RESUMEN
Flagella propel pathogens through their environments yet are expensive to synthesize and are immunogenic. Thus, complex hierarchical regulatory networks control flagellar gene expression. Spirochetes are highly motile bacteria, but peculiarly in the Lyme spirochete Borrelia burgdorferi, the archetypal flagellar regulator σ28 is absent. We rediscovered gene bb0268 in B. burgdorferi as flgV, a broadly-conserved gene in the flagellar superoperon alongside σ28 in many Spirochaetes, Firmicutes and other phyla, with distant homologs in Epsilonproteobacteria. We found that B. burgdorferi FlgV is localized within flagellar motors. B. burgdorferi lacking flgV construct fewer and shorter flagellar filaments and are defective in cell division and motility. During the enzootic cycle, B. burgdorferi lacking flgV survive and replicate in Ixodes ticks but are attenuated for dissemination and infection in mice. Our work defines infection timepoints when spirochete motility is most crucial and implicates FlgV as a broadly distributed structural flagellar component that modulates flagellar assembly.
RESUMEN
Colonization of a localized area of human skin by Borrelia burgdorferi after a bite from an infected tick is the first step in the development of Lyme disease. The initial interaction between the pathogen and the human host cells is suggested to impact later outcomes of the infection. MicroRNAs (miRNAs) are well known to be important regulators of host inflammatory and immune responses. While miRNAs have been shown to play a role in the inflammatory response to B. burgdorferi at late stages of infection in the joints, the contributions of miRNAs to early B. burgdorferi infection have yet to be explored. To address this knowledge gap, we used the published host transcriptional responses to B. burgdorferi in erythema migrans skin lesions of early Lyme disease patients and a human dermal fibroblasts (HDFs)/B. burgdorferi co-culture model to predict putative upstream regulator miRNAs. This analysis predicted a role for miR146a-5p in both, B. burgdorferi-infected skin and -stimulated HDFs. miR146a-5p was confirmed to be significantly upregulated in HDF stimulated with B. burgdorferi for 24 hours compared to uninfected control cells. Furthermore, manipulation of miR146a-5p expression (overexpression or inhibition) altered the B. burgdorferi driven inflammatory profile of HDF cells. Our results suggest that miR146a-5p is an important upstream regulator of the transcriptional and immune early response to early B. burgdorferi infection.
Asunto(s)
Borrelia burgdorferi , Enfermedad de Lyme , MicroARNs , Humanos , Borrelia burgdorferi/genética , Piel/patología , MicroARNs/genética , Fibroblastos/patologíaRESUMEN
Vector-borne diseases transmitted through the bites of hematophagous arthropods, such as mosquitoes, continue to be a significant threat to human health globally. Transmission of disease by biting arthropod vectors includes interactions between (1) saliva expectorated by a vector during blood meal acquisition from a human host, (2) the transmitted vector-borne pathogens, and (3) host cells present at the skin bite site. Currently, the investigation of bite-site biology is challenged by the lack of model 3D human skin tissues for in vitro analyses. To help fill this gap, we have used a tissue engineering approach to develop new stylized human dermal microvascular bed tissue approximates-complete with warm blood-built with 3D capillary alginate gel (Capgel) biomaterial scaffolds. These engineered tissues, termed a Biologic Interfacial Tissue-Engineered System (BITES), were cellularized with either human dermal fibroblasts (HDFs) or human umbilical vein endothelial cells (HUVECs). Both cell types formed tubular microvessel-like tissue structures of oriented cells (82% and 54% for HDFs and HUVECs, respectively) lining the unique Capgel parallel capillary microstructures. Female Aedes (Ae.) aegypti mosquitoes, a prototypic hematophagous biting vector arthropod, swarmed, bit, and probed blood-loaded HDF BITES microvessel bed tissues that were warmed (34-37 °C), acquiring blood meals in 151 ± 46 s on average, with some ingesting â³4 µL or more of blood. Further, these tissue-engineered constructs could be cultured for at least three (3) days following blood meal acquisitions. Altogether, these studies serve as a powerful proof-of-concept demonstration of the innovative BITES platform and indicate its potential for the future investigation of arthropod bite-site cellular and molecular biology.
RESUMEN
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.
