scholarly journals Borrelia burgdorferi bba74 Is Expressed Exclusively during Tick Feeding and Is Regulated by Both Arthropod- and Mammalian Host-Specific Signals

2009 ◽  
Vol 191 (8) ◽  
pp. 2783-2794 ◽  
Author(s):  
Vishwaroop B. Mulay ◽  
Melissa J. Caimano ◽  
Radha Iyer ◽  
Star Dunham-Ems ◽  
Dionysios Liveris ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Elevated Temperatures ◽  
Temperature Shift ◽  
Mammalian Host ◽  
Wild Type ◽  
Tick Feeding ◽  
Reciprocal Regulation ◽  
Heterogeneous Expression ◽  
Membrane Spanning

ABSTRACT Although BBA74 initially was described as a 28-kDa virulence-associated outer-membrane-spanning protein with porin-like function, subsequent studies revealed that it is periplasmic and downregulated in mammalian host-adapted spirochetes. To further elucidate the role of this protein in the Borrelia burgdorferi tick-mammal cycle, we conducted a thorough examination of its expression profile in comparison with the profiles of three well-characterized, differentially expressed borrelial genes (ospA, ospC, and ospE) and their proteins. In vitro, transcripts for bba74 were expressed at 23°C and further enhanced by a temperature shift (37°C), whereas BBA74 protein diminished at elevated temperatures; in contrast, neither transcript nor protein was expressed by spirochetes grown in dialysis membrane chambers (DMCs). Primer extension of wild-type B. burgdorferi grown in vitro, in conjunction with expression analysis of DMC-cultivated wild-type and rpoS mutant spirochetes, revealed that, like ospA, bba74 is transcribed by σ70 and is subject to RpoS-mediated repression within the mammalian host. A series of experiments utilizing wild-type and rpoS mutant spirochetes was conducted to determine the transcriptional and translational profiles of bba74 during the tick-mouse cycle. Results from these studies revealed (i) that bba74 is transcribed by σ70 exclusively during the larval and nymphal blood meals and (ii) that transcription of bba74 is bracketed by RpoS-independent and -dependent forms of repression that are induced by arthropod- and mammalian host-specific signals, respectively. Although loss of BBA74 does not impair the ability of B. burgdorferi to complete its infectious life cycle, the temporal compartmentalization of this gene's transcription suggests that BBA74 facilitates fitness of the spirochete within a narrow window of its tick phase. A reexamination of the paradigm for reciprocal regulation of ospA and ospC, performed herein, revealed that the heterogeneous expression of OspA and OspC displayed by spirochete populations during the nymphal blood meal results from the intricate sequence of transcriptional and translational changes that ensue as B. burgdorferi transitions between its arthropod vector and mammalian host.

scholarly journals Expression of a luxS Gene Is Not Required for Borrelia burgdorferi Infection of Mice via Needle Inoculation

2003 ◽  
Vol 71 (5) ◽  
pp. 2892-2896 ◽  
Author(s):  
Anette Hübner ◽  
Andrew T. Revel ◽  
Dena M. Nolen ◽  
Kayla E. Hagman ◽  
Michael V. Norgard
Keyword(s):  
Quorum Sensing ◽  
Borrelia Burgdorferi ◽  
Mammalian Host ◽  
Wild Type ◽  
Sensing System ◽  
Autoinducer 2 ◽  
Sensing Systems ◽  
Quorum Sensing System ◽  
Culture Supernatants

ABSTRACT The luxS gene product is an integral component of LuxS/autoinducer-2 (AI-2) quorum-sensing systems in bacteria. A putative luxS gene was expressed at comparable levels by Borrelia burgdorferi strain 297 cultivated either in vitro or in dialysis membrane chambers implanted in rat peritoneal cavities. Although the borrelial luxS gene functionally complemented a LuxS deficiency in Escherichia coli DH5α, AI-2-like activity could not be detected within B. burgdorferi culture supernatants or concentrated cell lysates. Finally, a luxS-deficient mutant of B. burgdorferi was infectious at wild-type levels when it was intradermally needle inoculated into mice, indicating that expression of luxS probably is not required for infectivity but, at the very least, is not essential for mammalian host adaptation. Our findings also challenge the notion that a LuxS/AI-2 quorum-sensing system is operative in B. burgdorferi.

