Hybrid cell lines producing monoclonal antibodies directed against Treponema

Continuous hybrid cell lines for producing monoclonal antibodies directed against antigens of Treponema pallidum have been developed. The hybrid cell lines were established by fusing differentiated lymphoid cells primed with antigens of Treponema pallidum with hybridoma cells. The resulting fused cells were cloned, isolated, cultured and characterized as to antibody specificity against antigenic determinants of Treponema pallidum.Board of Regents, University of Texas Syste

Assessment of decorin-binding protein A to the infectivity of Borrelia burgdorferi in the murine models of needle and tick infection

<p>Abstract</p> <p>Background</p> <p>Decorin-binding proteins (Dbps) A and B of <it>Borrelia burgdorferi</it>, the agent of Lyme disease, are surface-exposed lipoproteins that presumably bind to the extracellular matrix proteoglycan, decorin. <it>B. burgdorferi </it>infects various tissues including the bladder, heart, joints, skin and the central nervous system, and the ability of <it>B. burgdorferi </it>to bind decorin has been hypothesized to be important for this disseminatory pathogenic strategy.</p> <p>Results</p> <p>To determine the role of DbpBA in the infectious lifecycle of <it>B. burgdorferi</it>, we created a DbpBA-deficient mutant of <it>B. burgdorferi </it>strain 297 and compared the infectious phenotype of the mutant to the wild-type strain in the experimental murine model of Lyme borreliosis. The mutant strain exhibited a 4-log decrease in infectivity, relative to the wild-type strain, when needle inoculated into mice. Upon complementation of the DbpBA-mutant strain with DbpA, the wild-type level of infectivity was restored. In addition, we demonstrated that the DbpBA-deficient mutant was able to colonize <it>Ixodes scapularis </it>larval ticks after feeding on infected mice and persist within the ticks during the molt to the nymphal state. Moreover, surprisingly, the DbpBA-mutant strain was capable of being transmitted to naïve mice via tick bite, giving rise to infected mice.</p> <p>Conclusion</p> <p>These results suggest that DbpBA is not required for the natural tick-transmission process to mammals, despite inferences from needle-inoculation experiments implying a requirement for DbpBA during mammalian infection. The combined findings also send a cautionary note regarding how results from needle-inoculation experiments with mice should be interpreted.</p

Essential Role for OspA/B in the Life Cycle of the Lyme Disease Spirochete

The molecular basis of how Borrelia burgdorferi (Bb), the Lyme disease spirochete, maintains itself in nature via a complex life cycle in ticks and mammals is poorly understood. Outer surface (lipo)protein A (OspA) of Bb has been the most intensively studied of all borrelial molecular constituents, and hence, much has been speculated about the potential role(s) of OspA in the life cycle of Bb. However, the precise function of OspA (along with that of its close relative and operonic partner, outer surface [lipo]protein B [OspB]) heretofore has not been directly determined, due primarily to the inability to generate an OspA/B-deficient mutant from a virulent strain of Bb. In this study, we created an OspA/B-deficient mutant of an infectious human isolate of Bb (strain 297) and found that OspA/B function was not required for either Bb infection of mice or accompanying tissue pathology. However, OspA/B function was essential for Bb colonization of and survival within tick midguts, events crucial for sustaining Bb in its natural enzootic life cycle

Activation of the RpoN-RpoS regulatory pathway during the enzootic life cycle of Borrelia burgdorferi

<p>Abstract</p> <p>Background</p> <p>The maintenance of <it>Borrelia burgdorferi </it>in its complex tick-mammalian enzootic life cycle is dependent on the organism's adaptation to its diverse niches. To this end, the RpoN-RpoS regulatory pathway in <it>B. burgdorferi </it>plays a central role in microbial survival and Lyme disease pathogenesis by up- or down-regulating the expression of a number of virulence-associated outer membrane lipoproteins in response to key environmental stimuli. Whereas a number of studies have reported on the expression of RpoS and its target genes, a more comprehensive understanding of when activation of the RpoN-RpoS pathway occurs, and when induction of the pathway is most relevant to specific stage(s) in the life cycle of <it>B. burgdorferi</it>, has been lacking.</p> <p>Results</p> <p>Herein, we examined the expression of <it>rpoS </it>and key lipoprotein genes regulated by RpoS, including <it>ospC</it>, <it>ospA</it>, and <it>dbpA</it>, throughout the entire tick-mammal infectious cycle of <it>B. burgdorferi</it>. Our data revealed that transcription of <it>rpoS</it>, <it>ospC</it>, and <it>dbpA </it>is highly induced in nymphal ticks when taking a blood meal. The RpoN-RpoS pathway remains active during the mammalian infection phase, as indicated by the sustained transcription of <it>rpoS </it>and <it>dbpA </it>in <it>B. burgdorferi </it>within mouse tissues following borrelial dissemination. However, <it>dbpA </it>transcription levels in fed larvae and intermolt larvae suggested that an additional layer of control likely is involved in the expression of the <it>dbpBA </it>operon. Our results also provide further evidence for the downregulation of <it>ospA </it>expression during mammalian infection, and the repression of <it>ospC </it>at later phases of mammalian infection by <it>B. burgdorferi</it>.</p> <p>Conclusion</p> <p>Our study demonstrates that the RpoN-RpoS regulatory pathway is initially activated during the tick transmission of <it>B. burgdorferi </it>to its mammalian host, and is sustained during mammalian infection.</p

