The Lyme disease spirochete
Borrelia burgdorferi
relies on uptake of essential nutrients from its host environments for survival and infection. Therefore, nutrient acquisition mechanisms constitute ...key virulence properties of the pathogen, yet these mechanisms remain largely unknown.
In vivo
expression technology applied to
B
.
burgdorferi
(BbIVET) during mammalian infection identified gene
bb0562
, which encodes a hypothetical protein comprised of a conserved domain of unknown function, DUF3996. DUF3996 is also found across adjacent encoded hypothetical proteins BB0563 and BB0564, suggesting the possibility that the three proteins could be functionally related. Deletion of
bb0562
,
bb0563
and
bb0564
individually and together demonstrated that
bb0562
alone was important for optimal disseminated infection in immunocompetent and immunocompromised mice by needle inoculation and tick bite transmission. Moreover,
bb0562
promoted spirochete survival during the blood dissemination phase of infection. Gene
bb0562
was also found to be important for spirochete growth in low serum media and the growth defect of Δ
bb0562 B
.
burgdorferi
was rescued with the addition of various long chain fatty acids, particularly oleic acid. In mammals, fatty acids are primarily stored in fat droplets in the form of triglycerides. Strikingly, addition of glyceryl trioleate, the triglyceride form of oleic acid, to the low serum media did not rescue the growth defect of the mutant, suggesting
bb0562
may be important for the release of fatty acids from triglycerides. Therefore, we searched for and identified two canonical GXSXG lipase motifs within BB0562, despite the lack of homology to known bacterial lipases. Purified BB0562 demonstrated lipolytic activity dependent on the catalytic serine residues within the two motifs. In sum, we have established that
bb0562
is a novel nutritional virulence determinant, encoding a lipase that contributes to fatty acid scavenge for spirochete survival in environments deficient in free fatty acids including the mammalian host.
Lyme Disease Pathogenesis Coburn, Jenifer; Garcia, Brandon; Hu, Linden T ...
Current Issues in Molecular Biology,
2021, Letnik:
42
Journal Article
Recenzirano
Odprti dostop
Lyme disease
are obligately parasitic, tick- transmitted, invasive, persistent bacterial pathogens that cause disease in humans and non-reservoir vertebrates primarily through the induction of ...inflammation. During transmission from the infected tick, the bacteria undergo significant changes in gene expression, resulting in adaptation to the mammalian environment. The organisms multiply and spread locally and induce inflammatory responses that, in humans, result in clinical signs and symptoms.
virulence involves a multiplicity of mechanisms for dissemination and colonization of multiple tissues and evasion of host immune responses. Most of the tissue damage, which is seen in non-reservoir hosts, appears to result from host inflammatory reactions, despite the low numbers of bacteria in affected sites. This host response to the Lyme disease
can cause neurologic, cardiovascular, arthritic, and dermatologic manifestations during the disseminated and persistent stages of infection. The mechanisms by which a paucity of organisms (in comparison to many other infectious diseases) can cause varied and in some cases profound inflammation and symptoms remains mysterious but are the subjects of diverse ongoing investigations. In this review, we provide an overview of virulence mechanisms and determinants for which roles have been demonstrated
, primarily in mouse models of infection.
Lyme disease is a multistage inflammatory disease caused by the spirochete Borrelia burgdorferi transmitted through the bite of an infected Ixodes scapularis tick. We previously discovered a B. ...burgdorferi infectivity gene, bbk13, that facilitates mammalian infection by promoting spirochete population expansion in the skin inoculation site. Initial characterization of bbk13 was carried out using an intradermal needle inoculation model of mouse infection, which does not capture the complex interplay of the pathogen-vector-host triad of natural transmission. Here, we aimed to understand the role of bbk13 in the enzootic cycle of B. burgdorferi. B. burgdorferi spirochetes lacking bbk13 were unable to be acquired by naive larvae fed on needle-inoculated mice. Using a capsule feeding approach to restrict tick feeding activity to a defined skin site, we determined that delivery by tick bite alleviated the population expansion defect in the skin observed after needle inoculation of Δbbk13 B. burgdorferi. Despite overcoming the early barrier in the skin, Δbbk13 B. burgdorferi remained attenuated for distal tissue colonization after tick transmission. Disseminated infection by Δbbk13 B. burgdorferi was improved in needle-inoculated immunocompromised mice. Together, we established that bbk13 is crucial to the maintenance of B. burgdorferi in the enzootic cycle and that bbk13 is necessary beyond early infection in the skin, likely contributing to host immune evasion. Moreover, our data highlight the critical interplay between the pathogen, vector, and host as well as the distinct molecular genetic requirements for B. burgdorferi to survive at the pathogen-vector-host interface and achieve productive disseminated infection.
