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1.
Wolbachia endosymbiotic bacteria are widespread in filarial nematodes and are directly involved in the immune response of the host. In addition, antibiotics which disrupt Wolbachia interfere with filarial nematode development thus, Wolbachia provide an excellent target for control of filariasis. A 63.1 kb bacterial artificial chromosome insert, from the Wolbachia endosymbiont of the human filarial parasite Brugia malayi, has been sequenced using the New England Biolabs Inc. Genome Priming System() transposition kit in conjunction with primer walking methods. The bacterial artificial chromosome insert contains approximately 57 potential ORFs which have been compared by individual protein BLAST analysis with the 35 published complete microbial genomes in the Comprehensive Microbial Resource database at The Institute for Genomic Research and in the NCBI GenBank database, as well as to data from 22 incomplete genomes from the DOE Joint Genome Institute. Twenty five of the putative ORFs have significant similarity to genes from the alpha-proteobacteria Rickettsia prowazekii, the most closely related completed genome, as well as to the newly sequenced alpha-proteobacteria endosymbiont Sinorhizobium meliloti. The bacterial artificial chromosome insert sequence however has little conserved synteny with the R. prowazekii and S. meliloti genomes. Significant sequence similarity was also found in comparisons with the currently available sequence data from the Wolbachia endosymbiont of Drosophila melanogaster. Analysis of this bacterial artificial chromosome insert provides useful gene density and comparative genomic data that will contribute to whole genome sequencing of Wolbachia from the B. malayi host. This will also lead to a better understanding of the interactions between the endosymbiont and its host and will offer novel approaches and drug targets for elimination of filarial disease.  相似文献   

2.
Utilising three hypervariable microsatellite markers we have previously shown that scabies mites on people are genetically distinct from those on dogs in sympatric populations in northern Australia. This had important ramifications on the formulation of public health control policies. In contrast phylogenetic analyses using mitochondrial markers on scabies mites infecting multiple animal hosts elsewhere in the world could not differentiate any genetic variation between mite haplotype and host species. Here we further analyse the intra-specific relationship of Sarcoptes scabiei var. hominis with S. scabiei var. canis by using both mitochondrial DNA and an expanded nuclear microsatellite marker system. Phylogenetic studies using sequences from the mitochondrial genes coding for 16S rRNA and Cytochrome Oxidase subunit I demonstrated significant relationships between S. scabiei MtDNA haplotypes, host species and geographical location. Multi-locus genotyping using 15 microsatellite markers substantiated previous data that gene flow between scabies mite populations on human and dog hosts is extremely rare in northern Australia. These data clearly support our previous contention that control programs for human scabies in endemic areas with sympatric S. scabiei var. hominis and var. canis populations must focus on human-to-human transmission. The genetic division of dog and human derived scabies mites also has important implications in vaccine and diagnostic test development as well as the emergence and monitoring of drug resistance in S. scabiei in northern Australia.  相似文献   

3.

Background

Most filarial nematodes contain Wolbachia symbionts. The purpose of this study was to examine the effects of doxycycline on gene expression in Wolbachia and adult female Brugia malayi.

Methods

Brugia malayi infected gerbils were treated with doxycycline for 6-weeks. This treatment largely cleared Wolbachia and arrested worm reproduction. RNA recovered from treated and control female worms was labeled by random priming and hybridized to the Version 2- filarial microarray to obtain expression profiles.

Results and discussion

Results showed significant changes in expression for 200 Wolbachia (29% of Wolbachia genes with expression signals in untreated worms) and 546 B. malayi array elements after treatment. These elements correspond to known genes and also to novel genes with unknown biological functions. Most differentially expressed Wolbachia genes were down-regulated after treatment (98.5%). In contrast, doxycycline had a mixed effect on B. malayi gene expression with many more genes being significantly up-regulated after treatment (85% of differentially expressed genes). Genes and processes involved in reproduction (gender-regulated genes, collagen, amino acid metabolism, ribosomal processes, and cytoskeleton) were down-regulated after doxycycline while up-regulated genes and pathways suggest adaptations for survival in response to stress (energy metabolism, electron transport, anti-oxidants, nutrient transport, bacterial signaling pathways, and immune evasion).