scholarly journals RpoS Is Not Central to the General Stress Response in Borrelia burgdorferi but Does Control Expression of One or More Essential Virulence Determinants

2004 ◽  
Vol 72 (11) ◽  
pp. 6433-6445 ◽  
Author(s):  
Melissa J. Caimano ◽  
Christian H. Eggers ◽  
Karsten R. O. Hazlett ◽  
Justin D. Radolf
Keyword(s):  
Escherichia Coli ◽  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Temperature Shift ◽  
Mammalian Host ◽  
Virulence Determinants ◽  
Wild Type ◽  
Rt Pcr ◽  
Oxygen Intermediates ◽  
Wide Range

ABSTRACT Borrelia burgdorferi, the Lyme disease spirochete, undergoes dramatic changes in antigenic composition as it cycles between its arthropod and mammalian hosts. A growing body of evidence suggests that these changes reflect, at least in part, the need for spirochetes to adapt to the physiological stresses imposed by abrupt changes in environmental conditions and nutrient availability. In many microorganisms, global responses are mediated by master regulators such as alternative sigma factors, with Escherichia coli RpoS (σS) serving as a prototype. The importance of this transcriptional activator in other bacteria, coupled with the report by Hübner et al. (A. Hübner, X. Yang, D. M. Nolen, T. G. Popova, F. C. Cabello, and M. V. Norgard, Proc. Natl. Acad. Sci. USA 98:12724-12729, 2001) demonstrating that the borrelial RpoS ortholog controls expression of OspC and decorin-binding protein A (DbpA), prompted us to examine more closely the roles of RpoS-dependent and -independent differential gene expression in physiological adaptation by the Lyme disease spirochete. We observed that B. burgdorferi rpoS (rpoSBb ) was induced following temperature shift and transcript levels were further enhanced by reduced pH (pH 6.8). Using quantitative real-time reverse transcription-PCR (RT-PCR), we demonstrated that, in contrast to its ortholog (rpoSEc ) in Escherichia coli, rpoSBb was expressed at significant levels in B. burgdorferi throughout all phases of growth following temperature shift. By comparing a B. burgdorferi strain 297 rpoSBb mutant to its wild-type counterpart, we determined that RpoS Bb was not required for survival following exposure to a wide range of environmental stresses (i.e., temperature shift, serum starvation, increased osmolality, reactive oxygen intermediates, and increased or reduced oxygen tension), although the mutant was more sensitive to extremes of pH. While B. burgdorferi strains lacking RpoS were able to survive within intraperitoneal dialysis membrane chambers at a level equivalent to that of the wild type, they were avirulent in mice. Lastly, RT-PCR analysis of the ospE-ospF-elp paralogous lipoprotein families complements earlier findings that many temperature-inducible borrelial loci are controlled in an RpoS Bb -independent manner. Together, these data point to fundamental differences between the role(s) of RpoS in B. burgdorferi and that in E. coli. Rather than functioning as a master regulator, RpoS Bb appears to serve as a stress-responsive activator of a subset of virulence determinants that, together with the RpoS-independent, differentially expressed regulon, encompass the spirochete's genetic programs required for mammalian host adaptation.

scholarly journals Changes in Temporal and Spatial Patterns of Outer Surface Lipoprotein Expression Generate Population Heterogeneity and Antigenic Diversity in the Lyme Disease Spirochete, Borrelia burgdorferi

2002 ◽  
Vol 70 (7) ◽  
pp. 3468-3478 ◽  
Author(s):  
P. Scott Hefty ◽  
Sarah E. Jolliff ◽  
Melissa J. Caimano ◽  
Stephen K. Wikel ◽  
Darrin R. Akins
Keyword(s):  
Borrelia Burgdorferi ◽  
Salivary Glands ◽  
Cellular Localization ◽  
Expression Patterns ◽  
Population Heterogeneity ◽  
Mammalian Host ◽  
Tick Feeding ◽  
Temporal And Spatial ◽  
Cultivation In Vitro

