of Outer surface protein C (OspC), Decorin binding protein A (DbpA), fibronectin binding protein (BBK32), RpoS and BosR in the sodA mutant compared to the control strains. Viable sodA mutant spirochetes could not be recovered from both gp91/phox−⁄− and iNOS deficient mice while borrelial DNA was detected in multiple tissues samples from infected mice at significantly lower levels compared to the parental strain. Taken together, these observations indicate that the increased oxidation of select borrelial determinants and reduced levels of critical pathogenesis-associated lipoproteins contribute to the in vivo deficit of the sodA mutant in the mouse model of Lyme disease. This study, utilizing the sodA mutant, has provided insights into adaptive capabilities critical for survival of B. burgdorferi in its hosts

Implications of climate change on the distribution of the tick vector Ixodes scapularis and risk for Lyme disease in the Texas-Mexico transboundary region

BACKGROUND: Disease risk maps are important tools that help ascertain the likelihood of exposure to specific infectious agents. Understanding how climate change may affect the suitability of habitats for ticks will improve the accuracy of risk maps of tick-borne pathogen transmission in humans and domestic animal populations. Lyme disease (LD) is the most prevalent arthropod borne disease in the US and Europe. The bacterium Borrelia burgdorferi causes LD and it is transmitted to humans and other mammalian hosts through the bite of infected Ixodes ticks. LD risk maps in the transboundary region between the U.S. and Mexico are lacking. Moreover, none of the published studies that evaluated the effect of climate change in the spatial and temporal distribution of I. scapularis have focused on this region. METHODS: The area of study included Texas and a portion of northeast Mexico. This area is referred herein as the Texas-Mexico transboundary region. Tick samples were obtained from various vertebrate hosts in the region under study. Ticks identified as I. scapularis were processed to obtain DNA and to determine if they were infected with B. burgdorferi using PCR. A maximum entropy approach (MAXENT) was used to forecast the present and future (2050) distribution of B. burgdorferi-infected I. scapularis in the Texas-Mexico transboundary region by correlating geographic data with climatic variables. RESULTS: Of the 1235 tick samples collected, 109 were identified as I. scapularis. Infection with B. burgdorferi was detected in 45% of the I. scapularis ticks collected. The model presented here indicates a wide distribution for I. scapularis, with higher probability of occurrence along the Gulf of Mexico coast. Results of the modeling approach applied predict that habitat suitable for the distribution of I. scapularis in the Texas-Mexico transboundary region will remain relatively stable until 2050. CONCLUSIONS: The Texas-Mexico transboundary region appears to be part of a continuum in the pathogenic landscape of LD. Forecasting based on climate trends provides a tool to adapt strategies in the near future to mitigate the impact of LD related to its distribution and risk for transmission to human populations in the Mexico-US transboundary region

Immunization with a Borrelia burgdorferi BB0172-Derived Peptide Protects Mice against Lyme Disease

Lyme disease is the most prevalent arthropod borne disease in the US and it is caused by the bacterial spirochete Borrelia burgdorferi (Bb), which is acquired through the bite of an infected Ixodes tick. Vaccine development efforts focused on the von Willebrand factor A domain of the borrelial protein BB0172 from which four peptides (A, B, C and D) were synthesized and conjugated to Keyhole Limpet Hemocyanin, formulated in Titer Max® adjuvant and used to immunize C3H/HeN mice subcutaneously at days 0, 14 and 21. Sera were collected to evaluate antibody responses and some mice were sacrificed for histopathology to evaluate vaccine safety. Twenty-eight days post-priming, protection was evaluated by needle inoculation of half the mice in each group with 103 Bb/mouse, whereas the rest were challenged with 105Bb/mouse. Eight weeks post-priming, another four groups of similarly immunized mice were challenged using infected ticks. In both experiments, twenty-one days post-challenge, the mice were sacrificed to determine antibody responses, bacterial burdens and conduct histopathology. Results showed that only mice immunized with peptide B were protected against challenge with Bb. In addition, compared to the other the treatment groups, peptide B-immunized mice showed very limited inflammation in the heart and joint tissues. Peptide B-specific antibody titers peaked at 8 weeks post-priming and surprisingly, the anti-peptide B antibodies did not cross-react with Bb lysates. These findings strongly suggest that peptide B is a promising candidate for the development of a new DIVA vaccine (Differentiate between Infected and Vaccinated Animals) for protection against Lyme disease.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

