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

Microbiology ◽  
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.

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

2011 ◽  
Vol 18 (6) ◽  
pp. 901-906 ◽  
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.

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

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
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.

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

2013 ◽  
Vol 20 (3) ◽  
pp. 350-357 ◽  
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.

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

2013 ◽  
Vol 82 (1) ◽  
pp. 380-392 ◽  
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.

scholarly journals Interleukin-10 Alters Effector Functions of Multiple Genes Induced by Borrelia burgdorferi in Macrophages To Regulate Lyme Disease Inflammation

2011 ◽  
Vol 79 (12) ◽  
pp. 4876-4892 ◽  
Author(s):  
Aarti Gautam ◽  
Saurabh Dixit ◽  
Mario T. Philipp ◽  
Shree R. Singh ◽  
Lisa A. Morici ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Inflammatory Responses ◽  
Interleukin 10 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Effector Functions ◽  
Multiple Genes

ABSTRACTInterleukin-10 (IL-10) modulates inflammatory responses elicitedin vitroandin vivobyBorrelia burgdorferi, the Lyme disease spirochete. How IL-10 modulates these inflammatory responses still remains elusive. We hypothesize that IL-10 inhibits effector functions of multiple genes induced byB. burgdorferiin macrophages to control concomitantly elicited inflammation. Because macrophages are essential in the initiation of inflammation, we used mouse J774 macrophages and liveB. burgdorferispirochetes as the model target cell and stimulant, respectively. First, we employed transcriptome profiling to identify genes that were induced by stimulation of cells with live spirochetes and that were perturbed by addition of IL-10 to spirochete cultures. Spirochetes significantly induced upregulation of 347 genes at both the 4-h and 24-h time points. IL-10 inhibited the expression levels, respectively, of 53 and 65 of the 4-h and 24-h genes, and potentiated, respectively, at 4 h and 24 h, 65 and 50 genes. Prominent among the novel identified IL-10-inhibited genes also validated by quantitative real-time PCR (qRT-PCR) were Toll-like receptor 1 (TLR1), TLR2, IRAK3, TRAF1, IRG1, PTGS2, MMP9, IFI44, IFIT1, and CD40. Proteome analysis using a multiplex enzyme-linked immunosorbent assay (ELISA) revealed the IL-10 modulation/and or potentiation of RANTES/CCL5, macrophage inflammatory protein 2 (MIP-2)/CXCL2, IP-10/CXCL10, MIP-1α/CCL3, granulocyte colony-stimulating factor (G-CSF)/CSF3, CXCL1, CXCL5, CCL2, CCL4, IL-6, tumor necrosis factor alpha (TNF-α), IL-1α, IL-1β, gamma interferon (IFN-γ), and IL-9. Similar results were obtained using sonicated spirochetes or lipoprotein as stimulants. Our data show that IL-10 alters effectors induced byB. burgdorferiin macrophages to control concomitantly elicited inflammatory responses. Moreover, for the first time, this study provides global insight into potential mechanisms used by IL-10 to control Lyme disease inflammation.

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

2016 ◽  
Vol 23 (8) ◽  
pp. 725-731 ◽  
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.

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

2011 ◽  
Vol 80 (1) ◽  
pp. 82-90 ◽  
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.

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

2014 ◽  
Vol 81 (3) ◽  
pp. 1038-1046 ◽  
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.

Evaluation of in vivo expressed Borrelia burgdorferi antigens for improved IgM serodiagnosis of early Lyme disease

2019 ◽  
Vol 93 (3) ◽  
pp. 196-202 ◽  
Author(s):  
Kevin S. Brandt ◽  
Amy J. Ullmann ◽  
Claudia R. Molins ◽  
Kalanthe Horiuchi ◽  
Brad J. Biggerstaff ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi

scholarly journals Genetic regulation, biochemical properties and physiological importance of arginase from Sinorhizobium meliloti

Microbiology ◽  
2020 ◽  
Vol 166 (5) ◽  
pp. 484-497 ◽  
Author(s):  
Alejandra Arteaga Ide ◽  
Victor M. Hernández ◽  
Liliana Medina-Aparicio ◽  
Edson Carcamo-Noriega ◽  
Lourdes Girard ◽  
...  
Keyword(s):  
Type Species ◽  
Sinorhizobium Meliloti ◽  
Arginase Activity ◽  
Wild Type ◽  
Mobility Shift ◽  
Content Type ◽  
Link Type ◽  
Arginine Catabolism

In bacteria, l-arginine is a precursor of various metabolites and can serve as a source of carbon and/or nitrogen. Arginine catabolism by arginase, which hydrolyzes arginine to l-ornithine and urea, is common in nature but has not been studied in symbiotic nitrogen-fixing rhizobia. The genome of the alfalfa microsymbiont Sinorhizobium meliloti 1021 has two genes annotated as arginases, argI1 (smc03091) and argI2 (sma1711). Biochemical assays with purified ArgI1 and ArgI2 (as 6His-Sumo-tagged proteins) showed that only ArgI1 had detectable arginase activity. A 1021 argI1 null mutant lacked arginase activity and grew at a drastically reduced rate with arginine as sole nitrogen source. Wild-type growth and arginase activity were restored in the argI1 mutant genetically complemented with a genomically integrated argI1 gene. In the wild-type, arginase activity and argI1 transcription were induced several fold by exogenous arginine. ArgI1 purified as a 6His-Sumo-tagged protein had its highest in vitro enzymatic activity at pH 7.5 with Ni2+ as cofactor. The enzyme was also active with Mn2+ and Co2+, both of which gave the enzyme the highest activities at a more alkaline pH. The 6His-Sumo-ArgI1 comprised three identical subunits based on the migration of the urea-dissociated protein in a native polyacrylamide gel. A Lrp-like regulator (smc03092) divergently transcribed from argI1 was required for arginase induction by arginine or ornithine. This regulator was designated ArgIR. Electrophoretic mobility shift assays showed that purified ArgIR bound to the argI1 promoter in a region preceding the predicted argI1 transcriptional start. Our results indicate that ArgI1 is the sole arginase in S. meliloti , that it contributes substantially to arginine catabolism in vivo and that argI1 induction by arginine is dependent on ArgIR.

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