RESUMO
Tail-anchored membrane proteins (TAMPs) are relatively simple membrane proteins characterized by a single transmembrane domain (TMD) at their C-terminus. Consequently, the hydrophobic TMD, which acts as a subcellular targeting signal, emerges from the ribosome only after termination of translation precluding canonical co-translational targeting and membrane insertion. In contrast to the well-studied eukaryotic TAMPs, surprisingly little is known about the cellular components that facilitate the biogenesis of bacterial TAMPs. In this study, we identify DjlC and Flk as bona fide Escherichia coli TAMPs and show that their TMDs are necessary and sufficient for authentic membrane targeting of the fluorescent reporter mNeonGreen. Using strains conditional for the expression of known E. coli membrane targeting and insertion factors, we demonstrate that the signal recognition particle (SRP), its receptor FtsY, the chaperone DnaK and insertase YidC are each required for efficient membrane localization of both TAMPs. A close association between the TMD of DjlC and Flk with both the Ffh subunit of SRP and YidC was confirmed by site-directed in vivo photo-crosslinking. In addition, our data suggest that the hydrophobicity of the TMD correlates with the dependency on SRP for efficient targeting.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Proteínas de Bactérias/análise , Escherichia coli/citologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/análise , Proteínas de Choque Térmico HSP70/análise , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana Transportadoras/análise , Receptores Citoplasmáticos e Nucleares/análise , Partícula de Reconhecimento de Sinal/análiseRESUMO
BACKGROUND: The determinants of parasite persistence or elimination after treatment and clinical resolution of cutaneous leishmaniasis (CL) are unknown. We investigated clinical and parasitological parameters associated with the presence and viability of Leishmania after treatment and resolution of CL caused by L. Viannia. METHODS: Seventy patients who were treated with meglumine antimoniate (n = 38) or miltefosine (n = 32) and cured, were included in this study. Leishmania persistence and viability were determined by detection of kDNA and 7SLRNA transcripts, respectively, before, at the end of treatment (EoT), and 13 weeks after initiation of treatment in lesions and swabs of nasal and tonsillar mucosa. RESULTS: Sixty percent of patients (42/70) had evidence of Leishmania persistence at EoT and 30% (9/30) 13 weeks after treatment initiation. A previous episode of CL was found to be a protective factor for detectable Leishmania persistence (OR: 0.16, 95%CI: 0.03-0.92). kDNA genotyping could not discern differences between parasite populations that persisted and those isolated at diagnosis. CONCLUSIONS: Leishmania persist in skin and mucosal tissues in a high proportion of patients who achieved therapeutic cure of CL. This finding prompts assessment of the contribution of persistent infection in transmission and endemicity of CL, and in disease reactivation and protective immunity.
Assuntos
Leishmania/isolamento & purificação , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/parasitologia , Adolescente , Adulto , Idoso , DNA de Cinetoplasto/análise , DNA de Protozoário/análise , Feminino , Seguimentos , Humanos , Leishmania/fisiologia , Masculino , Pessoa de Meia-Idade , Mucosa/parasitologia , Estudos Prospectivos , RNA de Protozoário/análise , RNA Citoplasmático Pequeno/análise , Partícula de Reconhecimento de Sinal/análise , Pele/parasitologia , Análise de Sobrevida , Fatores de Tempo , Resultado do Tratamento , Adulto JovemAssuntos
Autoanticorpos/sangue , Miosite/imunologia , Miosite/patologia , Partícula de Reconhecimento de Sinal/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Progressão da Doença , Feminino , Humanos , Imunossupressores , Pessoa de Meia-Idade , Miosite/tratamento farmacológico , Prognóstico , Estudos Retrospectivos , Medição de Risco , Estudos de Amostragem , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Partícula de Reconhecimento de Sinal/análise , Resultado do TratamentoRESUMO
BACKGROUND: The contribution of individuals with subclinical infection to the transmission and endemicity of cutaneous leishmaniasis (CL) is unknown. Immunological evidence of exposure to Leishmania in residents of endemic areas has been the basis for defining the human population with asymptomatic infection. However, parasitological confirmation of subclinical infection is lacking. METHODS: We investigated the presence and viability of Leishmania in blood and non-invasive mucosal tissue samples from individuals with immunological evidence of subclinical infection in endemic areas for CL caused by Leishmania (Viannia) in Colombia. Detection of Leishmania kDNA was conducted by PCR-Southern Blot, and parasite viability was confirmed by amplification of parasite 7SLRNA gene transcripts. A