RESUMO
BACKGROUND: Postinfectious Lyme arthritis (LA) is associated with dysregulated immunity and autoreactive T- and B-cell responses in joints. Here we explored the role of host genetic variation in this outcome. METHODS: The frequency of 253 702 single-nucleotide polymorphisms (SNPs) was determined in 147 patients with LA (87 with postinfectious LA and 60 with antibiotic-responsive LA), and for comparison in 90 patients with erythema migrans or the general population (n = 2504). Functional outcome of candidate SNPs was assessed by evaluating their impact on clinical outcome and on immune responses in blood and synovial fluid in patients with LA. RESULTS: Six SNPs associated with late cornified envelope (LCE3) genes were present at greater frequency in patients with postinfectious LA compared to those with antibiotic-responsive LA (70% vs 30%; odds ratio, 2; P < .01). These SNPs were associated with heightened levels of inflammatory Th17 cytokines in serum but lower levels of interleukin 27, a regulatory cytokine, implying that they may contribute to dysregulated Th17 immunity in blood. Moreover, in patients with postinfectious LA, the levels of these Th17 mediators correlated directly with autoantibody responses in synovial fluid, providing a possible link between LCE3 SNPs, maladaptive systemic Th17 immunity, and autoreactive responses in joints. CONCLUSIONS: Variation in the LCE3 locus, a known genetic risk factor in psoriasis and psoriatic arthritis, is associated with dysregulated systemic Th17 immunity and heightened autoantibody responses in joints. These findings underscore the importance of host genetic predisposition and systemic Th17 immunity in the pathogenesis of postinfectious (antibiotic-refractory) Lyme arthritis.
Assuntos
Doença de Lyme , Polimorfismo de Nucleotídeo Único , Células Th17 , Humanos , Doença de Lyme/genética , Doença de Lyme/imunologia , Células Th17/imunologia , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Líquido Sinovial/imunologia , Idoso , Citocinas/genética , Citocinas/metabolismo , Artrite Infecciosa/genética , Artrite Infecciosa/imunologia , Adulto JovemRESUMO
The incidence of Lyme borreliosis has risen, accompanied by persistent symptoms. The innate immune system and related cytokines are crucial in the host response and symptom development. We characterized cytokine production capacity before and after antibiotic treatment in 1,060 Lyme borreliosis patients. We observed a negative correlation between antibody production and IL-10 responses, as well as increased IL-1Ra responses in patients with disseminated disease. Genome-wide mapping the cytokine production allowed us to identify 34 cytokine quantitative trait loci (cQTLs), with 31 novel ones. We pinpointed the causal variant at the TLR1-6-10 locus and validated the regulation of IL-1Ra responses at transcritpome level using an independent cohort. We found that cQTLs contribute to Lyme borreliosis susceptibility and are relevant to other immune-mediated diseases. Our findings improve the understanding of cytokine responses in Lyme borreliosis and provide a genetic map of immune function as an expanded resource.
Assuntos
Citocinas , Doença de Lyme , Locos de Características Quantitativas , Doença de Lyme/imunologia , Doença de Lyme/genética , Doença de Lyme/microbiologia , Humanos , Citocinas/genética , Citocinas/metabolismo , Masculino , Feminino , Interleucina-10/genética , Adulto , Estudo de Associação Genômica Ampla , Pessoa de Meia-Idade , Proteína Antagonista do Receptor de Interleucina 1/genética , Borrelia burgdorferi/imunologia , Borrelia burgdorferi/genética , Antibacterianos , Polimorfismo de Nucleotídeo Único , Predisposição Genética para Doença , IdosoRESUMO
Lyme disease is a tick-borne infection caused by the spirochete Borrelia (Borreliella) burgdorferi. Borrelia species have highly fragmented genomes composed of a linear chromosome and a constellation of linear and circular plasmids some of which are required throughout the enzootic cycle. Included in this plasmid repertoire by almost all Lyme disease spirochetes are the 32-kb circular plasmid cp32 prophages that are capable of lytic replication to produce infectious virions called ÏBB-1. While the B. burgdorferi genome contains evidence of horizontal transfer, the mechanisms of gene transfer between strains remain unclear. While we know that ÏBB-1 transduces cp32 and shuttle vector DNA during in vitro cultivation, the extent of ÏBB-1 DNA transfer is not clear. Herein, we use proteomics and long-read sequencing to further characterize ÏBB-1 virions. Our studies identified the cp32 pac region and revealed that ÏBB-1 packages linear cp32s via a headful mechanism with preferential packaging of plasmids containing the cp32 pac region. Additionally, we find ÏBB-1 packages fragments of the linear chromosome and full-length plasmids including lp54, cp26, and others. Furthermore, sequencing of ÏBB-1 packaged DNA allowed us to resolve the covalently closed hairpin telomeres for the linear B. burgdorferi chromosome and most linear plasmids in strain CA-11.2A. Collectively, our results shed light on the biology of the ubiquitous ÏBB-1 phage and further implicates ÏBB-1 in the generalized transduction of diverse genes and the maintenance of genetic diversity in Lyme disease spirochetes.
