RESUMO
Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB) is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB). Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of ß-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active ß-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP). As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome function by complementation.
Assuntos
Chlamydia trachomatis/genética , Vetores Genéticos , Glicogênio/biossíntese , Plasmídeos , Transformação Bacteriana/fisiologia , Chlamydia trachomatis/crescimento & desenvolvimento , Corpos de Inclusão/microbiologia , Resistência às Penicilinas/genética , Penicilinas/farmacologia , beta-Lactamases/genéticaRESUMO
Chlamydiae are evolutionarily well-separated bacteria that live exclusively within eukaryotic host cells. They include important human pathogens such as Chlamydia trachomatis as well as symbionts of protozoa. As these bacteria are experimentally challenging and genetically intractable, our knowledge about them is still limited. In this study, we obtained the genome sequences of Simkania negevensis Z, Waddlia chondrophila 2032/99, and Parachlamydia acanthamoebae UV-7. This enabled us to perform the first comprehensive comparative and phylogenomic analysis of representative members of four major families of the Chlamydiae, including the Chlamydiaceae. We identified a surprisingly large core gene set present in all genomes and a high number of diverse accessory genes in those Chlamydiae that do not primarily infect humans or animals, including a chemosensory system in P. acanthamoebae and a type IV secretion system. In S. negevensis, the type IV secretion system is encoded on a large conjugative plasmid (pSn, 132 kb). Phylogenetic analyses suggested that a plasmid similar to the S. negevensis plasmid was originally acquired by the last common ancestor of all four families and that it was subsequently reduced, integrated into the chromosome, or lost during diversification, ultimately giving rise to the extant virulence-associated plasmid of pathogenic chlamydiae. Other virulence factors, including a type III secretion system, are conserved among the Chlamydiae to variable degrees and together with differences in the composition of the cell wall reflect adaptation to different host cells including convergent evolution among the four chlamydial families. Phylogenomic analysis focusing on chlamydial proteins with homology to plant proteins provided evidence for the acquisition of 53 chlamydial genes by a plant progenitor, lending further support for the hypothesis of an early interaction between a chlamydial ancestor and the primary photosynthetic eukaryote.
Assuntos
Chlamydia/genética , Chlamydiales/genética , DNA Bacteriano/genética , Genoma Bacteriano , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Sequência de Bases , Membrana Celular , Chlamydia/classificação , Chlamydia/patogenicidade , Chlamydiales/classificação , Chlamydiales/patogenicidade , DNA Bacteriano/análise , Evolução Molecular , Transferência Genética Horizontal , Variação Genética , Interações Hospedeiro-Patógeno , Dados de Sequência Molecular , Filogenia , Plasmídeos , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , SimbioseRESUMO
Exposure to Simkania negevensis (Sn), an intracellular microorganism that has been associated with respiratory tract infections in infants and adults, is prevalent. Sn can multiply within free-living amoebae and has been detected in domestic water supplies, which may constitute a source of infection with the organism. Its path of transport from its portal of entry to the body to its target organs is unknown. In this study, the possibility that monocytes/macrophages may serve as vehicles of transmission was examined. In vitro cocultivation of Sn-infected Acanthamoeba polyphaga with the monocyte/macrophage cell line U937 resulted in the death of the amoebae and infection of the U937 cells. Sn entered and multiplied in U937 cells within short periods of time, and the microorganism could be transferred from U937 cells to cell cultures of various origins. Uninfected monocyte/macrophages could become infected when in contact with either actively or persistently Sn-infected cell cultures. Persistently infected cultures in contact with uninfected U937 cells became actively infected. The results of this study provide a basis for determination of the molecular mechanisms of monocyte/macrophage-cell interactions in transfer of infection and may contribute to a better understanding of the pathogenesis of Sn infections in vivo.
Assuntos
Chlamydiales/imunologia , Macrófagos/microbiologia , Monócitos/microbiologia , Acanthamoeba/microbiologia , Animais , Linhagem Celular , Chlamydiales/crescimento & desenvolvimento , Técnicas de Cocultura , Humanos , Macrófagos/ultraestrutura , Microscopia Eletrônica de Transmissão , Monócitos/ultraestruturaRESUMO
BACKGROUND: Simkania negevensis is a novel organism closely related to chlamydiae. The organism has been associated with community acquired pneumonia and acute exacerbation of chronic obstructive pulmonary disease. The prevalence and pathogenic potential of S. negevensis is not known in lung transplant recipients. METHODS: In this multicenter study comparative analysis of bronchoalveolar lavage (BAL) in lung transplants (Tx) and kidney Tx, immunocompromised and nasopharyngeal (NP) washes of immunocompetent patients was done. The BAL specimens were tested by nested polymerase chain reaction (PCR) for C. pneumoniae and S. negevensis. Selected S. negevensis positive PCR cases were confirmed by culture. RESULTS: In the initial 41 BAL samples S. negevensis was detected in 97.5% (40/41) of lung transplant recipients as compared to 14.1% (1/7) in other organ transplant recipients (P<0.0001). In the sequential samples of 19 lung transplant recipients, 59% (24/41) had concomitant positive PCR and rejection as compared to 30% (3/10) who had negative PCR but had rejection (P=0.16). S. negevensis infection had hazard ratio of 3.29 (95% CI: 0.73-14.76; P=0.11) for developing acute rejection. CONCLUSION: S. negevensis is highly prevalent in liver Tx recipients and may be associated with acute rejection.
