Perpetuation of the Lyme Disease Spirochete Borrelia lusitaniae by Lizards

To determine whether the Lyme disease spirochete Borrelia lusitaniae is associated with lizards, we compared the prevalence and genospecies of spirochetes present in rodent- and lizard-associated ticks at a site where this spirochete frequently infects questing ticks. Whereas questing nymphal Ixodes ricinus ticks were infected mainly by Borrelia afzelii, one-half of the infected adult ticks harbored B. lusitaniae at our study site. Lyme disease spirochetes were more prevalent in sand lizards (Lacerta agilis) and common wall lizards (Podarcis muralis) than in small rodents. Although subadult ticks feeding on rodents acquired mainly B. afzelii, subadult ticks feeding on lizards became infected by B. lusitaniae. Genetic analysis confirmed that the spirochetes isolated from ticks feeding on lizards are members of the B. lusitaniae genospecies and resemble type strain PotiB2. At our central European study site, lizards, which were previously considered zooprophylactic for the agent of Lyme disease, appear to perpetuate B. lusitaniae.     

Transformation of the Lyme Disease Spirochete Borrelia burgdorferi with Heterologous DNA

Studies of the spirochete Borrelia burgdorferi have been hindered by the scarcity of genetic tools that can be used in these bacteria. For the first time, a method has been developed by which heterologous DNA (DNA without a naturally occurring B. burgdorferi homolog) can be introduced into and persistently maintained by B. burgdorferi. This technique uses integration of circular DNA into the bacterial genome via a single-crossover event. The ability to transform B. burgdorferi with heterologous DNA will now permit a wide range of experiments on the biology of these bacteria and their involvement in the many facets of Lyme disease.     

Suggested Role for G4 DNA in Recombinational Switching at the Antigenic Variation Locus of the Lyme Disease Spirochete

Antigenic variation through targeted DNA rearrangements provides a powerful diversity generating mechanism that allows a variety of pathogens to stay one step ahead of acquired immunity in their hosts. The Lyme disease spirochete encodes such a system that is required for persistent infection. The vls locus, carried on a 29 kb linear plasmid (lp28-1) in the type strain B31, carries 15 silent cassettes from which information is unidirectionally transferred into the expression locus, vlsE. Recent studies have surprisingly shown that, with the exception of the RuvAB branch migrase, no other known recombination/repair proteins appear to play a role in the recombinational switching process. In the work presented here we show that G4 DNA can be formed by sequences within the B31 vlsE locus, prompting us to investigate the presence of potential G4-forming DNA throughout the vls locus of several Lyme spirochete strains and species. We found that runs of G, three nucleotides and longer occur at a very high density, with a greater than 100-fold strand-specific distribution in the vls locus of three B. burgdorferi strains as well as in B. afzelii and B. garinii, in spite of the bias for the use of A-T rich codons in Borrelia species. Our findings suggest the possibility that G4 DNA may be a mediator of recombinational switching at the vlsE locus in the Lyme spirochetes.     

Variable VlsE Is Critical for Host Reinfection by the Lyme Disease Spirochete

Many pathogens make use of antigenic variation as a way to evade the host immune response. A key mechanism for immune evasion and persistent infection by the Lyme disease spirochete, Borrelia burgdorferi, is antigenic variation of the VlsE surface protein. Recombination results in changes in the VlsE surface protein that prevent recognition by VlsE-specific antibodies in the infected host. Despite the presence of a substantial number of additional proteins residing on the bacterial surface, VlsE is the only known antigen that exhibits ongoing variation of its surface epitopes. This suggests that B. burgdorferi may utilize a VlsE-mediated system for immune avoidance of its surface antigens. To address this, the requirement of VlsE for host reinfection by the Lyme disease pathogen was investigated. Host-adapted wild type and VlsE mutant spirochetes were used to reinfect immunocompetent mice that had naturally cleared an infection with a VlsE-deficient clone. Our results demonstrate that variable VlsE is necessary for reinfection by B. burgdorferi, and this ability is directly related to evasion of the host antibody response. Moreover, the data presented here raise the possibility that VlsE prevents recognition of B. burgdorferi surface antigens from host antibodies. Overall, our findings represent a significant advance in our knowledge of immune evasion by B. burgdorferi, and provide insight to the possible mechanisms involved in VlsE-mediated immune avoidance.     