Assuntos
Bacteriófagos , Borrelia burgdorferi , Doença de Lyme , Humanos , Borrelia burgdorferi/genética , Bacteriófagos/genética , Plasmídeos/genética , Doença de Lyme/genética , Genômica , DNARESUMO
BACKGROUND: Genetic variation underly inter-individual variation in host immune responses to infectious diseases, and may affect susceptibility or the course of signs and symptoms. METHODS: We performed genome-wide association studies in a prospective cohort of 1138 patients with physician-confirmed Lyme borreliosis (LB), the most common tick-borne disease in the Northern hemisphere caused by the bacterium Borrelia burgdorferi sensu lato. Genome-wide variants in LB patients-divided into a discovery and validation cohort-were compared to two healthy cohorts. Additionally, ex vivo monocyte-derived cytokine responses of peripheral blood mononuclear cells to several stimuli including Borrelia burgdorferi were performed in both LB patient and healthy control samples, as were stimulation experiments using mechanistic/mammalian target of rapamycin (mTOR) inhibitors. In addition, for LB patients, anti-Borrelia antibody responses were measured. Finally, in a subset of LB patients, gene expression was analysed using RNA-sequencing data from the ex vivo stimulation experiments. RESULTS: We identified a previously unknown genetic variant, rs1061632, that was associated with enhanced LB susceptibility. This polymorphism was an eQTL for KCTD20 and ETV7 genes, and its major risk allele was associated with upregulation of the mTOR pathway and cytokine responses, and lower anti-Borrelia antibody production. In addition, we replicated the recently reported SCGB1D2 locus that was suggested to have a protective effect on B. burgdorferi infection, and associated this locus with higher Borrelia burgdorferi antibody indexes and lower IL-10 responses. CONCLUSIONS: Susceptibility for LB was associated with higher anti-inflammatory responses and reduced anti-Borrelia antibody production, which in turn may negatively impact bacterial clearance. These findings provide important insights into the immunogenetic susceptibility for LB and may guide future studies on development of preventive or therapeutic measures. TRIAL REGISTRATION: The LymeProspect study was registered with the International Clinical Trials Registry Platform (NTR4998, registration date 2015-02-13).
Assuntos
Grupo Borrelia Burgdorferi , Borrelia burgdorferi , Borrelia , Doença de Lyme , Humanos , Estudo de Associação Genômica Ampla , Estudos Prospectivos , Leucócitos Mononucleares , Suscetibilidade a Doenças , Doença de Lyme/genética , Doença de Lyme/diagnóstico , Borrelia burgdorferi/genética , Citocinas/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/uso terapêutico , Grupo Borrelia Burgdorferi/genética , Secretoglobinas/genéticaRESUMO
The complement system serves as the first line of defense against invading pathogens by promoting opsonophagocytosis and bacteriolysis. Antibody-dependent activation of complement occurs through the classical pathway and relies on the activity of initiating complement proteases of the C1 complex, C1r and C1s. The causative agent of Lyme disease, Borrelia burgdorferi, expresses two paralogous outer surface lipoproteins of the OspEF-related protein family, ElpB and ElpQ, that act as specific inhibitors of classical pathway activation. We have previously shown that ElpB and ElpQ bind directly to C1r and C1s with high affinity and specifically inhibit C2 and C4 cleavage by C1s. To further understand how these novel protease inhibitors function, we carried out a series of hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments using ElpQ and full-length activated C1s as a model of Elp-protease interaction. Comparison of HDX-MS profiles between unbound ElpQ and the ElpQ/C1s complex revealed a putative C1s-binding site on ElpQ. HDX-MS-guided, site-directed ElpQ mutants were generated and tested for direct binding to C1r and C1s using surface plasmon resonance. Several residues within the C-terminal region of ElpQ were identified as important for protease binding, including a single conserved tyrosine residue that was required for ElpQ- and ElpB-mediated complement inhibition. Collectively, our study identifies key molecular determinants for classical pathway protease recognition by Elp proteins. This investigation improves our understanding of the unique complement inhibitory mechanism employed by Elp proteins which serve as part of a sophisticated complement evasion system present in Lyme disease spirochetes.