Overexpression of CsrA (BB0184) Alters the Morphology and Antigen Profiles of Borrelia burgdorferi▿

Borrelia burgdorferi, the agent of Lyme disease, alters its gene expression in response to highly disparate environmental signals encountered in its hosts. Among the relatively few regulators of adaptive gene expression present in the borrelial genome is an open reading frame (ORF), BB0184, annotated as CsrA (carbon storage regulator A). CsrA, in several bacterial species, has been characterized as a small RNA binding protein that functions as a global regulator affecting mRNA stability or levels of translation of multiple ORFs. Consistent with known functions of CsrA, overexpression of CsrA from B. burgdorferi (CsrABb) in Escherichia coli resulted in reduced accumulation of glycogen. We determined that csrABb is part of the flgK motility operon and that the synthesis of CsrABb was increased when B. burgdorferi was propagated under fed-tick conditions. Overexpression of CsrABb in B. burgdorferi strain B31 (ML23, lp25-negative clonal isolate) resulted in a clone, designated ES25, which exhibited alterations in colony morphology and a significant reduction in the levels of FlaB. Several lipoproteins previously characterized as playing a role in infectivity were also altered in ES25. Real-time reverse transcription-PCR analysis of RNA revealed significant differences in the transcriptional levels of ospC in ES25, while there were no such differences in the levels of other transcripts, suggesting posttranscriptional regulation of expression of these latter genes. These observations indicate that CsrABb plays a role in the regulation of expression of pathophysiological determinants of B. burgdorferi, and further characterization of CsrABb will help in better understanding of the regulators of gene expression in B. burgdorferi

CsrA Modulates Levels of Lipoproteins and Key Regulators of Gene Expression Critical for Pathogenic Mechanisms of Borrelia burgdorferi▿

Carbon storage regulator A (CsrA) is an RNA binding protein that has been characterized in many bacterial species to play a central regulatory role by modulating several metabolic processes. We recently showed that a homolog of CsrA in Borrelia burgdorferi (CsrABb, BB0184) was upregulated in response to propagation of B. burgdorferi under mammalian host-specific conditions. In order to further delineate the role of CsrABb, we generated a deletion mutant designated ES10 in a linear plasmid 25-negative isolate of B. burgdorferi strain B31 (ML23). The deletion mutant was screened by PCR and Southern blot hybridization, and a lack of synthesis of CsrABb in ES10 was confirmed by immunoblot analysis. Analysis of ES10 propagated at pH 6.8/37°C revealed a significant reduction in the levels of OspC, DbpA, BBK32, and BBA64 compared to those for the parental wild-type strain propagated under these conditions, while there were no significant changes in the levels of either OspA or P66. Moreover, the levels of two regulatory proteins, RpoS and BosR, were also found to be lower in ES10 than in the control strain. Quantitative real-time reverse transcription-PCR analysis of total RNA extracted from the parental strain and csrABb mutant revealed significant differences in gene expression consistent with the changes at the protein level. Neither the csrABb mutant nor the trans-complemented strain was capable of infection following intradermal needle inoculation in C3H/HeN mice at either 103 or 105 spirochetes per mouse. The further characterization of molecular basis of regulation mediated by CsrABb will provide significant insights into the pathophysiology of B. burgdorferi

Peptide B induces partial protection in mice infected by using the tick model.

<p>Bacterial burden in tissues was significantly lower in animal immunized with Peptide B especially in skin (<b>A</b>) and spleen (<b>B</b>). Lymph nodes (<b>C</b>) and joints (<b>D</b>) show lower bacterial burden in both Peptide B and D immunized mice. Nevertheless, the bacterial recovery in cultures (<b>E</b>) was significantly reduced in mice receiving the Peptide B formulation compared with Peptide D or the control group. * Denotes statistically significant differences (* <i>P</i> value <0.05; ** <i>P</i> value < 0.01) when compared with the control group.</p

Summary of the study design.

