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Daniel P Dulebohn, Crystal L Richards, Hua Su, Kevin A Lawrence, Frank C Gherardini
The spirochete Borrelia burgdorferi survives in its tick vector, Ixodes scapularis, or within various hosts. To transition between and survive in these distinct niches, B. burgdorferi changes its gene expression in response to environmental cues, both biochemical and physiological. Exposure of B. burgdorferi to weak monocarboxylic organic acids, including those detected in the blood meal of fed ticks, decreased the cytoplasmic pH of B. burgdorferi in vitro. A decrease in the cytoplasmic pH induced the expression of genes encoding enzymes that have been shown to restore pH homeostasis in other bacteria...
2017: Frontiers in Microbiology
Kai Zhang, Chunhao Li
Swimming plate, cell motion tracking, and capillary tube assays are very useful tools to quantitatively measure bacterial motility and chemotaxis. These methods were modified and applied to study Borrelia burgdorferi motility and chemotaxis. By using these methods, numerous motility and chemotaxis mutants have been characterized and several chemoattractants were identified. With the assistance of these tools, the role of motility and chemotaxis in the pathogenicity of B. burgdorferi has been established. In addition, these tools also facilitate the study of motility and chemotaxis in other spirochetes...
2018: Methods in Molecular Biology
Juan Anguita, Ana Carreras-González, Nicolás Navasa
Phagocytosis of Borrelia burgdorferi, the causative agent of Lyme disease, is a poorly understood process, despite its importance during the host immune response to infection. Thus, macrophages infiltrate the infected tissues, including the base of the heart and phagocytose the spirochete, therefore contributing to their elimination from infected tissues and leading to inflammation. An impaired bacterial clearance will result in bacterial persistence that may interfere with normal physiology of the heart, such as electrical signals from the heart, resulting in an impaired coordination of the beating of the heart or "heart block...
2018: Methods in Molecular Biology
Alexia A Belperron, Jialing Mao, Linda K Bockenstedt
Two-photon intravital microscopy is a powerful tool that allows visualization of cells in intact tissues in a live animal in real time. In recent years, this advanced technology has been applied to understand pathogen-host interactions using fluorescently labeled bacteria. In particular, infectious fluorescent transformants of the Lyme disease spirochete Borrelia burgdorferi, an Ixodes tick-transmitted pathogen, have been imaged by two-photon intravital microscopy to study bacterial motility and interactions of the pathogen with feeding ticks and host tissues...
2018: Methods in Molecular Biology
Xiuli Yang, Meghna Thakur, Juraj Koci, Alexis A Smith, Preeti Singh, Xuran Zhuang, Kamoltip Promnares, Yan Wang, Ozlem Buyuktanir, Utpal Pal
The proteome of Borrelia burgdorferi undergoes dynamic alterations as the microbe cycles through and persists in diverse host or vector environments. Therefore, studies of B. burgdorferi proteome and protein-protein interactions, which play central roles in biological processes in diverse organisms, are critical in understanding biology and infectivity of spirochetes. Here, we describe the proteomic analysis of B. burgdorferi by two-dimensional (2-D) gel electrophoresis followed by protein identification via liquid chromatography-mass spectrometry and database searching...
2018: Methods in Molecular Biology
Tao Lin, Lihui Gao
Signature-tagged mutagenesis (STM) is a functional genomics approach to identify bacterial virulence determinants and virulence factors by simultaneously screening multiple mutants in a single host animal, and has been utilized extensively for the study of bacterial pathogenesis, host-pathogen interactions, and spirochete and tick biology. The signature-tagged transposon mutagenesis has been developed to investigate virulence determinants and pathogenesis of Borrelia burgdorferi. Mutants in genes important in virulence are identified by negative selection in which the mutants fail to colonize or disseminate in the animal host and tick vector...
2018: Methods in Molecular Biology
D Scott Samuels, Dan Drecktrah, Laura S Hall
The disciplines of Borrelia (Borreliella) burgdorferi microbiology and Lyme disease pathogenesis have come to depend on the genetic manipulation of the spirochete. Generating mutants in these recalcitrant bacteria, while not straightforward, is routinely accomplished in numerous laboratories, although there are several crucial caveats to consider. This chapter describes the design of basic molecular genetic experiments as well as the detailed methodologies to prepare and transform competent cells, select for and isolate transformants, and complement or genetically restore mutants...
