Borrelia burgdorferi Alters Its Gene Expression and Antigenic Profile in Response to CO2 Levels

ABSTRACT The etiologic agent of Lyme disease, Borrelia burgdorferi, must adapt to the distinct environments of its arthropod vector and mammalian host during its complex life cycle. B. burgdorferi alters gene expression and protein synthesis in response to temperature, pH, and other uncharacterized environmental factors. The hypothesis tested in this study is that dissolved gases, including CO2, serve as a signal for B. burgdorferi to alter protein production and gene expression. In this study we focused on characterization of in vitro anaerobic (5% CO2, 3% H2, 0.087 ppm O2) and microaerophilic (1% CO2, 3.48 ppm O2) growth conditions and how they modulate protein synthesis and gene expression in B. burgdorferi. Higher levels of several immunoreactive proteins, including BosR, NapA, DbpA, OspC, BBK32, and RpoS, were synthesized under anaerobic conditions. Previous studies demonstrated that lower levels of NapA were produced when microaerophilic cultures were purged with nitrogen gas to displace oxygen and CO2. In this study we identified CO2 as a factor contributing to the observed change in NapA synthesis. Specifically, a reduction in the level of dissolved CO2, independent of O2 levels, resulted in reduced NapA synthesis. BosR, DbpA, OspC, and RpoS synthesis was also decreased with the displacement of CO2. Quantitative reverse transcription-PCR indicated that the levels of the dbpA, ospC, and BBK32 transcripts are increased in the presence of CO2, indicating that these putative borrelial virulence determinants are regulated at the transcriptional level. Thus, dissolved CO2 may be an additional cue for borrelial host adaptation and gene regulation.

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Profiling of Temperature-Induced Changes in Borrelia burgdorferi Gene Expression by Using Whole Genome Arrays

Infection and Immunity ◽

10.1128/iai.71.4.1689-1705.2003 ◽

2003 ◽

Vol 71 (4) ◽

pp. 1689-1705 ◽

Cited By ~ 209

Author(s):

Caroline Ojaimi ◽

Chad Brooks ◽

Sherwood Casjens ◽

Patricia Rosa ◽

Abdallah Elias ◽

...

Keyword(s):

Gene Expression ◽

Borrelia Burgdorferi ◽

The United States ◽

Etiologic Agent ◽

Open Reading Frames ◽

Differentially Expressed ◽

Mammalian Host ◽

Regulated Expression ◽

Two Temperatures ◽

Induced Changes

ABSTRACT Borrelia burgdorferi is the etiologic agent of Lyme disease, the most prevalent arthropod-borne disease in the United States. The genome of the type strain, B31, consists of a 910,725-bp linear chromosome and 21 linear and circular plasmids comprising 610,694 bp. During its life cycle, the spirochete exists in distinctly different environments, cycling between a tick vector and a mammalian host. Temperature is one environmental factor known to affect B. burgdorferi gene expression. To identify temperature-responsive genes, genome arrays containing 1,662 putative B. burgdorferi open reading frames (ORFs) were prepared on nylon membranes and employed to assess gene expression in B. burgdorferi B31 grown at 23 and 35°C. Differences in expression of more than 3.5 orders of magnitude could be readily discerned and quantitated. At least minimal expression from 91% of the arrayed ORFs could be detected. A total of 215 ORFs were differentially expressed at the two temperatures; 133 were expressed at significantly greater levels at 35°C, and 82 were more significantly expressed at 23°C. Of these 215 ORFs, 134 are characterized as genes of unknown function. One hundred thirty-six (63%) of the differentially expressed genes are plasmid encoded. Of particular interest is plasmid lp54 which contains 76 annotated putative genes; 31 of these exhibit temperature-regulated expression. These findings underscore the important role plasmid-encoded genes may play in adjustment of B. burgdorferi to growth under diverse environmental conditions.