Assuntos
Proteínas da Membrana Bacteriana Externa , Borrelia burgdorferi , Via Clássica do Complemento , Humanos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Borrelia burgdorferi/imunologia , Borrelia burgdorferi/metabolismo , Borrelia burgdorferi/genética , Complemento C1r/metabolismo , Complemento C1r/genética , Complemento C1s/metabolismo , Complemento C1s/genética , Complemento C1s/química , Via Clássica do Complemento/imunologia , Lipoproteínas/metabolismo , Lipoproteínas/genética , Lipoproteínas/química , Lipoproteínas/imunologia , Doença de Lyme/genética , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Ligação ProteicaRESUMO
The σ54-σS sigma factor cascade plays a central role in regulating differential gene expression during the enzootic cycle of Borreliella burgdorferi, the Lyme disease pathogen. In this pathway, the primary transcription of rpoS (which encodes σS) is under the control of σ54 which is activated by a bacterial enhancer-binding protein (EBP), Rrp2. The σ54-dependent activation in B. burgdorferi has long been thought to be unique, requiring an additional factor, BosR, a homologue of classical Fur/PerR repressor/activator. However, how BosR is involved in this σ54-dependent activation remains unclear and perplexing. In this study, we demonstrate that BosR does not function as a regulator for rpoS transcriptional activation. Instead, it functions as a novel RNA-binding protein that governs the turnover rate of rpoS mRNA. We further show that BosR directly binds to the 5' untranslated region (UTR) of rpoS mRNA, and the binding region overlaps with a region required for rpoS mRNA degradation. Mutations within this 5'UTR region result in BosR-independent RpoS production. Collectively, these results uncover a novel role of Fur/PerR family regulators as RNA-binding proteins and redefine the paradigm of the σ54-σS pathway in B. burgdorferi.
Assuntos
Proteínas de Bactérias , Borrelia burgdorferi , Regulação Bacteriana da Expressão Gênica , Estabilidade de RNA , Proteínas de Ligação a RNA , Fator sigma , Fator sigma/metabolismo , Fator sigma/genética , Borrelia burgdorferi/genética , Borrelia burgdorferi/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Estabilidade de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Regiões 5' não Traduzidas , Doença de Lyme/microbiologia , Doença de Lyme/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , RNA Polimerase Sigma 54/metabolismo , RNA Polimerase Sigma 54/genéticaRESUMO
Introduction: Lyme disease, the most common tick-borne infectious disease in the US, is caused by a spirochetal pathogen Borrelia burgdorferi (Bb). Distinct host responses are observed in susceptible and resistant strains of inbred of mice following infection with Bb reflecting a subset of inflammatory responses observed in human Lyme disease. The advent of post-genomic methodologies and genomic data sets enables dissecting the host responses to advance therapeutic options for limiting the pathogen transmission and/or treatment of Lyme disease. Methods: In this study, we used single-cell RNA-Seq analysis in conjunction with mouse genomics exploiting GFP-expressing Bb to sort GFP+ splenocytes and GFP- bystander cells to uncover novel molecular and cellular signatures that contribute to early stages of immune responses against Bb. Results: These data decoded the heterogeneity of splenic neutrophils, macrophages, NK cells, B cells, and T cells in C3H/HeN mice in response to Bb infection. Increased mRNA abundance of apoptosis-related genes was observed in neutrophils and macrophages clustered from GFP+ splenocytes. Moreover, complement-mediated phagocytosis-related genes such as C1q and Ficolin were elevated in an inflammatory macrophage subset, suggesting upregulation of these genes during the interaction of macrophages with Bb-infected neutrophils. In addition, the role of DUSP1 in regulating the expression of Casp3 and pro-inflammatory cytokines Cxcl1, Cxcl2, Il1b, and Ccl5 in Bb-infected neutrophils were identified. Discussion: These findings serve as a growing catalog of cell phenotypes/biomarkers among murine splenocytes that can be exploited for limiting spirochetal burden to limit the transmission of the agent of Lyme disease to humans via reservoir hosts.
