A new obligate intracellular bacterium which we called 'Z' was isolated as a cell culture... more A new obligate intracellular bacterium which we called 'Z' was isolated as a cell culture contaminant of unknown origin. The organism grew in a variety of cultured cells with a 5-7-day developmental cycle, within cytoplasmic phagosomes, similarly to Chlamydia and some Rickettsia spp. Two alternating developmental forms (elementary bodies and reticulate bodies) were observed by electron microscopy. SDS-PAGE electrophoresis, immunoblotting with chlamydia-specific antibodies, and polymerase chain reaction using chlamydial genus specific primers provided evidence that our bacterium differs significantly from chlamydiae. Further characterization of 'Z' including determination of 16S ribosomal RNA sequences will allow its taxonomic position to be established.
Simkania negevensis is a novel organism closely related to chlamydiae. The organism has been asso... more 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. 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. 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. S. negevensis is highly prevalent in liver Tx recipients and may be associated with acute rejection.
Scandinavian Journal of Infectious Diseases, Feb 1, 2003
To determine the prevalence of Simkania negevensis in causing pulmonary infections in children, n... more 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.
1. Membrane vesicles of Escherichiu coli K-12 CS7, a strain genetically derepressed for glutamate... more 1. Membrane vesicles of Escherichiu coli K-12 CS7, a strain genetically derepressed for glutamate permease, maintain low aspartate transport activity, like that of preparations of the wild-type parent. Growth of the parent CSlOl on aspartate as the source of carbon or nitrogen results in derepression of both aspartate and glutamate transport. Growth of strain CS7 on aspartate derepresses aspartate transport to the same extent as in strain CSlOl, but only slightly increases the derepressed level ofglutamate transport activity. 2. The affinity of the membrane transport system for glutamate is enhanced by sodium, while that for aspartate is not. 3. Although the affinities for glutamate (23 KM) and aspartate (12 pM) are similar, aspartate does not inhibit glutamate transport, while glutamate competitively inhibits aspartate transport. 4. Aspartate transport, but not glutamate transport, is competitively inhibited by Cq dicarboxylic acids, whereas 2-oxoglutarate competitively inhibits glutamate transport, but not aspartate transport. 5. Competitive inhibition of L-aspartate transport by L-glutamate and by the 5-methyl ester of L-glutamate is abolished in the presence of 2-oxoglutarate. However, 2-oxoglutarate does not affect the competitive inhibition of L-aspartate transport by D-aspartate and by ~~-threo-3-hydroxyaspartate. The relationship between the two dicarboxylic amino acid transport systems and the spatial characteristics of the aspartate carrier are discussed in the light of these findings.
Simkania negevensis, a bacterium formerly referred to as 'the micro-organism Z' or 'S... more 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 elect...
Chlamydia was the only genus in the order Chlamydiales until the recent characterization of Simka... more Chlamydia was the only genus in the order Chlamydiales until the recent characterization of Simkania negevensis Z(T) and Parachlamydia acanthamoebae strains. The present study of Chlamydiales 23S ribosomal DNA (rDNA) focuses on a naturally occurring group I intron in the I-CpaI target site of 23S rDNA from S. negevensis. The intron, SnLSU. 1, belonged to the IB4 structural subgroup and was most closely related to large ribosomal subunit introns that express single-motif, LAGLIDADG endonucleases in chloroplasts of algae and in mitochondria of amoebae. RT-PCR and electrophoresis of in vivo rRNA indicated that the intron was not spliced out of the 23S rRNA. The unspliced 658-nt intron is the first group I intron to be found in bacterial rDNA or rRNA, and it may delay the S. negevensis developmental replication cycle by affecting ribosomal function.
International journal of systematic bacteriology, 1999
Simkania negevensis is the type species of Simkaniaceae, a recently proposed family in the order ... more Simkania negevensis is the type species of Simkaniaceae, a recently proposed family in the order Chlamydiales. In the current study, growth, antigenic and genomic characteristics of this intracellular bacterium were investigated and compared to those of members of the family Chlamydiaceae. Growth of the organism, as assessed by infectivity assays, reached a plateau in 2-3 d although by light microscopy the cytopathic effect on the host cells increased for 12 or more days after infection. S. negevensis growth was unaffected by sulfadiazine. Cells infected by S. negevensis strain ZT were not recognized by either of two monoclonal antibodies specific for Chlamydiaceae LPS and several specific Chlamydiaceae ompA primers were unable to PCR amplify a S. negevensis gene. The S. negevensis genome contained one copy of the ribosomal operon. The genome size of S. negevensis strain ZT was determined by PFGE to be 1.7 Mbp, and the G + C content was 42.5 mol%. These data, taken together with oth...