Gut Microbiota of the Tick Vector Ixodes scapularis Modulate Colonization of the Lyme Disease Spirochete

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Variation in Streptococcus pyogenes NAD+ Glycohydrolase Is Associated with Tissue Tropism

Streptococcus pyogenes is an important pathogen that causes a variety of diseases. The most common infections involve the throat (pharyngitis) or skin (impetigo); however, the factors that determine tissue tropism and severity are incompletely understood. The S. pyogenes NAD⁺ glycohydrolase (SPN) is a virulence factor that has been implicated in contributing to the pathogenesis of severe infections. However, the role of SPN in determining the bacterium''s tissue tropism has not been evaluated. In this report, we examine the sequences of spn and its endogenous inhibitor ifs from a worldwide collection of S. pyogenes strains. Analysis of average pairwise nucleotide diversity, average number of nucleotide differences, and ratio of nonsynonymous to synonymous substitutions revealed significant diversity in spn and ifs. Application of established models of molecular evolution shows that SPN is evolving under positive selection and diverging into NAD⁺ glycohydrolase (NADase)-active and -inactive subtypes. Additionally, the NADase-inactive SPN subtypes maintain the characteristics of a functional gene while ifs becomes a pseudogene. Thus, NADase-inactive SPN continues to evolve under functional constraint. Furthermore, NADase activity did not correlate with invasive disease in our collection but was associated with tissue tropism. The ability to cause infection at both the pharynx and the skin (“generalist” strains) is correlated with NADase-active SPN, while the preference for causing infection at either the throat or the skin (“specialist” strains) is associated with NADase-inactive SPN. These findings suggest that SPN has a NADase-independent function and prompt a reevaluation of the role of SPN in streptococcal pathogenesis.Many bacterial pathogens that are capable of causing infection at multiple tissue sites have considerable underlying genetic diversity that is reflected by the presence or absence of different subsets of virulence genes or by the presence of alternative alleles of specific virulence genes (37, 44, 48). For the latter genes, variation in sequence may arise under pressure to avoid the immune response or reflect proteins whose functions are diverging. Horizontal gene transfer (HGT) events can initially increase diversity through the reassortment of these variant virulence genes and may result in altered pathogenicity or the ability to more efficiently exploit a given ecological niche (37). Continued selection of fitter variants adapted for infection of a specific niche can then lead to a subsequent purging of genetic diversity and a reduction in the types of clinical syndromes a particular lineage can cause (8). As a consequence, genetically discrete subpopulations with strong tropisms for different tissues emerge within the existing species, and this process may represent a key step in the formation of new species (6). Understanding the changes that occur during niche specialization can provide important insights into pathogenic mechanisms required for infection of a specific tissue.Analysis of tissue-specific adaptation is emerging as an important approach for understanding the pathogenesis of the numerous diseases caused by Streptococcus pyogenes (group A streptococcus [GAS]). This Gram-positive bacterium has a worldwide distribution and is a pathogen of humans exclusively, causing important diseases, which include those that are destructive of tissue and life-threatening (cellulitis, necrotizing fasciitis) and those associated with deregulation of immunity (glomerulonephritis, rheumatic fever) (6, 12). However, most cases of S. pyogenes disease are more superficial and self-limiting and occur at either the throat (pharyngitis) or the skin (impetigo). These two tissue sites also represent the primary reservoirs responsible for dissemination of the organism to new hosts. A large body of epidemiological evidence that suggests that there are distinct subpopulations of strains more adapted for infection of either the throat or the skin has accumulated, suggesting that specific adaptations to these two tissues are driving the evolution of its pan-genome (6). However, the specific adaptations responsible for niche specialization are not well understood.A frequently used approach for uncovering a common molecular basis behind bacterial phenotype has been to group strains based on sequence variation in housekeeping genes (18). In the case of niche specialization, continued selection for variants more highly adapted to a particular tissue will purge neutral gene diversity in the adapted population relative to the population as a whole. However, a complication in deciphering trends associated with tissue adaptation in S. pyogenes has been that despite some niche separation, there are high rates of recombination relative to mutation within the species as a whole, on par with that of Streptococcus pneumoniae, a species considered to be highly recombinogenic (6, 22, 57). Frequent recombination has resulted in a random segregation