Assuntos
Borrelia burgdorferi , Doença de Lyme , Camundongos , Humanos , Animais , Borrelia burgdorferi/genética , Transcriptoma , Baço , Análise da Expressão Gênica de Célula Única , Camundongos Endogâmicos C3H , Doença de Lyme/genéticaRESUMO
As an enzootic pathogen, the Lyme disease bacterium Borrelia burgdorferi possesses multiple copies of chemotaxis proteins, including two chemotaxis histidine kinases (CHK), CheA1 and CheA2. Our previous study showed that CheA2 is a genuine CHK that is required for chemotaxis; however, the role of CheA1 remains mysterious. This report first compares the structural features that differentiate CheA1 and CheA2 and then provides evidence to show that CheA1 is an atypical CHK that controls the virulence of B. burgdorferi through modulating the stability of RpoS, a key transcriptional regulator of the spirochete. First, microscopic analyses using green-fluorescence-protein (GFP) tags reveal that CheA1 has a unique and dynamic cellular localization. Second, loss-of-function studies indicate that CheA1 is not required for chemotaxis in vitro despite sharing a high sequence and structural similarity to its counterparts from other bacteria. Third, mouse infection studies using needle inoculations show that a deletion mutant of CheA1 (cheA1mut) is able to establish systemic infection in immune-deficient mice but fails to do so in immune-competent mice albeit the mutant can survive at the inoculation site for up to 28 days. Tick and mouse infection studies further demonstrate that CheA1 is dispensable for tick colonization and acquisition but essential for tick transmission. Lastly, mechanistic studies combining immunoblotting, protein turnover, mutagenesis, and RNA-seq analyses reveal that depletion of CheA1 affects RpoS stability, leading to reduced expression of several RpoS-regulated virulence factors (i.e., OspC, BBK32, and DbpA), likely due to dysregulated clpX and lon protease expression. Bulk RNA-seq analysis of infected mouse skin tissues further show that cheA1mut fails to elicit mouse tnf-α, il-10, il-1ß, and ccl2 expression, four important cytokines for Lyme disease development and B. burgdorferi transmigration. Collectively, these results reveal a unique role and regulatory mechanism of CheA1 in modulating virulence factor expression and add new insights into understanding the regulatory network of B. burgdorferi.
Assuntos
Borrelia burgdorferi , Doença de Lyme , Carrapatos , Animais , Camundongos , Histidina Quinase/genética , Histidina Quinase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Virulência , Quimiotaxia , Doença de Lyme/genética , Doença de Lyme/microbiologia , Carrapatos/microbiologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator sigma/genética , Fator sigma/metabolismoRESUMO
Ixodes scapularis, the black-legged tick, is the principal vector of the Lyme disease spirochete, Borrelia burgdorferi, and is responsible for most of the â¼470,000 estimated Lyme disease cases annually in the USA. Ixodes scapularis can transmit six additional pathogens of human health significance. Because of its medical importance, I. scapularis was the first tick genome to be sequenced and annotated. However, the first assembly, I. scapularis Wikel (IscaW), was highly fragmented because of the technical challenges posed by the long, repetitive genome sequences characteristic of arthropod genomes and the lack of long-read sequencing techniques. Although I. scapularis has emerged as a model for tick research because of the availability of new tools such as embryo injection and CRISPR-Cas9-mediated gene editing yet the lack of chromosome-scale scaffolds has slowed progress in tick biology and the development of tools for their control. Here we combine diverse technologies to produce the I. scapularis Gulia-Nuss (IscGN) genome assembly and gene set. We used DNA from eggs and male and female adult ticks and took advantage of Hi-C, PacBio HiFi sequencing, and Illumina short-read sequencing technologies to produce a chromosome-level assembly. In this work, we present the predicted pseudochromosomes consisting of 13 autosomes and the sex pseudochromosomes: X and Y, and a markedly improved genome annotation compared with the existing assemblies and annotations.