Treponema pallidum, the syphilis spirochete: making a living as a stealth pathogen

The past two decades have seen a worldwide resurgence in infections caused by Treponema pallidum subsp. pallidum, the syphilis spirochete. The well-recognized capacity of the syphilis spirochete for early dissemination and immune evasion has earned it the designation 'the stealth pathogen'. Despite the many hurdles to studying syphilis pathogenesis, most notably the inability to culture and to genetically manipulate T. pallidum, in recent years, considerable progress has been made in elucidating the structural, physiological, and regulatory facets of T. pallidum pathogenicity. In this Review, we integrate this eclectic body of information to garner fresh insights into the highly successful parasitic lifestyles of the syphilis spirochete and related pathogenic treponemes

BosR (BB0647) Controls the RpoN-RpoS Regulatory Pathway and Virulence Expression in Borrelia burgdorferi by a Novel DNA-Binding Mechanism

In Borrelia burgdorferi (Bb), the Lyme disease spirochete, the alternative σ factor σ54 (RpoN) directly activates transcription of another alternative σ factor, σS (RpoS) which, in turn, controls the expression of virulence-associated membrane lipoproteins. As is customary in σ54-dependent gene control, a putative NtrC-like enhancer-binding protein, Rrp2, is required to activate the RpoN-RpoS pathway. However, recently it was found that rpoS transcription in Bb also requires another regulator, BosR, which was previously designated as a Fur or PerR homolog. Given this unexpected requirement for a second activator to promote σ54-dependent gene transcription, and the fact that regulatory mechanisms among similar species of pathogenic bacteria can be strain-specific, we sought to confirm the regulatory role of BosR in a second virulent strain (strain 297) of Bb. Indeed, BosR displayed the same influence over lipoprotein expression and mammalian infectivity for strain Bb 297 that were previously noted for Bb strain B31. We subsequently found that recombinant BosR (rBosR) bound to the rpoS gene at three distinct sites, and that binding occurred despite the absence of consensus Fur or Per boxes. This led to the identification of a novel direct repeat sequence (TAAATTAAAT) critical for rBosR binding in vitro. Mutations in the repeat sequence markedly inhibited or abolished rBosR binding. Taken together, our studies provide new mechanistic insights into how BosR likely acts directly on rpoS as a positive transcriptional activator. Additional novelty is engendered by the facts that, although BosR is a Fur or PerR homolog and it contains zinc (like Fur and PerR), it has other unique features that clearly set it apart from these other regulators. Our findings also have broader implications regarding a previously unappreciated layer of control that can be involved in σ54–dependent gene regulation in bacteria

Cyclic di-GMP is Essential for the Survival of the Lyme Disease Spirochete in Ticks

Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that modulates many biological processes. Although its role in bacterial pathogenesis during mammalian infection has been documented, the role of c-di-GMP in a pathogen's life cycle within a vector host is less understood. The enzootic cycle of the Lyme disease pathogen Borrelia burgdorferi involves both a mammalian host and an Ixodes tick vector. The B. burgdorferi genome encodes a single copy of the diguanylate cyclase gene (rrp1), which is responsible for c-di-GMP synthesis. To determine the role of c-di-GMP in the life cycle of B. burgdorferi, an Rrp1-deficient B. burgdorferi strain was generated. The rrp1 mutant remains infectious in the mammalian host but cannot survive in the tick vector. Microarray analyses revealed that expression of a four-gene operon involved in glycerol transport and metabolism, bb0240-bb0243, was significantly downregulated by abrogation of Rrp1. In vitro, the rrp1 mutant is impaired in growth in the media containing glycerol as the carbon source (BSK-glycerol). To determine the contribution of the glycerol metabolic pathway to the rrp1 mutant phenotype, a glp mutant, in which the entire bb0240-bb0243 operon is not expressed, was generated. Similar to the rrp1 mutant, the glp mutant has a growth defect in BSK-glycerol medium. In vivo, the glp mutant is also infectious in mice but has reduced survival in ticks. Constitutive expression of the bb0240-bb0243 operon in the rrp1 mutant fully rescues the growth defect in BSK-glycerol medium and partially restores survival of the rrp1 mutant in ticks. Thus, c-di-GMP appears to govern a catabolic switch in B. burgdorferi and plays a vital role in the tick part of the spirochetal enzootic cycle. This work provides the first evidence that c-di-GMP is essential for a pathogen's survival in its vector host