of neutral housekeeping haplotypes between S. pyogenes strains from ecologically distinct subpopulations (6). Thus, standard approaches to establishing relationships between strains have been of only limited utility for understanding niche adaptation for S. pyogenes.A more productive approach for S. pyogenes has been to look for genetic variation outside neutral housekeeping genes that is strongly associated with ecological niche. In this regard, genotypes based on the gene encoding the M protein (emm) provide a significant correlation with tissue tropism (6). The M protein is a fibrillar surface molecule that plays multiple roles in promoting virulence, and serological typing based on M protein diversity has been the traditional method for classifying S. pyogenes strains (35). It is well established that strains with certain M types have a strong preference for infection at either the throat or the skin (9, 40). There are more than 200 known M types (50), which can be divided into 4 major subfamilies based on the sequence of the peptidoglycan-spanning domain at the 3′ end of emm (25). Furthermore, the emm locus can encode one gene or a combination of subfamily genes in a tandem arrangement (7). Analyses of large strain collections have revealed that in ∼99% of strains, the organization of emm genes in the locus can be assigned to one of five patterns (designated A to E) (6). Although strains with each emm pattern may colonize the same tissue types, there is a strong correlation between emm pattern and the ability of the organism to cause disease at specific tissue sites. Strains with emm patterns A to C generally cause pharyngitis; emm pattern D strains are typically the cause of skin diseases, such as impetigo; and emm pattern E strains are “generalists,” which can cause symptomatic infection at either tissue site at approximately equal fractions of the total (6). Since emm pattern is strongly associated with tissue tropism, it is likely that characteristics consistently coinherited with the emm pattern also play a role in determining the tissue tropism of the organism (6, 29).The S. pyogenes NAD⁺ glycohydrolase (SPN, also known as Nga) is a virulence factor with characteristics that merit evaluation for a possible role in tissue tropism. This secreted toxin has an enzymatic activity (NADase) that cleaves the glycosidic bond of β-NAD⁺ to produce nicotinamide and ADP-ribose. All S. pyogenes strains examined to date possess the gene that encodes SPN (spn), but some strains produce a SPN that lacks detectable NADase activity (1, 30, 36, 42). Since there is evidence that SPN′s robust NADase activity contributes to virulence (4, 43, 52, 56), the existence of NADase-deficient SPN has yet to be explained. Epidemiological studies conducted on several limited strain collections have not been informative, as these studies have both found (1, 52) and failed to find (15) an association between NADase activity and whether a lineage has the capacity to cause invasive disease. Whether or not SPN is associated with tissue tropism is not known.SPN also has multiple complex interactions with other proteins that suggest it has an important, yet incompletely understood role in disease pathogenesis. These interactions also imply that SPN is under considerable coevolutionary pressure with its partners (47). For example, the ability of S. pyogenes to produce NADase-active SPN is absolutely dependent on the presence of an endogenous inhibitor protein, immunity factor for SPN (IFS) (31, 42). IFS is a competitive inhibitor of SPN′s β-NAD⁺ substrate and apparently acts to inhibit self-toxicity resulting from any presecretory SPN molecules that adventitiously fold prior to their export from the streptococcal cell. In the absence of IFS, SPN is lethal for S. pyogenes. Interestingly, strains that produce NADase-inactive SPN also have a truncated form of IFS (42). Once secreted, both NADase-active SPN and NADase-inactive SPN are injected into the host cell cytoplasm by a process known as cytolysin-mediated translocation (CMT), which requires interaction between multiple domains of SPN and the pore-forming cytolysin streptolysin O (SLO) (11, 20, 39, 41). When in the cytoplasmic compartment, NADase-active SPN can trigger rapid cell death, which is associated with depletion of β-NAD⁺ pools (10, 11, 39). The genes for SPN (spn), IFS (ifs), and SLO (slo) are encoded in the same operon (31, 42), as is typical of coevolving virulence factor/inhibitor pairs (47). Thus, SPN has multiple complex interactions and is suspected of being important in pathogenesis; however, there is a considerable amount of genetic and functional variation that has yet to be fully defined.In the present study, we sought to clarify the role of SPN in the infectious process through analysis of the genetic diversity in spn and ifs and the relationship this diversity has with disease severity and ecologic niche. By examining a diverse, worldwide collection of S. pyogenes strains, we identify the SPN domains evolving under positive (diversifying) and negative (purifying) selection, correlate these sites with NADase activity, and demonstrate that NADase activity is associated with tissue tropism but not invasiveness of disease.     