Assuntos
Borrelia burgdorferi , Ixodes , Doença de Lyme , Animais , Masculino , Feminino , Humanos , Ixodes/genética , Doença de Lyme/genética , Borrelia burgdorferi/genética , Genoma/genética , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Transcription termination is an essential and dynamic process that can tune gene expression in response to diverse molecular signals. Yet, the genomic positions, molecular mechanisms, and regulatory consequences of termination have only been studied thoroughly in model bacteria. Here, we use several RNA-seq approaches to map RNA ends for the transcriptome of the spirochete Borrelia burgdorferi - the etiological agent of Lyme disease. We identify complex gene arrangements and operons, untranslated regions and small RNAs. We predict intrinsic terminators and experimentally test examples of Rho-dependent transcription termination. Remarkably, 63% of RNA 3' ends map upstream of or internal to open reading frames (ORFs), including genes involved in the unique infectious cycle of B. burgdorferi. We suggest these RNAs result from premature termination, processing and regulatory events such as cis-acting regulation. Furthermore, the polyamine spermidine globally influences the generation of truncated mRNAs. Collectively, our findings provide insights into transcription termination and uncover an abundance of potential RNA regulators in B. burgdorferi.
Assuntos
Borrelia burgdorferi , Doença de Lyme , Humanos , Borrelia burgdorferi/genética , Transcriptoma , RNA , Doença de Lyme/genética , Doença de Lyme/microbiologia , Transcrição Gênica , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
PlzA is a c-di-GMP-binding protein crucial for adaptation of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi during its enzootic life cycle. Unliganded apo-PlzA is important for vertebrate infection, while liganded holo-PlzA is important for survival in the tick; however, the biological function of PlzA has remained enigmatic. Here, we report that PlzA has RNA chaperone activity that is inhibited by c-di-GMP binding. Holo- and apo-PlzA bind RNA and accelerate RNA annealing, while only apo-PlzA can strand displace and unwind double-stranded RNA. Guided by the crystal structure of PlzA, we identified several key aromatic amino acids protruding from the N- and C-terminal domains that are required for RNA-binding and unwinding activity. Our findings illuminate c-di-GMP as a switch controlling the RNA chaperone activity of PlzA, and we propose that complex RNA-mediated modulatory mechanisms allow PlzA to regulate gene expression during both the vector and host phases of the B. burgdorferi life cycle.
Assuntos
Grupo Borrelia Burgdorferi , Borrelia burgdorferi , Ixodes , Doença de Lyme , Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/metabolismo , Grupo Borrelia Burgdorferi/genética , Doença de Lyme/genética , RNA/metabolismoRESUMO
The blacklegged tick (Ixodes scapularis (Journal of the Academy of Natural Sciences of Philadelphia, 1821, 2, 59)) is a vector of Borrelia burgdorferi sensu stricto (s.s.) (International Journal of Systematic Bacteriology, 1984, 34, 496), the causative bacterial agent of Lyme disease, part of a slow-moving epidemic of Lyme borreliosis spreading across the northern hemisphere. Well-known geographical differences in the vectorial capacity of these ticks are associated with genetic variation. Despite the need for detailed genetic information in this disease system, previous phylogeographical studies of these ticks have been restricted to relatively few populations or few genetic loci. Here we present the most comprehensive phylogeographical study of genome-wide markers in I. scapularis, conducted by using 3RAD (triple-enzyme restriction-site associated sequencing) and surveying 353 ticks from 33 counties throughout the species' range. We found limited genetic variation among populations from the Northeast and Upper Midwest, where Lyme disease is most common, and higher genetic variation among populations from the South. We identify five spatially associated genetic clusters of I. scapularis. In regions where Lyme disease is increasing in frequency, the I. scapularis populations genetically group with ticks from historically highly Lyme-endemic regions. Finally, we identify 10 variable DNA sites that contribute the most to population differentiation. These variable sites cluster on one of the chromosome-scale scaffolds for I. scapularis and are within identified genes. Our findings illuminate the need for additional research to identify loci causing variation in the vectorial capacity of I. scapularis and where additional tick sampling would be most valuable to further understand disease trends caused by pathogens transmitted by I. scapularis.