Conclusions

Doxycycline reduced Wolbachia and significantly decreased bacterial gene expression. Wolbachia ribosomes are believed to be the primary biological target for doxycycline in filarial worms. B. malayi genes essential for reproduction, growth and development were also down-regulated; these changes are consistent with doxycycline effects on embryo development and reproduction. On the other hand, many B. malayi genes involved in energy production, electron-transport, metabolism, anti-oxidants, and others with unknown functions had increased expression signals after doxycycline treatment. These results suggest that female worms are able to compensate in part for the loss of Wolbachia so that they can survive, albeit without reproductive capacity. This study of doxycycline induced changes in gene expression has provided new clues regarding the symbiotic relationship between Wolbachia and B. malayi.  相似文献   

4.
In search of Wolbachia in human parasites, Wolbachia were identified in the sand flea Tunga penetrans. PCR and DNA sequencing of the bacterial 16S rDNA, the ftsZ cell division protein, the Wolbachia surface protein (wsp) and the Wolbachia aspartate aminotransferase genes revealed a high similarity to the respective sequences of endosymbionts of filarial nematodes. Using these sequences a phylogenetic tree was generated, that indicates a close relationship between Wolbachia from T. penetrans and from filarial parasites, but possibly as a member of a new supergroup. Ultrastructural studies showed that Wolbachia are abundant in the ovaries of neosomic fleas, whereas other, smaller and morphologically distinct, bacteria were observed in the lumen of the intestine. Wolbachia were labeled by immunohistology and immunogold electron microscopy using polyclonal antibodies against wsp of Drosophila, of the filarial parasite Dirofilaria immitis, or against hsp 60 from Yersinia enterocolitica. These results show that as in filariasis, humans with tungiasis are exposed to Wolbachia. Furthermore, antisera raised against proteins of Wolbachia from arthropods or from filarial parasites can be immunologically cross-reactive.  相似文献   

5.
The mite Sarcoptes scabiei causes sarcoptic mange, or scabies, a disease that affects both animals and humans worldwide. Our interest in S. scabiei led us to further characterise a glutathione S-transferase. This multifunctional enzyme is a target for vaccine and drug development in several parasitic diseases. The S. scabiei glutathione S-transferase open reading frame reported here is 684 nucleotides long and yields a protein with a predicted molecular mass of 26 kDa. Through phylogenetic analysis the enzyme was classified as a delta-class glutathione S-transferase, and our paper is the first to report that delta-class glutathione S-transferases occur in organisms other than insects. The recombinant S. scabiei glutathione S-transferase was expressed in Escherichia coli via three different constructs and purified for biochemical analysis. The S. scabiei glutathione S-transferase was active towards the substrate 1-chloro-2,4-dinitrobenzene, though the positioning of fusion partners influenced the kinetic activity of the enzyme. Polyclonal antibodies raised against S. scabiei glutathione S-transferase specifically localised the enzyme to the integument of the epidermis and cavities surrounding internal organs in adult parasites. However, some minor staining of parasite intestines was observed. No staining was seen in host tissues, nor could we detect any antibody response against S. scabiei glutathione S-transferase in sera from naturally S. scabiei infected dogs or pigs. Additionally, the polyclonal sera raised against recombinant S. scabiei glutathione S-transferase readily detected a protein from mites, corresponding to the predicted size of native glutathione S-transferase.  相似文献   

6.
Wolbachia are bacteria present within the tissues of most filarial nematodes. Filarial nematode survival is known to be affected by immune responses generated during filarial nematode infection and immune responses to Wolbachia can be found in different species harbouring filarial nematode infections, including humans. Using the rodent filarial model Litomosoides sigmodontis, we show that pre-exposure to wolbachia surface protein in a Th1 context (but not in a Th2-context) enhances worm survival on subsequent challenge. This study suggests that despite abundant evidence that pro-inflammatory reactions to the endosymbiont have detrimental effects on the both the nematode and mammalian host, they may under some circumstances be beneficial to the nematode.  相似文献   