Borrelia burgdorferi is the causative agent of Lyme disease, the leading tick-borne illness in the United States. However, due to, in part, to the significant number of proteins of unknown function ...encoded across the complex fragmented genome, the molecular mechanisms of B. burgdorferi infection remain largely undefined. Previous work identified the virulence determinant gene, bbk13, which is critical for B. burgdorferi’s ability to establish a productive disseminated infection. BBK13 is an immunogenic, non-surface exposed protein of unknown function predicted to harbor an N-terminal transmembrane domain and annotated as a member of the SIMPL domain protein superfamily (PF04402). In eukaryotes, SIMPL domain proteins have been shown to contribute to NF-kappa-B signaling but have no known functions in prokaryotes. Herein we investigated the biochemical and biophysical properties of BBK13 toward elucidation of its function. Bioinformatics analysis revealed secondary and tertiary structural homology between BBK13 and two other prokaryotic SIMPL domain proteins for which the crystal structures have been solved, Brucella abortus BP26 and Campylobacter jejuni cjSLP. Furthermore, comparable to BP26, recombinant BBK13 self-assembled into multimeric complexes in vitro and endogenous BBK13 was found in large oligomeric complexes in the spirochete membrane. Together these data suggest that the oligomeric structure of BBK13 may be important for the molecular function of this critical infection protein.
•BBK13 is SIMPL domain protein critical for B. burgdorferi infection.•BBK13 demonstrates structural homology with Brucella BP26.•Recombinant BBK13 self-associates and forms multimers in solution.•Endogenous BBK13 forms higher order oligomers in the spirochete membrane.
Complex genetic networks consist of structural modules that determine the levels and timing of a cellular response. While the functional properties of the regulatory architectures that make up these ...modules have been extensively studied, the evolutionary history of regulatory architectures has remained largely unexplored. Here, we investigate the transition between direct and indirect regulatory pathways governing inducible resistance to the antibiotic polymyxin B in enteric bacteria. We identify a novel regulatory architecture -- designated feedforward connector loop -- that relies on a regulatory protein that connects signal transduction systems post-translationally, allowing one system to respond to a signal activating another system. The feedforward connector loop is characterized by rapid activation, slow deactivation, and elevated mRNA expression levels in comparison with the direct regulation circuit. Our results suggest that, both functionally and evolutionarily, the feedforward connector loop is the transitional stage between direct transcriptional control and indirect regulation.
Borrelia burgdorferi, the bacterial pathogen responsible for Lyme disease, modulates its gene expression profile in response to the environments encountered throughout its tick-mammal infectious ...cycle. To begin to characterize the B. burgdorferi transcriptome during murine infection, we previously employed an in vivo expression technology-based approach (BbIVET). This identified 233 putative promoters, many of which mapped to un-annotated regions of the complex, segmented genome. Herein, we globally identify the 5' end transcriptome of B. burgdorferi grown in culture as a means to validate non-ORF associated promoters discovered through BbIVET. We demonstrate that 119 BbIVET promoters are associated with transcription start sites (TSSs) and validate novel RNA transcripts using Northern blots and luciferase promoter fusions. Strikingly, 49% of BbIVET promoters were not found to associate with TSSs. This finding suggests that these sequences may be primarily active in the mammalian host. Furthermore, characterization of the 6042 B. burgdorferi TSSs reveals a variety of RNAs including numerous antisense and intragenic transcripts, leaderless RNAs, long untranslated regions and a unique nucleotide frequency for initiating intragenic transcription. Collectively, this is the first comprehensive map of TSSs in B. burgdorferi and characterization of previously un-annotated RNA transcripts expressed by the spirochete during murine infection.
This study demonstrates a strict temporal requirement for a virulence determinant of the Lyme disease spirochete Borrelia burgdorferi during a unique point in its natural infection cycle, which ...alternates between ticks and small mammals. OspC is a major surface protein produced by B. burgdorferi when infected ticks feed but whose synthesis decreases after transmission to a mammalian host. We have previously shown that spirochetes lacking OspC are competent to replicate in and migrate to the salivary glands of the tick vector but do not infect mice. Here we assessed the timing of the requirement for OspC by using an ospC mutant complemented with an unstable copy of the ospC gene and show that B. burgdorferi's requirement for OspC is specific to the mammal and limited to a critical early stage of mammalian infection. By using this unique system, we found that most bacterial reisolates from mice persistently infected with the initially complemented ospC mutant strain no longer carried the wild-type copy of ospC. Such spirochetes were acquired by feeding ticks and migrated to the tick salivary glands during subsequent feeding. Despite normal behavior in ticks, these ospC mutant spirochetes did not infect naive mice. ospC mutant spirochetes from persistently infected mice also failed to infect naive mice by tissue transplantation. We conclude that OspC is indispensable for establishing infection by B. burgdorferi in mammals but is not required at any other point of the mouse-tick infection cycle.