Nuclear Markers Reveal Predominantly North to South Gene Flow in Ixodes scapularis,the Tick Vector of the Lyme Disease Spirochete

Ixodes scapularis, the tick vector of the Lyme disease spirochete, is distributed over most of the eastern United States, but >80% of all Lyme disease cases occur in the northeast. The role that genetic differences between northern and southern tick populations play in explaining this disparate distribution of Lyme disease cases is unclear. The present study was conducted with 1,155 SNP markers in eight nuclear genes; the 16S mitochondrial gene was examined for comparison with earlier studies. We examined 350 I. scapularis from 7 states covering a representative area of the species. A demographic analysis using Bayesian Extended Skyline Analysis suggested that I. scapularis populations in Mississippi and Georgia began expanding 500,000 years ago, those in Florida and North Carolina 200,000 years ago and those from Maryland and New Jersey only during the past 50,000 years with an accompanying bottleneck. Wisconsin populations only began expanding in the last 20,000 years. Analysis of current migration patterns suggests large amounts of gene flow in northern collections and equally high rates of gene flow among southern collections. In contrast there is restricted and unidirectional gene flow between northern and southern collections, mostly occurring from northern into southern populations. Northern populations are characterized by nymphs that quest above the leaf litter, are easy to collect by flagging, frequently feed on mammals such as rodents and shrews, commonly attach to people, and about 25% of which are infected with B. burgdorferi. If there is a genetic basis for these behaviors, then the patterns detected in this study are of concern because they suggest that northern I. scapularis populations with a greater ability to vector B. burgdorferi to humans are expanding south.     

What Ticks Do Under Your Skin: Two-Photon Intravital Imaging of Ixodes Scapularis Feeding in the Presence of the Lyme Disease Spirochete

Lyme disease, due to infection with the Ixodes-tick transmitted spirochete Borrelia burgdorferi, is the most common tick-transmitted disease in the northern hemisphere. Our understanding of the tick-pathogen-vertebrate host interactions that sustain an enzootic cycle for B. burgdorferi is incomplete. In this article, we describe a method for imaging the feeding of Ixodes scapularis nymphs in real-time using two-photon intravital microscopy and show how this technology can be applied to view the response of Lyme borrelia in the skin of an infected host to tick feeding.     

