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1.  Nest box-deployed bait for delivering oral vaccines to white-footed mice 
Ticks and tick-borne diseases  2011;2(3):151-155.
Although a wide range of interventions are available for use in reducing the public health burden of Lyme disease, additional tools are needed. Vaccinating mouse reservoirs may reduce the prevalence of spirochetal infection due to the powerful vector and reservoir competence-modulating effects of anti-outer surface protein A (OspA) antibody. A delivery system for an oral immunogen would be required for field trials of any candidate vaccine. Accordingly, we tested candidate bait preparations that were designed to be environmentally stable, attractive to mice, and non-nutritive. In addition, we determined whether delivery of such baits within nest boxes could effectively target white-footed mice. A peanut butter-scented bait was preferred by mice over a blueberry-scented one. At a deployment rate of 12.5 nest boxes per hectare, more than half of resident mice ingested a rhodamine-containing bait, as demonstrated by fluorescent staining of their vibrissae. We conclude that a peanut butter-scented hardened bait placed within simple wood nest boxes would effectively deliver vaccine to white-footed mice, thereby providing baseline information critical for designing field trials of a candidate oral vaccine.
doi:10.1016/j.ttbdis.2011.06.001
PMCID: PMC4018734  PMID: 21890068
Lyme disease; White-footed mice; Borrelia burgdorferi; Bait; Oral vaccination; Deer ticks
2.  Oral Vaccination With Vaccinia Virus Expressing the Tick Antigen Subolesin Inhibits Tick Feeding and Transmission of Borrelia burgdorferi Vaccination 
Vaccine  2012;30(42):6040-6046.
Immunization with the Ixodes scapularis protein, subolesin, has previously been shown to protect hosts against tick infestation and to decrease acquisition of Anaplsma marginale and Babesia bigemina. Here we report the efficacy of subolesin expressed from Vaccinia virus for use as an orally delivered reservoir–targeted vaccine for prevention of tick infestation and acquisition/transmission of Borrelia burgdorferi to its tick and mouse hosts. We cloned subolesin into Vaccinia virus and showed that it is expressed from mammalian cells infected with the recombinant virus in vitro. We then vaccinated mice by oral gavage. A single dose of the vaccine was sufficient for mice to generate antibody response to subolesin. Vaccination with the subolesin expressing Vaccinia virus inhibited tick infestation by 52% compared to control vaccination with Vaccinia virus and reduced uptake of B. burgdorferi among the surviving ticks that fed to repletion by 34%. There was a reduction in transmission of B. burgdorferi to uninfected vaccinated mice of 40% compared to controls. These results suggest that subolesin has potential as a component of a reservoir targeted vaccine to decrease B. burgdorferi, Babesia and Anaplasma species infections in their natural hosts.
doi:10.1016/j.vaccine.2012.07.053
PMCID: PMC3938954  PMID: 22864146
Vaccinia virus; Borrelia burgdorferi; Lyme disease; Reservoir-targeted vaccine; Ixodes scapularis; Tick; OspA; Subolesin
3.  Understanding Barriers to Borrelia burgdorferi Dissemination during Infection Using Massively Parallel Sequencing 
Infection and Immunity  2013;81(7):2347-2357.
Borrelia burgdorferi is an invasive spirochete that can cause acute and chronic infections in the skin, heart, joints, and central nervous system of infected mammalian hosts. Little is understood about where the bacteria encounter the strongest barriers to infection and how different components of the host immune system influence the population as the infection progresses. To identify population bottlenecks in a murine host, we utilized Tn-seq to monitor the composition of mixed populations of B. burgdorferi during infection. Both wild-type mice and mice lacking the Toll-like receptor adapter molecule MyD88 were infected with a pool of infectious B. burgdorferi transposon mutants with insertions in the same gene. At multiple time points postinfection, bacteria were isolated from the mice and the compositions of the B. burgdorferi populations at the injection site and in distal tissues determined. We identified a population bottleneck at the site of infection that significantly altered the composition of the population. The magnitude of this bottleneck was reduced in MyD88−/− mice, indicating a role for innate immunity in limiting early establishment of B. burgdorferi infection. There is not a significant bottleneck during the colonization of distal tissues, suggesting that founder effects are limited and there is not a strict limitation on the number of organisms able to initiate populations at distal sites. These findings further our understanding of the interactions between B. burgdorferi and its murine host in the establishment of infection and dissemination of the organism.
