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Andrew T Revel

Publications and source records attributed to Andrew T Revel.

7 recordsLinked to original sources
PubMed2007

Adaptation of a luciferase gene reporter and lac expression system to Borrelia burgdorferi.

The development of new genetic systems for studying the complex regulatory events that occur within Borrelia burgdorferi is an important goal of contemporary Lyme disease research. Although recent advancements have been made in the genetic manipulation of B. burgdorferi, there still remains a paucity of basic molecular systems for assessing differential gene expression in this pathogen. Herein, we describe the adaptation of two powerful genetic tools for use in B. burgdorferi. The first is a Photinus pyralis firefly luciferase gene reporter that was codon optimized to enhance translation in B. burgdorferi. Using this modified reporter, we demonstrated an increase in luciferase expression when B. burgdorferi transformed with a shuttle vector encoding the outer surface protein C (OspC) promoter fused to the luciferase reporter was cultivated in the presence of fresh rabbit blood. The second is a lac operator/repressor system that was optimized to achieve the tightest degree of regulation. Using the aforementioned luciferase reporter, we assessed the kinetics and maximal level of isopropyl-beta-D-thiogalactopyranoside (IPTG)-dependent gene expression. This lac-inducible expression system also was used to express the gene carried on lp25 required for borrelial persistence in ticks (bptA). These advancements should be generally applicable for assessing further the regulation of other genes potentially involved in virulence expression by B. burgdorferi.

Animals↗
PubMed2005

bptA (bbe16) is essential for the persistence of the Lyme disease spirochete, Borrelia burgdorferi, in its natural tick vector.

Borrelia burgdorferi (Bb), the agent of Lyme disease, is a zoonotic spirochetal bacterium that depends on arthropod (Ixodes ticks) and mammalian (rodent) hosts for its persistence in nature. The quest to identify borrelial genes responsible for Bb's parasitic dependence on these two diverse hosts has been hampered by limitations in the ability to genetically manipulate virulent strains of Bb. Despite this constraint, we report herein the inactivation and genetic complementation of a linear plasmid-25-encoded gene (bbe16) to assess its role in the virulence, pathogenesis, and survival of Bb during its natural life cycle. bbe16 was found to potentiate the virulence of Bb in the murine model of Lyme borreliosis and was essential for the persistence of Bb in Ixodes scapularis ticks. As such, we have renamed bbe16 a gene encoding borrelial persistence in ticks (bpt)A. Although protease accessibility experiments suggested that BptA as a putative lipoprotein is surface-exposed on the outer membrane of Bb, the molecular mechanism(s) by which BptA promotes Bb persistence within its tick vector remains to be elucidated. BptA also was shown to be highly conserved (>88% similarity and >74% identity at the deduced amino acid levels) in all Bb sensu lato strains tested, suggesting that BptA may be widely used by Lyme borreliosis spirochetes for persistence in nature. Given Bb's absolute dependence on and intimate association with its arthropod and mammalian hosts, BptA should be considered a virulence factor critical for Bb's overall parasitic strategy.

Animals↗
PubMed2005

Analysis of the ospC regulatory element controlled by the RpoN-RpoS regulatory pathway in Borrelia burgdorferi.

Outer surface lipoprotein C (OspC) is a key virulence factor of Borrelia burgdorferi. ospC is differentially regulated during borrelial transmission from ticks to rodents, and such regulation is essential for maintaining the spirochete in its natural enzootic cycle. Recently, we showed that the expression of ospC in B. burgdorferi is governed by a novel alternative sigma factor regulatory network, the RpoN-RpoS pathway. However, the precise mechanism by which the RpoN-RpoS pathway controls ospC expression has been unclear. In particular, there has been uncertainty regarding whether ospC is controlled directly by RpoS (sigma(s)) or indirectly through a transactivator (induced by RpoS). Using deletion analyses and genetic complementation in an OspC-deficient mutant of B. burgdorferi, we analyzed the cis element(s) required for the expression of ospC in its native borrelial background. Two highly conserved upstream inverted repeat elements, previously implicated in ospC regulation, were not required for ospC expression in B. burgdorferi. Using similar approaches, a minimal promoter that contained a canonical -35/-10 sequence necessary and sufficient for sigma(s)-dependent regulation of ospC was identified. Further, targeted mutagenesis of a C at position -15 within the extended -10 region of ospC, which is postulated to function like the strategic C residue important for Esigma(s) binding in Escherichia coli, abolished ospC expression. The minimal ospC promoter also was responsive to coumermycin A(1), further supporting its sigma(s) character. The combined data constitute a body of evidence that the RpoN-RpoS regulatory network controls ospC expression by direct binding of sigma(s) to a sigma(s)-dependent promoter of ospC. The implication of our findings to understanding how B. burgdorferi differentially regulates ospC and other ospC-like genes via the RpoN-RpoS regulatory pathway is discussed.

Antigens, Bacterial↗
PubMed2003

Expression of a luxS gene is not required for Borrelia burgdorferi infection of mice via needle inoculation.

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 DH5 alpha, 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.

Animals↗
PubMed2002

DNA microarray analysis of differential gene expression in Borrelia burgdorferi, the Lyme disease spirochete.

DNA microarrays were used to survey the adaptive genetic responses of Borrelia burgdorferi (Bb) B31, the Lyme disease spirochete, when grown under conditions analogous to those found in unfed ticks (UTs), fed ticks (FTs), or during mammalian host adaptation (Bb in dialysis membrane chambers implanted in rats). Microarrays contained 95.4% of the predicted B31 genes, 150 (8.6%) of which were differentially regulated (changes of > or = 1.8-fold) among the three growth conditions. A substantial proportion (46%) of the differentially regulated genes encoded proteins with predicted export signals (29% from predicted lipoproteins), emphasizing the importance to Bb of modulating its extracellular proteome. For B31 cultivated at the more restrictive UT condition, microarray data provided evidence of a bacterial stringent response and factors that restrict cell division. A large proportion of genes were responsive to the FT growth condition, wherein increased temperature and reduced pH were prominent environmental parameters. A surprising theme, supported by cluster analysis, was that many of the gene expression changes induced during the FT growth condition were transient and largely tempered as B31 adapted to the mammalian host, suggesting that once Bb gains entry and adapts to mammalian tissues, fewer differentially regulated genes are exploited. It therefore would seem that although widely dissimilar, the UT and dialysis membrane chamber growth conditions promote more static patterns of gene expression in Bb. The microarray data thus provide a basis for formulating new testable hypotheses regarding the life cycle of Bb and attaining a more complete understanding of many aspects of Bb's complex parasitic strategies.

Animals↗