<p><b>(A)</b> Schematic representation of the efficacy study. C3H/HeN mice were immunized with peptide B or D derived from the VWFA domain of BB0172 conjugated to KLH and administered at 50 µg/mouse with equal volume of TiterMax® Gold (Sigma-Aldrich) at days 0, 14 and 21. Eight weeks post-priming, a subgroup of mice (4/treatment) were sampled to determine antibody levels and pathological side effects. Twelve weeks post-priming a second subgroup of mice (4/treatment) were euthanized and sampled for antibody levels in blood, T-cell activity (from draining lymph nodes and spleens) and tissue damage. At the same time, a final group of 4 mice/treatment was infected by tick challenge, utilizing 8 infected <i>Ixodes scapulars</i> nymphs/mouse (containing around 150 <i>Borrelia</i>/nymph). Sixteen weeks post-priming mice were euthanized and protection evaluated by determining bacterial recovery from tissues as well as bacterial burden, tissue damage and antibody levels in blood. <b>(B)</b> Schematic representation of the passive transfer experiment conducted during phase II. Donor C3H/HeN were immunized with peptide B or D administered at days 0, 14, and 21. Eight weeks post-priming, donor mice were euthanized and blood and spleens were collected. Serum and splenocytes were isolated and passively transferred to recipient mice. Two-days after transfer mice were infected with either a low (10³ spriochetes/mouse) or a high (10⁵ spirochetes/mouse) dose of <i>B. burgdorferi</i> B31 by subcutaneous inoculation. Four weeks post-challenge mice were euthanized and protection was evaluated.</p

Schematic representation of the target identification phase.

<p>C3H/HeN mice were immunized with peptides derived from the VWFA domain of BB0172 (A, B, C and D) conjugated to KLH and administered at 50 µg/mouse with equal volume of TiterMax® Gold (Sigma-Aldrich) at days 0, 14, and 21. Four weeks post-priming, 4 mice per treatment were sampled to evaluate vaccine safety and antibody levels to each one of the peptides used. The other eight mice were infected with either 10³ (n = 4) or 10⁵ (n = 4) spirochetes/mouse. Four weeks post-challenge, mice were euthanized and blood collected to determine antibody levels. Tissues were sampled to determine bacterial burden by growth and qPCR as well as to determine any pathology by histology.</p

Representative histological images of the average level of inflammation observed in each treatment group (control, pepA, pepB, pepC, and pepD) after immunization and/or infection with the low (10³ spirochetes/mouse) or high (10⁵ spirochetes/mouse) doses in the tibiotarsal joint (A) and the heart (B).

<p>Tissues were histologically evaluated at four weeks post priming, as well as four weeks post needle inoculation. Average scores for areas of inflammation were classified as 0 =  none; 1 =  minimal; 2 =  mild; 3 =  moderate; 4 =  severe. Peptide B induces minimal inflammation in hearts and tibiotarsal joints after administration in the mouse model for Lyme disease. Of all the peptides evaluated after immunization, only peptide B showed inflammation comparable to the negative control group in both heart and joints. Similar results were observed after infection with low doses of <i>B. burgdorferi</i>. Images were captured using an Olympus BX41 microscope at 200X magnification. Average ± SD are presented in the graphs.</p

Low IgM and IgG antibodies were detected 4-weeks post priming in all groups.

<p>Antibody levels were evaluated 4-weeks post-priming as well as 4-weeks post needle infection. (<b>A</b>) Peptide-specific IgM antibodies. (<b>B</b>) <i>B. burgdorferi</i>-specific IgM antibodies. (<b>C</b>) Peptide-specific IgG antibodies. (<b>D</b>) <i>B. burgdorferi</i>-specific IgG antibodies. * Denotes statistically significant differences (* <i>P</i> value <0.05; ** <i>P</i> value < 0.01; *** <i>P</i> value < 0.001) when compared with the control group.</p