doi:10.1128/IAI.00266-13
PMCID: PMC3697624  PMID: 23608706
4.  Transposon mutagenesis as an approach to improved understanding of Borrelia pathogenesis and biology 
Transposon insertion provides a method for near-random mutation of bacterial genomes, and has been utilized extensively for the study of bacterial pathogenesis and biology. This approach is particularly useful for organisms that are relatively refractory to genetic manipulation, including Lyme disease Borrelia. In this review, progress to date in the application of transposon mutagenesis to the study of Borrelia burgdorferi is reported. An effective Himar1-based transposon vector has been developed and used to acquire a sequence-defined library of nearly 4500 mutants in the infectious, moderately transformable B. burgdorferi B31 derivative 5A18NP1. Analysis of these transposon mutants using signature-tagged mutagenesis (STM) and Tn-seq approaches has begun to yield valuable information regarding the genes important in the pathogenesis and biology of this organism.
doi:10.3389/fcimb.2014.00063
PMCID: PMC4033020  PMID: 24904839
Borrelia burgdorferi; Lyme disease; transposon mutagenesis; microbial pathogenesis; bacterial physiology; mouse models
5.  A Two-Component System Regulates Hemin Acquisition in Porphyromonas gingivalis 
PLoS ONE  2013;8(9):e73351.
Porphyromonas gingivalis is a Gram-negative oral anaerobe associated with infection of the periodontia. The organism has a small number of two-component signal transduction systems, and after comparing genome sequences of strains W83 and ATCC 33277 we discovered that the latter was mutant in histidine kinase (PGN_0752), while the cognate response regulator (PGN_0753) remained intact. Microarray-based transcriptional profiling and ChIP-seq assays were carried out with an ATCC 33277 transconjugant containing the functional histidine kinase from strain W83 (PG0719). The data showed that the regulon of this signal transduction system contained genes that were involved in hemin acquisition, including gingipains, at least three transport systems, as well as being self-regulated. Direct regulation by the response regulator was confirmed by electrophoretic mobility shift assays. In addition, the system appears to be activated by hemin and the regulator acts as both an activator and repressor.
doi:10.1371/journal.pone.0073351
PMCID: PMC3764172  PMID: 24039921
6.  TRIF Mediates Toll-Like Receptor 2-Dependent Inflammatory Responses to Borrelia burgdorferi 
Infection and Immunity  2013;81(2):402-410.
TRIF is an adaptor molecule important in transducing signals from intracellularly signaling Toll-like receptor 3 (TLR3) and TLR4. Recently, TLR2 was found to signal from intracellular compartments. Using a synthetic ligand for TLR2/1 heterodimers, as well as Borrelia burgdorferi, which is a strong activator of TLR2/1, we found that TLR2 signaling can utilize TRIF. Unlike TRIF signaling by other TLRs, TLR2-mediated TRIF signaling is dependent on the presence of another adaptor molecule, MyD88. However, unlike MyD88 deficiency, TRIF deficiency does not result in diminished control of infection with B. burgdorferi in a murine model of disease. This appears to be due to the effects of MyD88 on phagocytosis via scavenger receptors, such as MARCO, which are not affected by the loss of TRIF. In mice, TRIF deficiency did have an effect on the production of inflammatory cytokines, suggesting that regulation of inflammatory cytokines and control of bacterial growth may be uncoupled, in part through transduction of TLR2 signaling through TRIF.
doi:10.1128/IAI.00890-12
PMCID: PMC3553797  PMID: 23166161
7.  Development of a baited oral vaccine for use in reservoir-targeted strategies against Lyme disease 
Vaccine  2011;29(44):7818-7825.
Lyme disease is a major human health problem which continues to increase in incidence and geographic distribution. As a vector-borne zoonotic disease, Lyme disease may be amenable to reservoir targeted strategies for control. We have previously reported that a vaccinia virus (VV) based vaccine expressing outer surface protein A (OspA) of Borrelia burgdorferi, the causative agent of Lyme disease, protects inbred strains of laboratory mice against infection by feeding ticks and clears the ticks of infection when administered by gavage. Here we extend these studies to develop an effective bait formulation for delivery of the VV based vaccine and test its characteristics under simulated environmental conditions. We show that this vaccine is efficacious in decreasing acquisition of B. burgdorferi by uninfected larval ticks as well as in decreasing transmission from infected ticks to its natural reservoir, Peromyscus leucopus, when fed to mice in oral baits. Using live, in vivo imaging techniques, we describe the distribution of vaccinia virus infection after ingestion of the baited vaccines and establish the use of in vivo imaging technology for optimization of bait delivery. In summary, a VV based OspA vaccine is stable in an oral bait preparation and provides protection against infection for both the natural reservoir and the tick vector of Lyme disease.