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Changes in Gene Expression during Drying in a Desiccation-Tolerant Grass Sporobolus stapfianus and a Desiccation-Sensitive Grass Sporobolus pyramidalis

Functional Plant Biology ◽

10.1071/pp96073 ◽

1997 ◽

Vol 24 (5) ◽

pp. 617 ◽

Cited By ~ 6

Author(s):

D.F. Gaff ◽

D. Bartels ◽

J.L. Gaff

Keyword(s):

Gene Expression ◽

Desiccation Tolerance ◽

Transcriptional Level ◽

Novel Proteins ◽

Sporobolus Stapfianus ◽

Induced Changes ◽

First Time ◽

Dimensional Electrophoresis ◽

Water Contents

For the first time in the grasses, a desiccation-tolerant species (Sporobolus stapfianus) was examined for evidence of drought-induced changes in gene transcription. Desiccation tolerance (the ability of this species to recover from a water potential of –540 MPa) is induced in the resurrection grass during the drying process itself. Specific mRNA was compared in extracts of air-dry, drying and fully hydrated leaves by comparisons of the encoded proteins translated in vitro and partitioned by 2- dimensional electrophoresis. Forty-one genes, that were not expressed in hydrated leaves, were transcribed during drying, whereas only 25 novel polypeptides (translated in vitro) were detected; this suggests that gene expression was controlled mainly at the transcriptional level, but possibly also at the translational level. Leaves of S. stapfianus become desiccation tolerant as they dry on intact plants with mechanically undisturbed roots, whereas leaves on plants whose roots have been disturbed die during drying. Complements of mRNA from live S. stapfianus leaves changed markedly from full hydration to 70% RWC and to air-dryness; they also differed markedly from drought-sensitive leaves (on plants with disturbed roots) at 70% RWC and dead air-dry S. stapfianus leaves and from leaves of the desiccation sensitive grass S. pyramidalis at the same water contents. Drought-induced injury could not be attributed to low abundance of mRNA in either species. Five criteria which might be involved in desiccation tolerance were applied to specific in vitro proteins of S. stapfianus; 12 novel proteins correlated with desiccation tolerance in a least four of the five criteria.

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Role of the BBA64 Locus of Borrelia burgdorferi in Early Stages of Infectivity in a Murine Model of Lyme Disease

Infection and Immunity ◽

10.1128/iai.01118-07 ◽

2007 ◽

Vol 76 (1) ◽

pp. 391-402 ◽

Cited By ~ 37

Author(s):

Mahulena Maruskova ◽

M. Dolores Esteve-Gassent ◽

Valerie L. Sexton ◽

J. Seshu

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Immunoblot Analysis ◽

Open Reading Frames ◽

Mammalian Host ◽

Environmental Signals ◽

Significant Difference ◽

Linear Plasmid 54

ABSTRACT Borrelia burgdorferi, the causative agent of Lyme disease, undergoes rapid adaptive gene expression in response to environmental signals encountered during different stages of its life cycle in the arthropod vector or the mammalian host. Among all the plasmid-encoded genes of B. burgdorferi, several linear plasmid 54 (lp54)-encoded open reading frames (ORFs) exhibit the greatest differential expression in response to mammalian host-specific temperature, pH, and other uncharacterized signals. These ORFs include members of the paralogous gene family 54 (pgf 54), such as BBA64, BBA65, and BBA66, present on lp54. In an attempt to correlate transcriptional up-regulation of these pgf 54 members to their role in infectivity, we inactivated BBA64 and characterized the phenotype of this mutant both in vitro and in vivo. There were no major differences in the protein profiles between the BBA64 mutant and the control strains, while immunoblot analysis indicated that inactivation of BBA64 resulted in increased levels of BBA65. Moreover, there was no significant difference in the ability of the BBA64 mutant to infect C3H/HeN mice compared to that of its parental or complemented control strains as determined by culturing of viable spirochetes from infected tissues. However, enumeration of spirochetes using quantitative real-time PCR revealed tissue-specific differences, suggesting a minimal role for BBA64 in the survival of B. burgdorferi in select tissues. Infectivity analysis of the BBA64 mutant suggests that B. burgdorferi may utilize multiple determinants to establish infection in mammalian hosts.

