Immunolocalization of a 22 kDa protein (IPLA7, P22) of<i>Borrelia burgdorferi</i>

Grewe, Carola; Nuske, Joachim H.

doi:10.1111/j.1574-6968.1996.tb08160.x

Cited by 5 publications

(2 citation statements)

References 17 publications

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“…Immunoblot analysis of OspD production in all engineered strains indicated an increase in OspD protein in strains transformed with a shuttle vector encoding either a single tetracysteine motif, A34-SV-OspDTC, or two tetracysteine motifs, A34-SV-OspD2xT (Supplementary Figure 1A). LA7, originally proposed to be an outer membrane lipoprotein (Grewe and Nuske, 1996), but subsequently shown to localize primarily to the inner membrane (von Lackum et al, 2007; Yang et al, 2013), was chosen as a target to assess the ability of biarsenical dyes to freely diffuse across the outer membrane of living B. burgdorferi cells. Allelic exchange transformants were screened by PCR using a primer set spanning la7 and the kanamycin resistance cassette (Figure 2B, lower panel), and spirochetes containing a tetracysteine motif engineered on LA7 (strain A3-LA7TC) produced comparable levels of protein as their wild-type counterpart (Supplementary Figure 1B).…”

Section: Resultsmentioning

confidence: 99%

Visualization of Spirochetes by Labeling Membrane Proteins With Fluorescent Biarsenical Dyes

Hillman

Stewart

Strnad

et al. 2019

Front. Cell. Infect. Microbiol.

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Numerous methods exist for fluorescently labeling proteins either as direct fusion proteins (GFP, RFP, YFP, etc.—attached to the protein of interest) or utilizing accessory proteins to produce fluorescence (SNAP-tag, CLIP-tag), but the significant increase in size that these accompanying proteins add may hinder or impede proper protein folding, cellular localization, or oligomerization. Fluorescently labeling proteins with biarsenical dyes, like FlAsH, circumvents this issue by using a short 6-amino acid tetracysteine motif that binds the membrane-permeable dye and allows visualization of living cells. Here, we report the successful adaptation of FlAsH dye for live-cell imaging of two genera of spirochetes, Leptospira and Borrelia , by labeling inner or outer membrane proteins tagged with tetracysteine motifs. Visualization of labeled spirochetes was possible by fluorescence microscopy and flow cytometry. A subsequent increase in fluorescent signal intensity, including prolonged detection, was achieved by concatenating two copies of the 6-amino acid motif. Overall, we demonstrate several positive attributes of the biarsenical dye system in that the technique is broadly applicable across spirochete genera, the tetracysteine motif is stably retained and does not interfere with protein function throughout the B. burgdorferi infectious cycle, and the membrane-permeable nature of the dyes permits fluorescent detection of proteins in different cellular locations without the need for fixation or permeabilization. Using this method, new avenues of investigation into spirochete morphology and motility, previously inaccessible with large fluorescent proteins, can now be explored.

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Section: Resultsmentioning

confidence: 99%

Visualization of Spirochetes by Labeling Membrane Proteins With Fluorescent Biarsenical Dyes

Hillman

Stewart

Strnad

et al. 2019

Front. Cell. Infect. Microbiol.

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“…B. burgdorferi gene la7, annotated as bb0365, encodes P22, which is also known as lipoprotein LA7 (Lam et al, 1994;Grewe and Nuske, 1996). la7 is produced in the mammalian host (von Lackum et al, 2007) and is highly upregulated in spirochetes upon their entry into feeding ticks (Pal et al, 2008a).…”