doi:10.1016/j.vaccine.2011.07.100
PMCID: PMC3577423  PMID: 21816190
Vaccinia virus; Borrelia burgdorferi; Lyme disease; Reservoir-targeted vaccine; Tick; OspA
8.  Identification of essential genes of the periodontal pathogen Porphyromonas gingivalis 
BMC Genomics  2012;13:578.
Background
Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with periodontal disease onset and progression. Genetic tools for the manipulation of bacterial genomes allow for in-depth mechanistic studies of metabolism, physiology, interspecies and host-pathogen interactions. Analysis of the essential genes, protein-coding sequences necessary for survival of P. gingivalis by transposon mutagenesis has not previously been attempted due to the limitations of available transposon systems for the organism. We adapted a Mariner transposon system for mutagenesis of P. gingivalis and created an insertion mutant library. By analyzing the location of insertions using massively-parallel sequencing technology we used this mutant library to define genes essential for P. gingivalis survival under in vitro conditions.
Results
In mutagenesis experiments we identified 463 genes in P. gingivalis strain ATCC 33277 that are putatively essential for viability in vitro. Comparing the 463 P. gingivalis essential genes with previous essential gene studies, 364 of the 463 are homologues to essential genes in other species; 339 are shared with more than one other species. Twenty-five genes are known to be essential in P. gingivalis and B. thetaiotaomicron only. Significant enrichment of essential genes within Cluster of Orthologous Groups ‘D’ (cell division), ‘I’ (lipid transport and metabolism) and ‘J’ (translation/ribosome) were identified. Previously, the P. gingivalis core genome was shown to encode 1,476 proteins out of a possible 1,909; 434 of 463 essential genes are contained within the core genome. Thus, for the species P. gingivalis twenty-two, seventy-seven and twenty-three percent of the genome respectively are devoted to essential, core and accessory functions.
Conclusions
A Mariner transposon system can be adapted to create mutant libraries in P. gingivalis amenable to analysis by next-generation sequencing technologies. In silico analysis of genes essential for in vitro growth demonstrates that although the majority are homologous across bacterial species as a whole, species and strain-specific subsets are apparent. Understanding the putative essential genes of P. gingivalis will provide insights into metabolic pathways and niche adaptations as well as clinical therapeutic strategies.
doi:10.1186/1471-2164-13-578
PMCID: PMC3547785  PMID: 23114059
Porphyromonas gingivalis; Transposon mutagenesis; Essential genes; Tn-seq; Periodontal disease
9.  Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes 
Nature Reviews. Microbiology  2012;10(2):87-99.
In little more than 30 years, Lyme disease, which is caused by the spirochaete Borrelia burgdorferi, has risen from relative obscurity to become a global public health problem and a prototype of an emerging infection. During this period, there has been an extraordinary accumulation of knowledge on the phylogenetic diversity, molecular biology, genetics and host interactions of B. burgdorferi. In this Review, we integrate this large body of information into a cohesive picture of the molecular and cellular events that transpire as Lyme disease spirochaetes transit between their arthropod and vertebrate hosts during the enzootic cycle.
doi:10.1038/nrmicro2714
PMCID: PMC3313462  PMID: 22230951
10.  Development of a vaccinia virus based reservoir-targeted vaccine against Yersinia pestis 
Vaccine  2010;28(48):7683-7689.
Yersinia pestis, the causative organism of plague, is a zoonotic organism with a worldwide distribution. Although the last plague epidemic occurred in early 1900s, human cases continue to occur due to contact with infected wild animals. In this study, we have developed a reservoir-targeted vaccine against Y. pestis, to interrupt transmission of disease in wild animals as a potential strategy for decreasing human disease. A vaccinia virus delivery system was used to express the F1 capsular protein and the LcrV type III secretion component of Y. pestis as a fusion protein. Here we show that a single dose of this vaccine administered orally, generates a dose-dependent antibody response in mice. Antibody titers peak by 3 weeks after administration and remain elevated for a minimum of 45 weeks. Vaccination provided up to 100% protection against challenge with Y. pestis administered by intranasal challenge at 10 times the lethal dose with protection lasting a minimum of 45 weeks. An orally available, vaccinia virus expressed vaccine against Y. pestis may be a suitable vaccine for a reservoir targeted strategy for the prevention of enzootic plague.