ABSTRACT Borrelia burgdorferi differentially expresses many of the OspE/F/Elp paralogs during tick feeding. These findings, combined with the recent report that stable B. burgdorferi infection of mammals occurs only after 53 h of tick attachment, prompted us to further analyze the expression of the OspE/F/Elp paralogs during this critical period of transmission. Indirect immunofluorescence analysis revealed that OspE, p21, ElpB1, ElpB2, and OspF/BbK2.11 are expressed in the salivary glands of ticks allowed to feed on mice for 53 to 58 h. Interestingly, many of the spirochetes in the salivary glands that expressed abundant amounts of these antigens were negative for OspA and OspC. Although prior reports have indicated that OspE/F/Elp orthologs are surface exposed, none of the individual lipoproteins or combinations of the lipoproteins protected mice from challenge infections. To examine why these apparently surface-exposed lipoproteins were not protective, we analyzed their genetic stability during infection and their cellular locations after cultivation in vitro and within dialysis membrane chambers, mimicking a mammalian host-adapted state. Combined restriction fragment length polymorphism and nucleotide sequence analyses revealed that the genes encoding these lipoproteins are stable for at least 8 months postinfection. Interestingly, cellular localization experiments revealed that while all of these proteins can be surface localized, there were significant populations of spirochetes that expressed these lipoproteins only in the periplasm. Furthermore, host-specific signals were found to alter the expression patterns and final cellular location of these lipoproteins. The combined data revealed a remarkable heterogeneity in populations of B. burgdorferi during tick transmission and mammalian infection. The diversity is generated not only by temporal changes in antigen expression but also by modulation of the surface lipoproteins during infection. The ability to regulate the temporal and spatial expression patterns of lipoproteins throughout infection likely contributes to persistent infection of mammals by B. burgdorferi.

scholarly journals Regulation of OspE-Related, OspF-Related, and Elp Lipoproteins of Borrelia burgdorferi Strain 297 by Mammalian Host-Specific Signals

2001 ◽  
Vol 69 (6) ◽  
pp. 3618-3627 ◽  
Author(s):  
P. Scott Hefty ◽  
Sarah E. Jolliff ◽  
Melissa J. Caimano ◽  
Stephen K. Wikel ◽  
Justin D. Radolf ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Posttranscriptional Regulation ◽  
Expression Profiles ◽  
Pcr Analysis ◽  
Mammalian Host ◽  
Promoter Regions ◽  
Tick Feeding ◽  
Reverse Transcription Pcr ◽  
Encoding Genes

ABSTRACT In previous studies we have characterized the cp32/18 loci inBorrelia burgdorferi 297 which encode OspE and OspF orthologs and a third group of lipoproteins which possess OspE/F-like leader peptides (Elps). To further these studies, we have comprehensively analyzed their patterns of expression throughout the borrelial enzootic cycle. Serial dilution reverse transcription-PCR analysis indicated that although a shift in temperature from 23 to 37°C induced transcription for all nine genes analyzed, this effect was often markedly enhanced in mammalian host-adapted organisms cultivated within dialysis membrane chambers (DMCs) implanted within the peritoneal cavities of rats. Indirect immunofluorescence assays performed on temperature-shifted, in vitro-cultivated spirochetes and organisms in the midguts of unfed and fed ticks revealed distinct expression profiles for many of the OspE-related, OspF-related, and Elp proteins. Other than BbK2.10 and ElpA1, all were expressed by temperature-shifted organisms, while only OspE, ElpB1, OspF, and BbK2.11 were expressed in the midguts of fed ticks. Additionally, although mRNA was detected for all nine lipoprotein-encoding genes, two of these proteins (BbK2.10 and ElpA1) were not expressed by spirochetes cultivated in vitro, within DMCs, or by spirochetes within tick midguts. However, the observation that B. burgdorferi-infected mice generated specific antibodies against BbK2.10 and ElpA1 indicated that these antigens are expressed only in the mammalian host and that a form of posttranscriptional regulation is involved. Analysis of the upstream regions of these genes revealed several differences between their promoter regions, the majority of which were found in the −10 and −35 hexamers and the spacer regions between them. Also, rather than undergoing simultaneous upregulation during tick feeding, these genes and the corresponding lipoproteins appear to be subject to progressive recruitment or enhancement of expression as B. burgdorferi is transmitted from its tick vector to the mammalian host. These findings underscore the potential relevance of these molecules to the pathogenic events of early Lyme disease.