A highly specific energy-dependent glutamate transport system was demonstrated in membrane vesicl... more A highly specific energy-dependent glutamate transport system was demonstrated in membrane vesicles of glutamate-utilizing Escherichia coli K-12 mutants. The glutamate transport activity of membranes from the parent strain, unable to grow on glutamate, was very low. With ascorbate-phenazine methosulfate as the electron donor, mutant preparations displayed 17 to 20 times higher activity than did the wild type. However, the affinity of the mutant carrier for L-glutamate remained the same as in the parent strain. Comparative inhibition analysis of glutamate transport in whole cells and membrane vesicles and of in vitro binding of glutamate to a specific periplasmic-binding protein suggests that under certain conditions the latter may be a component of the E. coli K-12 glutamate transport system.
Simkania negevensis is a novel organism closely related to chlamydiae. The organism has been asso... more 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. 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. 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. S. negevensis is highly prevalent in liver Tx recipients and may be associated with acute rejection.
To determine the prevalence of Simkania negevensis in causing pulmonary infections in children, n... more 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.
Chlamydia trachomatis remains one of the few major human pathogens for which there is no transfor... more 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 b-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 b-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.
Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical... more 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, plasmidbased 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 b-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 b-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 bgalactosidase 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.
Simkania negevensis is a Chlamydia-like intracellular organism that is prevalent in populations f... more 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. To study the association between infection with S. negevensis and bronchiolitis, pneumonia or asthma in Brooklyn, New York. 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. 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. S. negevensis was not a significant respiratory pathogen in Brooklyn, NY, during the period of the study.
Chlamydiae are evolutionarily well-separated bacteria that live exclusively within eukaryotic hos... more 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.
Although evidence for the existence of numerous chlamydia-like microorganisms has been discovered... more 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.
A new obligate intracellular bacterium which we called 'Z' was isolated as a cell culture... more A new obligate intracellular bacterium which we called 'Z' was isolated as a cell culture contaminant of unknown origin. The organism grew in a variety of cultured cells with a 5-7-day developmental cycle, within cytoplasmic phagosomes, similarly to Chlamydia and some Rickettsia spp. Two alternating developmental forms (elementary bodies and reticulate bodies) were observed by electron microscopy. SDS-PAGE electrophoresis, immunoblotting with chlamydia-specific antibodies, and polymerase chain reaction using chlamydial genus specific primers provided evidence that our bacterium differs significantly from chlamydiae. Further characterization of 'Z' including determination of 16S ribosomal RNA sequences will allow its taxonomic position to be established.
Simkania negevensis is a novel organism closely related to chlamydiae. The organism has been asso... more 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. 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. 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. S. negevensis is highly prevalent in liver Tx recipients and may be associated with acute rejection.
Scandinavian Journal of Infectious Diseases, Feb 1, 2003
To determine the prevalence of Simkania negevensis in causing pulmonary infections in children, n... more 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.
1. Membrane vesicles of Escherichiu coli K-12 CS7, a strain genetically derepressed for glutamate... more 1. Membrane vesicles of Escherichiu coli K-12 CS7, a strain genetically derepressed for glutamate permease, maintain low aspartate transport activity, like that of preparations of the wild-type parent. Growth of the parent CSlOl on aspartate as the source of carbon or nitrogen results in derepression of both aspartate and glutamate transport. Growth of strain CS7 on aspartate derepresses aspartate transport to the same extent as in strain CSlOl, but only slightly increases the derepressed level ofglutamate transport activity. 2. The affinity of the membrane transport system for glutamate is enhanced by sodium, while that for aspartate is not. 3. Although the affinities for glutamate (23 KM) and aspartate (12 pM) are similar, aspartate does not inhibit glutamate transport, while glutamate competitively inhibits aspartate transport. 4. Aspartate transport, but not glutamate transport, is competitively inhibited by Cq dicarboxylic acids, whereas 2-oxoglutarate competitively inhibits glutamate transport, but not aspartate transport. 5. Competitive inhibition of L-aspartate transport by L-glutamate and by the 5-methyl ester of L-glutamate is abolished in the presence of 2-oxoglutarate. However, 2-oxoglutarate does not affect the competitive inhibition of L-aspartate transport by D-aspartate and by ~~-threo-3-hydroxyaspartate. The relationship between the two dicarboxylic amino acid transport systems and the spatial characteristics of the aspartate carrier are discussed in the light of these findings.