7.
The parasitic nematode, Brugia malayi, causes lymphatic filariasis in humans, which in severe cases leads to the condition known as elephantiasis. The parasite contains an endosymbiotic alpha-proteobacterium of the genus Wolbachia that is required for normal worm development and fecundity and is also implicated in the pathology associated with infections by these filarial nematodes. Bacterial artificial chromosome libraries were constructed from B. malayi DNA and provide over 11-fold coverage of the nematode genome. Wolbachia genomic fragments were simultaneously cloned into the libraries giving over 5-fold coverage of the 1.1 Mb bacterial genome. A physical framework for the Wolbachia genome was developed by construction of a plasmid library enriched for Wolbachia DNA as a source of sequences to hybridise to high-density bacterial artificial chromosome colony filters. Bacterial artificial chromosome end sequencing provided additional Wolbachia probe sequences to facilitate assembly of a contig that spanned the entire genome. The Wolbachia sequences provided a marker approximately every 10 kb. Four rare-cutting restriction endonucleases were used to restriction map the genome to a resolution of approximately 60 kb and demonstrate concordance between the bacterial artificial chromosome clones and native Wolbachia genomic DNA. Comparison of Wolbachia sequences to public databases using BLAST algorithms under stringent conditions allowed confident prediction of 69 Wolbachia peptide functions and two rRNA genes. Comparison to closely related complete genomes revealed that while most sequences had orthologs in the genome of the Wolbachia endosymbiont from Drosophila melanogaster, there was no evidence for long-range synteny. Rather, there were a few cases of short-range conservation of gene order extending over regions of less than 10 kb. The molecular scaffold produced for the genome of the Wolbachia from B. malayi forms the basis of a genomic sequencing effort for this bacterium, circumventing the difficult challenge of purifying sufficient endosymbiont DNA from a tropical parasite for a whole genome shotgun sequencing strategy.  相似文献   

8.
Wolbachia sp. was first reported in filarial nematodes over 25 yr ago. Today, much research is focused on the role of these bacteria in filarial worm biology. The filarial symbionts are closely related to arthropod symbionts, which are known to modify host reproduction and biology through various mechanisms. Similarly, it has been suggested that Wolbachia sp. is essential for long-term survival and reproduction of filariae. We report that Wolbachia sp. 16S rDNA was not found in the equine filarial nematode Setaria equina, using either polymerase chain reaction (PCR) or DNA hybridization. In addition, ultrastructural analysis of adult worms did not reveal the presence of Wolbachia sp. in hypodermal cords or reproductive tissues. These data suggest that like Onchocerca flexuosa and Acanthocheilonema vitae, S. equina may not be dependent on Wolbachia sp. for survival.  相似文献   

9.
Filarial nematodes harbour intracellular symbiotic bacteria belonging to the genus Wolbachia. Wolbachia is thought to play an important role in the biology of the nematode. Moreover, Wolbachia appears to be involved in the immunopathogenesis of filariasis and in the onset of the side-effects of antifilarial therapy. Investigations in these research areas require reliable methods to quantify Wolbachia both in nematodes and in vertebrate tissues. To this purpose, we designed a quantitative real-time PCR targeted on the ftsZ gene of the Wolbachia of Brugia pahangi, a model filarial species maintained in gerbils. The method was applied to quantify Wolbachia in Brugia pahangi, from animals with or without tetracycline treatment. Our results show that tetracycline treatment leads to dramatic reduction or clearance of Wolbachia from the nematode. Results obtained from different replicates were reproducible and the method appeared very sensitive compared to other PCR protocols for Wolbachia detection. Real-time PCR is thus an appropriate method for investigations on the biological role of Wolbachia and on the implication of these bacteria in the pathogenesis of filariasis. With slight modifications of the primers and probe, the protocol we have developed could be applied in studies of the human pathogen Brugia malayi and on the model filarial species Litomosoides sigmodontis.  相似文献   