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An enhanced GFP reporter system to monitor gene expression in Borrelia burgdorferi

Microbiology ◽

10.1099/mic.0.26165-0 ◽

2003 ◽

Vol 149 (7) ◽

pp. 1819-1828 ◽

Cited By ~ 49

Author(s):

James A. Carroll ◽

Philip E. Stewart ◽

Patricia Rosa ◽

Abdallah F. Elias ◽

Claude F. Garon

Keyword(s):

Gene Expression ◽

Borrelia Burgdorferi ◽

Fluorescent Protein ◽

Regulation Of Gene Expression ◽

Epifluorescence Microscopy ◽

Reporter System ◽

Environmental Signals ◽

Gfp Reporter ◽

Green Fluorescent

Borrelia burgdorferi regulates genes in response to a number of environmental signals such as temperature and pH. A green fluorescent protein (GFP) reporter system using the ospC, ospA and flaB promoters from B. burgdorferi B31 was introduced into infectious clonal isolates of strains B31 and N40 to monitor and compare gene expression in response to pH and temperature in vitro. GFP could be assayed by epifluorescence microscopy, immunoblotting or spectrofluorometry and was an accurate reporter of target gene expression. It was determined that only 179 bp 5′ of ospC was sufficient to regulate the reporter gfp in vitro in response to pH and temperature in B. burgdorferi B31. The loss of linear plasmid (lp) 25, lp28-1, lp36 and lp56 had no effect on the ability of B. burgdorferi B31 to regulate ospC in response to pH or temperature. The amount of OspC in N40 transformants was unaffected by changes in pH or temperature of the culture medium. This suggests that regulation of gene expression in response to pH and temperature may vary between these two B. burgdorferi strains.

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

Infection and Immunity ◽

10.1128/iai.71.5.2892-2896.2003 ◽

2003 ◽

Vol 71 (5) ◽

pp. 2892-2896 ◽

Cited By ~ 44

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.

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

Infection and Immunity ◽

10.1128/iai.72.11.6433-6445.2004 ◽

2004 ◽

Vol 72 (11) ◽

pp. 6433-6445 ◽

Cited By ~ 126

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.

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Dissolved Oxygen Levels Alter Gene Expression and Antigen Profiles in Borrelia burgdorferi

Infection and Immunity ◽

10.1128/iai.72.3.1580-1586.2004 ◽

2004 ◽

Vol 72 (3) ◽

pp. 1580-1586 ◽

Cited By ~ 65

Author(s):

J. Seshu ◽

Julie A. Boylan ◽

Frank C. Gherardini ◽

Jonathan T. Skare

Keyword(s):

Gene Expression ◽

Borrelia Burgdorferi ◽

Dissolved Oxygen ◽

Reverse Transcription ◽

Host Factors ◽

Pcr Analysis ◽

Transcriptional Level ◽

Environmental Signals ◽

Reverse Transcription Pcr ◽

Genes Encoding

ABSTRACT The Lyme disease spirochete, Borrelia burgdorferi, encounters many environmental signals as it cycles between the arthropod vector and mammalian hosts, including temperature, pH, and other host factors. To test the possibility that dissolved oxygen modulates gene expression in B. burgdorferi, spirochetes were exposed to differential levels of dissolved oxygen, and distinct alterations were observed at both the transcriptional and translational levels. Specifically NapA, a Dps/Dpr homologue involved in the oxidative stress response in other bacteria, was reduced when B. burgdorferi was grown under oxygen-limiting conditions. In contrast, several immunoreactive proteins were altered when tested with infection-derived sera from different hosts. Specifically, OspC, DbpA, and VlsE were synthesized at greater levels when cells were grown in limiting oxygen, whereas VraA was reduced. The levels of oxygen in the medium did not affect OspA production. Real-time reverse transcription-PCR analysis of RNA isolated from infectious isolates of strains B31 and cN40 indicated that the expression of ospC, dbpA, and vlsE increased while napA expression decreased under dissolved-oxygen-limiting conditions, whereas flaB was not affected. The reverse transcription-PCR results corroborated the immunoblot analyses and indicated that the increase in OspC, DbpA, and VlsE was due to regulation at the transcriptional level of the genes encoding these antigens. These results indicate that dissolved oxygen modulates gene expression in B. burgdorferi and imply that the redox environment may be an additional regulatory cue that spirochetes exploit to adapt to the disparate niches that they occupy in nature.

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

Infection and Immunity ◽

10.1128/iai.70.7.3468-3478.2002 ◽

2002 ◽

Vol 70 (7) ◽

pp. 3468-3478 ◽

Cited By ~ 70

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.

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