molecular tool for genetic diversity analysis of parasite populations causing persistent subclinical infection based on PCR amplification and sequence analysis of an 82bp region between kDNA conserved blocks 1 and 2 was developed. PRINCIPAL FINDINGS: Persistent Leishmania infection was demonstrated in 40% (46 of 114) of leishmanin skin test (LST) positive individuals without active disease; parasite viability was established in 59% of these (27 of 46; 24% of total). Parasite burden quantified from circulating blood monocytes, nasal, conjunctival or tonsil mucosal swab samples was comparable, and ranged between 0.2 to 22 parasites per reaction. kDNA sequences were obtained from samples from 2 individuals with asymptomatic infection and from 26 with history of CL, allowing genetic distance analysis that revealed diversity among sequences and clustering within the L. (Viannia) subgenus. CONCLUSIONS: Our results provide parasitological confirmation of persistent infection among residents of endemic areas of L. (Viannia) transmission who have experienced asymptomatic infection or recovered from CL, revealing a reservoir of infection that potentially contributes to the endemicity and transmission of disease. kDNA genotyping establishes proof-of-principle of the feasibility of genetic diversity analysis in previously inaccessible and unexplored parasite populations in subclinically infected individuals.
Assuntos
Variação Genética , Leishmania/classificação , Leishmania/genética , Leishmaniose Cutânea/parasitologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções Assintomáticas , Southern Blotting , Criança , Análise por Conglomerados , Colômbia , DNA de Helmintos/genética , DNA de Cinetoplasto/química , DNA de Cinetoplasto/genética , Feminino , Genótipo , Humanos , Leishmania/isolamento & purificação , Leishmania/fisiologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , RNA Citoplasmático Pequeno/análise , RNA Citoplasmático Pequeno/genética , Análise de Sequência de DNA , Partícula de Reconhecimento de Sinal/análise , Partícula de Reconhecimento de Sinal/genética , Adulto JovemRESUMO
North African gundis (Ctenodactylus gundi) were trapped in the Leishmania (L.) tropica focus of cutaneous leishmaniasis, situated in southeast Tunisia and evaluated for Leishmania infection by real-time kinetoplast DNA polymerase chain reaction (PCR). Species identification was performed by internal transcribed spacer one (ITS1)-PCR-restriction fragment length polymorphism (RFLP) and high-resolution melting (HRM) analysis of the 7SL RNA gene. Real-time PCR on blood was positive in 6 of 13 (46.2%) tested gundis. Leishmania tropica was identified in five infected gundis and Leishmania major in one specimen. Alignments of the ITS-1 DNA sequences and 7S-HRM curves analysis indicated that similar genotypes were present in humans, a sandfly, and gundis from the same region suggesting a potential role of this rodent as reservoir host of L. tropica in southeast Tunisia.
Assuntos
Reservatórios de Doenças/parasitologia , Leishmania major/isolamento & purificação , Leishmania tropica/isolamento & purificação , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/veterinária , Roedores/parasitologia , Animais , DNA de Cinetoplasto/isolamento & purificação , Reservatórios de Doenças/veterinária , Leishmania major/patogenicidade , Leishmania tropica/patogenicidade , Polimorfismo de Fragmento de Restrição , RNA Citoplasmático Pequeno/análise , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Partícula de Reconhecimento de Sinal/análise , Tunísia/epidemiologiaRESUMO
BACKGROUND: Leishmaniasis is a vector-borne disease, which is still endemic in the west and northwest area of China. Canines are the major reservoirs of Leishmania, the etiological agent of human visceral leishmaniasis. Phlebotomus chinensis is the main transmission vector of zoonotic visceral leishmaniasis (ZVL). METHODS: In this study, rK39 dip-stick, ELISA and PCR methods were used to investigate the prevalence of canine leishmaniasis (CanL) in Beichuan County, Sichuan Province, China. RESULTS: Among the 86 dogs which were included in the study, 13 dogs were positive using the dip-stick test (15.12%), while 8 dogs were positive using ELISA (9.30%) and 19 dogs were positive for PCR (22.03%). In total, 32 dogs were positive for one or more tests (37.21%). Interestingly, phylogenetic analysis based on the partial 7SL RNA fragment provided evidence that an undescribed Leishmania species, which is clearly a causative agent of CanL and human visceral leishmaniasis, does exist in China. This result is consistent with our previous study. CONCLUSIONS: Our work confirmed that canine leishmaniasis is still prevalent in Beichuan County. Further control is urgently needed, as canine leishmaniasis is of great public health importance. The phylogenetic analysis based on 7SL RNA segment provides evidence for the existence of an undescribed Leishmania sp. in China.