Simkania negevensis, a bacterium formerly referred to as 'the micro-organism Z' or 'S... more 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 elect...
Chlamydia was the only genus in the order Chlamydiales until the recent characterization of Simka... more Chlamydia was the only genus in the order Chlamydiales until the recent characterization of Simkania negevensis Z(T) and Parachlamydia acanthamoebae strains. The present study of Chlamydiales 23S ribosomal DNA (rDNA) focuses on a naturally occurring group I intron in the I-CpaI target site of 23S rDNA from S. negevensis. The intron, SnLSU. 1, belonged to the IB4 structural subgroup and was most closely related to large ribosomal subunit introns that express single-motif, LAGLIDADG endonucleases in chloroplasts of algae and in mitochondria of amoebae. RT-PCR and electrophoresis of in vivo rRNA indicated that the intron was not spliced out of the 23S rRNA. The unspliced 658-nt intron is the first group I intron to be found in bacterial rDNA or rRNA, and it may delay the S. negevensis developmental replication cycle by affecting ribosomal function.
International journal of systematic bacteriology, 1999
Simkania negevensis is the type species of Simkaniaceae, a recently proposed family in the order ... more Simkania negevensis is the type species of Simkaniaceae, a recently proposed family in the order Chlamydiales. In the current study, growth, antigenic and genomic characteristics of this intracellular bacterium were investigated and compared to those of members of the family Chlamydiaceae. Growth of the organism, as assessed by infectivity assays, reached a plateau in 2-3 d although by light microscopy the cytopathic effect on the host cells increased for 12 or more days after infection. S. negevensis growth was unaffected by sulfadiazine. Cells infected by S. negevensis strain ZT were not recognized by either of two monoclonal antibodies specific for Chlamydiaceae LPS and several specific Chlamydiaceae ompA primers were unable to PCR amplify a S. negevensis gene. The S. negevensis genome contained one copy of the ribosomal operon. The genome size of S. negevensis strain ZT was determined by PFGE to be 1.7 Mbp, and the G + C content was 42.5 mol%. These data, taken together with oth...
A highly specific energy-dependent glutamate transport system was demonstrated in membrane vesicl... more A highly specific energy-dependent glutamate transport system was demonstrated in membrane vesicles of glutamate-utilizing Escherichia coli K-12 mutants. The glutamate transport activity of membranes from the parent strain, unable to grow on glutamate, was very low. With ascorbate-phenazine methosulfate as the electron donor, mutant preparations displayed 17 to 20 times higher activity than did the wild type. However, the affinity of the mutant carrier for L-glutamate remained the same as in the parent strain. Comparative inhibition analysis of glutamate transport in whole cells and membrane vesicles and of in vitro binding of glutamate to a specific periplasmic-binding protein suggests that under certain conditions the latter may be a component of the E. coli K-12 glutamate transport system.
Simkania negevensis is a novel organism closely related to chlamydiae. The organism has been asso... more 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. 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. 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. S. negevensis is highly prevalent in liver Tx recipients and may be associated with acute rejection.
To determine the prevalence of Simkania negevensis in causing pulmonary infections in children, n... more 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.
Chlamydia trachomatis remains one of the few major human pathogens for which there is no transfor... more 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 b-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 b-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.
Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical... more 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, plasmidbased 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 b-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 b-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 bgalactosidase 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.
Simkania negevensis is a Chlamydia-like intracellular organism that is prevalent in populations f... more 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. To study the association between infection with S. negevensis and bronchiolitis, pneumonia or asthma in Brooklyn, New York. 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. 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. S. negevensis was not a significant respiratory pathogen in Brooklyn, NY, during the period of the study.
Chlamydiae are evolutionarily well-separated bacteria that live exclusively within eukaryotic hos... more 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.
Although evidence for the existence of numerous chlamydia-like microorganisms has been discovered... more 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.
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Papers by Simona Kahane