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1.
Int J Syst Evol Microbiol ; 71(10)2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34672919

RESUMO

A Gram-stain-negative, obligatory anaerobic spirochaete (RCC2812T) was isolated from a faecal sample obtained from an individual residing in a remote Amazonian community in Peru. The bacterium showed highest 16S rRNA gene sequence similarity to the pig intestinal spirochete Treponema succinifaciens (89.48 %). Average nucleotide identity values between strain RCC2812T and all available Treponema genomes from validated type strains were all <73 %, thus clearly lower than the species delineation threshold. The DNA G+C content of RCC2812T was 41.24 mol%. Phenotypic characterization using the API-ZYM and API 20A systems confirmed the divergent position of this bacterium within the genus Treponema. Strain RCC2812T could be differentiated from the phylogenetically most closely related T. succinifaciens by the presence of alkaline phosphatase and α -glucosidase activities. Unlike T. succinifaciens, strain RCC2812T grew equally well with or without serum. Strain RCC2812T is the first commensal Treponema isolated from the human faecal microbiota of remote populations, and based on the collected data represents a novel Treponema species for which the name Treponema peruense sp. nov. is proposed. The type strain is RCC2812T (=LMG 31794T=CIP 111910T).


Assuntos
Ácidos Graxos , Spirochaetales , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Fezes , Humanos , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Suínos , Treponema/genética
2.
Int Ophthalmol ; 41(12): 4111-4126, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34297303

RESUMO

PURPOSE: Analysis of cases with spirochetal uveitis related to spirochetes in a tertiary referral academic center. METHODS: Retrospective study of patients diagnosed with uveitis attributed to Treponema pallidum, Leptospira spp. and Borrelia burgdorferi from June 1991 until December 2019. RESULTS: A total of 57 cases of spirochetal uveitis (22 patients with T. pallidum, 26 with Leptospira spp., and 9 with B. burgdorferi) that consisted 1% of the overall number of uveitics were recorded. All these cases presented with a wide spectrum of clinical presentations (anterior uveitis, posterior uveitis, panuveitis, vasculitis, papillitis, and in some rare cases concomitant posterior scleritis). The treatment included mainly penicillin or doxycycline, while corticosteroids were administered systematically in some cases with Borrelia or Leptospira infection. The final visual outcome was favorable (> 6/10 in Snellen visual acuity) in approximately 76% of our patients. CONCLUSION: Despite being rare, spirochetal uveitis can be detrimental for the vision and must always be included in the differential diagnosis.


Assuntos
Esclerite , Sífilis , Uveíte , Humanos , Estudos Retrospectivos , Spirochaetales , Uveíte/diagnóstico , Uveíte/tratamento farmacológico , Uveíte/epidemiologia
3.
PLoS Pathog ; 17(7): e1009612, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34228757

RESUMO

Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.


Assuntos
Engenharia Genética/métodos , Treponema/genética , Animais , Masculino , Coelhos , Spirochaetales/genética
4.
Ticks Tick Borne Dis ; 12(5): 101735, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33989985

RESUMO

Many local anaesthetics, including lidocaine, procaine and ropivacaine inhibit bacterial growth. This study investigates potential effects of these local anaesthetics on growth of Borrelia burgdorferi sensu stricto (Bbss), Borrelia bavariensis (Bbav) and Borrelia afzelii (Ba). For this purpose, Borrelia spp. organisms were either continuously or temporarily exposed to one of four local anaesthetics preparations: 20 mg/ml procaine hydrochloride (P); 10 mg/ml ropivacaine hydrochloride (R); 20 mg/ml lidocaine hydrochloride (L1, L2). L2 also contained the preservatives methyl-benzoate and propyl-benzoate, whereas P, R and L1 did not. All four local anaesthetic preparations inhibited in vitro growth of Borrelia spp. depending on concentration and exposure time. There are differences in sensitivity among the Borrelia spp. with Bbav being more susceptible to growth inhibition than Bbss and Ba. When comparing the different local anaesthetic preparations with their regard to inhibition of growth of Borrelia spp. organisms, P showed the lowest impact. It cannot be completely excluded that preservatives present in L2, methyl-benzoate and propyl-benzoate, may be a reason for further inhibition of Borrelia spp. organisms. Concentrations of local anaesthetics used in these experiments may also be present in the skin of patients during regular medical procedures. These are preliminary findings and further experiments, preferably in vivo, are necessary. To minimize the risk to produce false negative results with cultures, we recommend using procaine in a preparation without preservatives for local anaesthesia prior to skin sampling.