scholarly journals The BB0345 Hypothetical Protein of Borrelia burgdorferi Is Essential for Mammalian Infection

2020 ◽  
Vol 88 (12) ◽  
Author(s):  
Danielle E. Graham ◽  
Ashley M. Groshong ◽  
Clay D. Jackson-Litteken ◽  
Brendan P. Moore ◽  
Melissa J. Caimano ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Transcriptional Regulator ◽  
Hypothetical Protein ◽  
Proteinase K ◽  
Mammalian Host ◽  
Wild Type ◽  
Content Type ◽  
Cell Partitioning ◽  
Immunocompetent Mice

ABSTRACT During the natural enzootic life cycle of Borrelia burgdorferi (also known as Borreliella burgdorferi), the bacteria must sense conditions within the vertebrate and arthropod and appropriately regulate expression of genes necessary to persist within these distinct environments. bb0345 of B. burgdorferi encodes a hypothetical protein of unknown function that is predicted to contain an N-terminal helix-turn-helix (HTH) domain. Because HTH domains can mediate protein-DNA interactions, we hypothesized that BB0345 might represent a previously unidentified borrelial transcriptional regulator with the ability to regulate events critical for the B. burgdorferi enzootic cycle. To study the role of BB0345 within mammals, we generated a bb0345 mutant and assessed its virulence potential in immunocompetent mice. The bb0345 mutant was able to initiate localized infection and disseminate to distal tissues but was cleared from all sites by 14 days postinfection. In vitro growth curve analyses revealed that the bb0345 mutant grew similar to wild-type bacteria in standard Barbour-Stoenner-Kelley II (BSK-II) medium; however, the mutant was not able to grow in dilute BSK-II medium or dialysis membrane chambers (DMCs) implanted in rats. Proteinase K accessibility assays and whole-cell partitioning indicated that BB0345 was intracellular and partially membrane associated. Comparison of protein production profiles between the wild-type parent and the bb0345 mutant revealed no major differences, suggesting BB0345 may not be a global transcriptional regulator. Taken together, these data show that BB0345 is essential for B. burgdorferi survival in the mammalian host, potentially by aiding the spirochete with a physiological function that is required by the bacterium during infection.

scholarly journals Competitive Advantage of Borrelia burgdorferi with Outer Surface Protein BBA03 during Tick-Mediated Infection of the Mammalian Host

2012 ◽  
Vol 80 (10) ◽  
pp. 3501-3511 ◽  
Author(s):  
Aaron Bestor ◽  
Ryan O. M. Rego ◽  
Kit Tilly ◽  
Patricia A. Rosa
Keyword(s):  
Borrelia Burgdorferi ◽  
Competitive Advantage ◽  
Surface Protein ◽  
Culture Conditions ◽  
Mammalian Host ◽  
Wild Type ◽  
Tick Feeding ◽  
Transmitted Infection ◽  
Content Type ◽  
Tick Transmission

ABSTRACTLinear plasmid lp54 is one of the most highly conserved and differentially expressed elements of the segmented genome of the Lyme disease spirocheteBorrelia burgdorferi. We previously reported that deletion of a 4.1-kb region of lp54 (bba01tobba07[bba01-bba07]) led to a slight attenuation of tick-transmitted infection in mice following challenge with a large number of infected ticks. In the current study, we reduced the number of ticks in the challenge to more closely mimic the natural dose and found a profound defect in tick-transmitted infection of thebba01-bba07mutant relative to wild-typeB. burgdorferi. We next focused on deletion ofbba03as the most likely cause of this mutant phenotype, as previous studies have shown that expression ofbba03is increased by culture conditions that simulate tick feeding. Consistent with this hypothesis, we demonstrated increased expression ofbba03by spirochetes in fed relative to unfed ticks. We also observed that abba03deletion mutant, although fully competent by itself, did not efficiently infect mice when transmitted by ticks that were simultaneously coinfected with wild-typeB. burgdorferi. These results suggest that BBA03 provides a competitive advantage to spirochetes carrying this protein during tick transmission to a mammalian host in the natural infectious cycle.