2018: Methods in Molecular Biology
Philip P Adams, Mollie W Jewett
In vivo expression technology (IVET) has been applied to a variety of organisms to identify active promoters in specific environments or growth conditions of interest. Here, we describe modifications to employ this genome-wide screening method for Borrelia burgdorferi, the Lyme disease spirochete, during an active murine infection. Utilization of this technique provides valuable insights into the B. burgdorferi transcriptome during infection, despite the low bacterial numbers in the mammalian host environment...
2018: Methods in Molecular Biology
Alexis A Smith, Xiuli Yang, Erol Fikrig, Utpal Pal
Borrelia burgdorferi is maintained in nature by a tick-rodent infection cycle where it traverses and colonizes a variety of host and vector tissues. A tick-borne murine model has been developed to study Lyme disease in the laboratory, which has a substantial impact in advancing our knowledge of spirochete infectivity and pathogenesis. Here, we detail a microinjection-based method for rapid and efficient infection of ticks with B. burgdorferi. While laboratory generation of B. burgdorferi-infected nymphs via natural larval engorgement on infected hosts and subsequent molting could take several weeks to months, the microinjection-based infection procedure requires only a few hours to generate infected ticks and allows introduction of defined quantities of spirochetes, including mutant isolates that are attenuated for infection in mice and thus cannot be naturally acquired by ticks...
2018: Methods in Molecular Biology
Peter Kraiczy
Acquisition of host-derived proteins possessing key regulatory function is a hallmark of Borrelia burgdorferi, and an important step to successfully infect the human host, inhibiting activation of complement as innate immunity's first line of defense. Hence, the identification and characterization of interacting ligands is a prerequisite to gain deeper insights into the molecular principles of how spirochetes overcome the detrimental effects of complement. Far western blotting enables the detection of protein-protein interactions in vitro using cell lysates containing the prey proteins and purified complement proteins or human serum as a source for soluble complement proteins as bait proteins...
2018: Methods in Molecular Biology
Xin Li, Peng Wang, Anthony Lutton, John Olesik
Transition metal ions play important structural, regulatory, and catalytic roles in all biological systems by serving as cofactors for proteins. Due to their relatively low levels in the cell compared to abundant metal ions such as potassium and magnesium, transition metals are often considered micronutrients and referred to as trace elements. Manganese (Mn), iron (Fe), copper (Cu), and zinc (Zn) are the most prevalent transition metals in the Lyme disease spirochete Borrelia burgdorferi. Here, we describe a method for the accurate measurement of these trace elements in B...
2018: Methods in Molecular Biology
Yi-Pin Lin, John M Leong
Lyme disease Borreliae produces outer surface adhesins to confer bacterial attachment to the extracellular matrix (ECM) components on the surface of mammalian cells. Here, we describe protocols to characterize the activity and specificity of these adhesins by flow cytometry or measurement of the binding of radiolabeled spirochetes to immobilized ECM or mammalian cells.
2018: Methods in Molecular Biology
Henna Iqbal, Darrin R Akins, Melisha R Kenedy
Co-immunoprecipitation can be utilized to study protein-protein interactions from various environments, cell types, or tissues. Herein, we describe a co-immunoprecipitation protocol that can be used to examine protein complexes found in the pathogenic spirochete Borrelia burgdorferi. The method outlined here has successfully identified known and unknown members of borrelial protein complexes and is an efficient method for studying protein interactions in this pathogenic spirochete.
2018: Methods in Molecular Biology
Melissa J Caimano
The transmission, survival, and virulence of Borrelia burgdorferi depend upon the spirochete's ability to modulate its transcriptome as it cycles between its arthropod vector and reservoir host. This complex adaptive process is collectively referred to as "host-adaptation." The paucibacillary nature of borrelial infections precludes the detailed analysis of host adaptation within infected mammalian tissues. To circumvent this limitation, we (J Clin Invest 101:2240-2250, 1998) developed a model system whereby spirochetes are cultivated within dialysis membrane chambers (DMCs) surgically implanted within the peritoneal cavity of a rat...