Assuntos
Anestésicos Locais/farmacologia , Borrelia/efeitos dos fármacos , Animais , Anti-Infecciosos/farmacologia , Borrelia burgdorferi/efeitos dos fármacos , Grupo Borrelia Burgdorferi/efeitos dos fármacos , Humanos , Doença de Lyme/microbiologia , Spirochaetales/efeitos dos fármacos
5.
Ann Agric Environ Med ; 28(1): 49-55, 2021 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-33775067

RESUMO

INTRODUCTION AND OBJECTIVE: Ixodes ricinus (I. ricinus) and Dermacentor reticulatus (D. reticulatus) are the most common ticks in Poland. These ticks contain many bacteria, which compose a microbiome with potential impact on humans. The aim of the study was to discover the microbiome of ticks in Poland. MATERIAL AND METHODS: Ticks were collected in The Protected Landscape Area of the Bug and Nurzec Valley, Poland, in 2016-2018 by flagging. They were cleaned in 70% ethanol and damaged in mortar with PBS (without Ca2+ and Mg2+ ions). DNA was extracted from the homogenates with spin columns kits, and used as a matrix in end-point PCR for bacterial 16S rRNA fragments amplifications, and further for next generation sequencing (NGS) by ILLUMINA. RESULTS: In 22 ticks (3 I. ricinus and 19 D. reticulatus) 38 microorganisms were detected. The most common were Francisella hispaniensis and Francisella novicida. In 17 ticks, Sphingomonas oligophenolica, and in 12 Rickettsia aeshlimanii were found. In 2, I. ricinus specific DNA of Borrelia americana and Borrelia carolinensis were found. In one female, D. reticulatus Anaplasma phagocytophilum and Anaplasma centrale were found. Pseudomonas lutea and Ps. moraviensis were detected in 9 and 8 ticks, respectively. CONCLUSIONS: Polish ticks microbiome contains not only well-known tick-borne pathogens, but also other pathogenic microorganisms. For the first time in Poland, Borrelia americana and Borrelia carolinensis in I. ricinus collected from the environment were detected. The dominant pathogenic microorganisms for humans were Francisella spp. and Rickettsia spp., and non-pathogenic - Sphingomonas oligophenolica. Knowledge of a tick microbiome might be useful in tick-borne biocontrol and tick-borne diseases prevention.


Assuntos
Dermacentor/microbiologia , Ixodes/microbiologia , Microbiota , Spirochaetales/genética , Animais , Francisella/classificação , Francisella/genética , Francisella/isolamento & purificação , Genoma Bacteriano , Metagenômica , Polônia , Pseudomonas/classificação , Pseudomonas/isolamento & purificação , Sphingomonas/classificação , Sphingomonas/genética , Sphingomonas/isolamento & purificação , Spirochaetales/classificação , Spirochaetales/isolamento & purificação
6.
Curr Protoc ; 1(2): e44, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33599121

RESUMO

For over a century, investigation of Treponema pallidum subsp. pallidum, the spiral-shaped bacterium that causes syphilis, was hindered by an inability to culture the organism in vitro. A recent breakthrough has enabled continuous in vitro growth of this organism in co-culture with mammalian tissue culture cells. This article contains the protocols needed to culture T. pallidum in the standard laboratory environment. In addition, protocols for growing and maintaining the required tissue culture cells, for generating isogenic strains by limiting dilution, and for quantitating T. pallidum by darkfield microscopy are included. © 2021 Wiley Periodicals LLC. Basic Protocol 1: In vitro cultivation of Treponema pallidum Basic Protocol 2: Generation of isogenic strains Support Protocol 1: Alternate harvest procedure Support Protocol 2: Culture of Sf1Ep cells Support Protocol 3: Assessment of T. pallidum number and viability.


