Live Attenuated Borrelia burgdorferi Targeted Mutants in an Infectious Strain Background Protect Mice from Challenge Infection

ABSTRACTBorrelia burgdorferi,B. garinii, andB. afzeliiare all agents of Lyme disease in different geographic locations. If left untreated, Lyme disease can cause significant and long-term morbidity, which may continue after appropriate antibiotic therapy has been administered and live bacteria are no longer detectable. The increasing incidence and geographic spread of Lyme disease are renewing interest in the vaccination of at-risk populations. We took the approach of vaccinating mice with two targeted mutant strains ofB. burgdorferithat, unlike the parental strain, are avirulent in mice. Mice vaccinated with both strains were protected against a challenge with the parental strain and a heterologousB. burgdorferistrain by either needle inoculation or tick bite. In ticks, the homologous strain was eliminated but the heterologous strain was not, suggesting that the vaccines generated a response to antigens that are produced by the bacteria both early in mammalian infection and in the tick. Partial protection againstB. gariniiinfection was also conferred. Protection was antibody mediated, and reactivity to a variety of proteins was observed. These experiments suggest that live attenuatedB. burgdorferistrains may be informative regarding the identification of protective antigens produced by the bacteria and recognized by the mouse immune systemin vivo. Further work may illuminate new candidates that are effective and safe for the development of Lyme disease vaccines.

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Evaluation of RevA, a Fibronectin-Binding Protein of Borrelia burgdorferi, as a Potential Vaccine Candidate for Lyme Disease

Clinical and Vaccine Immunology ◽

10.1128/cvi.00758-12 ◽

2013 ◽

Vol 20 (6) ◽

pp. 892-899 ◽

Cited By ~ 12

Author(s):

Angela M. Floden ◽

Tammy Gonzalez ◽

Robert A. Gaultney ◽

Catherine A. Brissette

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Passive Immunization ◽

Surface Protein ◽

Surface Expression ◽

Content Type ◽

Good Target ◽

Potential Vaccine ◽

Mammalian Infection

ABSTRACTPrevious studies indicated that the Lyme disease spirocheteBorrelia burgdorferiexpresses the RevA outer surface protein during mammalian infection. As an adhesin that promotes bacterial interaction with fibronectin, RevA appears to be a good target for preventive therapies. RevA proteins are highly conserved across all Lyme borreliae, and antibodies against RevA protein are cross-reactive among RevA proteins from diverse strains. Mice infected withB. burgdorferimounted a rapid IgM response to RevA, followed by a strong IgG response that generally remained elevated for more than 12 months, suggesting continued exposure of RevA protein to the immune system. RevA antibodies were bactericidalin vitro. To evaluate the RevA antigen as a potential vaccine, mice were vaccinated with recombinant RevA and challenged withB. burgdorferiby inoculation with a needle or by a tick bite. Cultured tissues from all treatment groups were positive forB. burgdorferi. Vaccinated animals also appeared to have similar levels ofB. burgdorferiDNA compared to nonvaccinated controls. Despite its antigenicity, surface expression, and the production of bactericidal antibodies against it, RevA does not protect againstBorrelia burgdorferiinfection in a mouse model. However, passive immunization with anti-RevA antibodies did prevent infection, suggesting the possible utility of RevA-based immunotherapeutics or vaccine.

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Disulfide-Mediated Oligomer Formation in Borrelia burgdorferi Outer Surface Protein C, a Critical Virulence Factor and Potential Lyme Disease Vaccine Candidate

Clinical and Vaccine Immunology ◽

10.1128/cvi.05004-11 ◽

2011 ◽

Vol 18 (6) ◽

pp. 901-906 ◽

Cited By ~ 10

Author(s):

