Simultaneous Isolation of Ixodidae and Bacterial (Borrelia spp.) Genomic DNA

Jutras, Brandon L.; Liu, Zhiwei; Brissette, Catherine A.

doi:10.1002/9780471729259.mc01e02s19

Cited by 8 publications

(6 citation statements)

References 28 publications

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“…pneumoniae , a 5′ biotin modification 218-bp DNA probe (C2 probe;313542-313760 nt; 400 ng/μl) was PCR amplified from the chromosomal DNA of D39-WT strain using the primer pairs of P cps –F and P cps –R (Moscoso and Garcia, 2009; Jutras et al, 2010). Procedures for DNA affinity chromatography were reported previously (Jutras et al, 2012).…”

Section: Methodsmentioning

confidence: 99%

ComE, an Essential Response Regulator, Negatively Regulates the Expression of the Capsular Polysaccharide Locus and Attenuates the Bacterial Virulence in Streptococcus pneumoniae

et al. 2017

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The capsular polysaccharide (CPS) of Streptococcus pneumoniae is the main virulence factors required for effective colonization and invasive disease. The capacity to regulate CPS production at the transcriptional level is critical for the survival of S. pneumoniae in different host niches, but little is known about the transcription regulators of cps locus. In the present study, we isolated and identified the response regulator ComE, the master competence switch in transformation of S. pneumoniae, as a transcriptional regulator of cps locus by DNA affinity chromatography-pulldown, MALDI-TOF mass spectrometry (MS) and electrophoretic mobility shift assay (EMSA). Our results showed that phosphorylated mimetic of ComE (ComED58E) bound specifically to the cps locus prompter in vitro, and phosphorylated ComE negatively impacted both cps locus transcription and CPS production attenuating the pneumococcal virulence in vivo. Compared with D39-WT strain, D39ΔcomE mutant exhibited much thicker capsule, attenuated nasopharyngeal colonization and enhanced virulence in both pneumonia and bacteremia models of Balb/c mice. Furthermore, it was demonstrated that CSP-ComD/E competence system involved in regulating negatively the CPS production during the progress of transformation in D39. Our CSP1 induction experiment results showed that the expression of ComE in D39-WT strain increased powerfully by 120% after 10 min of CSP1 induction, but the CPS production in D39-WT strain decreased sharply by 67.1% after 15 min of CSP1 induction. However, the CPS production in D39ΔcomE mutant was almost constant during the whole stage of induction. Additionally, we found that extracellular glucose concentration could affect both the expression of ComE and CPS production of D39 in vitro. Taken together, for the first time, we report that ComE, as a transcriptional regulator of cps locus, plays an important role in transcriptional regulation of cps locus and capsular production level.

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Section: Methodsmentioning

confidence: 99%

ComE, an Essential Response Regulator, Negatively Regulates the Expression of the Capsular Polysaccharide Locus and Attenuates the Bacterial Virulence in Streptococcus pneumoniae

et al. 2017

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“…Prior to tick feeding, a sample of 10 ticks was tested to ensure they were harboring B. burgdorferi (47). Mice, which were culture-and serology-positive for B. burgdorferi, were euthanized 1 year after tick transmission.…”

Section: Methodsmentioning

confidence: 99%

BBA70 of Borrelia burgdorferi Is a Novel Plasminogen-binding Protein

Koenigs

Hammerschmidt

Jutras

et al. 2013

Journal of Biological Chemistry

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Background: Recruitment of plasminogen is important for efficient dissemination of Borrelia burgdorferi. Results: BBA70 of B. burgdorferi binds plasminogen, and following activation, bound plasmin can cleave fibrinogen and inactivate the key complement components C3b and C5. Conclusion: BBA70 is a potent plasminogen-binding protein.Significance: Investigation suggests that binding of plasminogen may aid in pathogen dissemination and inhibit bacteriolytic effects of the host complement system.