Assuntos
Doenças do Cão/parasitologia , Leishmania/isolamento & purificação , Leishmaniose Visceral/veterinária , RNA Citoplasmático Pequeno/análise , Partícula de Reconhecimento de Sinal/análise , Animais , China/epidemiologia , Doenças do Cão/epidemiologia , Cães , Doenças Endêmicas , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Insetos Vetores/parasitologia , Leishmania/genética , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Masculino , Filogenia , Reação em Cadeia da Polimerase , Prevalência , Fitas Reagentes , Zoonoses/epidemiologia , Zoonoses/parasitologiaRESUMO
HIV-1 is known to package several small cellular RNAs in addition to its genome. Previous work consistently demonstrated that the host structural RNA 7SL is abundant in HIV-1 virions but has yielded conflicting results regarding whether 7SL is present in minimal, assembly-competent virus-like particles (VLPs). Here, we demonstrate that minimal HIV-1 VLPs retain 7SL RNA primarily as an endoribonucleolytic fragment, referred to as 7SL remnant (7SLrem). Nuclease mapping showed that 7SLrem is a 111-nucleotide internal portion of 7SL, with 5' and 3' ends corresponding to unpaired loops in the 7SL two-dimensional structure. Analysis of VLPs comprised of different subsets of Gag domains revealed that all NC-positive VLPs contained intact 7SL while the presence of 7SLrem correlated with the absence of the NC domain. Because 7SLrem, which maps to the 7SL S domain, was not detectable in infected cells, we propose a model whereby the species recruited to assembling VLPs is intact 7SL RNA, with 7SLrem produced by an endoribonuclease in the absence of NC. Since recruitment of 7SL RNA was a conserved feature of all tested minimal VLPs, our model further suggests that 7SL's recruitment is mediated, either directly or indirectly, through interactions with conserved features of all tested VLPs, such as the C-terminal domain of CA.
Assuntos
HIV-1/química , RNA Citoplasmático Pequeno/análise , Partícula de Reconhecimento de Sinal/análise , Vírion/química , Virossomos/química , Sequência de Bases , Linhagem Celular , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ribonucleases/metabolismoRESUMO
The virion incorporation of 7SL, the RNA component of the host signal recognition particle (SRP), has been shown for several simple retroviruses. Data here demonstrate that 7SL is also packaged by HIV-1, in sevenfold molar excess of genomic RNA. Viral determinants of HIV-1 genome and primer tRNA packaging were not required for 7SL incorporation, as virus-like particles with only minimal assembly components efficiently packaged 7SL. The majority of 7SL within cells resides in ribonucleoprotein complexes bound by SRP proteins, and most SRP protein exists in signal recognition particles. However, Western blot comparison of virion and cell samples revealed that there is at least 25-fold less SRP p54 protein per 7SL RNA in HIV-1 particles than in cells. Comparing 7SL:actin mRNA ratios in virions and cells revealed that 7SL RNA appears selectively enriched in virions.