10.
Wolbachia are intracellular bacteria of many filarial nematodes. A mutualistic interaction between the endobacteria and the filarial host is likely, because the clearance of Wolbachia by tetracycline leads to the obstruction of embryogenesis and larval development. Databases were searched for exported molecules to identify candidates involved in this mutualism. Fragments of a Wolbachia serine protease from the human filarial parasite Onchocerca volvulus were obtained (Wol-Ov-HtrA) by the use of a PCR technique and primers based on the Rickettsia prowazekii genome. The deduced amino acid sequence exhibited 87% and 81% identity to the homologous Wolbachia proteases identified from Brugia malayi and Drosophila melanogaster, respectively. The full-length cDNA encodes 494 amino acids with a calculated mass of 54 kDa. Three characteristic features, (i) a catalytic triad of serine proteases, (ii) two PDZ domains and (iii) a putative signal peptide, classify the endobacterial protein as a member of the periplasmic HtrA family of proteases known to express chaperone and regulator activity of apoptosis. Using a rabbit antiserum raised against a recombinantly expressed 33-kDa fragment of Wol-Ov-HtrA, strong labelling of the antigen was found associated with endobacteria in hypodermis, oocytes, zygotes, all embryonic stages and microfilariae of O. volvulus. Staining of hypodermal cytoplasm surrounding the endobacteria indicated a possible release of the protein from the Wolbachia. The demonstration of Wol-Ov-HtrA-reactive IgG1 antibodies in sera of O. volvulus-infected persons indicated the exposure to the protein and its recognition by the human immune system. Wol-Ov-HtrA is a candidate for an exported Wolbachia protein that may interact with the filarial host metabolism.  相似文献   

11.
Wolbachia are intracellular bacteria that infect arthropods and filarial nematodes. These bacteria play an important role in the immunology and pathogenesis of filarial diseases through their proteins and, possibly, other molecules. GroEL is a constitutively expressed bacterial protein; it is highly conserved among bacteria and is involved in the correct folding of newly synthesized proteins. Here we report the production of recombinant GroEL from the Wolbachia of Dirofilaria immitis. Our goal is to test the hypothesis that GroEL is involved in the immunopathology of filariases. The complete groel gene was PCR-amplified, sequenced and cloned into an expression vector. The recombinant GroEL was purified by affinity chromatography by using high-performance liquid chromatography (HPLC).  相似文献   

12.
Wolbachia harbored by most filarial parasites, is critical to both embryogenesis and microfilarial development, and may lead to inflammation and pathogenesis in infected hosts. Based on alignment of the sequences from the wsp, ftsZ, and 16S rRNA genes, Wolbachia was demonstrated to exist in Angiostrongylus cantonensis, a non-filaroid nematode. Although the wsp gene may not be the best candidate for evolutionary analysis of Wolbachia, this gene has been sequenced from a broader coverage of the host species, making it feasible to be used for phylogenetic analysis in this study. The results from both Neighbor-joining and Maximum parsimony methods showed that this novel Wolbachia does not belong to any of the known groups (C or D) of nematode-derived Wolbachia. In addition, the wsp gene sequence of this newly identified endosymbiont revealed a high degree of identity (98%) with that from Diaea circumlita c2, tentatively classified into the putative group G. This suggests that Wolbachia from A. cantonensis could represent a deeply branched lineage in Wolbachia evolution or the occurrence of horizontal transfer between infected hosts. In conclusion, the findings provide some insights into our understanding of the evolution of Wolbachia, particularly the isolate from A. cantonensis.  相似文献   

13.
The discovery that endosymbiotic Wolbachia bacteria play an important role in the pathophysiology of diseases caused by filarial nematodes, including lymphatic filariasis and onchocerciasis (river blindness) has transformed our approach to these disabling diseases. Because these parasites infect hundreds of millions of individuals worldwide, understanding host factors involved in the pathogenesis of filarial-induced diseases is paramount. However, the role of early innate responses to filarial and Wolbachia ligands in the development of filarial diseases has not been fully elucidated. To determine the role of TLRs, we used cell lines transfected with human TLRs and macrophages from TLR and adaptor molecule-deficient mice and evaluated macrophage recruitment in vivo. Extracts of Brugia malayi and Onchocerca volvulus, which contain Wolbachia, directly stimulated human embryonic kidney cells expressing TLR2, but not TLR3 or TLR4. Wolbachia containing filarial extracts stimulated cytokine production in macrophages from C57BL/6 and TLR4(-/-) mice, but not from TLR2(-/-) or TLR6(-/-) mice. Similarly, macrophages from mice deficient in adaptor molecules Toll/IL-1R domain-containing adaptor-inducing IFN-beta and Toll/IL-1R domain-containing adaptor-inducing IFN-beta-related adaptor molecule produced equivalent cytokines as wild-type cells, whereas responses were absent in macrophages from MyD88(-/-) and Toll/IL-1R domain-containing adaptor protein (TIRAP)/MyD88 adaptor-like (Mal) deficient mice. Isolated Wolbachia bacteria demonstrated similar TLR and adaptor molecule requirements. In vivo, macrophage migration to the cornea in response to filarial extracts containing Wolbachia was dependent on TLR2 but not TLR4. These results establish that the innate inflammatory pathways activated by endosymbiotic Wolbachia in B. malayi and O. volvulus filaria are dependent on TLR2-TLR6 interactions and are mediated by adaptor molecules MyD88 and TIRAP/Mal.  相似文献   