Assuntos
Líquido da Lavagem Broncoalveolar/microbiologia , Chlamydiales/fisiologia , Rejeição de Enxerto/microbiologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Transplante de Pulmão , Adulto , Líquido da Lavagem Broncoalveolar/imunologia , Feminino , Infecções por Bactérias Gram-Negativas/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Fatores de TempoRESUMO
BACKGROUND: Simkania negevensis is a Chlamydia-like intracellular organism that is prevalent in populations from a wide range of geographic areas. The role of the organism in respiratory disease in the United States is unknown. OBJECTIVE: To study the association between infection with S. negevensis and bronchiolitis, pneumonia or asthma in Brooklyn, New York. MATERIALS AND METHODS: Pediatric and adult inpatients/outpatients with bronchiolitis, pneumonia or asthma were recruited, and a similar number of healthy control subjects were enrolled. Nasopharyngeal swabs were obtained for culture of S. negevensis and Chlamydia pneumoniae and polymerase chain reaction detection of S. negevensis. Sera were obtained for measurement of antibodies to S. negevensis and C. pneumoniae. RESULTS: One hundred eighty-eight patients and 110 healthy control subjects were enrolled. S. negevensis serologic assays were positive for 18% of patients, compared with 29% of control subjects (P = 0.09). S. negevensis DNA was detected by PCR for 17% of case subjects and 23% of control subjects (P = 0.25). S. negevensis was isolated by culture for 1 patient with bronchiolitis. C. pneumoniae IgG and S. negevensis IgG were found to increase with increasing age, ie, 14%, 50% and 78% (C. pneumoniae) and 13%, 17% and 33% (S. negevensis) for subjects 0-18 months, 18 months-18 years and older than 18 years of age, respectively. CONCLUSION: S. negevensis was not a significant respiratory pathogen in Brooklyn, NY, during the period of the study.
Assuntos
Chlamydiales/isolamento & purificação , Infecções por Bactérias Gram-Negativas/epidemiologia , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/microbiologia , Adolescente , Adulto , Anticorpos Antibacterianos/sangue , Criança , Pré-Escolar , Chlamydiales/patogenicidade , DNA Bacteriano/análise , Feminino , Infecções por Bactérias Gram-Negativas/diagnóstico , Humanos , Lactente , Recém-Nascido , Masculino , Cidade de Nova Iorque/epidemiologia , Infecções Respiratórias/diagnóstico , Estudos SoroepidemiológicosRESUMO
Although evidence for the existence of numerous chlamydia-like microorganisms has been discovered in both environmental samples and clinical specimens, very few have been grown in vitro, and little is known of their pathogenic potential. Of all such organisms, Simkania negevensis is probably the most extensively studied. This review summarizes current knowledge about this intracellular bacterium, focusing especially on human infections.
Assuntos
Infecções por Chlamydiaceae/microbiologia , Chlamydiales/isolamento & purificação , Doenças Transmissíveis Emergentes/microbiologia , Infecções por Chlamydiaceae/imunologia , Chlamydiales/imunologia , Chlamydiales/ultraestrutura , Transmissão de Doença Infecciosa , Humanos , Prevalência , Estudos SoroepidemiológicosRESUMO
Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide "proof of principle" that it is possible to "knock out" selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb ß-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed ß-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active ß-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need for plaquing, fluorescence or antibody staining.
Assuntos
Chlamydia trachomatis/genética , Técnicas de Inativação de Genes/métodos , Engenharia Genética/métodos , Transformação Bacteriana/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Southern Blotting , Primers do DNA/genética , Galactosídeos , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/metabolismo , Corpos de Inclusão/metabolismo , Indóis , Microscopia de Fluorescência , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo Real , beta-Galactosidase/metabolismoRESUMO
OBJECTIVE: Simkania negevensis (Sn) is an intracellular microorganism belonging to the family Simkaniaceae in the order Chlamydiales and has been associated with respiratory tract infections in infants and adults. The aim of this study was to analyze the outcome of Sn infection in different cell types. METHODS: The results of Sn infection were examined by infectivity assays, PCR and EM. The cellular response to infection was evaluated by following the synthesis of mRNA for inflammatory cytokines and cytokine secretion. RESULTS: Infections could be active, with production of progeny and cytopathic effects (CPE); persistent, induced by iron depletion or in minimally permissive cell types, with small numbers of infectious progeny; or cryptic, with no CPE or infectious progeny, but with Sn DNA detected. EM showed an abundance of EB and multiplying RB in active infection, small inclusions with mainly single RB particles in persistent infection, and aberrant inclusions in cryptic infection. We report reversion to active infection of iron-induced or spontaneous persistence; attempts to "cure" persistence with antibiotic treatment resulted in the absence of infectivity but not in the eradication of Sn DNA. CONCLUSION: Sn infections are versatile and induce a host cell inflammatory response, which may be relevant to potential Sn pathologies in vivo.