Assuntos
Sífilis , Treponema pallidum , Animais , Spirochaetales , Treponema
7.
J Med Entomol ; 58(3): 1219-1233, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33600574

RESUMO

Lyme disease is the most common vector-borne disease in the United States. While Lyme disease vectors are widespread, high incidence states are concentrated in the Northeast, North Central and Mid-Atlantic regions. Mapping the distribution of Lyme disease spirochetes in ticks may aid in providing data-driven explanations of epidemiological trends and recommendations for targeting prevention strategies to communities at risk. We compiled data from the literature, publicly available tickborne pathogen surveillance databases, and internal CDC pathogen testing databases to map the county-level distribution of Lyme disease spirochetes reported in host-seeking Ixodes pacificus and Ixodes scapularis across the contiguous United States. We report B. burgdorferi s.s.-infected I. scapularis from 384 counties spanning 26 eastern states located primarily in the North Central, Northeastern, and Mid-Atlantic regions, and in I. pacificus from 20 counties spanning 2 western states, with most records reported from northern and north-coastal California. Borrelia mayonii was reported in I. scapularis in 10 counties in Minnesota and Wisconsin in the North Central United States, where records of B. burgdorferi s.s. were also reported. In comparison to a broad distribution of vector ticks, the resulting map shows a more limited distribution of Lyme disease spirochetes.


Assuntos
Vetores Aracnídeos/microbiologia , Borrelia burgdorferi/isolamento & purificação , Ixodes/microbiologia , Spirochaetales/isolamento & purificação , Animais , Especificidade da Espécie , Estados Unidos
8.
Sci Rep ; 11(1): 1926, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33479281

RESUMO

Borrelia spirochetes are the causative agents of Lyme borreliosis (LB) and relapsing fever (RF). Despite the steady rise in infections and the identification of new species causing human illness over the last decade, isolation of borreliae in culture has become increasingly rare. A modified Barbour-Stoenner-Kelly (BSK) media formulation, BSK-R, was developed for isolation of the emerging RF pathogen, Borrelia miyamotoi. BSK-R is a diluted BSK-II derivative supplemented with Lebovitz's L-15, mouse and fetal calf serum. Decreasing the concentration of CMRL 1066 and other components was essential for growth of North American B. miyamotoi. Sixteen B. miyamotoi isolates, originating from Ixodes scapularis ticks, rodent and human blood collected in the eastern and upper midwestern United States, were isolated and propagated to densities > 108 spirochetes/mL. Growth of five other RF and ten different LB borreliae readily occurred in BSK-R. Additionally, primary culture recovery of 20 isolates of Borrelia hermsii, Borrelia turicatae, Borrelia burgdorferi and Borrelia mayonii was achieved in BSK-R using whole blood from infected patients. These data indicate this broadly encompassing borreliae media can aid in in vitro culture recovery of RF and LB spirochetes, including the direct isolation of new and emerging human pathogens.


Assuntos
Borrelia/isolamento & purificação , Ixodes/microbiologia , Doença de Lyme/microbiologia , Febre Recorrente/microbiologia , Animais , Borrelia/patogenicidade , Borrelia burgdorferi/isolamento & purificação , Borrelia burgdorferi/patogenicidade , Meios de Cultura , Humanos , Doença de Lyme/transmissão , Camundongos , Febre Recorrente/transmissão , Spirochaetales/isolamento & purificação , Spirochaetales/patogenicidade
9.
FEMS Microbiol Lett ; 368(3)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33417693

RESUMO

Sampling of different body regions can reveal highly specialized bacterial associations within the holobiont and facilitate identification of core microbial symbionts that would otherwise be overlooked by bulk sampling methods. Here, we characterized compartment-specific associations present within the model cnidarian Nematostella vectensis by dividing its morphology into three distinct microhabitats. This sampling design allowed us to uncover a capitulum-specific dominance of spirochetes within N. vectensis. Bacteria from the family Spirochaetaceae made up 66% of the community in the capitulum, while only representing 1.2% and 0.1% of the communities in the mesenteries and physa, respectively. A phylogenetic analysis of the predominant spirochete sequence recovered from N. vectensis showed a close relation to spirochetes previously recovered from wild N. vectensis. These sequences clustered closer to the recently described genus Oceanispirochaeta, rather than Spirochaeta perfilievii, supporting them as members of this clade. This suggests a prevalent and yet uncharacterized association between N. vectensis and spirochetes from the order Spirochaetales.