Assuntos
HIV-1/genética , RNA Citoplasmático Pequeno/análise , Partícula de Reconhecimento de Sinal/análise , Vírion/genética , Sequência de Bases , Linhagem Celular , Primers do DNA , Humanos , Reação em Cadeia da PolimeraseRESUMO
Moloney murine leukemia virus (MLV) particles contain both viral genomic RNA and an assortment of host cell RNAs. Packaging of virus-encoded RNA is selective, with virions virtually devoid of spliced env mRNA and highly enriched for unspliced genome. Except for primer tRNA, it is unclear whether packaged host RNAs are randomly sampled from the cell or specifically encapsidated. To address possible biases in host RNA sampling, the relative abundances of several host RNAs in MLV particles and in producer cells were compared. Using 7SL RNA as a standard, some cellular RNAs, such as those of the Ro RNP, were found to be enriched in MLV particles in that their ratios relative to 7SL differed little, if at all, from their ratios in cells. Some RNAs were underrepresented, with ratios relative to 7SL several orders of magnitude lower in virions than in cells, while others displayed intermediate values. At least some enriched RNAs were encapsidated by genome-defective nucleocapsid mutants. Virion RNAs were not a random sample of the cytosol as a whole, since some cytoplasmic RNAs like tRNA(Met) were vastly underrepresented, while U6 spliceosomal RNA, which functions in the nucleus, was enriched. Real-time PCR demonstrated that env mRNA, although several orders of magnitude less abundant than unspliced viral RNA, was slightly enriched relative to actin mRNA in virions. These data demonstrate that certain host RNAs are nearly as enriched in virions as genomic RNA and suggest that Psi- mRNAs and some other host RNAs may be specifically excluded from assembly sites.
Assuntos
Vírus da Leucemia Murina de Moloney/fisiologia , RNA Mensageiro/genética , RNA Ribossômico 5S/genética , RNA Citoplasmático Pequeno/genética , Partícula de Reconhecimento de Sinal/genética , Vírion/genética , Northern Blotting , Linhagem Celular , Humanos , Vírus da Leucemia Murina de Moloney/genética , RNA Mensageiro/análise , RNA Ribossômico 5S/análise , RNA Citoplasmático Pequeno/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Partícula de Reconhecimento de Sinal/análise , Replicação ViralRESUMO
OBJECTIVE: Autoantibodies against signal recognition particle (SRP) are detected in patients with polymyositis/dermatomyositis (PM/DM). The SRP consists of 7SL RNA and 6 protein components. We examined autoantibodies against deproteinized 7SL RNA in PM/DM patients with anti-SRP antibodies and evaluated the association of anti-7SL RNA antibodies with PM/DM clinically and serologically. METHODS: Sera from 10 Japanese and 22 North American PM/DM patients with anti-SRP antibodies were tested for the presence of anti-7SL RNA antibodies, using the sera to immunoprecipitate deproteinized RNA extracts derived from HeLa cells. RESULTS: The immunoprecipitation analysis indicated that 5 Japanese (50%) and one North American (5%) patient with anti-SRP antibodies had novel autoantibodies against deproteinized 7SL RNA. The frequency of anti-7SL RNA antibodies was significantly higher in Japanese than North American patients (p = 0.006). The presence of anti-7SL RNA antibodies appeared to be associated with DM (2 patients) and finger swelling (2 PM patients). The seasonal onset of the disease was different (p = 0.008) for Japanese PM/DM patients with anti-7SL RNA antibodies, who developed the disease between October and January (mean month November; p = 0.01) from that of patients without these antibodies, who developed it between June and August (mean month July; p = 0.01). CONCLUSION: Novel autoantibodies against 7SL RNA were identified in patients with PM/DM, and the presence of these antibodies was correlated to ethnic background, clinical features, and season of disease onset. These findings indicated that autoantibodies against 7SL RNA are a novel serological marker for a subset of PM/DM cases.