2018: Methods in Molecular Biology
Alan G Barbour, Vanessa J Cook
The tick-borne spirochetes that cause Lyme disease in North America and Eurasia display strong linkage disequilibrium between certain chromosomal and plasmid loci within each three major geographic areas of their distribution. For strain typing for epidemiologic and ecologic purposes, the commonly used genotypes based on a single locus are the spacer between the 16S-23S ribosomal RNA and the ospC gene of a plasmid. A simple genotyping scheme based on the two loci allows for discrimination between strains representing all the areas of distribution...
2018: Methods in Molecular Biology
Vaclav Honig, Heather E Carolan, Zuzana Vavruskova, Christian Massire, Michael R Mosel, Christopher D Crowder, Megan A Rounds, David J Ecker, Daniel Ruzek, Libor Grubhoffer, Benjamin J Luft, Mark W Eshoo
Ixodes ricinus ticks are vectors of numerous human and animal pathogens. They are host generalists able to feed on more than 300 vertebrate species. The prevalence of tick-borne pathogens is influenced by host-vector-pathogen interactions that results in spatial distribution of infection risk. Broad-range PCR and electrospray ionization mass-spectrometry (PCR/ESI-MS) was used to analyze 435 I. ricinus nymphs from four localities in the south of the Czech Republic for the species identification of tick-borne pathogens...
October 3, 2017: FEMS Microbiology Ecology
Spinello Antinori, Cristina Tonello, Sophie Edouard, Carlo Parravicini, Daniela Gastaldi, Romualdo Grande, Laura Milazzo, Davide Ricaboni, Florence Fenollar, Didier Raoult, Mario Corbellino, Oleg Mediannikov
We report two cases of louse-borne relapsing fever observed at our Institution in June 2016. Both patients were young asylum seekers from Africa who had recently arrived in Milan, Italy. Notably, direct microscopic examination of peripheral blood smears was repeatedly negative for the presence of spirochetes and the diagnosis, supported by clinical and epidemiologic evidence, required molecular confirmation by polymerase chain reaction amplification of DNA extracted from blood and sequencing of the amplified products...
September 25, 2017: American Journal of Tropical Medicine and Hygiene
Tadeusz Płusa
From the beginning of the twentieth century interest in tick-borne borreliosis continues to be an important diagnostic and therapeutic problem. In 1977, in Lyme and Old Lyme, Cunnecticut, in the United States, arthritis and skin lesions were described in 12 children, which were associated with tick bites. The name of the place was called a syndrome of Lyme disease. Dr Willy Burgdorfer observed microaerophilic Gram-negative bacteria (Spirochetes) with a diameter of 0.3-0.5 µm and a length of about 20-30 µm, which were isolated from the tick colon...
September 29, 2017: Polski Merkuriusz Lekarski: Organ Polskiego Towarzystwa Lekarskiego
R C C Melo, V Antonello, M C Ramos
Syphilis is a sexually transmitted infection (STI) caused by the spirochete Treponema pallidum (1). Low and medium-income countries are overburdened, nonetheless high-income countries also face an increasing incidence since the year 2000 (2). In Brazil, syphilis is a grave public health problem, with an incidence of 42.7 per 100.000 in 2015 (3). This article is protected by copyright. All rights reserved.
October 6, 2017: Journal of the European Academy of Dermatology and Venereology: JEADV
Bilal Aslam, Muhammad Atif Nisar, Mohsin Khurshid, Muhammad Khalid Farooq Salamat
The borrelial resurge demonstrates that Borrelia burgdorferi is a persistent health problem. This spirochete is responsible for a global public health concern called Lyme disease. B. burgdorferi faces diverse environmental conditions of its vector and host during its life cycle. To circumvent the host immune system is a prominent feature of B. burgdorferi. To date, numerous studies have reported on the various mechanisms used by this pathogen to evade the host defense mechanisms. This current review attempts to consolidate this information to describe the immunological and molecular methods used by B...
October 2017: Future Microbiology
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