Christopher G. Earnhart ◽

DeLacy V. L. Rhodes ◽

Richard T. Marconi

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Disulfide Bonds ◽

Vaccine Candidate ◽

Site Directed Mutagenesis ◽

Yield Strain ◽

Content Type ◽

Oligomer Formation ◽

Lyme Disease Vaccine

ABSTRACTBorrelia burgdorferiOspC is an outer membrane lipoprotein required for the establishment of infection in mammals. Due to its universal distribution amongB. burgdorferisensu lato strains and high antigenicity, it is being explored for the development of a next-generation Lyme disease vaccine. An understanding of the surface presentation of OspC will facilitate efforts to maximize its potential as a vaccine candidate. OspC forms homodimers at the cell surface, and it has been hypothesized that it may also form oligomeric arrays. Here, we employ site-directed mutagenesis to test the hypothesis that interdimeric disulfide bonds at cysteine 130 (C130) mediate oligomerization.B. burgdorferiB31ospCwas replaced with a C130A substitution mutant to yield strain B31::ospC(C130A). Recombinant protein was also generated. Disulfide-bond-dependent oligomer formation was demonstrated and determined to be dependent on C130. Oligomerization was not required forin vivofunction, as B31::ospC(C130A) retained infectivity and disseminated normally. The total IgG response and the induced isotype pattern were similar between mice infected with untransformed B31 and those infected with the B31::ospC(C130A) strain. These data indicate that the immune response to OspC is not significantly altered by formation of OspC oligomers, a finding that has significant implications in Lyme disease vaccine design.

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Borrelia burgdorferi RevA Significantly Affects Pathogenicity and Host Response in the Mouse Model of Lyme Disease

Infection and Immunity ◽

10.1128/iai.00530-15 ◽

2015 ◽

Vol 83 (9) ◽

pp. 3675-3683 ◽

Cited By ~ 8

Author(s):

Rebecca Byram ◽

Robert A. Gaultney ◽

Angela M. Floden ◽

Christopher Hellekson ◽

Brandee L. Stone ◽

...

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Host Response ◽

Collagen Deposition ◽

Wild Type ◽

Content Type ◽

Cardiac Tissues ◽

Monocyte Chemoattractant ◽

Bacterial Interaction ◽

Mammalian Infection

The Lyme disease spirochete,Borrelia burgdorferi, expresses RevA and numerous outer surface lipoproteins during mammalian infection. As an adhesin that promotes bacterial interaction with fibronectin, RevA is poised to interact with the extracellular matrix of the host. To further define the role(s) of RevA during mammalian infection, we created a mutant that is unable to produce RevA. The mutant was still infectious to mice, although it was significantly less well able to infect cardiac tissues. Complementation of the mutant with a wild-typerevAgene restored heart infectivity to wild-type levels. Additionally,revAmutants led to increased evidence of arthritis, with increased fibrotic collagen deposition in tibiotarsal joints. The mutants also induced increased levels of the chemokine CCL2, a monocyte chemoattractant, in serum, and this increase was abolished in the complemented strain. Therefore, whilerevAis not absolutely essential for infection, deletion ofrevAhad distinct effects on dissemination, arthritis severity, and host response.

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Regulatory Protein BBD18 of the Lyme Disease Spirochete: Essential Role During Tick Acquisition?

mBio ◽

10.1128/mbio.01017-14 ◽

2014 ◽

Vol 5 (2) ◽

Cited By ~ 4

Author(s):

Beth M. Hayes ◽

Daniel P. Dulebohn ◽

Amit Sarkar ◽

Kit Tilly ◽

Aaron Bestor ◽

...