Assuntos
Borrelia burgdorferi , Ixodes , Doença de Lyme , Animais , Ixodes/genética , Ixodes/microbiologia , Filogeografia , Doença de Lyme/genética , Doença de Lyme/microbiologia , Borrelia burgdorferi/genética , BactériasRESUMO
Borreliella (syn. Borrelia) burgdorferi is a spirochete bacterium that causes tick-borne Lyme disease. Along its lifecycle B. burgdorferi develops several pleomorphic forms with unclear biological and medical relevance. Surprisingly, these morphotypes have never been compared at the global transcriptome level. To fill this void, we grew B. burgdorferi spirochete, round body, bleb, and biofilm-dominated cultures and recovered their transcriptomes by RNAseq profiling. We found that round bodies share similar expression profiles with spirochetes, despite their morphological differences. This sharply contrasts to blebs and biofilms that showed unique transcriptomes, profoundly distinct from spirochetes and round bodies. To better characterize differentially expressed genes in non-spirochete morphotypes, we performed functional, positional, and evolutionary enrichment analyses. Our results suggest that spirochete to round body transition relies on the delicate regulation of a relatively small number of highly conserved genes, which are located on the main chromosome and involved in translation. In contrast, spirochete to bleb or biofilm transition includes substantial reshaping of transcription profiles towards plasmids-residing and evolutionary young genes, which originated in the ancestor of Borreliaceae. Despite their abundance the function of these Borreliaceae-specific genes is largely unknown. However, many known Lyme disease virulence genes implicated in immune evasion and tissue adhesion originated in this evolutionary period. Taken together, these regularities point to the possibility that bleb and biofilm morphotypes might be important in the dissemination and persistence of B. burgdorferi inside the mammalian host. On the other hand, they prioritize the large pool of unstudied Borreliaceae-specific genes for functional characterization because this subset likely contains undiscovered Lyme disease pathogenesis genes.
Assuntos
Borrelia burgdorferi , Doença de Lyme , Animais , Humanos , Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/genética , Borrelia burgdorferi/metabolismo , Doença de Lyme/genética , Mamíferos/metabolismo , TranscriptomaRESUMO
The RNA polymerase alternative σ factor RpoS in Borrelia burgdorferi (Bb), the Lyme disease pathogen, is responsible for programmatic-positive and -negative gene regulation essential for the spirochete's dual-host enzootic cycle. RpoS is expressed during tick-to-mammal transmission and throughout mammalian infection. Although the mammalian-phase RpoS regulon is well described, its counterpart during the transmission blood meal is unknown. Here, we used Bb-specific transcript enrichment by tick-borne disease capture sequencing (TBDCapSeq) to compare the transcriptomes of WT and ΔrpoS Bb in engorged nymphs and following mammalian host-adaptation within dialysis membrane chambers. TBDCapSeq revealed dramatic changes in the contours of the RpoS regulon within ticks and mammals and further confirmed that RpoS-mediated repression is specific to the mammalian-phase of Bb's enzootic cycle. We also provide evidence that RpoS-dependent gene regulation, including repression of tick-phase genes, is required for persistence in mice. Comparative transcriptomics of engineered Bb strains revealed that the Borrelia oxidative stress response regulator (BosR), a noncanonical Fur family member, and the cyclic diguanosine monophosphate (c-di-GMP) effector PlzA reciprocally regulate the function of RNA polymerase complexed with RpoS. BosR is required for RpoS-mediated transcription activation and repression in addition to its well-defined role promoting transcription of rpoS by the RNA polymerase alternative σ factor RpoN. During transmission, ligand-bound PlzA antagonizes RpoS-mediated repression, presumably acting through BosR.
Assuntos
Borrelia burgdorferi , Borrelia , Doença de Lyme , Carrapatos , Camundongos , Animais , Borrelia burgdorferi/genética , Borrelia/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carrapatos/genética , Fator sigma/genética , Fator sigma/metabolismo , Doença de Lyme/genética , Mamíferos/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
Borrelia burgdorferi, the tick-transmitted spirochete agent of Lyme disease, has a highly segmented genome with a linear chromosome and various linear or circular plasmids. Here, by imaging several chromosomal loci and 16 distinct plasmids, we show that B. burgdorferi is polyploid during growth in culture and that the number of genome copies decreases during stationary phase. B. burgdorferi is also polyploid inside fed ticks and chromosome copies are regularly spaced along the spirochete's length in both growing cultures and ticks. This patterning involves the conserved DNA partitioning protein ParA whose localization is controlled by a potentially phage-derived protein, ParZ, instead of its usual partner ParB. ParZ binds its own coding region and acts as a centromere-binding protein. While ParA works with ParZ, ParB controls the localization of the condensin, SMC. Together, the ParA/ParZ and ParB/SMC pairs ensure faithful chromosome inheritance. Our findings underscore the plasticity of cellular functions, even those as fundamental as chromosome segregation.
