Simona Kahane

Development of a Transformation System for Chlamydia trachomatis: Restoration of Glycogen Biosynthesis by Acquisition of a Plasmid Shuttle Vector

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 β-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 β-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.

Unity in Variety—The Pan-Genome of the Chlamydiae

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.

Infection of Acanthamoeba polyphaga with Simkania negevensis and S. negevensis Survival within Amoebal Cysts

ABSTRACT Simkania negevensis, a novel microorganism belonging to the family Simkaniaceae in the orderChlamydiales, has an intracellular developmental cycle during which two morphological entities, elementary bodies (EB) and reticulate bodies (RB), are seen by electron microscopy. Rates of seropositivity to the organism are high in certain population groups, and S. negevensis has been associated with respiratory illness in humans. This study reports for the first time the ability ofS. negevensis to survive and grow insideAcanthamoeba polyphaga in addition to its known ability to grow in cell cultures of human or simian origin. Infectivity ofS. negevensis and growth in amoebae were monitored by immunoperoxidase assays. Long-term persistence and exponential growth of S. negevensis in amoebal trophozoites were demonstrated by infectivity assays and by electron microscopy. EB and dividing RB of S. negevensis were observed within inclusion bodies inside A. polyphaga. When S. negevensis-infected A. polyphaga amoebae were exposed to adverse conditions resulting in encystation of the amoebae, several possible outcomes were observed: cysts containing both normal amoebic cytoplasm and S. negevensis; cysts in whichS. negevensis cells were relegated to the space between cyst walls; and cysts containing S. negevensis, but apparently lacking amoebal cytoplasm. S. negevensiswithin dried amoebal cysts was capable of long-term survival. The possibility that amoebae may have a role in natural transmission ofS. negevensis needs to be investigated.

Infections with the chlamydia-like microorganism Simkania negevensis, a possible emerging pathogen

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.

High Rate of Simkania negevensis among Canadian Inuit Infants Hospitalized with Lower Respiratory Tract Infections

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.

Simkania negevensis strain ZT: growth, antigenic and genome characteristics.

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 other published data, are consistent with the proposal that S. negevensis belongs to a distinct family in the order Chlamydiales.

Serological Evidence of Acute Infection with the Chlamydia-Like Microorganism Simkania negevensis (Z) in Acute Exacerbation of Chronic Obstructive Pulmonary Disease

Abstract.The aims of this study were twofold: (i) to test for possible associations between serological evidence of acute Simkania negevensis (Sn) infection and acute exacerbation of chronic obstructive pulmonary disease and (ii) to examine the prevalence of past infections with Sn in patients with chronic obstructive pulmonary disease. In 120 patients (63%) there was serological evidence of past infection with Sn, which was not significantly different from the rate in a control population. In five hospitalizations serological evidence existed of acute infection with Sn around the time of the exacerbation of chronic obstructive pulmonary disease. In four of these cases, there was serological evidence of acute infection with at least one other respiratory pathogen. It is concluded that Sn can be associated serologically with exacerbation of chronic obstructive pulmonary disease, in most cases together with other respiratory pathogens. The implications of these findings should be investigated further.

Genetic Transformation of a Clinical (Genital Tract), Plasmid-Free Isolate of Chlamydia trachomatis: Engineering the Plasmid as a Cloning Vector

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 β-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 β-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 β-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.

Detection of Simkania negevensis by culture, PCR, and serology in respiratory tract infection in Cornwall, UK

Respiratory tract infections are often treated empirically without investigation to detect the aetiological agent, which may be a virus or a bacterium, including atypical pathogens such as Chlamydophila pneumoniae or Mycoplasma pneumoniae. Recently, several types Chlamydia-like intracellular bacteria have been detected in environmental samples and clinical specimens. Little is known of their geographical distribution and potential pathogenicity. We describe the detection, by PCR and isolation in cell culture, of Simkania negevensis in nasopharyngeal aspirates of paediatric patients with bronchiolitis in Cornwall, UK. We also present serological evidence of exposure to the organism in 62% of adult patients and 46% of a sample of pregnant women. Empirical treatment of serious respiratory tract infection should consider the possible contribution of these organisms.

Infection with Simkania negevensis in Brooklyn, New York.

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.