Asunto(s)
Borrelia burgdorferi , Ixodes , Enfermedad de Lyme , Animales , Ratones , Adenosina Trifosfatasas , Borrelia burgdorferi/genética , Carbono , Mononucleótido de Flavina , Flavina-Adenina Dinucleótido , Mamíferos , Oxidorreductasas , RiboflavinaRESUMEN
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 Δbb0562 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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ácidos Grasos/deficiencia , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-Patógeno , Lipasa/metabolismo , Enfermedad de Lyme/microbiología , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Borrelia burgdorferi/fisiología , Femenino , Ixodes/microbiología , Enfermedad de Lyme/inmunología , Enfermedad de Lyme/metabolismo , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos NOD , Factores de Virulencia/genéticaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Borrelia burgdorferi/genética , Borrelia burgdorferi/patogenicidad , Enfermedad de Lyme/microbiología , Animales , Ixodes/microbiología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos NOD , Piel/microbiología , Mordeduras de Garrapatas/microbiologíaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Borrelia burgdorferi/metabolismo , Membrana Celular/metabolismo , Enfermedad de Lyme/metabolismo , Enfermedad de Lyme/microbiología , Multimerización de Proteína , Secuencia de Aminoácidos , Dominios Proteicos , Mapas de Interacción de Proteínas , Proteínas Recombinantes/química , Homología Estructural de ProteínaRESUMEN
Genetic studies in Borrelia 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 Borrelia, 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 Borrelia. We have summarized published methods and highlighted their critical elements, but we are not providing detailed protocols. Although many advances have been made since B. burgdorferi was first transformed over 25 years ago, some standard genetic tools remain elusive for Borrelia. 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 Borrelia.
Asunto(s)
Infecciones por Borrelia/microbiología , Borrelia/genética , Ingeniería Genética , Animales , Susceptibilidad a Enfermedades , Ingeniería Genética/métodos , Interacciones Huésped-Patógeno , Humanos , Enfermedad de Lyme/microbiologíaRESUMEN
Lyme disease Borrelia 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. Borrelia 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 Borrelia 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 in vivo, primarily in mouse models of infection.
Asunto(s)
Borrelia , Susceptibilidad a Enfermedades , Enfermedad de Lyme/microbiología , Animales , Vectores Artrópodos/microbiología , Borrelia/genética , Modelos Animales de Enfermedad , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Enfermedad de Lyme/transmisión , Garrapatas/microbiología , Virulencia , Factores de Virulencia/genéticaRESUMEN
Receptor-interacting protein 2 (RIP2) is a kinase that mediates signaling downstream of the bacterial peptidoglycan sensors NOD1 and NOD2. Genetic loss or pharmaceutical inhibition of RIP2 has been shown to be beneficial in multiple inflammatory disease models with the effects largely attributed to reducing proinflammatory signaling downstream of peptidoglycan recognition. However, given the widespread expression of this kinase and its reported interactions with numerous other proteins, it is possible that RIP2 may also function in roles outside of peptidoglycan sensing. In this work, we show that RIP2 undergoes tyrosine phosphorylation and activation in response to engagement of the Fc γ receptor (FcγR). Using bone marrow-derived macrophages from WT and RIP2-KO mice, we show that loss of RIP2 leads to deficient FcγR signaling and reactive oxygen species (ROS) production upon FcγR cross-linking without affecting cytokine secretion, phagocytosis, or nitrate/nitrite production. The FcγR-induced ROS response was still dependent on NOD2, as macrophages deficient in this receptor showed similar defects. Mechanistically, we found that different members of the Src family kinases (SFKs) can promote RIP2 tyrosine phosphorylation and activation. Altogether, our findings suggest that RIP2 is functionally important in pathways outside of bacterial peptidoglycan sensing and that involvement in such pathways may depend on the actions of SFKs. These findings will have important implications for future therapies designed to target this kinase.
Asunto(s)
Macrófagos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/fisiología , Receptores de IgG/metabolismo , Animales , Citocinas/metabolismo , Inmunidad Innata/inmunología , Macrófagos/inmunología , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis , Fosforilación , Receptores de IgG/genética , Transducción de SeñalRESUMEN
Lyme disease is caused by the spirochete Borrelia burgdorferi and is transmitted via the bite of an infected tick. B. burgdorferi enters the skin, disseminates via the bloodstream, and infects various distal tissues, leading to inflammatory sequelae, such as Lyme arthritis and Lyme carditis. B. burgdorferi 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, bbk13, which encodes an immunogenic, non-surface-exposed membrane protein that is important for efficient mammalian infection. Loss of bbk13 resulted in reduced spirochete loads in distal tissues in a mouse model of infection. Through a detailed analysis of B. burgdorferi infection kinetics, we discovered that bbk13 is important for promoting spirochete proliferation in the skin inoculation site. The attenuated ability of Δbbk13 spirochetes to proliferate in the inoculation site was followed by reduced numbers of B. burgdorferi spirochetes in the bloodstream and, ultimately, consistently reduced spirochete loads in distal tissues. Together, our data indicate that bbk13 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 B. burgdorferi infection but also begins to define the genetic basis for B. burgdorferi 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.