An economic model of long-term use of celecoxib in patients with osteoarthritis

<p>Abstract</p> <p>Background</p> <p>Previous evaluations of the cost-effectiveness of the cyclooxygenase-2 selective inhibitor celecoxib (Celebrex, Pfizer Inc, USA) have produced conflicting results. The recent controversy over the cardiovascular (CV) risks of rofecoxib and other coxibs has renewed interest in the economic profile of celecoxib, the only coxib now available in the United States. The objective of our study was to evaluate the long-term cost-effectiveness of celecoxib compared with nonselective nonsteroidal anti-inflammatory drugs (nsNSAIDs) in a population of 60-year-old osteoarthritis (OA) patients with average risks of upper gastrointestinal (UGI) complications who require chronic daily NSAID therapy.</p> <p>Methods</p> <p>We used decision analysis based on data from the literature to evaluate cost-effectiveness from a modified societal perspective over patients' lifetimes, with outcomes expressed as incremental costs per quality-adjusted life-year (QALY) gained. Sensitivity tests were performed to evaluate the impacts of advancing age, CV thromboembolic event risk, different analytic horizons and alternate treatment strategies after UGI adverse events.</p> <p>Results</p> <p>Our main findings were: 1) the base model incremental cost-effectiveness ratio (ICER) for celecoxib versus nsNSAIDs was $31,097 per QALY; 2) the ICER per QALY was$19,309 for a model in which UGI ulcer and ulcer complication event risks increased with advancing age; 3) the ICER per QALY was $17,120 in sensitivity analyses combining serious CV thromboembolic event (myocardial infarction, stroke, CV death) risks with base model assumptions.</p> <p>Conclusion</p> <p>Our model suggests that chronic celecoxib is cost-effective versus nsNSAIDs in a population of 60-year-old OA patients with average risks of UGI events.</p

Analysis of the dbpBA Upstream Regulatory Region Controlled by RpoS in Borrelia burgdorferi▿

Decorin-binding proteins B and A (DbpB and DbpA) are thought to play important roles in Borrelia burgdorferi pathogenesis by serving as adhesins for the extracellular matrix. It has been established that the expression of DbpBA is governed by the Rrp2-RpoN-RpoS regulatory pathway. However, the precise mechanism underlying the control of DbpBA expression has been unclear. In particular, it has been unknown whether RpoS influences DbpBA expression directly or indirectly (through an additional regulatory molecule[s]). Here, employing a wild-type B. burgdorferi strain and a dbpBA-deficient mutant, we analyzed the 5′ genetic elements of the dbpBA operon using deletion analysis, coupled with luciferase reporter assays, quantitative reverse transcription PCR, and immunoblot analyses. A minimal promoter, encompassed within 70 bp upstream of the ATG start codon of dbpBA, was identified and found to be necessary and sufficient to initiate dbpBA transcription. The minimal dbpBA promoter was responsive to environmental stimuli such as temperature, pH, and whole blood. Two in silico-identified inverted repeat elements were not involved in the response of dbpBA expression to in vitro stimulation by environmental factors. The expression of dbpBA from the minimal promoter was abolished when rpoS was inactivated. In addition, the targeted mutagenesis of a C at position −14 within the extended −10 region of dbpBA, which has been postulated to be strategic for EσS binding in Escherichia coli, abolished dbpBA expression in B. burgdorferi. These combined data suggest that the Rrp2-RpoN-RpoS pathway controls dbpBA expression by the direct binding of RpoS to an RpoS-dependent promoter. However, given that there remains a distinct difference between the expression of DbpBA and other genes under the direct control of RpoS (e.g., OspC), our findings do not preclude the existence of another layer of gene regulation that may contribute to the modulation of DbpBA expression via an as-yet unknown mechanism