scholarly journals Role of the Surface Lipoprotein BBA07 in the Enzootic Cycle of Borrelia burgdorferi

2010 ◽  
Vol 78 (7) ◽  
pp. 2910-2918 ◽  
Author(s):  
Haijun Xu ◽  
Ming He ◽  
Jane Jingyuan He ◽  
X. Frank Yang
Keyword(s):  
Borrelia Burgdorferi ◽  
Protein Profile ◽  
Mammalian Host ◽  
Tick Feeding ◽  
Tick Transmission ◽  
Nymphal Tick ◽  
Encoding Gene ◽  
Enzootic Cycle

ABSTRACT Borrelia burgdorferi, the Lyme disease pathogen, dramatically alters its protein profile when it is transmitted between ticks and mammals. Several differentially expressed proteins have been shown to be critical for the enzootic cycle of B. burgdorferi. In this study, we demonstrated that expression of the surface lipoprotein-encoding gene bba07 is induced by an elevated temperature and a reduced pH during in vitro cultivation, as well as during nymphal tick feeding. Expression of bba07 is regulated by the Rrp2-RpoN-RpoS pathway, a central regulatory network that is activated during nymphal feeding. By generating a bba07 mutant of an infectious strain of B. burgdorferi, we demonstrated that although BBA07-deficient spirochetes were capable of infecting mice via needle inoculation and surviving in ticks, they were defective in infection of mammals via tick transmission. Complementation of the bba07 mutant with a wild-type copy of bba07 partially restored the transmission defect of the bba07 mutant. Based on these findings, we concluded that the surface lipoprotein BBA07 is produced during tick feeding and facilitates optimal transmission of B. burgdorferi from the tick vector to a mammalian host.

scholarly journals OspC-Independent Infection and Dissemination by Host-Adapted Borrelia burgdorferi

2009 ◽  
Vol 77 (7) ◽  
pp. 2672-2682 ◽  
Author(s):  
Kit Tilly ◽  
Aaron Bestor ◽  
Daniel P. Dulebohn ◽  
Patricia A. Rosa
Keyword(s):  
Borrelia Burgdorferi ◽  
Mouse Skin ◽  
Surface Protein ◽  
Mammalian Host ◽  
Recipient Mouse ◽  
Wild Type ◽  
Surface Protein Gene ◽  
Tick Transmission ◽  
Mammalian Infection

ABSTRACTBorrelia burgdorferiOspC is required for the spirochete to establish infection in a mammal by tick transmission or needle inoculation. After a brief essential period, the protein no longer is required and the gene can be shut off. Using a system in which spirochetes contain only an unstable wild-type copy of theospCgene, we can obtain mice persistently infected with bacteria lacking OspC. We implanted pieces of infected mouse skin subcutaneously in naïve mice, using donors carrying wild-type orospCmutant spirochetes, and found that both could infect mice by this method, with similar numbers of wild-type orospCmutant spirochetes disseminated throughout the tissues of recipient mice. Recipient mouse immune responses to tissue transfer-mediated infection with wild-type orospCmutant spirochetes were similar. These experiments demonstrate that mammalian host-adapted spirochetes can infect and disseminate in mice in the absence of OspC, thereby circumventing this hallmark of tick-derived or in vitro-grown spirochetes. We propose a model in which OspC is one of a succession of functionally equivalent, essential proteins that are synthesized at different stages of mammalian infection. In this model, another protein uniquely present on host-adapted spirochetes performs the same essential function initially fulfilled by OspC. The strict temporal control ofB. burgdorferiouter surface protein gene expression may reflect immunological constraints rather than distinct functions.

scholarly journals Borrelia burgdorferi Lacking BBK32, a Fibronectin-Binding Protein, Retains Full Pathogenicity