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Critical Role ◽

Regulatory Protein ◽

Site Directed Mutagenesis ◽

Mammalian Host ◽

Content Type ◽

Block Transmission ◽

Infectious Cycle

ABSTRACTThe Lyme disease spirocheteBorrelia burgdorferisenses and responds to environmental cues as it transits between the tick vector and vertebrate host. Failure to properly adapt can block transmission of the spirochete and persistence in either vector or host. We previously identified BBD18, a novel plasmid-encoded protein ofB. burgdorferi, as a putative repressor of the host-essential factor OspC. In this study, we investigate thein vivorole of BBD18 as a regulatory protein, using an experimental mouse-tick model system that closely resembles the natural infectious cycle ofB. burgdorferi. We show that spirochetes that have been engineered to constitutively produce BBD18 can colonize and persist in ticks but do not infect mice when introduced by either tick bite or needle inoculation. Conversely, spirochetes lacking BBD18 can persistently infect mice but are not acquired by feeding ticks. Through site-directed mutagenesis, we have demonstrated that abrogation of spirochete infection in mice by overexpression of BBD18 occurs only withbbd18alleles that can suppress OspC synthesis. Finally, we demonstrate that BBD18-mediated regulation does not utilize a previously describedospCoperator sequence required byB. burgdorferifor persistence in immunocompetent mice. These data lead us to conclude that BBD18 does not represent the putative repressor utilized byB. burgdorferifor the specific downregulation of OspC in the mammalian host. Rather, we suggest that BBD18 exhibits features more consistent with those of a global regulatory protein whose critical role occurs during spirochete acquisition by feeding ticks.IMPORTANCELyme disease, caused byBorrelia burgdorferi, is the most common arthropod-borne disease in North America.B. burgdorferiis transmitted to humans and other vertebrate hosts by ticks as they take a blood meal. Transmission between vectors and hosts requires the bacterium to sense changes in the environment and adapt. However, the mechanisms involved in this process are not well understood. By determining howB. burgdorfericycles between two very different environments, we can potentially establish novel ways to interfere with transmission and limit infection of this vector-borne pathogen. We are studying a regulatory protein called BBD18 that we recently described. We found that too much BBD18 interferes with the spirochete’s ability to establish infection in mice, whereas too little BBD18 appears to prevent colonization in ticks. Our study provides new insight into key elements of the infectious cycle of the Lyme disease spirochete.

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BBK07, a Dominant In Vivo Antigen of Borrelia burgdorferi, Is a Potential Marker for Serodiagnosis of Lyme Disease

Clinical and Vaccine Immunology ◽

10.1128/cvi.00301-09 ◽

2009 ◽

Vol 16 (11) ◽

pp. 1569-1575 ◽

Cited By ~ 8

Author(s):

Adam S. Coleman ◽

Utpal Pal

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Amino Acid Identity ◽

Amino Terminal ◽

Terminal Domain ◽

Potential Marker ◽

Mammalian Infection ◽

Amino Terminal Domain

ABSTRACT One of the recently identified Borrelia burgdorferi immunogens, BBK07, is characterized for its expression in the spirochete infection cycle and evaluated for its potential use as a serodiagnostic marker for Lyme disease. We show that the BBK07 gene is expressed at extremely low levels in vitro and in ticks but is dramatically induced by spirochetes once introduced into the host and is highly expressed throughout mammalian infection. In contrast, the expression of BBK12, a paralog of BBK07 with 87% amino acid identity, although expressed in vitro, remained undetectable in vivo throughout murine infection and in ticks. BBK07 is localized in the outer membrane, and the amino-terminal domain of the antigen is exposed on the microbial surface. A truncated BBK07 protein representing the amino-terminal domain is able to effectively detect antibodies to B. burgdorferi, both in experimentally infected mice and in humans. Further characterization of the immunodominant antigens of B. burgdorferi, such as BBK07, could contribute to the development of novel serodiagnostic markers for detection of Lyme disease.

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BBB07 contributes to, but is not essential for, Borrelia burgdorferi infection in mice

Microbiology ◽

10.1099/mic.0.000972 ◽

2020 ◽

Vol 166 (10) ◽

pp. 988-994

Author(s):

Beth Hahn ◽

Phillip Anderson ◽

Zouyan Lu ◽

Rebecca Danner ◽

Zhipeng Zhou ◽

...

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Type Species ◽

Inflammatory Responses ◽

Synovial Cells ◽

Single Strain ◽

Content Type ◽

Transposon Mutants ◽

Integrin Ligand

Borrelia burgdorferi, a causative agent of Lyme disease, encodes a protein BBB07 on the genomic plasmid cp26. BBB07 was identified as a candidate integrin ligand based on the presence of an RGD tripeptide motif, which is present in a number of mammalian ligands for β1 and β3 integrins . Previous work demonstrated that BBB07 in recombinant form binds to β1 integrins and induces inflammatory responses in synovial cells in culture. Several transposon mutants in bbb07 were attenuated in an in vivo screen of the transposon library in mice. We therefore tested individual transposon mutant clones in single-strain infections in mice and found that they were attenuated in terms of ID50 but did not have significantly reduced tissue burdens in mice. Based on data presented here we conclude that BBB07 is not essential for, but does contribute to, B. burgdorferi infectivity in mice.