Assuntos
Infecções por Chlamydiaceae/imunologia , Chlamydiales/patogenicidade , Citocinas/biossíntese , Animais , Antibacterianos/uso terapêutico , Azitromicina/uso terapêutico , Células Cultivadas , Infecções por Chlamydiaceae/tratamento farmacológico , Infecções por Chlamydiaceae/metabolismo , Humanos , Técnicas In VitroRESUMO
OBJECTIVES: Simkania negevensis, a Chlamydia-like microorganism that has been associated with respiratory infections in children and adults, can multiply within free-living amoebae; moreover, it has been detected in domestic water supplies. The aim of this study was to determine whether there is similarity between Simkania organisms found in water and those detected in clinical samples. METHODS: PCR, membrane immunoassay for the detection of Simkania antigen, and isolation in cell culture were used for the detection of S. negevensis in nasopharyngeal wash samples (NPW) of 34 children with pneumonia and in domestic water samples from their homes. Sequencing of PCR amplicons was used for comparison of Simkania strains isolated from clinical samples and from water samples. RESULTS: In 26 cases (76%) both NPW and water were positive, and partial 16S rDNA sequences suggested that they may be the same organisms. CONCLUSION: Simkania found in domestic water supplies may be transmitted to children .
Assuntos
Chlamydiales/classificação , Chlamydiales/isolamento & purificação , Infecções por Bactérias Gram-Negativas/epidemiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Microbiologia da Água , Abastecimento de Água , Proteínas de Bactérias/análise , Proteínas de Bactérias/imunologia , Pré-Escolar , Chlamydiales/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Feminino , Humanos , Imunoensaio , Lactente , Israel , Masculino , Epidemiologia Molecular , Nasofaringe/microbiologia , Pneumonia Bacteriana/microbiologia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Simkania negevensis, a bacterium formerly referred to as 'the micro-organism Z' or 'Simkania Z', belongs to the order Chlamydiales, assigned to the family Simkaniaceae: The purpose of this study was to investigate the production of Simkania negevensis progeny in infected cells in comparison with the well-documented Chlamydiaceae developmental cycle. It was found that replicating Simkania negevensis in Vero cells resembled the reticulate bodies of all known chlamydial species: in electron micrographs they were reticulated, homogeneously staining, and often caught in the process of binary division. These replicative forms were found in low abundance shortly after infection, but by 3 days post-infection they were the most prevalent particles in host cells. Electron-dense forms of Simkania negevensis began to appear on the third day post-infection, but quantitatively did not account for the high titre of infectivity in extracts from these host cells. These had both electron-dense and electron-lucent areas, a characteristic seen only in a few chlamydial species. Simkania negevensis infectivity did not appreciably change during the ensuing 12 days required for host cell lysis, despite an eightfold increase in the proportion of electron-dense bacteria over this time. The emergence of electron-dense bodies, increase in infectivity and host-cell lysis were not synchronized developmental events. This is a novel finding in Chlamydiales spp. and suggests that Simkania negevensis will provide new perspectives in the mechanisms of chlamydial intracellular growth.
Assuntos
Chlamydiales/crescimento & desenvolvimento , Animais , Centrifugação com Gradiente de Concentração , Chlamydiales/patogenicidade , Chlamydiales/ultraestrutura , Chlorocebus aethiops , Microscopia Eletrônica , Células VeroRESUMO
To determine the prevalence of Simkania negevensis in causing pulmonary infections in children, nasopharyngeal washes were obtained from 22 infants hospitalized with acute bronchiolitis in the Baffin Island, Canada. 14 (63.6%) were positive for S. negevensis. Mixed infections with other respiratory viruses were common. All patients recovered without specific antibiotic treatment. Even though a high prevalence of S. negevensis was found, this organism may potentially well be an opportunistic agent rather than a true pathogen.
Assuntos
Chlamydiales/isolamento & purificação , Inuíte , Pneumonia Bacteriana/epidemiologia , Pneumonia Bacteriana/microbiologia , Canadá/epidemiologia , Estudos de Coortes , Feminino , Seguimentos , Hospitalização , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Probabilidade , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/microbiologia , Medição de Risco , Índice de Gravidade de DoençaRESUMO
Simkania negevensis is a recently discovered chlamydia-like intracellular microorganism which has been associated with bronchiolitis in infants and with community-acquired pneumonia in adults; a high seroprevalence of antibodies to the microorganism has been found in various population groups. S. negevensis can be grown in various cell lines as well as in free-living amoebae such as Acanthamoeba polyphaga. In this study, evidence for the existence of Simkania or Simkania-like microorganisms in drinking water and in reclaimed wastewater is presented for the first time. Detection of the microorganism was made possible by the development of a specific and sensitive filter membrane immunoassay and was confirmed by PCR detection of microbial DNA in the water samples. The common presence of S. negevensis in water sources together with the high seroprevalence of antibodies to it and early age of acquisition of infection may implicate water as a source of infection. The possible significance of this finding for public health and for municipal water testing and treatment needs to be further examined.