Asunto(s)
Proteínas Bacterianas/sangre , Borrelia burgdorferi/genética , Interacciones Huésped-Parásitos/genética , Enfermedad de Lyme/microbiología , Enfermedad de Lyme/patología , Proteínas Recombinantes/genética , Virulencia/genética , Animales , Proteínas Bacterianas/genética , Modelos Animales de Enfermedad , Ratones , Plásmidos , ConejosRESUMEN
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.
Asunto(s)
Borrelia burgdorferi/genética , Regulación Bacteriana de la Expresión Génica , Enfermedad de Lyme/microbiología , Regiones Promotoras Genéticas , Animales , Borrelia burgdorferi/patogenicidad , Clonación Molecular/métodos , Escherichia coli/genética , Expresión Génica , Biblioteca de Genes , Humanos , Enfermedad de Lyme/patología , Ratones , Plásmidos/genética , Activación Transcripcional , TranscriptomaRESUMEN
Lyme disease, the most commonly reported vector-borne disease in the United States, results from infection with Borrelia burgdorferi. Early clinical diagnosis of this disease is largely based on the presence of an erythematous skin lesion for individuals in high-risk regions. This, however, can be confused with other illnesses including southern tick-associated rash illness (STARI), an illness that lacks a defined etiological agent or laboratory diagnostic test, and is coprevalent with Lyme disease in portions of the eastern United States. By applying an unbiased metabolomics approach with sera retrospectively obtained from well-characterized patients, we defined biochemical and diagnostic differences between early Lyme disease and STARI. Specifically, a metabolic biosignature consisting of 261 molecular features (MFs) revealed that altered N-acyl ethanolamine and primary fatty acid amide metabolism discriminated early Lyme disease from STARI. Development of classification models with the 261-MF biosignature and testing against validation samples differentiated early Lyme disease from STARI with an accuracy of 85 to 98%. These findings revealed metabolic dissimilarity between early Lyme disease and STARI, and provide a powerful and new approach to inform patient management by objectively distinguishing early Lyme disease from an illness with nearly identical symptoms.
Asunto(s)
Exantema/diagnóstico , Exantema/parasitología , Enfermedad de Lyme/diagnóstico , Enfermedad de Lyme/metabolismo , Infestaciones por Garrapatas/diagnóstico , Infestaciones por Garrapatas/metabolismo , Animales , Estudios de Casos y Controles , Simulación por Computador , Diagnóstico Diferencial , Exantema/sangre , Femenino , Geografía , Humanos , Enfermedad de Lyme/sangre , Enfermedad de Lyme/clasificación , Masculino , Redes y Vías Metabólicas , Metaboloma , Metabolómica , Persona de Mediana Edad , Infestaciones por Garrapatas/sangre , Infestaciones por Garrapatas/clasificaciónRESUMEN
Knowledge of the transcriptional responses of vector-borne pathogens at the vector-pathogen interface is critical for understanding disease transmission. Borrelia (Borreliella) burgdorferi, the causative agent of Lyme disease in the United States, is transmitted by the bite of infected Ixodes sp. ticks. It is known that B. burgdorferi has altered patterns of gene expression during tick acquisition, persistence and transmission. Recently, we and others have discovered in vitro expression of RNAs found internal, overlapping, and antisense to annotated open reading frames in the B. burgdorferi genome. However, there is a lack of molecular genetic tools for B. burgdorferi for quantitative, strand-specific, comparative analysis of these transcripts in distinct environments such as the arthropod vector. To address this need, we have developed a dual luciferase reporter system to quantify B. burgdorferi promoter activities in a strand-specific manner. We demonstrate that constitutive expression of a B. burgdorferi codon-optimized Renilla reniformis luciferase gene (rlucBb ) allows normalization of the activity of a promoter of interest when fused to the B. burgdorferi codon-optimized Photinus pyralis luciferase gene (flucBb) on the same plasmid. Using the well characterized, differentially regulated, promoters for flagellin (flaBp), outer surface protein A (ospAp) and outer surface protein C (ospCp), we document the efficacy of the dual luciferase system for quantitation of promoter activities during in vitro growth and in infected ticks. Cumulatively, the dual luciferase method outlined herein is the first dual reporter system for B. burgdorferi, providing a novel and highly versatile approach for strand-specific molecular genetic analyses.
