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

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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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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The Factor H-Binding Site of CspZ as a Protective Target against Multistrain, Tick-Transmitted Lyme Disease

Infection and Immunity ◽

10.1128/iai.00956-19 ◽

2020 ◽

Vol 88 (5) ◽

Cited By ~ 2

Author(s):

Ashley L. Marcinkiewicz ◽

Ilva Lieknina ◽

Xiuli Yang ◽

Patricia L. Lederman ◽

Thomas M. Hart ◽

...

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Binding Site ◽

Causative Agent ◽

Vaccine Candidate ◽

Binding Activity ◽

Factor H ◽

Tick Feeding ◽

Content Type ◽

Complement Regulator

ABSTRACT The spirochete Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD). The spirochetes produce the CspZ protein that binds to a complement regulator, factor H (FH). Such binding downregulates activation of host complement to facilitate spirochete evasion of complement killing. However, vaccination with CspZ does not protect against LD infection. In this study, we demonstrated that immunization with CspZ-YA, a CspZ mutant protein with no FH-binding activity, protected mice from infection by several spirochete genotypes introduced via tick feeding. We found that the sera from CspZ-YA-vaccinated mice more efficiently eliminated spirochetes and blocked CspZ FH-binding activity than sera from CspZ-immunized mice. We also found that vaccination with CspZ, but not CspZ-YA, triggered the production of anti-FH antibodies, justifying CspZ-YA as an LD vaccine candidate. The mechanistic and efficacy information derived from this study provides insights into the development of a CspZ-based LD vaccine.

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

Clinical and Vaccine Immunology ◽

10.1128/cvi.00302-16 ◽

2016 ◽

Vol 23 (8) ◽

pp. 725-731 ◽

Cited By ~ 5

Author(s):

Beth L. Hahn ◽

Lavinia J. Padmore ◽

Laura C. Ristow ◽

Michael W. Curtis ◽

Jenifer Coburn

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Parental Strain ◽

Content Type ◽

Protective Antigens ◽

Homologous Strain ◽

Live Bacteria ◽

At Risk Populations ◽

Mammalian 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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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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Regulation of Swarming Motility and flhDCSm Expression by RssAB Signaling in Serratia marcescens

Journal of Bacteriology ◽

10.1128/jb.01670-07 ◽

2008 ◽

Vol 190 (7) ◽

pp. 2496-2504 ◽

Cited By ~ 25

Author(s):

Po-Chi Soo ◽

Yu-Tze Horng ◽

Jun-Rong Wei ◽

Jwu-Ching Shu ◽

Chia-Chen Lu ◽

...

Keyword(s):

Serratia Marcescens ◽

Binding Site ◽

Promoter Region ◽

Transcriptional Start Site ◽

Direct Interaction ◽

Underlying Mechanism ◽

Inhibitory Effect ◽

Start Codon ◽

Mobility Shift

ABSTRACT Serratia marcescens cells swarm at 30°C but not at 37°C, and the underlying mechanism is not characterized. Our previous studies had shown that a temperature upshift from 30 to 37°C reduced the expression levels of flhDCSm and hagSm in S. marcescens CH-1. Mutation in rssA or rssB, cognate genes that comprise a two-component system, also resulted in precocious swarming phenotypes at 37°C. To further characterize the underlying mechanism, in the present study, we report that expression of flhDCSm and synthesis of flagella are significantly increased in the rssA mutant strain at 37°C. Primer extension analysis for determination of the transcriptional start site(s) of flhDCSm revealed two transcriptional start sites, P1 and P2, in S. marcescens CH-1. Characterization of the phosphorylated RssB (RssB∼P) binding site by an electrophoretic mobility shift assay showed direct interaction of RssB∼P, but not unphosphorylated RssB [RssB(D51E)], with the P2 promoter region. A DNase I footprinting assay using a capillary electrophoresis approach further determined that the RssB∼P binding site is located between base pair positions −341 and −364 from the translation start codon ATG in the flhDCSm promoter region. The binding site overlaps with the P2 “−35” promoter region. A modified chromatin immunoprecipitation assay was subsequently performed to confirm that RssB∼P binds to the flhDCSm promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signaling directly inhibits flhDCSm promoter activity at 37°C. This inhibitory effect was comparatively alleviated at 30°C. This finding might explain, at least in part, the phenomenon of inhibition of S. marcescens swarming at 37°C.

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Use of an Endogenous Plasmid Locus for Stable intransComplementation in Borrelia burgdorferi

Applied and Environmental Microbiology ◽

10.1128/aem.03657-14 ◽

2014 ◽

Vol 81 (3) ◽

pp. 1038-1046 ◽

Cited By ~ 5

Author(s):

Irene N. Kasumba ◽

Aaron Bestor ◽

Kit Tilly ◽

Patricia A. Rosa

Keyword(s):

Borrelia Burgdorferi ◽

Shuttle Vector ◽

Targeted Mutagenesis ◽

Multiple Cloning Site ◽

Stable Gene ◽

Content Type ◽

Shuttle Vectors ◽

Stable Maintenance

ABSTRACTTargeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirocheteBorrelia burgdorferi. A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers thanB. burgdorferiplasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle.B. burgdorferihas over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochetein vivobut relatively unstable duringin vitrocultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number andin vivostability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into thebbe02locus, a site on lp25 that was previously shown to be nonessential during bothin vitroandin vivogrowth. We demonstrate the functional utility of this strategy by restoring infectivity to anospCmutant through complementation at this site on lp25 and stable maintenance of theospCgene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation inB. burgdorferi.

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