Assuntos
Bactérias/classificação , Interações entre Hospedeiro e Microrganismos/fisiologia , Anêmonas-do-Mar/microbiologia , Spirochaetales/genética , Animais , Bactérias/genética , Biodiversidade , Microbiota/genética , Filogenia
10.
Urologe A ; 60(5): 628-639, 2021 May.
Artigo em Alemão | MEDLINE | ID: mdl-32047955

RESUMO

The present contribution analyses the antivenereal theatre performances as a means of fighting sexually transmitted diseases in the former Soviet Union. It is asked which images, figures and actions regarding venereal knowledge were presented on stage, which genre traditions and communicative instruments were used, and how the audience and theatre critics perceived the events on stage. For this, the archive sources, selected texts of antivenereal mock trial plays and dramas, reports and reviews in the daily press have been evaluated.


Assuntos
Doenças Sexualmente Transmissíveis , Humanos , Higiene , Doenças Sexualmente Transmissíveis/prevenção & controle , Spirochaetales , U.R.S.S.
11.
Int J STD AIDS ; 32(1): 52-58, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33232216

RESUMO

Human intestinal spirochetosis (HIS) is a possible cause of chronic diarrhoea and affects mainly men who have sex with men (MSM) and people living with HIV. Diagnosis is based on colon biopsy, where spirochetes can be observed on the luminal surface, especially with the Warthin-Starry stain or similar silver stains. We conducted a retrospective descriptive study of all HIS cases diagnosed in two sexually transmitted infections (STI) centres in Barcelona from 2009 until 2018. The medical histories were reviewed to gather epidemiological, clinical, and diagnostic variables. Six patients were diagnosed with HIS. All the individuals were MSM, with a median age of 31.5 years (interquartile range [IQR] 29.5;49.25) and half of them were living with HIV. Five patients reported condomless anal intercourse and 4 patients had practised oro-anal sex previously. Concomitantly, two of them had rectal gonorrhoea, one had rectal Chlamydia trachomatis and none of them had syphilis. The predominant clinical symptom was diarrhoea (5 patients). All cases were diagnosed by a Warthin-Starry stain on a colon biopsy specimen, and mild inflammatory changes were found in 5 cases. Five patients were treated with metronidazole and one with benzathine penicillin G. Treatment was successful in all the patients. HIS should be considered in patients with chronic diarrhoea who report risky sexual practices and/or concomitant STI. HIS may also be sexually transmitted according to the context.


Assuntos
Diarreia/complicações , Homossexualidade Masculina , Infecções por Spirochaetales/diagnóstico , Spirochaetales/isolamento & purificação , Adulto , Biópsia , Colo/patologia , Humanos , Masculino , Metronidazol/uso terapêutico , Penicilina G Benzatina/uso terapêutico , Infecções por Spirochaetales/tratamento farmacológico , Resultado do Tratamento
12.
Methods Mol Biol ; 2210: 15-23, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32815123

RESUMO

There have been more than 60 different oral Treponema species identified in the oral cavity; however, only few species can be cultivated in vitro reliably. Among those cultivable species, due to its medical importance and genetic tractability, Treponema denticola, one of the keystone pathogens associated with human periodontitis, has emerged as a paradigm model organism to understanding the genetics, etiology, and pathophysiology of oral Treponema species. During the last two decades, several genetic tools have been developed, which have played an instrumental role in the study of T. denticola. This chapter describes the experimental design and procedure of genetic manipulations of T. denticola.