Asunto(s)
Proteínas Bacterianas/genética , Borrelia burgdorferi/genética , Borrelia burgdorferi/metabolismo , Interacciones Huésped-Patógeno/fisiología , Ixodes/microbiología , Luciferasas/genética , Animales , Antígenos Bacterianos/genética , Antígenos de Superficie/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/metabolismo , Vacunas Bacterianas/genética , Borrelia burgdorferi/crecimiento & desarrollo , Borrelia burgdorferi/patogenicidad , Pruebas de Enzimas , Femenino , Flagelina/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Genes Reporteros , Vectores Genéticos/genética , Ixodes/fisiología , Lipoproteínas/genética , Luciferasas/metabolismo , Enfermedad de Lyme/transmisión , Ratones , Ratones Endogámicos C3H , Plásmidos/genéticaRESUMEN
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.
Asunto(s)
Borrelia burgdorferi/genética , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Enfermedad de Lyme/microbiología , Transcriptoma , Animales , Expresión Génica , Genes Reporteros , Genoma Bacteriano , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Regiones Promotoras Genéticas , Reproducibilidad de los Resultados , Sitio de Iniciación de la Transcripción , Regiones no TraducidasRESUMEN
A greater understanding of the molecular mechanisms that Borrelia burgdorferi uses to survive during mammalian infection is critical for the development of novel diagnostic and therapeutic tools to improve the clinical management of Lyme disease. By use of an in vivo expression technology (IVET)-based approach to identify B. burgdorferi genes expressed in vivo, we discovered the bb0318 gene, which is thought to encode the ATPase component of a putative riboflavin ABC transport system. Riboflavin is a critical metabolite enabling all organisms to maintain redox homeostasis. B. burgdorferi appears to lack the metabolic capacity for de novo synthesis of riboflavin and so likely relies on scavenging riboflavin from the host environment. In this study, we sought to investigate the role of bb0318 in B. burgdorferi pathogenesis. No in vitro growth defect was observed for the Δbb0318 clone. However, the mutant spirochetes displayed reduced levels of survival when exposed to exogenous hydrogen peroxide or murine macrophages. Spirochetes lacking bb0318 were found to have a 100-fold-higher 50% infectious dose than spirochetes containing bb0318 In addition, at a high inoculum dose, bb0318 was found to be important for effective spirochete dissemination to deep tissues for as long as 3 weeks postinoculation and to be critical for B. burgdorferi infection of mouse hearts. Together, these data implicate bb0318 in the oxidative stress response of B. burgdorferi and indicate the contribution of bb0318 to B. burgdorferi mammalian infectivity.
Asunto(s)
Proteínas Bacterianas/genética , Borrelia burgdorferi/genética , Borrelia burgdorferi/patogenicidad , Estrés Oxidativo/genética , Factores de Virulencia/genética , Animales , Borrelia burgdorferi/efectos de los fármacos , Modelos Animales de Enfermedad , Regulación Bacteriana de la Expresión Génica , Peróxido de Hidrógeno/farmacología , Enfermedad de Lyme/genética , Ratones , Ratones Endogámicos C3H , Estrés Oxidativo/efectos de los fármacosRESUMEN
The Lyme disease spirochete Borrelia burgdorferi is dependent on purine salvage from the host environment for survival. The genes bbb22 and bbb23 encode purine permeases that are essential for B. burgdorferi mouse infectivity. We now demonstrate the unique contributions of each of these genes to purine transport and murine infection. The affinities of spirochetes carrying bbb22 alone for hypoxanthine and adenine were similar to those of spirochetes carrying both genes. Spirochetes carrying bbb22 alone were able to achieve wild-type levels of adenine saturation but not hypoxanthine saturation, suggesting that maximal hypoxanthine uptake requires the presence of bbb23. Moreover, the purine transport activity conferred by bbb22 was dependent on an additional distal transcriptional start site located within the bbb23 open reading frame. The initial rates of uptake of hypoxanthine and adenine by spirochetes carrying bbb23 alone were below the level of detection. However, these spirochetes demonstrated a measurable increase in hypoxanthine uptake over a 30-min time course. Our findings indicate that bbb22-dependent adenine transport is essential for B. burgdorferi survival in mice. The bbb23 gene was dispensable for B. burgdorferi mouse infectivity, yet its presence was required along with that of bbb22 for B. burgdorferi to achieve maximal spirochete loads in infected mouse tissues. These data demonstrate that both genes, bbb22 and bbb23, are critical for B. burgdorferi to achieve wild-type infection of mice and that the differences in the capabilities of the two transporters may reflect distinct purine salvage needs that the spirochete encounters throughout its natural infectious cycle.