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Enhanced Detection of Host Response Antibodies to Borrelia burgdorferi Using Immuno-PCR

Clinical and Vaccine Immunology ◽

10.1128/cvi.00630-12 ◽

2013 ◽

Vol 20 (3) ◽

pp. 350-357 ◽

Cited By ~ 21

Author(s):

Micah D. Halpern ◽

Sunny Jain ◽

Mollie W. Jewett

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Host Response ◽

Magnetic Beads ◽

Disease Patient ◽

Rank Correlation ◽

Protein Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Content Type

ABSTRACTLyme disease is the fastest-growing zoonotic disease in North America. Current methods for detection ofBorrelia burgdorferiinfection are challenged by analysis subjectivity and standardization of antigen source. In the present study, we developed an immuno-PCR (iPCR)-based approach employing recombinantin vivo-expressedB. burgdorferiantigens for objective detection of a host immune response toB. burgdorferiinfection. iPCR is a liquid-phase protein detection method that combines the sensitivity of PCR with the specificity and versatility of immunoassay-based protocols. Use of magnetic beads coated with intact spirochetes provided effective antigen presentation and allowed detection of host-generated antibodies in experimentally infected mice at day 11 postinoculation, whereas host-generated antibodies were detected at day 14 by enzyme-linked immunosorbent assay (ELISA) and day 21 by immunoblotting. Furthermore, magnetic beads coated with recombinantB. burgdorferi in vivo-expressed antigen OspC or BmpA demonstrated positive detection of host-generated antibodies in mice at day 7 postinoculation with markedly increased iPCR signals above the background, with the quantification cycle (Cq) value for each sample minus the mean backgroundCqplus 3 standard deviations (ΔCq) being 4 to 10, whereas ΔCqwas 2.5 for intact spirochete-coated beads. iPCR demonstrated a strong correlation (Spearman rank correlation = 0.895,P< 0.0001) with a commercial ELISA for detection of host antibodies in human Lyme disease patient sera using theB. burgdorferiVlsE C6 peptide. In addition, iPCR showed potential applicability for direct detection of spirochetes in blood. The results presented here indicate that our iPCR assay has the potential to provide an objective format that can be used for sensitive detection of multiple host response antibodies and isotypes toB. burgdorferiinfection.

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Manganese and Zinc Regulate Virulence Determinants in Borrelia burgdorferi

Infection and Immunity ◽

10.1128/iai.00507-13 ◽

2013 ◽

Vol 81 (8) ◽

pp. 2743-2752 ◽

Cited By ~ 23

Author(s):

Bryan Troxell ◽

Meiping Ye ◽

Youyun Yang ◽

Sebastian E. Carrasco ◽

Yongliang Lou ◽

...

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Critical Role ◽

Transition Metal Ions ◽

Mammalian Host ◽

Virulence Determinants ◽

Temperature Dependent ◽

Content Type ◽

Mammalian Infection

ABSTRACTBorrelia burgdorferi, the causative agent of Lyme disease, must adapt to two diverse niches, an arthropod vector and a mammalian host. RpoS, an alternative sigma factor, plays a central role in spirochetal adaptation to the mammalian host by governing expression of many genes important for mammalian infection.B. burgdorferiis known to be unique in metal utilization, and little is known of the role of biologically available metals inB. burgdorferi. Here, we identified two transition metal ions, manganese (Mn2+) and zinc (Zn2+), that influenced regulation of RpoS. The intracellular Mn2+level fluctuated approximately 20-fold under different conditions and inversely correlated with levels of RpoS and the major virulence factor OspC. Furthermore, an increase in intracellular Mn2+repressed temperature-dependent induction of RpoS and OspC; this repression was overcome by an excess of Zn2+. Conversely, a decrease of intracellular Mn2+by deletion of the Mn2+transporter gene,bmtA, resulted in elevated levels of RpoS and OspC. Mn2+affected RpoS through BosR, a Fur family homolog that is required forrpoSexpression: elevated intracellular Mn2+levels greatly reduced the level of BosR protein but not the level ofbosRmRNA. Thus, Mn2+and Zn2+appeared to be important in modulation of the RpoS pathway that is essential to the life cycle of the Lyme disease spirochete. This finding supports the emerging notion that transition metals such as Mn2+and Zn2+play a critical role in regulation of virulence in bacteria.