14.
Intracellular bacteria have been observed in various species of filarial nematodes (family Onchocercidae). The intracellular bacterium of the canine filaria Dirofilaria immitis has been shown to be closely related to Wolbachia, a rickettsia-like micro-organism that is widespread among arthropods. However, the relationships between endosymbionts of different filariae, and between these and the arthropod wolbachiae, appear not to have been studied. To address these issues we have examined ten species of filarial nematodes for the presence of Wolbachia. For nine species, all samples examined were PCR positive using primers specific for the ftsZ gene of Wolbachia. For one species, the examined samples were PCR negative. Sequences of the amplified ftsZ gene fragments of filarial wolbachiae fall into two clusters (C and D), which are distinct from the A and B clusters recognized for arthropod wolbachiae. These four lineages (A-D) are related in a star-like phylogeny, with higher nucleotide divergence observed between C and D wolbachiae than that observed between A and B wolbachiae. In addition, within each of the two lineages of filarial wolbachiae, the phylogeny of the symbionts is consistent with the host phylogeny. Thus, there is no evidence for recent Wolbachia transmission between arthropods and nematodes. Endosymbiont 16S ribosomal DNA sequences from a subset of filarial species support these findings.  相似文献   

15.
The primary parasitic relations between the itch mite Sarcoptes scabiei (L.) and man are confirmed indirectly by the presence in the family Sarcoptidae of close genera, members of which parasitize primates. Judging from the composition of domestic and wild animals-hosts, the source of their infection with S. scabiei was man that gave rise to numerous forms of mites specific to individual species of hosts or their related groups belonging to different taxonomic ranks. Three types of host relations and, accordingly, forms of sarcoptosis are suggested at the infection from an alien host: pseudosarcoptosis, temporary self-curable and typical lingering sarcoptosis. The ways of reinfection, invasiveness of stages of the mite and clinical picture for each form of the disease are analyzed.  相似文献   

16.
Wolbachia pipientis is a bacterial endosymbiont associated with arthropods and filarial nematodes. In filarial nematodes, W. pipientis has been shown to play an important role in the biology of the host and in the immuno-pathology of filariasis. Several species of filariae, including the most important parasites of humans and animals (e.g. Onchocerca volvulus, Wuchereria bancrofti and Dirofilaria immitis) have been shown to harbour these bacteria. Other filarial species, including an important rodent species (Acanthocheilonema viteae), which has been used as a model for the study of filariasis, do not appear to harbour these symbionts. There are still several open questions about the distribution of W. pipientis in filarial nematodes. Firstly the number of species examined is still limited. Secondly, it is not clear whether the absence of W. pipientis in negative species could represent an ancestral characteristic or the result of a secondary loss. Thirdly, several aspects of the phylogeny of filarial nematodes are still unclear and it is thus difficult to overlay the presence/absence of W. pipientis on a tree representing filarial evolution. Here we present the results of a PCR screening for W. pipientis in 16 species of filariae and related nematodes, representing different families/subfamilies. Evidence for the presence of W. pipientis is reported for five species examined for the first time (representing the genera Litomosoides, Litomosa and Dipetalonema); original results on the absence of this bacterium are reported for nine species; for the remaining two species, we have confirmed the absence of W. pipientis recently reported by other authors. In the positive species, the infecting W. pipientis bacteria have been identified through 16S rDNA gene sequence analysis. In addition to the screening for W. pipientis in 16 species, we have generated phylogenetic reconstructions based on mitochondrial gene sequences (12S rDNA; COI), including a total of 28 filarial species and related spirurid nematodes. The mapping of the presence/absence of W. pipientis on the trees generated indicates that these bacteria have possibly been lost during evolution along some lineages of filarial nematodes.  相似文献   