Assuntos
Borrelia burgdorferi , Doença de Lyme , Humanos , Borrelia burgdorferi/genética , Poliploidia , DNA , Doença de Lyme/genética , Segregação de CromossomosRESUMO
Intracellular compartmentalization of ligands, receptors and signaling molecules has been recognized as an important regulator of inflammation. The toll-like receptor (TLR) 2 pathway utilizes the trafficking molecule adaptor protein 3 (AP-3) to activate interleukin (IL)-6 signaling from within phagosomal compartments. To better understand the vesicular pathways that may contribute to intracellular signaling and cooperate with AP-3, we performed a vesicular siRNA screen. We identified Rab8 and Rab11 GTPases as important in IL-6 induction upon stimulation with the TLR2 ligand Pam3 CSK4 or the pathogen, Borrelia burgdorferi (Bb), the causative agent of Lyme disease. These Rabs were recruited to late and lysosomal stage phagosomes and co-transported with TLR2 signaling adaptors and effectors, such as MyD88, TRAM and TAK1, in an AP-3-dependent manner. Our data support a model where AP-3 mediates the recruitment of recycling and secretory vesicles and the assembly of signaling complexes at the phagosome.
Assuntos
Borrelia burgdorferi , Doença de Lyme , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Borrelia burgdorferi/metabolismo , Ligantes , Doença de Lyme/genética , Doença de Lyme/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Fagossomos/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Proteínas rab de Ligação ao GTP , Animais , CamundongosRESUMO
The acceleration of climate change has been associated with an alarming increase in the prevalence and geographic range of tick-borne diseases (TBD), many of which have severe and long-lasting effects-particularly when treatment is delayed principally due to inadequate diagnostics and lack of physician suspicion. Moreover, there is a paucity of treatment options for many TBDs that are complicated by diagnostic limitations for correctly identifying the offending pathogens. This review will focus on the biology, disease pathology, and detection methodologies used for the Borreliaceae family which includes the Lyme disease agent Borreliella burgdorferi. Previous work revealed that Borreliaceae genomes differ from most bacteria in that they are composed of large numbers of replicons, both linear and circular, with the main chromosome being the linear with telomeric-like termini. While these findings are novel, additional gene-specific analyses of each class of these multiple replicons are needed to better understand their respective roles in metabolism and pathogenesis of these enigmatic spirochetes. Historically, such studies were challenging due to a dearth of both analytic tools and a sufficient number of high-fidelity genomes among the various taxa within this family as a whole to provide for discriminative and functional genomic studies. Recent advances in long-read whole-genome sequencing, comparative genomics, and machine-learning have provided the tools to better understand the fundamental biology and phylogeny of these genomically-complex pathogens while also providing the data for the development of improved diagnostics and therapeutics.
Assuntos
Borrelia burgdorferi , Doença de Lyme , Borrelia burgdorferi/genética , Genoma Bacteriano , Genômica/métodos , Humanos , Doença de Lyme/genética , Doença de Lyme/microbiologia , FilogeniaRESUMO
Host association-the selective adaptation of pathogens to specific host species-evolves through constant interactions between host and pathogens, leaving a lot yet to be discovered on immunological mechanisms and genomic determinants. The causative agents of Lyme disease (LD) are spirochete bacteria composed of multiple species of the Borrelia burgdorferi sensu lato complex, including B. burgdorferi (Bb), the main LD pathogen in North America-a useful model for the study of mechanisms underlying host-pathogen association. Host adaptation requires pathogens' ability to evade host immune responses, such as complement, the first-line innate immune defense mechanism. We tested the hypothesis that different host-adapted phenotypes among Bb strains are linked to polymorphic loci that confer complement evasion traits in a host-specific manner. We first examined the survivability of 20 Bb strains in sera in vitro and/or bloodstream and tissues in vivo from rodent and avian LD models. Three groups of complement-dependent host-association phenotypes emerged. We analyzed complement-evasion genes, identified a priori among all strains and sequenced and compared genomes for individual strains representing each phenotype. The evolutionary history of ospC loci is correlated with host-specific complement-evasion phenotypes, while comparative genomics suggests that several gene families and loci are potentially involved in host association. This multidisciplinary work provides novel insights into the functional evolution of host-adapted phenotypes, building a foundation for further investigation of the immunological and genomic determinants of host association. IMPORTANCE Host association is the phenotype that is commonly found in many pathogens that preferential survive in particular hosts. The Lyme disease (LD)-causing agent, B. burgdorferi (Bb), is an ideal model to study host association, as Bb is mainly maintained in nature through rodent and avian hosts. A widespread yet untested concept posits that host association in Bb strains is linked to Bb functional genetic variation conferring evasion to complement, an innate defense mechanism in vertebrate sera. Here, we tested this concept by grouping 20 Bb strains into three complement-dependent host-association phenotypes based on their survivability in sera and/or bloodstream and distal tissues in rodent and avian LD models. Phylogenomic analysis of these strains further correlated several gene families and loci, including ospC, with host-specific complement-evasion phenotypes. Such multifaceted studies thus pave the road to further identify the determinants of host association, providing mechanistic insights into host-pathogen interaction.