Assuntos
Povo Asiático , Autoanticorpos/imunologia , Polimiosite/etnologia , Polimiosite/imunologia , RNA Citoplasmático Pequeno/imunologia , Partícula de Reconhecimento de Sinal/imunologia , População Branca , Adulto , Autoanticorpos/análise , Biomarcadores , Estudos de Coortes , Dermatomiosite/sangue , Dermatomiosite/etnologia , Dermatomiosite/imunologia , Feminino , Seguimentos , Humanos , Imunoprecipitação , Masculino , Pessoa de Meia-Idade , Polimiosite/sangue , Probabilidade , RNA Citoplasmático Pequeno/análise , Medição de Risco , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Partícula de Reconhecimento de Sinal/análiseRESUMO
The signal recognition particle (SRP) is a cytoplasmic RNA-protein complex that targets proteins to the rough endoplasmic reticulum. Although SRP functions in the cytoplasm, RNA microinjection and cDNA transfection experiments in animal cells, as well as genetic analyses in yeast, have indicated that SRP assembles in the nucleus. Nonetheless, the mechanisms responsible for nuclear-cytoplasmic transport of SRP RNA and SRP proteins are largely unknown. Here we show that the 19 kDa protein subunit of mammalian SRP, SRP19, was efficiently imported into the nucleus in vitro by two members of the importin beta superfamily of transport receptors, importin 8 and transportin; SRP19 was also imported less efficiently by several other members of the importin beta family. Although transportin is known to import a variety of proteins, SRP19 import is the first function assigned to importin 8. Furthermore, we show that a significant pool of endogenous SRP19 is located in the nucleus, as well as the nucleolus. Our results show that at least one mammalian SRP protein is specifically imported into the nucleus, by members of the importin beta family of transport receptors, and the findings add additional evidence for nuclear assembly of SRP.
Assuntos
Núcleo Celular/metabolismo , Carioferinas/metabolismo , Proteínas Nucleares/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Núcleo Celular/química , Células HeLa , Humanos , Carioferinas/análise , Proteínas Nucleares/análise , Receptores Citoplasmáticos e Nucleares , Partícula de Reconhecimento de Sinal/análise , beta Carioferinas/análise , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/análiseRESUMO
The signal recognition particle (SRP) of eukaryotic cells is a cytoplasmic ribonucleoprotein machine that arrests the translational elongation of nascent secretory and membrane proteins and facilitates their transport into the endoplasmic reticulum. The spatial pathway of SRP RNA processing and ribonucleoprotein assembly in the cell is not known. In the present investigation, microinjection of fluorescently tagged SRP RNA into the nucleus of mammalian cells was used to examine its intranuclear sites of localization. Microinjection of SRP RNA into the nuclei of normal rat kidney (NRK) epithelial cells maintained at 37 degreesC on the microscope stage resulted in a very rapid initial localization in nucleoli, followed by a progressive decline of nucleolar signal and an increase of fluorescence at discrete sites in the cytoplasm. Nuclear microinjection of a molecule corresponding to a major portion of the Alu domain of SRP RNA revealed a pattern of rapid nucleolar localization followed by cytoplasmic appearance of signal that was similar to the results obtained with full-length SRP RNA. In contrast, a molecule corresponding to the S domain of SRP RNA did not display nucleolar localization to the extent observed with full-length SRP RNA. An SRP RNA molecule lacking helix 6 of the S domain displayed normal nucleolar localization, whereas one lacking helix 8 of the S domain did not. These results, obtained by direct, real-time observation of fluorescent RNA molecules inside the nucleus of living mammalian cells, suggest that the processing of SRP RNA or its ribonucleoprotein assembly into the SRP involves a nucleolar phase.
Assuntos
Nucléolo Celular/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , RNA/análise , Partícula de Reconhecimento de Sinal/metabolismo , Animais , Sequência de Bases , Transporte Biológico , Linhagem Celular , Cães , Microinjeções , Dados de Sequência Molecular , Mutação , RNA/genética , Ratos , Partícula de Reconhecimento de Sinal/análise , Partícula de Reconhecimento de Sinal/genéticaRESUMO
We measured the concentrations of both 4.5S RNA and Ffh protein under a variety of growth conditions and found that there were 400 molecules of 4.5S RNA per 10,000 ribosomes in wild-type cells and that the concentration of Ffh protein was one-fourth of that. This difference in concentration is 1 order of magnitude less than that previously reported but still significant. Pulse-chase labeling experiments indicated that Ffh protein is unstable in cells carrying ffh on high-copy-number plasmids and that simultaneous overproduction of 4.5S RNA stabilizes Ffh protein. Our analyses show that free Ffh protein is degraded with a half-life of approximately 20 min. We also tested whether three previously isolated suppressors of 4.5S RNA deficiency could reduce the requirement for Ffh protein. Since the two sffE suppressors do not suppress the Ffh requirement, we suggest that 4.5S RNA either acts in a sequential reaction with Ffh or has two functions.