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.

scholarly journals Extracellular Glycanases of Rhizobium leguminosarum Are Activated on the Cell Surface by an Exopolysaccharide-Related Component

2000 ◽  
Vol 182 (5) ◽  
pp. 1304-1312 ◽  
Author(s):  
Angeles Zorreguieta ◽  
Christine Finnie ◽  
J. Allan Downie
Keyword(s):  
Cell Wall ◽  
Agrobacterium Tumefaciens ◽  
Cell Surface ◽  
Rhizobium Leguminosarum ◽  
Plant Cell Wall ◽  
Agar Medium ◽  
Wild Type ◽  
Close Proximity ◽  
Mutant Strains ◽  
Colony Surface

ABSTRACT Rhizobium leguminosarum secretes two extracellular glycanases, PlyA and PlyB, that can degrade exopolysaccharide (EPS) and carboxymethyl cellulose (CMC), which is used as a model substrate of plant cell wall cellulose polymers. When grown on agar medium, CMC degradation occurred only directly below colonies of R. leguminosarum, suggesting that the enzymes remain attached to the bacteria. Unexpectedly, when a PlyA-PlyB-secreting colony was grown in close proximity to mutants unable to produce or secrete PlyA and PlyB, CMC degradation occurred below that part of the mutant colonies closest to the wild type. There was no CMC degradation in the region between the colonies. By growing PlyB-secreting colonies on a lawn of CMC-nondegrading mutants, we could observe a halo of CMC degradation around the colony. Using various mutant strains, we demonstrate that PlyB diffuses beyond the edge of the colony but does not degrade CMC unless it is in contact with the appropriate colony surface. PlyA appears to remain attached to the cells since no such diffusion of PlyA activity was observed. EPS defective mutants could secrete both PlyA and PlyB, but these enzymes were inactive unless they came into contact with an EPS+ strain, indicating that EPS is required for activation of PlyA and PlyB. However, we were unable to activate CMC degradation with a crude EPS fraction, indicating that activation of CMC degradation may require an intermediate in EPS biosynthesis. Transfer of PlyB to Agrobacterium tumefaciens enabled it to degrade CMC, but this was only observed if it was grown on a lawn ofR. leguminosarum. This indicates that the surface ofA. tumefaciens is inappropriate to activate CMC degradation by PlyB. Analysis of CMC degradation by other rhizobia suggests that activation of secreted glycanases by surface components may occur in other species.

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 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 The unusual cell wall of the Lyme disease spirochaete Borrelia burgdorferi is shaped by a tick sugar

2021 ◽  
Vol 6 (12) ◽  
pp. 1583-1592
Author(s):  
Tanner G. DeHart ◽  
Mara R. Kushelman ◽  
Sherry B. Hildreth ◽  
Richard F. Helm ◽  
Brandon L. Jutras
Keyword(s):  
Cell Wall ◽  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Cell Walls ◽  
Structural Strength ◽  
Cell Envelope ◽  
Wild Type ◽  
Tick Vector ◽  
Cell Wall Alteration ◽  
Main Component

AbstractPeptidoglycan—a mesh sac of glycans that are linked by peptides—is the main component of bacterial cell walls. Peptidoglycan provides structural strength, protects cells from osmotic pressure and contributes to shape. All bacterial glycans are repeating disaccharides of N-acetylglucosamine (GlcNAc) β-(1–4)-linked to N-acetylmuramic acid (MurNAc). Borrelia burgdorferi, the tick-borne Lyme disease pathogen, produces glycan chains in which MurNAc is occasionally replaced with an unknown sugar. Nuclear magnetic resonance, liquid chromatography–mass spectroscopy and genetic analyses show that B. burgdorferi produces glycans that contain GlcNAc–GlcNAc. This unusual disaccharide is chitobiose, a component of its chitinous tick vector. Mutant bacteria that are auxotrophic for chitobiose have altered morphology, reduced motility and cell envelope defects that probably result from producing peptidoglycan that is stiffer than that in wild-type bacteria. We propose that the peptidoglycan of B. burgdorferi probably evolved by adaptation to obligate parasitization of a tick vector, resulting in a biophysical cell-wall alteration to withstand the atypical torque associated with twisting motility.

scholarly journals Borrelia burgdorferi RevA Significantly Affects Pathogenicity and Host Response in the Mouse Model of Lyme Disease

2015 ◽  
Vol 83 (9) ◽  
pp. 3675-3683 ◽  
Author(s):  
Rebecca Byram ◽  
Robert A. Gaultney ◽  
Angela M. Floden ◽  
Christopher Hellekson ◽  
Brandee L. Stone ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Host Response ◽  
Collagen Deposition ◽  
Wild Type ◽  
Content Type ◽  
Cardiac Tissues ◽  
Monocyte Chemoattractant ◽  
Bacterial Interaction ◽  
Mammalian Infection