Assuntos
Borrelia burgdorferi , Borrelia , Doença de Lyme , Humanos , Filogenia , Doença de Lyme/genética , Borrelia burgdorferi/genética , Proteínas do Sistema Complemento/genéticaRESUMO
The bacterial agent of Lyme disease, Borrelia burgdorferi, relies on an intricate gene regulatory network to transit between the disparate Ixodes tick vector and mammalian host environments. We recently reported that a B. burgdorferi mutant lacking a transcriptionally active intergenic region of lp17 displayed attenuated murine tissue colonization and pathogenesis due to altered expression of multiple antigens. In this study, a more detailed characterization of the putative regulatory factor encoded by the intergenic region was pursued. In cis complemented strains featuring mutations aimed at eliminating potential protein translation were capable of full tissue colonization, suggesting that the functional product encoded by the intergenic region is not a protein as previously predicted. In trans complementation of the intergenic region resulted in elevated transcription of the sequence compared to wild type and was found to completely abolish infectivity in both immunocompetent "and immunodeficient mice. Quantitative analysis of transcription of the intergenic region by wild-type B. burgdorferi showed it to be highly induced during murine infection relative to in vitro culture. Lastly, targeted deletion of this intergenic region resulted in significant changes to the transcriptome, including genes with potential roles in transmission and host adaptation. The findings reported herein strongly suggest that this segment of lp17 serves a potentially critical role in the regulation of genes required for adaptation and persistence of the pathogen in a mammalian host.
Assuntos
Borrelia burgdorferi , Ixodes , Doença de Lyme , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/genética , Borrelia burgdorferi/metabolismo , DNA Intergênico/genética , Adaptação ao Hospedeiro , Ixodes/microbiologia , Doença de Lyme/genética , Doença de Lyme/metabolismo , Doença de Lyme/microbiologia , Camundongos , Oligopeptídeos/genética , Oligopeptídeos/metabolismoRESUMO
Hematogenous dissemination is a critical step in the evolution of local infection to systemic disease. The Lyme disease (LD) spirochete, which efficiently disseminates to multiple tissues, has provided a model for this process, in particular for the key early event of pathogen adhesion to the host vasculature. This occurs under shear force mediated by interactions between bacterial adhesins and mammalian cell-surface proteins or extracellular matrix (ECM). Using real-time intravital imaging of the Lyme spirochete in living mice, we previously identified BBK32 as the first LD spirochetal adhesin demonstrated to mediate early vascular adhesion in a living mouse; however, deletion of bbk32 resulted in loss of only about half of the early interactions, suggesting the existence of at least one other adhesin (adhesin-X) that promotes early vascular interactions. VlsE, a surface lipoprotein, was identified long ago by its capacity to undergo rapid antigenic variation, is upregulated in the mammalian host and required for persistent infection in immunocompetent mice. In immunodeficient mice, VlsE shares functional overlap with OspC, a multi-functional protein that displays dermatan sulfate-binding activity and is required for joint invasion and colonization. In this research, using biochemical and genetic approaches as well as intravital imaging, we have identified VlsE as adhesin-X; it is a dermatan sulfate (DS) adhesin that efficiently promotes transient adhesion to the microvasculature under shear force via its DS binding pocket. Intravenous inoculation of mice with a low-passage infectious B. burgdorferi strain lacking both bbk32 and vlsE almost completely eliminated transient microvascular interactions. Comparative analysis of binding parameters of VlsE, BBK32 and OspC provides a possible explanation why these three DS adhesins display different functionality in terms of their ability to promote early microvascular interactions.