Relationships of a Novel Lyme Disease Spirochete, Borrelia spielmani sp. nov., with Its Hosts in Central Europe

To determine whether the pathogenic variant of Lyme disease spirochetes, isolate A14S, is perpetuated in a particular reservoir-vector relationship, we screened vector ticks in various Central European sites for a related spirochete and determined its host association. A14S-like spirochetes infect numerous questing ticks in the Petite Camargue Alsacienne (PC). They frequently infect dormice, but no mice or voles. Garden dormice appear to be better reservoir hosts for A14S-like spirochetes than for Borrelia afzelii, because these spirochetes are retained longer and infect ticks more readily. Spirochetes associated with garden dormice in the PC site form a homologous entity with those isolated from a human patient in The Netherlands. Its unique biological relationship together with previous genetic characterization justifies designating this dormouse-associated genospecies as a distinct entity. Garden dormice serve as the main reservoir hosts of a novel genospecies, Borrelia spielmani sp. nov., one of several that cause Lyme disease in people.     

Complex Population Structure of Lyme Borreliosis Group Spirochete Borrelia garinii in Subarctic Eurasia

Borrelia garinii, a causative agent of Lyme borreliosis in Europe and Asia, is naturally maintained in marine and terrestrial enzootic cycles, which primarily involve birds, including seabirds and migratory passerines. These bird groups associate with, correspondingly, Ixodes uriae and Ixodes ricinus ticks, of which the latter species may bite and transmit the infection to humans. Studies of the overlap between these two natural cycles of B. garinii have been limited, in part due to the absence of representative collections of this spirochete''s samples, as well as of the lack of reliable measure of the genetic heterogeneity of its strains. As a prerequisite for understanding the epidemiological correlates of the complex maintenance of B. garinii, the present study sought to assess the diversity and phylogenetic relationships of this species'' strains from its natural hosts and patients with Lyme borreliosis from subarctic Eurasia. We used sequence typing of the partial rrs-rrl intergenic spacer (IGS) of archived and prospective samples of B. garinii from I. uriae ticks collected predominantly on Commander Islands in North Pacific, as well as on the islands in northern Sweden and arctic Norway. We also typed B. garinii samples from patients with Lyme borreliosis and I. ricinus ticks infesting migratory birds in southern Sweden, or found questing in selected sites on the islands in the Baltic Sea and Lithuania. Fifty-two (68%) of 77 B. garinii samples representing wide geographical range and associated with I. ricinus and infection of humans contributed 12 (60%) of total 20 identified IGS variants. In contrast, the remaining 25 (32%) samples recovered from I. uriae ticks from a few islands accounted for as many as 10 (50%) IGS types, suggesting greater local diversity of B. garinii maintained by seabirds and their ticks. Two IGS variants of the spirochete in common for both tick species were found in I. ricinus larvae from migratory birds, an indication that B. garinii strains are exchanged between different ecological niches. Notably, B. garinii variants associated with I. uriae ticks were found in each of the six clusters, representing two phylogenetic lineages of this species identified among the studied samples. Our findings suggest that B. garinii in subarctic Eurasia comprises two partially overlapping populations with different levels of genetic heterogeneity, presumably, due to distinctive selective pressures on the spirochete in its marine and terrestrial enzootic cycles.     

Comparative Analysis of Tissue Tropism of Bipartite Geminiviruses

Abutilon mosaic virus (AbMV), a bipartite geminivirus of the genus Begomovirus, has been vegetatively propagated for many years in Abutilon sellovianum in which it is strictly phloem-restricted. Using in situ hybridization and immunological analyses, the tissue tropism of AbMV in the laboratory host Nicotiana benthamiana was compared with that of two other bipartite begomoviruses, African cassava mosaic virus (ACMV) and tomato golden mosaic virus (TGMV). Analysis of the first systemically infected leaves and longitudinal sections of axillary and flower buds revealed that all three viruses are initially confined to the vascular traces, although both ACMV and TGMV are later detectable in nearly all tissue types. In contrast, AbMV remained strictly phloem-limited in this host throughout the course of infection. The ability of ACMV and TGMV to move out of N. benthamiana phloem tissues is correlated with the development of severe symptoms in comparison with the mild symptoms associated with AbMV infection. It was also demonstrated that Sida micrantha mosaic virus, a virus that is closely related to AbMV, is phloem-limited in Malva parviflora even though it induces severe leaf curl, stunting and necrosis in this host. The present data demonstrate that bipartite begomoviruses can exhibit strikingly different patterns of tissue tropism.     

