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 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 Manganese and Zinc Regulate Virulence Determinants in Borrelia burgdorferi

2013 ◽  
Vol 81 (8) ◽  
pp. 2743-2752 ◽  
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.

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 The luxS Gene Is Not Required for Borrelia burgdorferi Tick Colonization, Transmission to a Mammalian Host, or Induction of Disease

2004 ◽  
Vol 72 (8) ◽  
pp. 4864-4867 ◽  
Author(s):  
Jon S. Blevins ◽  
Andrew T. Revel ◽  
Melissa J. Caimano ◽  
Xiaofeng F. Yang ◽  
James A. Richardson ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Ixodes Scapularis ◽  
Mammalian Host ◽  
Luxs Gene ◽  
Wild Type ◽  
Molting Process

ABSTRACT luxS mutants of Borrelia burgdorferi strain 297 naturally colonized their arthropod (Ixodes scapularis) vector, were maintained in ticks throughout the molting process (larvae to nymphs), were tick transmitted to uninfected mice, and elicited histopathology in mice indistinguishable from that induced by wild-type B. burgdorferi.

scholarly journals Membrane directed expression in Escherichia coli of BBA57 and other virulence factors from the Lyme disease agent Borrelia burgdorferi

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Karie E. Robertson ◽  
Chloe D. Truong ◽  
Felicia M. Craciunescu ◽  
Jay-How Yang ◽  
Po-Lin Chiu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Virulence Factors ◽  
Protein Complexes ◽  
Signal Sequence ◽  
Surface Proteins ◽  
Disease Agent ◽  
And Function ◽  
Lyme Disease Agent

AbstractMembrane-embedded proteins are critical to the establishment, survival and persistence in the host of the Lyme disease bacterium Borrelia burgdorferi (Bb), but to date, there are no solved structures of transmembrane proteins representing these attractive therapeutic targets. All available structures from the genus Borrelia represent proteins expressed without a membrane-targeting signal peptide, thus avoiding conserved pathways that modify, fold and assemble membrane protein complexes. Towards elucidating structure and function of these critical proteins, we directed translocation of eleven expression-optimized Bb virulence factors, including the signal sequence, to the Escherichia coli membrane, of which five, BBA57, HtrA, BB0238, BB0323, and DipA, were expressed with C-terminal His-tags. P66 was also expressed using the PelB signal sequence fused to maltose binding protein. Membrane-associated BBA57 lipoprotein was solubilized by non-ionic and zwitterionic detergents. We show BBA57 translocation to the outer membrane, purification at a level sufficient for structural studies, and evidence for an α-helical multimer. Previous studies showed multiple critical roles of BBA57 in transmission, joint arthritis, carditis, weakening immune responses, and regulating other Bb outer surface proteins. In describing the first purification of membrane-translocated BBA57, this work will support subsequent studies that reveal the precise mechanisms of this important Lyme disease virulence factor.

scholarly journals Use of rpsL as a Counterselectable Marker in Borrelia burgdorferi

2009 ◽  
Vol 76 (3) ◽  
pp. 985-987 ◽  
Author(s):  
Dan Drecktrah ◽  
J. Miles Douglas ◽  
D. Scott Samuels
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Causative Agent ◽  
Ribosomal Subunit ◽  
Streptomycin Resistance ◽  
Wild Type ◽  
Small Ribosomal Subunit ◽  
Disease Mutations

ABSTRACT We have demonstrated that rpsL, encoding the S12 protein of the small ribosomal subunit, can be used as a counterselectable marker in Borrelia burgdorferi, the causative agent of Lyme disease. Mutations in rpsL confer streptomycin resistance. Streptomycin susceptibility is dominant in an rpsL merodiploid, and streptomycin selects for the loss of wild-type rpsL carried in trans. This is the first description of a counterselectable marker in B. burgdorferi.

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

2007 ◽  
Vol 76 (1) ◽  
pp. 391-402 ◽  
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.

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

2006 ◽  
Vol 189 (2) ◽  
pp. 437-445 ◽  
Author(s):  
Jenny A. Hyde ◽  
Jerome P. Trzeciakowski ◽  
Jonathan T. Skare
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Borrelia Burgdorferi ◽  
Growth Conditions ◽  
Etiologic Agent ◽  
Complex Life Cycle ◽  
Mammalian Host ◽  
Transcriptional Level ◽  
Virulence Determinants

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.

scholarly journals Comparative Transcriptional Profiling of Borrelia burgdorferi Clinical Isolates Differing in Capacities for Hematogenous Dissemination

2005 ◽  
Vol 73 (10) ◽  
pp. 6791-6802 ◽  
Author(s):  
Caroline Ojaimi ◽  
Vishwaroop Mulay ◽  
Dionysios Liveris ◽  
Radha Iyer ◽  
Ira Schwartz
Keyword(s):  
Lyme Disease ◽  
Cell Surface ◽  
Borrelia Burgdorferi ◽  
Nutrient Uptake ◽  
Transcriptional Profiling ◽  
Clinical Isolates ◽  
Wild Type ◽  
Phase Growth ◽  
Hematogenous Dissemination ◽  
Mutant Strains

ABSTRACT Borrelia burgdorferi, the etiologic agent of Lyme disease, is genetically heterogeneous. Previous studies have shown a significant association between the frequency of hematogenous dissemination in Lyme disease patients and the genotype of the infecting B. burgdorferi strain. Comparative transcriptional profiling of two representative clinical isolates with distinct genotypes (BL206 and B356) was undertaken. A total of 78 open reading frames (ORFs) had expression levels that differed significantly between the two isolates. A number of genes with potential involvement in nutrient uptake (BB0603, BBA74, BB0329, BB0330, and BBB29) have significantly higher expression levels in isolate B356. Moreover, nearly 25% of the differentially expressed genes are predicted to be localized on the cell surface, implying that these two isolates have cell surface properties that differ considerably. One of these genes, BBA74, encodes a protein of 257 amino acid residues that has been shown to possess porin activity. BBA74 transcript level was >20-fold higher in B356 than in BL206, and strain B356 contained three- to fivefold more BBA74 protein. BBA74 was disrupted by the insertion of a kanamycin resistance cassette into the coding region. The growth rates of both wild-type and mutant strains were essentially identical, and cultures reached the same final cell densities. However, the mutant strains consistently showed prolonged lags of 2 to 5 days prior to the induction of log-phase growth compared to wild-type strains. It is tempting to speculate that the absence of BBA74 interferes with the enhanced nutrient uptake that may be required for the entry of cells into log-phase growth. These studies demonstrate the value of comparative transcriptional profiling for identifying differences in the transcriptomes of B. burgdorferi clinical isolates that may provide clues to pathogenesis. The 78 ORFs identified here are a good starting point for the investigation of factors involved in the hematogenous dissemination of B. burgdorferi.

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