Section: La7mentioning

confidence: 99%

Interactions Between Ticks and Lyme Disease Spirochetes

Pal¹,

Kitsou²,

Drecktrah³

et al. 2022

Current Issues in Molecular Biology

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Borrelia burgdorferi sensu lato causes Lyme borreliosis in a variety of animals and humans. These atypical bacterial pathogens are maintained in a complex enzootic life cycle that primarily involves a vertebrate host and Ixodes spp. ticks. In the Northeastern United States, I. scapularis is the main vector, while wild rodents serve as the mammalian reservoir host. As B. burgdorferi is transmitted only by I. scapularis and closely related ticks, the spirochete-tick interactions are thought to be highly specific. Various borrelial and arthropod proteins that directly or indirectly contribute to the natural cycle of B. burgdorferi infection have been identified. Discrete molecular interactions between spirochetes and tick components also have been discovered, which often play critical roles in pathogen persistence and transmission by the arthropod vector. This review will focus on the past discoveries and future challenges that are relevant to our understanding of the molecular interactions between B. burgdorferi and Ixodes ticks. This information will not only impact scientific advancements in the research of ticktransmitted infections but will also contribute to the development of novel preventive measures that interfere with the B. burgdorferi life cycle. caister.com/cimb 113 Curr. Issues Mol. Biol. Vol. 42 Curr. Issues Mol. Biol. 42: 113-144. caister.com/cimb Interactions Between Ticks and Spirochetes Pal et al.scapularis are highly specific. Limited studies have shed light on these complex pathogen-tick interactions (Munderloh and Kurtti, 1995;Fikrig and Narasimhan, 2006); a recent review summarizes our most current state of knowledge about the interactions between B. burgdorferi and I. scapularis ticks (Kurokawa et al., 2020). Vector competence, which depends on genetic determinants influencing the vector's ability to transmit a pathogen, also shapes interactions involving the tick, pathogen, and host (de la Fuente et al., 2017). With the advent of genetic tools for creating Borrelia mutants (Samuels et al., 2018) (see also Radolf and Samuels, 2021) and the current availability of the I. scapularis genome (Gulia-Nuss et al., 2016;Miller et al., 2018), investigators will have increasingly robust tools for studying Borrelia-tick interactions, both during the arthropod phase of the spirochete life cycle and at the interface with the mammalian host. A few borrelial proteins already have been demonstrated as prerequisites for spirochete-vector interactions, particularly with receptors in select tick organs, such as the gut or salivary gland (

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The lipoprotein La7 contributes to Borrelia burgdorferi persistence in ticks and their transmission to naïve hosts

Yang

Hegde

Shroder

et al. 2013

Microbes and Infection

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La7, an immunogenic outer membrane lipoprotein of Borrelia burgdorferi, produced during infection, has been shown to play a redundant role in mammalian infectivity. Here we show that La7 facilitates pathogen survival in all tested phases of the vector-specific spirochete life cycle, including tick-to-host transmission. Unlike wild type or la7-complemented isolates, isogenic La7-deficient spirochetes are severely impaired in their ability to persist within feeding ticks during acquisition from mice, in quiescent ticks during larval-nymphal inter-molt, and in subsequent pathogen transmission from ticks to naïve hosts. Analysis of gene expression during the major stages of the tick-rodent infection cycle showed increased expression of la7 in the vector and a swift downregulation in the mammalian hosts. Co-immunoprecipitation studies coupled with liquid chromatography-mass spectrometry analysis further suggested that La7, a highly conserved and abundant inner membrane protein, is involved in protein-protein interaction with a discrete set of borrelial ligands although biological significance of such interactions remains unclear. Further characterization of vector-induced membrane antigens like La7 and its interacting partners will likely aid in our understanding of the molecular details of B. burgdorferi persistence and transmission through a complex enzootic cycle.

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Immunolocalization of a 22 kDa protein (IPLA7, P22) ofBorrelia burgdorferi

Cited by 5 publications

References 17 publications

Visualization of Spirochetes by Labeling Membrane Proteins With Fluorescent Biarsenical Dyes

Visualization of Spirochetes by Labeling Membrane Proteins With Fluorescent Biarsenical Dyes

Interactions Between Ticks and Lyme Disease Spirochetes

The lipoprotein La7 contributes to Borrelia burgdorferi persistence in ticks and their transmission to naïve hosts

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