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Versatile Roles of CspA Orthologs in Complement Inactivation of Serum-Resistant Lyme Disease Spirochetes

Infection and Immunity ◽

10.1128/iai.01094-13 ◽

2013 ◽

Vol 82 (1) ◽

pp. 380-392 ◽

Cited By ~ 30

Author(s):

Claudia Hammerschmidt ◽

Arno Koenigs ◽

Corinna Siegel ◽

Teresia Hallström ◽

Christine Skerka ◽

...

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Complement Activation ◽

Direct Interaction ◽

Inhibitory Effect ◽

Factor H ◽

Content Type ◽

Terminal Complement Complex ◽

Terminal Complement

ABSTRACTCspA of the Lyme disease spirocheteBorrelia burgdorferirepresents a key molecule in immune evasion, protecting borrelial cells from complement-mediated killing. As previous studies focused almost exclusively on CspA ofB. burgdorferi, here we investigate the different binding capacities of CspA orthologs ofBorrelia burgdorferi,B. afzelii, andB. spielmaniifor complement regulator factor H and plasminogen and their ability to inhibit complement activation by either binding these host-derived plasma proteins or independently by direct interaction with components involved in formation of the lethal, pore-like terminal complement complex. To further examine their function in serum resistancein vivo, a serum-sensitiveB. gariniistrain was used to generate spirochetes, ectopically producing functional CspA orthologs. Irrespective of their species origin, all three CspA orthologs impart resistance to complement-mediated killing when produced in a serum-sensitiveB. gariniisurrogate strain. To analyze the inhibitory effect on complement activation and to assess the potential to inactivate C3b by binding of factor H and plasminogen, recombinant CspA orthologs were also investigated. All three CspA orthologs simultaneously bound factor H and plasminogen but differed in regard to their capacity to inactivate C3b via bound plasmin(ogen) and inhibit formation of the terminal complement complex. CspA ofB. afzeliibinds plasmin(ogen) and inhibits the terminal complement complex more efficiently than CspA ofB. burgdorferiandB. spielmanii. Taken together, CspA orthologs of serum-resistant Lyme disease spirochetes act as multifunctional evasion molecules that inhibit complement on two central activation levels, C3b generation and assembly of the terminal complement complex.

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A Surface Enolase Participates in Borrelia burgdorferi-Plasminogen Interaction and Contributes to Pathogen Survival within Feeding Ticks

Infection and Immunity ◽

10.1128/iai.05671-11 ◽

2011 ◽

Vol 80 (1) ◽

pp. 82-90 ◽

Cited By ~ 46

Author(s):

Sarah Veloso Nogueira ◽

Alexis A. Smith ◽

Jin-Hong Qin ◽

Utpal Pal

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Bacterial Pathogen ◽

Active Immunization ◽

Surface Proteins ◽

Content Type ◽

Multiple Organs ◽

Membrane Bound ◽

Native Antigen

ABSTRACTBorrelia burgdorferi, a tick-borne bacterial pathogen, causes a disseminated infection involving multiple organs known as Lyme disease. Surface proteins can directly participate in microbial virulence by facilitating pathogen dissemination via interaction with host factors. We show here that a fraction of theB. burgdorferichromosomal gene product BB0337, annotated as enolase or phosphopyruvate dehydratase, is associated with spirochete outer membrane and is surface exposed.B. burgdorferienolase, either in a recombinant form or as a membrane-bound native antigen, displays enzymatic activities intrinsic to the glycolytic pathway. However, the protein also interacts with host plasminogen, potentially leading to its activation and resulting inB. burgdorferi-induced fibrinolysis. As expected, enolase displayed consistent expressionin vivo, however, with a variable temporal and spatial expression during spirochete infection in mice and ticks. Despite an extracellular exposure of the antigen and a potential role in host-pathogen interaction, active immunization of mice with recombinant enolase failed to evoke protective immunity against subsequentB. burgdorferiinfection. In contrast, enolase immunization of murine hosts significantly reduced the acquisition of spirochetes by feeding ticks, suggesting that the protein could have a stage-specific role inB. burgdorferisurvival in the feeding vector. Strategies to interfere with the function of surface enolase could contribute to the development of novel preventive measures to interrupt the spirochete infection cycle and reduce the incidences of Lyme disease.

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