Genetic Manipulation of Borrelia Rosa, Patricia A; Jewett, Mollie W
Current Issues in Molecular Biology,
2021, Letnik:
42
Journal Article
Recenzirano
Odprti dostop
Genetic studies in
require special consideration of the highly segmented genome, complex growth requirements and evolutionary distance of spirochetes from other genetically tractable bacteria. ...Despite these challenges, a robust molecular genetic toolbox has been constructed to investigate the biology and pathogenic potential of these important human pathogens. In this review we summarize the tools and techniques that are currently available for the genetic manipulation of
, including the relapsing fever spirochetes, viewing them in the context of their utility and shortcomings. Our primary objective is to help researchers discern what is feasible and what is not practical when thinking about potential genetic experiments in
. We have summarized published methods and highlighted their critical elements, but we are not providing detailed protocols. Although many advances have been made since
was first transformed over 25 years ago, some standard genetic tools remain elusive for
. We mention these limitations and why they persist, if known. We hope to encourage investigators to explore what might be possible, in addition to optimizing what currently can be achieved, through genetic manipulation of
.
Lyme disease is caused by the spirochete
and is transmitted via the bite of an infected tick.
enters the skin, disseminates via the bloodstream, and infects various distal tissues, leading to ...inflammatory sequelae, such as Lyme arthritis and Lyme carditis.
linear plasmid 36 (lp36) is critical for mammalian infectivity; however, the full complement of genes on lp36 that contribute to this process remains unknown. Through a targeted mutagenesis screen of the genes on lp36, we identified a novel infectivity gene of unknown function,
, which encodes an immunogenic, non-surface-exposed membrane protein that is important for efficient mammalian infection. Loss of
resulted in reduced spirochete loads in distal tissues in a mouse model of infection. Through a detailed analysis of
infection kinetics, we discovered that
is important for promoting spirochete proliferation in the skin inoculation site. The attenuated ability of Δ
spirochetes to proliferate in the inoculation site was followed by reduced numbers of
spirochetes in the bloodstream and, ultimately, consistently reduced spirochete loads in distal tissues. Together, our data indicate that
contributes to disseminated infection by promoting spirochete proliferation in the early phase of infection in the skin. This work not only increases the understanding of the contribution of the genes on lp36 to
infection but also begins to define the genetic basis for
expansion in the skin during localized infection and highlights the influence of the early expansion of spirochetes in the skin on the outcome of infection.
The Lyme disease agent, Borrelia burgdorferi, harbors a significantly reduced genome and relies on the scavenging of critical nutrients from its tick and mammalian hosts for survival. Riboflavin ...salvage has been shown to be important for B. burgdorferi infection of mice, yet the contributions of riboflavin to B. burgdorferi metabolism and survival in the tick remain unknown. Using a targeted mass spectrometry approach, we confirmed the importance of bb0318, the putative ATPase component of an ABC‐type riboflavin transporter, for riboflavin salvage and the production of FMN and FAD. This analysis further revealed that Δbb0318 B. burgdorferi displayed increased levels of glycerol 3‐phosphate compared to the wild‐type. The glycerol 3‐phosphate dehydrogenase activity of GlpD was found to be FAD‐dependent and the transcription and translation of glpD were significantly decreased in Δbb0318 B. burgdorferi. Finally, gene bb0318 was found to be important for maximal spirochete burden in unfed larvae and essential for survival in feeding ticks. Together, these data demonstrate the importance of riboflavin salvage for B. burgdorferi carbon metabolism and survival in ticks.
The Lyme disease agent Borrelia burgdorferi relies on riboflavin (RF) salvage through the BB0316‐BB0319 transport system for the biosynthesis of FMN and FAD. Disruption of riboflavin salvage has consequences for multiple metabolic systems. Riboflavin salvage and maintenance of the pathogen's intracellular flavin pools contribute to the complex regulation of the glycerol utilization (glp) operon and are critical for the glycerol 3‐phosphate dehydrogenase activity of GlpD. Riboflavin salvage is essential for B. burgdorferi survival in ticks.
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