Assuntos
Spirochaetales/genética , Treponema denticola/genética , Humanos , Periodontite/microbiologia
13.
Mediators Inflamm ; 2020: 8829072, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33162832

RESUMO

This study was conducted to analyze plasma reproductive hormone and biochemical parameter changes, as well as fecal microbiota composition and metabolites in sows, at different pregnancy and lactation stages, using Bama mini pig as an experimental animal model. We found that plasma prolactin (PRL), progesterone, follicle-stimulating hormone (FSH), and estrogen levels decreased from day 45 to day 105 of pregnancy. Plasma total protein and albumin levels were lower in pregnant sows, while glucose, urea nitrogen, total cholesterol, and high-density lipoprotein-cholesterol, as well as fecal acetate, butyrate, valerate, total short-chain fatty acids, skatole, and tyramine levels, were higher in lactating sows. Interestingly, the lactating sows showed lower α-diversity and Spirochaetes and Verrucomicrobia relative abundances, while pregnant sows showed a higher Proteobacteria relative abundance. Notably, the Akkermansia relative abundance was highest on day 7 of lactation. Spearman analysis showed a positive correlation between plasma triglyceride and cholinesterase levels and Akkermansia and Streptococcus relative abundances. Moreover, Oscillospira and Desulfovibrio relative abundances were also positively correlated with plasma FSH, LH, and E2 levels, as well as PRL and LH with Bacteroides. Collectively, plasma reproductive hormones, biochemical parameters, and fecal microbiota composition and metabolite levels could alter along with pregnancy and lactation, which might contribute to the growth and development demands of fetuses and newborns.


Assuntos
Fezes/microbiologia , Lactação , Microbiota , Akkermansia , Albuminas/biossíntese , Animais , Bacteroides , Proteínas Sanguíneas/análise , Clostridiales , Desulfovibrio , Estrogênios/sangue , Feminino , Hormônio Foliculoestimulante/sangue , Hormônio Luteinizante/sangue , Gravidez , Prenhez , Progesterona/sangue , Prolactina/sangue , Proteobactérias , Spirochaetales , Streptococcus , Suínos , Porco Miniatura , Verrucomicrobia
14.
BMC Genomics ; 21(1): 702, 2020 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-33032522

RESUMO

BACKGROUND: Borrelia bavariensis is one of the agents of Lyme Borreliosis (or Lyme disease) in Eurasia. The genome of the Borrelia burgdorferi sensu lato species complex, that includes B. bavariensis, is known to be very complex and fragmented making the assembly of whole genomes with next-generation sequencing data a challenge. RESULTS: We present a genome reconstruction for 33 B. bavariensis isolates from Eurasia based on long-read (Pacific Bioscience, for three isolates) and short-read (Illumina) data. We show that the combination of both sequencing techniques allows proper genome reconstruction of all plasmids in most cases but use of a very close reference is necessary when only short-read sequencing data is available. B. bavariensis genomes combine a high degree of genetic conservation with high plasticity: all isolates share the main chromosome and five plasmids, but the repertoire of other plasmids is highly variable. In addition to plasmid losses and gains through horizontal transfer, we also observe several fusions between plasmids. Although European isolates of B. bavariensis have little diversity in genome content, there is some geographic structure to this variation. In contrast, each Asian isolate has a unique plasmid repertoire and we observe no geographically based differences between Japanese and Russian isolates. Comparing the genomes of Asian and European populations of B. bavariensis suggests that some genes which are markedly different between the two populations may be good candidates for adaptation to the tick vector, (Ixodes ricinus in Europe and I. persulcatus in Asia). CONCLUSIONS: We present the characterization of genomes of a large sample of B. bavariensis isolates and show that their plasmid content is highly variable. This study opens the way for genomic studies seeking to understand host and vector adaptation as well as human pathogenicity in Eurasian Lyme Borreliosis agents.