doi:10.1016/j.vaccine.2010.09.039
PMCID: PMC3131225  PMID: 20875494
11.  Prevention of Lyme Disease (and other tick borne infections) 
Summary
Prevention is the best method for avoiding potentially serious complications of Lyme disease. In this chapter, we will discuss preventative measures that can be employed by individuals and/or communities. Among the topics discussed are personal protective measures, tick reduction, reservoir reduction and vaccination. Additionally, new preventative measures that are in development including new Lyme disease vaccines, anti-tick vaccines and reservoir-targeted vaccination are discussed.
doi:10.1016/j.idc.2008.03.007
PMCID: PMC3195518  PMID: 18755380
Lyme; Borrelia burgdorferi; prevention; Ixodes; vaccine
12.  Role of Adrenomedullin in Lyme Disease▿  
Infection and Immunity  2010;78(12):5307-5313.
Borrelia burgdorferi stimulates a strong inflammatory response during infection of a mammalian host. To understand the mechanisms of immune regulation employed by the host to control this inflammatory response, we focused our studies on adrenomedullin, a peptide produced in response to bacterial stimuli that exhibits antimicrobial activity and regulates inflammatory responses by modulating the expression of inflammatory cytokines. Specifically, we investigated the effect of B. burgdorferi on the expression of adrenomedullin as well as the ability of adrenomedullin to dampen host inflammatory responses to the spirochete. The concentration of adrenomedullin in the synovial fluid of untreated Lyme arthritis patients was elevated compared with that in control osteoarthritis patient samples. In addition, coculture with B. burgdorferi significantly increased the expression of adrenomedullin in RAW264.7 macrophages through MyD88-, phosphatidylinositol 3-kinase (PI3-K)-, and p38-dependent signaling cascades. Furthermore, the addition of exogenous adrenomedullin to B. burgdorferi-stimulated RAW264.7 macrophages resulted in a significant decrease in the induction of proinflammatory cytokines. Taken together, these results suggest that B. burgdorferi increases the production of adrenomedullin, which in turn negatively regulates the B. burgdorferi-stimulated inflammatory response.
doi:10.1128/IAI.00630-10
PMCID: PMC2981333  PMID: 20921145
13.  Nod2 Suppresses Borrelia burgdorferi Mediated Murine Lyme Arthritis and Carditis through the Induction of Tolerance 
PLoS ONE  2011;6(2):e17414.
The internalization of Borrelia burgdorferi, the causative agent of Lyme disease, by phagocytes is essential for an effective activation of the immune response to this pathogen. The intracellular, cytosolic receptor Nod2 has been shown to play varying roles in either enhancing or attenuating inflammation in response to different infectious agents. We examined the role of Nod2 in responses to B. burgdorferi. In vitro stimulation of Nod2 deficient bone marrow derived macrophages (BMDM) resulted in decreased induction of multiple cytokines, interferons and interferon regulated genes compared with wild-type cells. However, B. burgdorferi infection of Nod2 deficient mice resulted in increased rather than decreased arthritis and carditis compared to control mice. We explored multiple potential mechanisms for the paradoxical response in in vivo versus in vitro systems and found that prolonged stimulation with a Nod2 ligand, muramyl dipeptide (MDP), resulted in tolerance to stimulation by B. burgdorferi. This tolerance was lost with stimulation of Nod2 deficient cells that cannot respond to MDP. Cytokine patterns in the tolerance model closely paralleled cytokine profiles in infected Nod2 deficient mice. We propose a model where Nod2 has an enhancing role in activating inflammation in early infection, but moderates inflammation after prolonged exposure to the organism through induction of tolerance.
doi:10.1371/journal.pone.0017414
PMCID: PMC3046161  PMID: 21387014
14.  Identification of interspecies interactions affecting Porphyromonas gingivalis virulence phenotypes 
Journal of Oral Microbiology  2011;3:10.3402/jom.v3i0.8396.
Background
Periodontitis is recognized as a complex polymicrobial disease, however, the impact of the bacterial interactions among the 700–1,000 different species of the oral microbiota remains poorly understood. We conducted an in vitro screen for oral bacteria that mitigate selected virulence phenotypes of the important periodontal pathogen, Porphyromonas gingivalis.