The Lyme disease spirochete,Borrelia burgdorferi, expresses RevA and numerous outer surface lipoproteins during mammalian infection. As an adhesin that promotes bacterial interaction with fibronectin, RevA is poised to interact with the extracellular matrix of the host. To further define the role(s) of RevA during mammalian infection, we created a mutant that is unable to produce RevA. The mutant was still infectious to mice, although it was significantly less well able to infect cardiac tissues. Complementation of the mutant with a wild-typerevAgene restored heart infectivity to wild-type levels. Additionally,revAmutants led to increased evidence of arthritis, with increased fibrotic collagen deposition in tibiotarsal joints. The mutants also induced increased levels of the chemokine CCL2, a monocyte chemoattractant, in serum, and this increase was abolished in the complemented strain. Therefore, whilerevAis not absolutely essential for infection, deletion ofrevAhad distinct effects on dissemination, arthritis severity, and host response.

scholarly journals Borrelia burgdorferi Binding of Host Complement Regulator Factor H Is Not Required for Efficient Mammalian Infection

2007 ◽  
Vol 75 (6) ◽  
pp. 3131-3139 ◽  
Author(s):  
Michael E. Woodman ◽  
Anne E. Cooley ◽  
Jennifer C. Miller ◽  
John J. Lazarus ◽  
Kathryn Tucker ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Alternative Pathway ◽  
Host Factor ◽  
Factor H ◽  
Surface Proteins ◽  
Wild Type ◽  
Factor B ◽  
Mammalian Infection ◽  
Deficient Mice

ABSTRACT The causative agent of Lyme disease, Borrelia burgdorferi, is naturally resistant to its host's alternative pathway of complement-mediated killing. Several different borrelial outer surface proteins have been identified as being able to bind host factor H, a regulator of the alternative pathway, leading to a hypothesis that such binding is important for borrelial resistance to complement. To test this hypothesis, the development of B. burgdorferi infection was compared between factor H-deficient and wild-type mice. Factor B- and C3-deficient mice were also studied to determine the relative roles of the alternative and classical/lectin pathways in B. burgdorferi survival during mammalian infection. While it was predicted that B. burgdorferi should be impaired in its ability to infect factor H-deficient animals, quantitative analyses of bacterial loads indicated that those mice were infected at levels similar to those of wild-type and factor B- and C3-deficient mice. Ticks fed on infected factor H-deficient or wild-type mice all acquired similar numbers of bacteria. Indirect immunofluorescence analysis of B. burgdorferi acquired by feeding ticks from the blood of infected mice indicated that none of the bacteria had detectable levels of factor H on their outer surfaces, even though such bacteria express high levels of surface proteins capable of binding factor H. These findings demonstrate that the acquisition of host factor H is not essential for mammalian infection by B. burgdorferi and indicate that additional mechanisms are employed by the Lyme disease spirochete to evade complement-mediated killing.

scholarly journals Infectivity of the Highly Transformable BBE02− lp56− Mutant of Borrelia burgdorferi, the Lyme Disease Spirochete, via Ticks

2006 ◽  
Vol 74 (6) ◽  
pp. 3678-3681 ◽  
Author(s):  
Mary B. Jacobs ◽  
Steven J. Norris ◽  
Kathrine M. Phillippi-Falkenstein ◽  
Mario T. Philipp
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Ixodes Scapularis ◽  
Shuttle Vector ◽  
Lower Number ◽  
Linear Plasmid ◽  
Wild Type ◽  
Gene Encoding ◽  
Restriction Modification ◽  
Feeding Tick

ABSTRACT Infectious Borrelia burgdorferi strains that have increased transformability with the shuttle vector pBSV2 were recently constructed by inactivating the gene encoding BBE02, a putative restriction-modification gene product expressed by the linear plasmid lp25 (Kawabata et al., Infect. Immun. 72:7147-7154, 2004). The absence of the linear plasmid lp56, which carries another putative restriction-modification gene, further enhanced transformation rates. The infectivity of these mutants was assessed previously in mice that were inoculated with needle and syringe and was found to be equivalent to that of wild-type spirochetes. Here we examined the infectivity of spirochetes to ticks after capillary inoculation of Ixodes scapularis nymphs and the subsequent spirochetal infectivity to mice via ticks by using B. burgdorferi B31 clonal isolates lacking lp56 and/or BBE02. The absence of lp56 (but not BBE02) correlated with a lower number of spirochetes in ticks after feeding on mice; this plasmid thus may play a role, albeit not an essential one, in supporting spirochetal survival in the feeding tick. Importantly, however, the absence of lp56 and BBE02 did not detectably influence infectivity to mice via ticks.

scholarly journals Linear and Circular Plasmid Content in Borrelia burgdorferi Clinical Isolates