2006 ◽  
Vol 74 (6) ◽  
pp. 3305-3313 ◽  
Author(s):  
Xin Li ◽  
Xianzhong Liu ◽  
Deborah S. Beck ◽  
Fred S. Kantor ◽  
Erol Fikrig
Keyword(s):  
Life Cycle ◽  
Borrelia Burgdorferi ◽  
Deletion Mutant ◽  
Binding Protein ◽  
Kanamycin Resistance ◽  
Binding Activity ◽  
Mammalian Host ◽  
Wild Type ◽  
Fibronectin Binding Protein ◽  
Fibronectin Binding

ABSTRACT BBK32, a fibronectin-binding protein of Borrelia burgdorferi, is one of many surface lipoproteins that are differentially expressed by the Lyme disease spirochete at various stages of its life cycle. The level of BBK32 expression in B. burgdorferi is highest during infection of the mammalian host and lowest in flat ticks. This temporal expression profile, along with its fibronectin-binding activity, strongly suggests that BBK32 may play an important role in Lyme pathogenesis in the host. To test this hypothesis, we constructed an isogenic BBK32 deletion mutant from wild-type B. burgdorferi B31 by replacing the BBK32 gene with a kanamycin resistance cassette through homologous recombination. We examined both the wild-type strain and the BBK32 deletion mutant extensively in the experimental mouse-tick model of the Borrelia life cycle. Our data indicated that B. burgdorferi lacking BBK32 retained full pathogenicity in mice, regardless of whether mice were infected artificially by syringe inoculation or naturally by tick bite. The loss of BBK32 expression in the mutant had no adverse effect on spirochete acquisition (mouse-to-tick) and transmission (tick-to-mouse) processes. These results suggest that additional B. burgdorferi proteins can complement the function of BBK32, fibronectin binding or otherwise, during the natural spirochete life cycle.

scholarly journals Mutations Conferring Aminoglycoside and Spectinomycin Resistance in Borrelia burgdorferi

2006 ◽  
Vol 50 (2) ◽  
pp. 445-452 ◽  
Author(s):  
Daniel Criswell ◽  
Virginia L. Tobiason ◽  
J. Stephen Lodmell ◽  
D. Scott Samuels
Keyword(s):  
16S Rrna ◽  
Borrelia Burgdorferi ◽  
Type Strain ◽  
Wild Type Strain ◽  
Small Subunit ◽  
Wild Type ◽  
Spectinomycin Resistance ◽  
E Coli ◽  
Resistant Mutants

ABSTRACT We have isolated and characterized in vitro mutants of the Lyme disease agent Borrelia burgdorferi that are resistant to spectinomycin, kanamycin, gentamicin, or streptomycin, antibiotics that target the small subunit of the ribosome. 16S rRNA mutations A1185G and C1186U, homologous to Escherichia coli nucleotides A1191 and C1192, conferred >2,200-fold and 1,300-fold resistance to spectinomycin, respectively. A 16S rRNA A1402G mutation, homologous to E. coli A1408, conferred >90-fold resistance to kanamycin and >240-fold resistance to gentamicin. Two mutations were identified in the gene for ribosomal protein S12, at a site homologous to E. coli residue Lys-87, in mutants selected in streptomycin. Substitutions at codon 88, K88R and K88E, conferred 7-fold resistance and 10-fold resistance, respectively, to streptomycin on B. burgdorferi. The 16S rRNA A1185G and C1186U mutations, associated with spectinomycin resistance, appeared in a population of B. burgdorferi parental strain B31 at a high frequency of 6 × 10−6. These spectinomycin-resistant mutants successfully competed with the wild-type strain during 100 generations of coculture in vitro. The aminoglycoside-resistant mutants appeared at a frequency of 3 × 10−9 to 1 ×10−7 in a population and were unable to compete with wild-type strain B31 after 100 generations. This is the first description of mutations in the B. burgdorferi ribosome that confer resistance to antibiotics. These results have implications for the evolution of antibiotic resistance, because the 16S rRNA mutations conferring spectinomycin resistance have no significant fitness cost in vitro, and for the development of new selectable markers.

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