Method
We isolated and identified oral anaerobic bacteria from subgingival plaque of dental patients. When cocultured with P. gingivalis W83, specific isolates reduced the cytopathogenic effects of P. gingivalis on oral epithelial cells.
Result
In an initial screen of 103 subgingival isolates, we identified 19 distinct strains from nine species of bacteria (including Actinomyces naeslundii, Streptococcus oralis, Streptococcus mitis, and Veilonella dispar) that protect oral epithelial cells from P. gingivalis-induced cytotoxicity. We found that some of these strains inhibited P. gingivalis growth in plate assays through the production of organic acids, whereas some decreased the gingipain activity of P. gingivalis in coculture or mixing experiments.
Conclusion
In summary, we identified 19 strains isolated from human subgingival plaque that interacted with P. gingivalis, resulting in mitigation of its cytotoxicity to oral epithelial cells, inhibition of growth, and/or reduction of gingipain activity. Understanding the mechanisms of interaction between bacteria in the oral microbial community may lead to the development of new probiotic agents and new strategies for interrupting the development of periodontal disease.
doi:10.3402/jom.v3i0.8396
PMCID: PMC3198504  PMID: 22022641
oral pathogen; bacterial interactions; virulence; gingipain; cytotoxicity
15.  Human Integrin α3β1 Regulates TLR2 Recognition of Lipopeptides from Endosomal Compartments 
PLoS ONE  2010;5(9):e12871.
Background
Toll-like receptor (TLR)-2/TLR1 heterodimers recognize bacterial lipopeptides and initiate the production of inflammatory mediators. Adaptors and co-receptors that mediate this process, as well as the mechanisms by which these adaptors and co-receptors function, are still being discovered.
Methodology/Principal Findings
Using shRNA, blocking antibodies, and fluorescent microscopy, we show that U937 macrophage responses to the TLR2/1 ligand, Pam3CSK4, are dependent upon an integrin, α3β1. The mechanism for integrin α3β1 involvement in TLR2/1 signaling is through its role in endocytosis of lipopeptides. Using inhibitors of endosomal acidification/maturation and physical tethering of the ligand, we show that the endocytosis of Pam3CSK4 is necessary for the complete TLR2/1-mediated pro-inflammatory cytokine response. We also show that TLR2/1 signaling from the endosome results in the induction of different inflammatory mediators than TLR2/1 signaling from the plasma membrane.
Conclusion/Significance
Here we identify integrin α3β1 as a novel regulator for the recognition of bacterial lipopeptides. We demonstrate that induction of a specific subset of cytokines is dependent upon integrin α3β1-mediated endocytosis of the ligand. In addition, we address an ongoing controversy regarding endosomal recognition of bacterial lipopeptides by demonstrating that TLR2/1 signals from within endosomal compartments as well as the plasma membrane, and that downstream responses may differ depending upon receptor localization. We propose that the regulation of endosomal TLR2/1 signaling by integrin α3β1 serves as a mechanism for modulating inflammatory responses.
doi:10.1371/journal.pone.0012871
PMCID: PMC2943923  PMID: 20877569
16.  Downstream signals for MyD88-mediated phagocytosis of B. burgdorferi can be initiated by TRIF and are dependent upon PI3K 
We previously have shown that MyD88 is important for uptake of B. burgdorferi by bone marrow derived macrophages (BMDMs). The mechanism by which MyD88 is involved in uptake of B. burgdorferi is currently is not well characterized. Here, we report that MyD88-mediated defect in the phagocytosis of B. burgdorferi can be complemented by TLR3/TRIF activation in BMDMs from MyD88−/− mice. This effect of TLR3/TRIF activation was not due to its induction of type I interferons, suggesting instead a convergence of signaling pathways downstream of MyD88 and TRIF. In order to characterize signaling pathways involved in MyD88-mediated phagocytosis of B. burgdorferi, BMDMs were treated with specific inhibitors of MAPK, PKC, JAK/STAT, or PI3K. Only inhibition of PI3K resulted in a significant decrease of B. burgdorferi uptake. Consistent with this, B. burgdorferi activation of MyD88- or TLR3-/TRIF-signaling resulted in increased activity of PI3K. In addition, association of B. burgdorferi with actin related protein (Arp2/3) complexes, which facilitate actin rearrangements during phagocytosis, was similarly reduced in MyD88−/− BMDMs and BMDMs treated with a PI3K inhibitor. Taken together, these findings define an essential pathway whereby downstream signals from MyD88 or TRIF converge on PI3K, which triggers actin polymerization to initiate the phagocytosis of B. burgdorferi.
doi:10.4049/jimmunol.0900724
PMCID: PMC2772066  PMID: 19542460
17.  Matrix Metalloproteinase 9 Plays a Key Role in Lyme Arthritis but Not in Dissemination of Borrelia burgdorferi▿  
Infection and Immunity  2009;77(7):2643-2649.