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“…Frozen mouse tissue samples (20 mg) were first minced with sterile single-use razor blades on a DNA/ DNase-free glass surface and were resuspended in Buffer ATL with proteinase K for overnight digestion at 56°C as recommended by the manufacturer (Qiagen). For ticks, cohorts of 30 fed larvae were processed according to the method of Jutras et al (48). qPCR was performed using a Bio-Rad MyiQ2 thermal cycler and Bio-Rad SYBR green Supermix.…”

Section: Methodsmentioning

confidence: 99%

“…Each run included a sample that lacked a template in order to test for DNA contamination of reagents. The oligonucleotide primers used for amplification of B. burgdorferi recA (nTM17F and nTM17R) (49), Ixodes scapularis 16S rRNA, B. burgdorferi flaB (48), and mouse nidogen are given in Table 1. Reaction conditions consisted of a 10-min initial denaturation step at 95°C; 40 cycles of 95°C for 15 s and 55°C (for recA) or 60°C (for nidogen) for 1 min; 95°C for 1 min; 60°C for 1 min; and melting analysis starting at 60°C and increasing by increments of 0.5°C, with a hold at each temperature for 10 s. Tenfold serial dilutions of B. burgdorferi genomic DNA, mouse genomic DNA, or Ixodes genomic DNA were included in every assay for each primer set.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2015

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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-type revA gene restored heart infectivity to wild-type levels. Additionally, revA mutants 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, while revA is not absolutely essential for infection, deletion of revA had distinct effects on dissemination, arthritis severity, and host response. Borrelia burgdorferi, the causative agent of Lyme disease, is a vector-borne pathogen that successfully colonizes both ticks and a variety of vertebrate hosts. In mammals, B. burgdorferi is frequently found associated with connective tissues (1-13), and the bacterium is often detected in cartilaginous or collagen-rich tissues, such as skin and joints (6,7,9,(11)(12)(13)(14)(15)(16)(17)(18). Adhesins expressed by B. burgdorferi facilitate interactions with these tissues. B. burgdorferi expresses a plethora of outer surface proteins that interact with numerous components of the extracellular matrix (ECM), such as fibronectin, decorin, and integrins (19-23). The attachment of B. burgdorferi to the host ECM is likely critical for pathogenesis and persistence in mammals. Indeed, for many of these adhesins, deletion mutants have significant defects in infectivity, fail to disseminate widely in the host, or have other effects on disease, such as alterations in the severity of arthritis (24-27).The interactions of B. burgdorferi with fibronectin are facilitated by several distinct bacterial surface proteins, including BBK32, RevA, BB0347, and CspA (BbCRASP-1) (28-31). The redundancy in adhesins makes it difficult to dissect the role of each B. burgdorferi fibronectin binding protein in the pathogenesis of Lyme disease. For example, real-time microscopic imaging in live mice revealed a significant role for BBK32 in interactions with host vasculature, yet bbk32 mutants are still able to infect mammals (32).RevA is a 19-kDa surface protein encoded on the circular plasmid 32 (cp32) family of plasmids (33). RevA expression is elevated during mammalian infection over its expression in the tick vector, suggesting a role in pathogenesis (30,(34)(35)(36). RevA binds to fibronectin, and anti-RevA antibodies block the binding of whole B. burgdorferi bacteria to fibronectin (30). RevA is antigenic, as evidenced by the fact that both mice and human Lyme disease patients produce antibodies...

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Simultaneous Isolation of Ixodidae and Bacterial (Borrelia spp.) Genomic DNA

Cited by 8 publications

References 28 publications

ComE, an Essential Response Regulator, Negatively Regulates the Expression of the Capsular Polysaccharide Locus and Attenuates the Bacterial Virulence in Streptococcus pneumoniae

ComE, an Essential Response Regulator, Negatively Regulates the Expression of the Capsular Polysaccharide Locus and Attenuates the Bacterial Virulence in Streptococcus pneumoniae

BBA70 of Borrelia burgdorferi Is a Novel Plasminogen-binding Protein

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

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