Asunto(s)
Borrelia burgdorferi/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Enfermedad de Lyme/microbiología , Purinas/metabolismo , Animales , Carga Bacteriana , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , Femenino , Ratones , Plásmidos/genéticaRESUMEN
Analysis of the transcriptome of Borrelia burgdorferi, the causative agent of Lyme disease, during infection has proven difficult due to the low spirochete loads in the mammalian tissues. To overcome this challenge, we have developed an In Vivo Expression Technology (IVET) system for identification of B. burgdorferi genes expressed during an active murine infection. Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable.Mouse infection can be restored to these spirochetes by expression of the essential lp25-encoded pnc A gene alone. Therefore, this IVET-based approach selects for in vivo-expressed promoters that drive expression of pncA resulting in the recovery of infectious spirochetes lacking lp25 following a three week infection in mice.Screening of approximately 15,000 clones in mice identified 289 unique in vivo-expressed DNA fragments from across all 22 replicons of the B. burgdorferi B31 genome. The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions. Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent. The bbk46 gene was dispensable for B. burgdorferi infection in mice. Our findings highlight the power of the IVET-based approach for identification of B. burgdorferi in vivo-expressed genes, which might not be discovered using other genome-wide gene expression methods. Further investigation of the novel in vivo-expressed candidate genes will contribute to advancing the understanding of molecular mechanisms of B.burgdorferi survival and pathogenicity in the mammalian host.
Asunto(s)
Borrelia burgdorferi/inmunología , Borrelia burgdorferi/patogenicidad , Enfermedad de Lyme/inmunología , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Virulencia/genética , Secuencia de Aminoácidos , Animales , Borrelia burgdorferi/genética , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano/genética , Genotipo , Enfermedad de Lyme/genética , Enfermedad de Lyme/patología , Ratones , Ratones Endogámicos C3H , Ratones SCID , Sistemas de Lectura Abierta/genética , Fenotipo , Alineación de Secuencia , Transcriptoma/genética , Factores de Virulencia/biosíntesis , Factores de Virulencia/inmunologíaRESUMEN
A serology-based tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease, but it lacks sensitivity in the early stages of disease and is often dependent on subjectively scored immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detecting Borrelia burgdorferi antibodies in patient serum samples that were positive for Lyme disease. To better understand the performance of the Lyme disease iPCR assay, the repeatability and variability of the background of the assay across samples from a healthy population (n = 36) were analyzed. Both of these parameters were found to have coefficients of variation of <3%. Using eight antigen-specific iPCR assays and positive call thresholds established for each assay, iPCR IgM and/or IgG diagnosis from Lyme disease patient serum samples (n = 12) demonstrated a strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach using a single hybrid antigen and detecting only IgG antibodies confirmed the 2-tier diagnosis in the Lyme disease patient serum samples (n = 12). Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient serum samples (n = 92) resulted in a sensitivity of 69% (95% confidence interval [CI], 50% to 84%), compared to that of the 2-tier analysis at 59% (95% CI, 41% to 76%), and a specificity of 98% (95% CI, 91% to 100%) compared to that of the 2-tier analysis at 97% (95% CI, 88% to 100%). A single-tier hybrid antigen iPCR assay has the potential to be an improved method for detecting host-generated antibodies against B. burgdorferi.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Enfermedad de Lyme/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Borrelia burgdorferi/inmunología , Línea Celular , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Enfermedad de Lyme/sangre , Enfermedad de Lyme/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Sensibilidad y EspecificidadRESUMEN
Analysis of the transcriptome of Borrelia burgdorferi, the causative agent of Lyme disease, during infection has proven difficult due to the low spirochete loads in the mammalian tissues. To overcome this challenge, we have developed an In Vivo Expression Technology (IVET) system for identification of B. burgdorferi genes expressed during an active murine infection. Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable. Mouse infection can be restored to these spirochetes by expression of the essential lp25-encoded pncA gene alone. Therefore, this IVET-based approach selects for in vivo-expressed promoters that drive expression of pncA resulting in the recovery of infectious spirochetes lacking lp25 following a three week infection in mice. Screening of approximately 15,000 clones in mice identified 289 unique in vivo-expressed DNA fragments from across all 22 replicons of the B. burgdorferi B31 genome. The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions. Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent. Immunocompetent mice inoculated with spirochetes lacking bbk46 seroconverted but no spirochetes were recovered from mouse tissues three weeks post inoculation. However, the bbk46 gene was not required for B. burgdorferi infection of immunodeficient mice. Therefore, through an initial IVET screen in B. burgdorferi we have identified a novel in vivo-induced virulence factor critical for the ability of the spirochete to evade the humoral immune response and persistently infect mice.