Borrelia burgdorferi, the causative agent of Lyme arthritis, does not produce any exported proteases capable of degrading extracellular matrix despite the fact that it is able to disseminate from a skin insertion site to infect multiple organs. Prior studies have shown that B. burgdorferi induces the host protease, matrix metalloproteinase 9 (MMP-9), and suggested that the induction of MMP-9 may allow the organism to disseminate and produce local tissue destruction. We examined the role of MMP-9 in dissemination of B. burgdorferi and pathogenesis of Lyme arthritis. In a MMP-9−/− mouse model, MMP-9 was not required for the dissemination of the spirochete to distant sites. However, MMP-9−/− exhibited significantly decreased arthritis compared to wild-type mice. The decrease in arthritis was not due to an inability to control infection since the spirochete numbers in the joints were identical. Levels of inflammatory chemokines and cytokines were also similar in MMP-9−/− and wild-type mice. We examined whether decreased inflammation in MMP-9−/− mice may be the result of decreased production of neoattractants by MMP-9-dependent cleavage of collagen. MMP-9 cleavage of type I collagen results in increased monocyte chemoattraction. MMP-9 plays an important role in regulating inflammation in Lyme arthritis, potentially through the cleavage of type I collagen.
doi:10.1128/IAI.00214-09
PMCID: PMC2708580  PMID: 19364840
18.  Role of Novel Protein Kinase C (PKC) isoforms in Lyme Arthritis 
Cellular microbiology  2007;9(8):1987-1996.
Inflammation caused by Borrelia burgdorferi infection occurs as a result of induction of pro-inflammatory cytokines from activation of multiple signaling pathways. It has previously been shown that MAPK and JAK/STAT signaling pathways are activated by B. burgdorferi in cultured human chondrocytes. Protein Kinase C (PKC) signaling pathways are potential candidates that may control these downstream signaling pathways. Here we show that B. burgdorferi infection leads to phosphorylation and activation of novel PKC isoforms (PKC δ, ε, η, and θ) in a time-dependent manner. A specific inhibitor of novel PKC isoforms blocked the induction of pro-inflammatory molecules in response to B. burgdorferi infection as did transient transfection of novel PKC dominant-negative plasmids into chondrocytes. B. burgdorferi-induced p38 MAPK phosphorylation was also significantly inhibited by an inhibitor of novel PKC isoforms, suggesting that PKC activation occurs upstream of p38 activation. In vivo, administration of an inhibitor of classical and novel PKC isoforms to C3H/HeN mice infected with B. burgdorferi resulted in significantly reduced ankle inflammation and swelling. In conclusion, these data suggest that novel PKC isoforms are specifically activated by B. burgdorferi infection and this can contribute to the regulation of inflammation in vitro and in vivo.
doi:10.1111/j.1462-5822.2007.00929.x
PMCID: PMC2643348  PMID: 17394560
19.  Borrelia burgdorferi BBB07 Interaction With Integrin α3β1 Stimulates Production of Pro-Inflammatory Mediators in Primary Human Chondrocytes 
Cellular microbiology  2007;10(2):320-331.
Summary
Borrelia burgdorferi, the causative agent of Lyme disease, activates multiple signaling pathways leading to induction of pro-inflammatory mediators at sites of inflammation. Binding of B. burgdorferi to integrin α3β1on human chondrocytes activates signaling leading to release of several pro-inflammatory mediators, but the B. burgdorferi protein that binds integrin α3β1and elicits this response has remained unknown. A search of the B. burgdorferi genome for a canonical integrin-binding motif, the RGD (Arg-Gly-Asp) tripeptide, revealed several candidate ligands for integrins. In this study we show that one of these candidates, BBB07, binds to integrin α3β1 and inhibits attachment of intact B. burgdorferi to the same integrin. BBB07 is expressed during murine infection as demonstrated by recognition by infected mouse sera. Recombinant purified BBB07 induces pro-inflammatory mediators in primary human chondrocyte cells by interaction with integrin α3β1. This interaction is specific, as P66, another integrin ligand of B. burgdorferi, does not activate signaling through α3β1. In summary, we have identified a B. burgdorferi protein, BBB07 that interacts with integrin α3β1 and stimulates production of pro-inflammatory mediators in primary human chondrocyte cells.