Assuntos
Sequência Conservada , Genoma Bacteriano , Ixodes , Filogenia , Spirochaetales , Animais , Ásia , Grupo Borrelia Burgdorferi , Sequência Conservada/genética , Europa (Continente) , Genoma Bacteriano/genética , Genômica , Humanos , Doença de Lyme/microbiologia , Plasmídeos/genética , Federação Russa , Spirochaetales/classificação , Spirochaetales/genética
15.
Sci Rep ; 10(1): 17053, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-33051478

RESUMO

Spirochetal bacteria were successfully isolated from mosquitoes (Culex pipiens, Aedes cinereus) in the Czech Republic between 1999 and 2002. Preliminary 16S rRNA phylogenetic sequence analysis showed that these strains differed significantly from other spirochetal genera within the family Spirochaetaceae and suggested a novel bacterial genus in this family. To obtain more comprehensive genomic information of these isolates, we used Illumina MiSeq and Oxford Nanopore technologies to sequence four genomes of these spirochetes (BR151, BR149, BR193, BR208). The overall size of the genomes varied between 1.68 and 1.78 Mb; the GC content ranged from 38.5 to 45.8%. Draft genomes were compared to 36 publicly available genomes encompassing eight genera from the class Spirochaetes. A phylogeny generated from orthologous genes across all taxa and the percentage of conserved proteins (POCP) confirmed the genus status of these novel spirochetes. The genus Entomospira gen. nov. is proposed with BR151 selected as type species of the genus. For this isolate and the closest related isolate, BR149, we propose the species name Entomospira culicis sp. nov. The two other isolates BR208 and BR193 are named Entomospira nematocera sp. nov. (BR208) and Entomospira entomophilus sp. nov. (BR193). Finally, we discuss their interesting phylogenetic positioning.


Assuntos
Spirochaetales/classificação , Spirochaetales/genética , Spirochaetales/isolamento & purificação , Animais , Artrópodes/genética , Técnicas de Tipagem Bacteriana/métodos , Composição de Bases/genética , DNA Bacteriano/genética , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA/métodos , Spirochaeta/genética
17.
Infect Immun ; 88(12)2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-32928964

RESUMO

Borrelia burgdorferi causes Lyme disease, the most common tick-transmitted illness in North America. When Ixodes scapularis feed on an infected vertebrate host, spirochetes enter the tick gut along with the bloodmeal and colonize the vector. Here, we show that a secreted tick protein, I. scapularis protein disulfide isomerase A3 (IsPDIA3), enhances B. burgdorferi colonization of the tick gut. I. scapularis ticks in which ispdiA3 has been knocked down using RNA interference have decreased spirochete colonization of the tick gut after engorging on B. burgdorferi-infected mice. Moreover, administration of IsPDIA3 antiserum to B. burgdorferi-infected mice reduced the ability of spirochetes to colonize the tick when feeding on these animals. We show that IsPDIA3 modulates inflammatory responses at the tick bite site, potentially facilitating spirochete survival at the vector-host interface as it exits the vertebrate host to enter the tick gut. These data provide functional insights into the complex interactions between B. burgdorferi and its arthropod vector and suggest additional targets to interfere with the spirochete life cycle.


Assuntos
Borrelia burgdorferi/fisiologia , Ixodes/metabolismo , Doença de Lyme/transmissão , Isomerases de Dissulfetos de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Vetores Aracnídeos/microbiologia , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Imunidade Humoral , Inflamação/enzimologia , Inflamação/genética , Inflamação/metabolismo , Ixodes/enzimologia , Ixodes/genética , Proteínas de Membrana/metabolismo , Camundongos , Filogenia , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/imunologia , Interferência de RNA , Proteínas Recombinantes , Alinhamento de Sequência , Spirochaetales/fisiologia
18.
Infect Immun ; 88(12)2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-32928963