The Role of Interferon in Influenza Virus Tissue Tropism

We have studied the pathogenesis of influenza virus infection in mice that are unable to respond to type I or II interferons due to a targeted disruption of the STAT1 gene. STAT1−/− animals are 100-fold more sensitive to lethal infection with influenza A/WSN/33 virus than are their wild-type (WT) counterparts. Virus replicated only in the lungs of WT animals following intranasal (i.n.) virus inoculation, while STAT1−/− mice developed a fulminant systemic influenza virus infection following either i.n. or intraperitoneal inoculation. We investigated the mechanism underlying this altered virus tropism by comparing levels of virus replication in fibroblast cell lines and murine embryonic fibroblasts derived from WT mice, STAT−/− mice, and mice lacking gamma interferon (IFNγ−/− mice) or the IFN-α receptor (IFNαR−/− mice). Influenza A/WSN/33 virus replicates to high titers in STAT1−/− or IFNαR−/− fibroblasts, while cells derived from WT or IFNγ−/− animals are resistant to influenza virus infection. Immunofluorescence studies using WT fibroblast cell lines demonstrated that only a small subpopulation of WT cells can be infected and that in the few infected WT cells, virus replication is aborted at an early, nuclear phase. In all organs examined except the lung, influenza A WSN/33 virus infection is apparently prevented by an intact type I interferon response. Our results demonstrate that type I interferon plays an important role in determining the pathogenicity and tissue restriction of influenza A/WSN/33 virus in vivo and in vitro.     

A Dityrosine Network Mediated by Dual Oxidase and Peroxidase Influences the Persistence of Lyme Disease Pathogens within the Vector

Ixodes scapularis ticks transmit a wide array of human and animal pathogens including Borrelia burgdorferi; however, how tick immune components influence the persistence of invading pathogens remains unknown. As originally demonstrated in Caenorhabditis elegans and later in Anopheles gambiae, we show here that an acellular gut barrier, resulting from the tyrosine cross-linking of the extracellular matrix, also exists in I. scapularis ticks. This dityrosine network (DTN) is dependent upon a dual oxidase (Duox), which is a member of the NADPH oxidase family. The Ixodes genome encodes for a single Duox and at least 16 potential peroxidase proteins, one of which, annotated as ISCW017368, together with Duox has been found to be indispensible for DTN formation. This barrier influences pathogen survival in the gut, as an impaired DTN in Doux knockdown or in specific peroxidase knockdown ticks, results in reduced levels of B. burgdorferi persistence within ticks. Absence of a complete DTN formation in knockdown ticks leads to the activation of specific tick innate immune pathway genes that potentially resulted in the reduction of spirochete levels. Together, these results highlighted the evolution of the DTN in a diverse set of arthropod vectors, including ticks, and its role in protecting invading pathogens like B. burgdorferi. Further understanding of the molecular basis of tick innate immune responses, vector-pathogen interaction, and their contributions in microbial persistence may help the development of new targets for disrupting the pathogen life cycle.     

莱姆病致病机理研究进展

莱姆病是20世纪70年代发现的一种由硬蜱叮咬传播的自然疫源性人兽共患传染病,严重威胁人类健康,引起全球性的关注。伯氏疏螺旋体是莱姆病的病原体。目前莱姆病在很多方面的研究已经取得显著进展,但其致病机理尚不完全清楚,主要原因可能是莱姆病病原体及其成分直接作用、炎性细胞因子作用以及自身免疫因素的影响。对近年有关莱姆病致病机理的研究进展进行概述。