doi:10.1111/j.1462-5822.2007.01043.x
PMCID: PMC2586958  PMID: 17822440
20.  Taking a Bite Out of Vector-Transmitted Infectious Diseases 
The New England journal of medicine  2007;356(25):2567-2569.
doi:10.1056/NEJMp078081
PMCID: PMC2577026  PMID: 17582066
21.  Distinct Roles for MyD88 and Toll-Like Receptors 2, 5, and 9 in Phagocytosis of Borrelia burgdorferi and Cytokine Induction ▿  
Infection and Immunity  2008;76(6):2341-2351.
The contribution of Toll-like receptors (TLRs) to phagocytosis of Borrelia burgdorferi has not been extensively studied. We show that bone marrow-derived macrophages (BMDM) from MyD88−/− mice or Raw cells transfected with a dominant-negative MyD88 were unable to efficiently internalize B. burgdorferi. Knockouts of TLR2 and TLR9 or knockdown of TLR5 by small interfering RNA produced no defects in phagocytosis of B. burgdorferi. Production of inflammatory cytokines was greatly diminished in MyD88−/− BMDM but only partially affected in TLR2−/− BMDM or knockdown of TLR5 and unaffected in TLR9−/− BMDM. Cytochalasin D reduced cytokine induction, but not to the level of the MyD88−/− BMDM. Addition of cytochalasin D to TLR2−/− BMDM inhibited inflammatory responses to B. burgdorferi to the level of MyD88−/− BMDM, consistent with a role for TLR2 in both recognition of extracellular products and lysosomal sampling by TLR2 after processing of the organism. Cytochalasin D had no impact on cytokine production in cells undergoing TLR5 knockdown. These results suggest that MyD88, but not TLR2, TLR5, and TLR9, is important for the uptake of B. burgdorferi and that MyD88 affects inflammatory responses through both its effects on phagocytosis and its role in transducing signals from TLR2 and TLR5.
doi:10.1128/IAI.01600-07
PMCID: PMC2423091  PMID: 18378636
22.  Protective efficacy of an oral vaccine to reduce carriage of Borrelia burgdorferi (strain N40) in mouse and tick reservoirs 
Vaccine  2005;24(11):1949-1957.
Lyme disease is caused by the spirochete Borrelia burgdorferi, which is transmitted through the bite of infected Ixodes ticks. Vaccination of mice with outer surface protein A (OspA) of B. burgdorferi has been shown to both protect mice against B. burgdorferi infection and reduce carriage of the organism in feeding ticks. Here we report the development of a murine-targeted OspA vaccine utilizing Vaccinia virus to interrupt transmission of disease in the reservoir hosts, thus reducing incidence of human disease. Oral vaccination of mice with a single dose of Vaccinia expressing OspA resulted in high antibody titers to OspA, 100% protection of vaccinated mice from infection with B. burgdorferi, and significant clearance of B. burgdorferi from infected ticks fed on vaccinated animals. The results indicate the vaccine is effective and may provide a manner to reduce incidence of Lyme disease.
doi:10.1016/j.vaccine.2005.10.044
PMCID: PMC1388312  PMID: 16300863
Lyme disease; Vaccinia virus; OspA
23.  Regulators of Expression of the Oligopeptide Permease A Proteins of Borrelia burgdorferi▿  
Journal of Bacteriology  2007;189(7):2653-2659.