2003 ◽  
Vol 71 (7) ◽  
pp. 3699-3706 ◽  
Author(s):  
Radha Iyer ◽  
Ogori Kalu ◽  
Joye Purser ◽  
Steven Norris ◽  
Brian Stevenson ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Southern Hybridization ◽  
Plasmid Profile ◽  
Etiologic Agent ◽  
Clinical Isolates ◽  
Pcr Analysis ◽  
Plasmid Content ◽  
Pulsed Field ◽  
Specific Pcr

ABSTRACT The genome of Borrelia burgdorferi, the etiologic agent of Lyme disease, is composed of a linear chromosome and more than 20 linear and circular plasmids. Typically, plasmid content analysis has been carried out by pulsed-field gel electrophoresis and confirmed by Southern hybridization. However, multiple plasmids of virtually identical sizes (e.g., lp28 and cp32) complicate the interpretation of such data. The present study was undertaken to investigate the complete plasmid complements of B. burgdorferi clinical isolates cultivated from patients from a single region where early Lyme disease is endemic. A total of 21 isolates obtained from the skin biopsy or blood samples of Lyme disease patients were examined for their complete plasmid complements by Southern hybridization and plasmid-specific PCR analysis. All clinical isolates harbored at least six of the nine previously characterized cp32s. Fourteen isolates harbored all B31-like linear plasmids, and seven isolates simultaneously lacked lp56, lp38, and some segments of lp28-1. The distinctive plasmid profile observed in these seven isolates was specific to organisms that had ribosomal spacer type 2 and pulsed-field gel type A, which implies a clonal origin for this genotype. The presence of nearly identical complements of multiple linear and circular plasmids in all of the human isolates suggests that these plasmids may be particularly necessary for infection, adaptation, and/or maintenance in the infected host.

scholarly journals groEL Expression in gyrB Mutants of Borrelia burgdorferi

2002 ◽  
Vol 184 (21) ◽  
pp. 6069-6072 ◽  
Author(s):  
Janet Alverson ◽  
D. Scott Samuels
Keyword(s):  
Gene Expression ◽  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Dna Topology ◽  
Mrna Transcript ◽  
Wild Type ◽  
Linear Chromosome ◽  
Transcript Levels ◽  
Circular Dna ◽  
Dna Molecule

ABSTRACT GroEL protein and groEL mRNA transcript were up-regulated in gyrB mutants of Borrelia burgdorferi, a causative agent of Lyme disease. Furthermore, the protein and transcript levels in gyrB mutants were greater than those in experimentally heat-shocked cultures of wild-type B. burgdorferi. Circular DNA in the gyrB mutants was more relaxed than in wild-type cells, although groEL is on the linear chromosome of B. burgdorferi. To our knowledge, this is the first evidence, albeit indirect, for the effect of DNA topology on gene expression from a linear DNA molecule in a bacterium.

Identification ofPseudomonas aeruginosaflagellin as an adhesin for Muc1 mucin

2002 ◽  
Vol 282 (4) ◽  
pp. L751-L756 ◽  
Author(s):  
Erik P. Lillehoj ◽  
Beom T. Kim ◽  
K. Chul Kim
Keyword(s):  
Epithelial Cell ◽  
Cell Surface ◽  
Chinese Hamster ◽  
Wild Type ◽  
Muc1 Mucin ◽  
Bacterial Binding ◽  
Empty Plasmid ◽  
Mutant Strains ◽  
Epithelial Cell Surface ◽  
X Cells

We reported previously that Muc1 mucin on the epithelial cell surface is an adhesion site for Pseudomonas aeruginosa (Lillehoj EP, Hyun SW, Kim BT, Zhang XG, Lee DI, Rowland S, and Kim KC. Am J Physiol Lung Cell Mol Physiol 280: L181–L187, 2001). The present study was designed to identify the adhesin(s) responsible for bacterial binding to Muc1 mucin using genetic and biochemical approaches. Chinese hamster ovary (CHO) cells stably transfected with a Muc1 cDNA (CHO-Muc1) or empty plasmid (CHO-X) were compared for adhesion of P. aeruginosa strain PAK. Our results showed that 1) wild-type PAK and isogenic mutant strains lacking pili (PAK/NP) or flagella cap protein (PAK/ fliD) demonstrated significantly increased binding to CHO-Muc1 cells, whereas flagellin-deficient (PAK/ fliC) bacteria were no more adherent to CHO-Muc1 than CHO-X cells, and 2) P. aeruginosa adhesion was blocked by pretreatment of bacteria with antibody to flagellin or pretreatment of CHO-Muc1 cells with purified flagellin. We conclude that flagellin is an adhesin of P. aeruginosa responsible for its binding to Muc1 mucin on the epithelial cell surface.

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