17.
Symbiotic relationships underlie the evolutionary success of many different life forms. The filarial worms are long, slender nematode parasites that cause considerable pathology in large segments of the world's population. About 25 years ago, investigators first reported the presence of bacterial organisms living inside these parasitic worms. Recent molecular biological studies have indicated that these bacteria belong to the genus Wolbachia, members of which have been known to be associated with numerous species of insects. Elimination of the Wolbachia from the nematodes (by, for instance, treatment with broad spectrum antibiotics) results in profound disturbances in the physiology of the latter, including a complete block in reproduction. This observation, taken together with the fact that every individual worm examined to date contains Wolbachia, validates the classification of the latter as "endosymbionts." Many studies indicate that the Wolbachia may also play an important role in the pathology caused by the nematode worms, and that it might be possible to target therapy against the Wolbachia to treat the filarial disease. Intriguingly, the intense recent interest in Wolbachia is in complete contrast with the virtually complete indifference evoked by the original discovery of these organisms.  相似文献   

18.
Wolbachia are well known as bacterial symbionts of arthropods, where they are reproductive parasites, but have also been described from nematode hosts, where the symbiotic interaction has features of mutualism. The majority of arthropod Wolbachia belong to clades A and B, while nematode Wolbachia mostly belong to clades C and D, but these relationships have been based on analysis of a small number of genes. To investigate the evolution and relationships of Wolbachia symbionts we have sequenced over 70 kb of the genome of wOvo, a Wolbachia from the human-parasitic nematode Onchocerca volvulus, and compared the genes identified to orthologues in other sequenced Wolbachia genomes. In comparisons of conserved local synteny, we find that wBm, from the nematode Brugia malayi, and wMel, from Drosophila melanogaster, are more similar to each other than either is to wOvo. Phylogenetic analysis of the protein-coding and ribosomal RNA genes on the sequenced fragments supports reciprocal monophyly of nematode and arthropod Wolbachia. The nematode Wolbachia did not arise from within the A clade of arthropod Wolbachia, and the root of the Wolbachia clade lies between the nematode and arthropod symbionts. Using the wOvo sequence, we identified a lateral transfer event whereby segments of the Wolbachia genome were inserted into the Onchocerca nuclear genome. This event predated the separation of the human parasite O. volvulus from its cattle-parasitic sister species, O. ochengi. The long association between filarial nematodes and Wolbachia symbionts may permit more frequent genetic exchange between their genomes.  相似文献   

19.
Filariasis is a major public health problem throughout many regions of the tropics. The disease is caused by several species of filarial nematode including Wuchereria bancrofti and Brugia malayi, the agents of lymphatic filariasis, and Onchocerca volvulus, the cause of 'riverblindness'. Disease caused by these worms varies depending on the tissue location of the parasite, and is associated with episodes of acute and chronic inflammation. These pathologies, including elephantiasis and blindness, rank among the most disabling in the world. Studies aimed at characterizing the molecular nature of the inflammatory stimuli derived from filarial nematodes uncovered a long forgotten secret, their symbiont Wolbachia. LPS-like molecules from these intracellular bacteria are responsible for potent inflammatory responses from macrophages and in animal models of filarial disease. Wolbachia has also been associated with severe inflammatory reactions to filarial chemotherapy, being released into the blood following the death of the parasite. Recent studies in animal models even implicate Wolbachia in the onset of lymphodema and blindness. Taken together these studies suggest a major role for Wolbachia in the pathogenesis of filarial disease. It may be possible, through the use of antibiotic therapy, to clear worms of their bacteria, in the hope that this will prevent the onset and development of filarial pathology.  相似文献   

20.
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