Borrelia burgdorferi undergoes an infectious cycle that requires adaptation to different hosts and marked differences in environment. B. burgdorferi copes with its different environments by regulating the expression of proteins required for survival in specific settings. The B. burgdorferi oligopeptide permease (Opp) is one of only a few transporters encoded by the B. burgdorferi genome. Opp proteins in other bacteria serve multiple environmental adaptation functions. B. burgdorferi appears to broaden the usage of this transporter by utilizing five different substrate binding proteins (OppA proteins) that interact with the integral membrane components of the transporter. Expression of the OppA proteins is individually regulated and may play different roles in adaptation to host environments. Very little is known about the mechanisms used by B. burgdorferi to regulate the expression of different OppA proteins. Here we show that the alternative sigma factors, RpoS and RpoN, regulate the expression of oppA5 but not that of other oppA genes. Using a reporter assay with Escherichia coli and gel shift binding assays, we also show that the B. burgdorferi BosR/Fur homologue interacts with the oppA4 promoter and that another candidate transcription factor, EbfC, interacts with the oppA5 promoter. Binding to the promoters was confirmed by gel shift assays. Expression of BosR/Fur in its different hosts does appear to parallel the expression of oppA4. A better understanding of the factors involved in gene regulation in B. burgdorferi will help to identify coregulated proteins that may cooperate to allow the organism to survive in a specific environment.
doi:10.1128/JB.01760-06
PMCID: PMC1855802  PMID: 17237172
24.  MyD88 Deficiency Results in Tissue-Specific Changes in Cytokine Induction and Inflammation in Interleukin-18-Independent Mice Infected with Borrelia burgdorferi  
Infection and Immunity  2006;74(3):1462-1470.
Toll-like receptors (TLRs) play an important role in the control of infection with Borrelia burgdorferi. Deficiencies in TLR-2 or the shared TLR adapter molecule MyD88 have been shown to result in greatly increased bacterial burdens in mice. However, although in vitro studies have shown that the activation of TLR pathways by B. burgdorferi results in the release of inflammatory cytokines, studies in deficient mice have shown either no change or increased rather than decreased inflammation in infected animals. In this study, we looked at mechanisms to explain the increase in inflammation in the absence of MyD88. We found that MyD88-deficient mice infected with B. burgdorferi did not show increased inflammation at sites typically associated with Lyme disease (joints and heart). However, there was markedly increased inflammation in the muscles, kidneys, pancreas, and lungs of deficient animals. Muscle inflammation was typically seen perivascularly and perineuronally similar to that seen in infected humans. Chemotactic chemokines and cytokines were greatly increased in the muscle and kidneys of MyD88-deficient animals but not in the joints or heart tissue, suggesting that MyD88-independent pathways for recognizing B. burgdorferi and inducing these chemokines are present in the muscle and kidneys. Interleukin-18 signaling through MyD88 does not appear to play a role in either control of infection or inflammation.
doi:10.1128/IAI.74.3.1462-1470.2006
PMCID: PMC1418660  PMID: 16495516
25.  Induction of Host Matrix Metalloproteinases by Borrelia burgdorferi Differs in Human and Murine Lyme Arthritis  
Infection and Immunity  2005;73(1):126-134.
Matrix metalloproteinases (MMPs) are induced from host tissues in response to Borrelia burgdorferi. Upregulation of MMPs may play a role in the dissemination of the organism through extracellular matrix tissues, but it can also result in destructive pathology. Although mice are a well-accepted model for Lyme arthritis, there are significant differences compared to human disease. We sought to determine whether MMP expression could account for some of these differences. MMP expression patterns following B. burgdorferi infection were analyzed in primary human chondrocytes, synovial fluid samples from patients with Lyme arthritis, and cartilage tissue from Lyme arthritis-susceptible and -resistant mice by using a gene array, real-time PCR, an enzyme-linked immunosorbent assay, and immunohistochemistry. B. burgdorferi infection significantly induced transcription of MMP-1, -3, -13, and -19 from primary human chondrocyte cells. Transcription of MMP-10 and tissue inhibitor of metalloprotease 1 was increased with B. burgdorferi infection, but protein expression was only minimally increased. The synovial fluid levels of MMPs from patients with high and low spirochete burdens were consistent with results seen in the in vitro studies. B. burgdorferi-susceptible C3H/HeN mice infected with B. burgdorferi showed induction of MMP-3 and MMP-19 but no other MMP or tissue inhibitor of metalloprotease. As determined by immunohistochemistry, MMP-3 expression was increased only in chondrocytes near the articular surface. The levels of MMPs were significantly lower in the more Lyme arthritis-resistant BALB/c and C57BL/6 mice. Differences between human and murine Lyme arthritis may be related to the lack of induction of collagenases, such MMP-1 and MMP-13, in mouse joints.
doi:10.1128/IAI.73.1.126-134.2005
PMCID: PMC539001  PMID: 15618147

Results 1-25 (29)