RESUMO

During the natural enzootic life cycle of Borrelia burgdorferi (also known as Borreliella burgdorferi), the bacteria must sense conditions within the vertebrate and arthropod and appropriately regulate expression of genes necessary to persist within these distinct environments. bb0345 of B. burgdorferi encodes a hypothetical protein of unknown function that is predicted to contain an N-terminal helix-turn-helix (HTH) domain. Because HTH domains can mediate protein-DNA interactions, we hypothesized that BB0345 might represent a previously unidentified borrelial transcriptional regulator with the ability to regulate events critical for the B. burgdorferi enzootic cycle. To study the role of BB0345 within mammals, we generated a bb0345 mutant and assessed its virulence potential in immunocompetent mice. The bb0345 mutant was able to initiate localized infection and disseminate to distal tissues but was cleared from all sites by 14 days postinfection. In vitro growth curve analyses revealed that the bb0345 mutant grew similar to wild-type bacteria in standard Barbour-Stoenner-Kelley II (BSK-II) medium; however, the mutant was not able to grow in dilute BSK-II medium or dialysis membrane chambers (DMCs) implanted in rats. Proteinase K accessibility assays and whole-cell partitioning indicated that BB0345 was intracellular and partially membrane associated. Comparison of protein production profiles between the wild-type parent and the bb0345 mutant revealed no major differences, suggesting BB0345 may not be a global transcriptional regulator. Taken together, these data show that BB0345 is essential for B. burgdorferi survival in the mammalian host, potentially by aiding the spirochete with a physiological function that is required by the bacterium during infection.


Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Interações entre Hospedeiro e Microrganismos/genética , Lipoproteínas/metabolismo , Doença de Lyme/microbiologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Borrelia burgdorferi/genética , Borrelia burgdorferi/crescimento & desenvolvimento , Borrelia burgdorferi/patogenicidade , Biologia Computacional , Feminino , Lipoproteínas/química , Lipoproteínas/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes , Spirochaetales/genética , Spirochaetales/metabolismo , Spirochaetales/patogenicidade
19.
Sci Rep ; 10(1): 13937, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811890

RESUMO

The spirochete Leptospira spp. can move in liquid and on a solid surface using two periplasmic flagella (PFs), and its motility is an essential virulence factor for the pathogenic species. Mammals are infected with the spirochete through the wounded dermis, which implies the importance of behaviors on the boundary with such viscoelastic milieu; however, the leptospiral pathogenicity involving motility remains unclear. We used a glass chamber containing a gel area adjoining the leptospiral suspension to resemble host dermis exposed to contaminated water and analyzed the motility of individual cells at the liquid-gel border. Insertion of one end of the cell body to the gel increased switching of the swimming direction. Moreover, the swimming force of Leptospira was also measured by trapping single cells using an optical tweezer. It was found that they can generate [Formula: see text] 17 pN of force, which is [Formula: see text] 30 times of the swimming force of Escherichia coli. The force-speed relationship suggested the load-dependent force enhancement and showed that the power (the work per unit time) for the propulsion is [Formula: see text] 3.1 × 10-16 W, which is two-order of magnitudes larger than the propulsive power of E. coli. The powerful and efficient propulsion of Leptospira using back-and-forth movements could facilitate their invasion.


Assuntos
Leptospira/metabolismo , Movimento/fisiologia , Infecções por Spirochaetales/metabolismo , Fenômenos Biofísicos/fisiologia , Flagelos/fisiologia , Leptospira/patogenicidade , Movimento (Física) , Pinças Ópticas , Spirochaeta/metabolismo , Spirochaeta/patogenicidade , Spirochaetales/metabolismo , Spirochaetales/patogenicidade , Fatores de Virulência
20.
Methods Mol Biol ; 2134: 131-138, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32632865

RESUMO

Spirochetes such as Treponema, Borrelia, and Leptospira species can rotate their bodies to swim in liquid environments by rotating periplasmic flagella or endoflagella, which are present inside the cell. Electron cryotomography (ECT) is an imaging technique that directly provides three-dimensional (3D) structures of cells and molecular complexes in their cellular environment at nanometer resolution. Here, I present a general protocol of ECT that covers the sample preparation, data collection, tilt series alignment, and tomographic reconstruction for visualization of intact periplasmic flagella in Leptospira spp. This protocol is capable of determining protein structures at resolutions high enough to visualize their individual domains and secondary structures in their cellular environment.


Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Leptospira/citologia , Flagelos/fisiologia , Estrutura Secundária de Proteína , Spirochaetales/citologia
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