A C. trachomatis Cloning Vector and the Generation of C. trachomatis Strains Expressing Fluorescent Proteins under the Control of a C. trachomatis Promoter

Agaisse, Hervé; Derré, Isabelle

doi:10.1371/journal.pone.0057090

Cited by 109 publications

(123 citation statements)

References 53 publications

(53 reference statements)

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“…Such events might occur within the confines of infected epithelial cells, and several studies have demonstrated that more than one C. trachomatis strain can simultaneously infect the same cell and form a single mixed intracellular inclusion (20,141,142). Once inside the host cell, coinfecting strains may exchange DNA, presumably while coinhabiting the same inclusion, although inclusion fusion might not be an absolute requirement for DNA exchange, because C. trachomatis isolates with nonfusogenic inclusions can exchange DNA in vitro (143).…”

Section: Evidence Of Lgt Events Among Chlamydia Clinical Isolatesmentioning

confidence: 99%

“…1 and 2). For example, the plasmid p2TK2-SW2 (20) and the pBOMB4 series of plasmids (21) combined a ␤-lactamase-encoding gene (bla) and a multiple-cloning site into the L2 pSW2 and pL2 (L2/434/Bu) plasmids, respectively. The pBOMB4 series offers the additional benefit of utilizing an intact L2 plasmid (pL2) rather than the SW2 variant plasmid, which harbors a 377-bp deletion in CDS1 (148).…”

Section: Ectopic Gene Expressionmentioning

confidence: 99%

“…Transformation of exogenous DNA into Chlamydia has been achieved via several methods, including electroporation, dendrimer-based delivery, and a calcium chloride-based treatment (14)(15)(16)(17)(18)(19). Successful and stable transformation of C. trachomatis LGV L2 with an Escherichia coli-C. trachomatis L2 shuttle plasmid conferring ␤-lactam resistance led to the development of expression vectors for expressing Chlamydia open reading frames (ORFs), fluorescent proteins (green fluorescent protein [GFP], cyan fluorescent protein [CFP], mCherry, and mKate2), and reporter proteins (␤-galactosidase, adenylate cyclase, and glycogen synthase kinase [GSK]-tagged proteins) (19)(20)(21)(22)(23)(24)(25)(26). A conditional expression vector was also developed using a tetracycline-inducible system as well as vectors conferring chloramphenicol and blasticidin resistance (21,(26)(27)(28)(29).…”

mentioning

confidence: 99%

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Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

Bastidas

Valdivia

2016

Microbiol Mol Biol Rev

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SUMMARYChlamydiaspecies infect millions of individuals worldwide and are important etiological agents of sexually transmitted disease, infertility, and blinding trachoma. Historically, the genetic intractability of this intracellular pathogen has hindered the molecular dissection of virulence factors contributing to its pathogenesis. The obligate intracellular life cycle ofChlamydiaand restrictions on the use of antibiotics as selectable markers have impeded the development of molecular tools to genetically manipulate these pathogens. However, recent developments in the field have resulted in significant gains in our ability to alter the genome ofChlamydia, which will expedite the elucidation of virulence mechanisms. In this review, we discuss the challenges affecting the development of molecular genetic tools forChlamydiaand the work that laid the foundation for recent advancements in the genetic analysis of this recalcitrant pathogen.

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Section: Evidence Of Lgt Events Among Chlamydia Clinical Isolatesmentioning

confidence: 99%

Section: Ectopic Gene Expressionmentioning

confidence: 99%

mentioning

confidence: 99%

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Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

Bastidas

Valdivia

2016

Microbiol Mol Biol Rev

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“…A novel application of this approach is for derivation of meaningful quantitative data about the inclusion, for example inclusion area, volume and shape. Finally, this visualization method is highly adaptable and can be used in combination with other fluorescent markers to reveal additional insight into the biology of Chlamydia infected host cells, for example GFP-or mKate2-expressing Chlamydia 6,11 , fluorescent actin 12 or GFP tagged Inc proteins 13 .…”

Section: Discussionmentioning

confidence: 99%

Using Fluorescent Proteins to Visualize and Quantitate <em>Chlamydia </em>Vacuole Growth Dynamics in Living Cells

Zuck

Feng

Hybiske

2015

JoVE

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The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C. trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP-or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains. Video LinkThe video component of this article can be found at

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“…One of these limitations was removed when a novel method for introducing DNA and modificating the native plasmid to allow effective selection of transformants has enabled the development of key molecular tools for studying Chlamydia. This transformation system was employed to test the expression of a diverse set of fluorescent proteins and to demonstrate the utility of subcellular localization studies [52]. The C. trachomatis developmental cycle incorporates numerous poorly understood processes, but a method for transforming Chlamydia has recently enabled the development of essential molecular tools to better study the biology and pathogenesis of these bacteria.…”

Section: Determining the Antibiotics Resistance Of Pathogen Std Agentsmentioning

confidence: 99%

Determining the Antibiotic Resistance of Bacterial Pathogens in Sexually Transmitted Diseases

Laura¹,

Vladi²,

Vasile³

2017

Antibacterial Agents

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Sexually transmitted diseases (STD) are among the most common infections worldwide. These bacterial infections have spread predominantly in the developing/underdeveloped countries, the most common being syphilis, gonorrhea and those induced by Chlamydia trachomatis, Ureaplasma urealyticum or Mycoplasma spp. Due to extensive usage of antibiotics in the recent past, these bacteria developed resistance to those commonly used for treatment, such resistant strains becoming a public health problem in a number of countries. It is well documented that bacterial STD agents are difficult to detect using standard culture media because these methods require special conditions and adequate nutrients. Antimicrobial susceptibility testing is, therefore, difficult to obtain in such cases. In recent years, genetic tests have been frequently employed in STD diagnosis. The study of genes that induce resistance to antibiotics using DNA isolated from these bacteria may prove to be a viable alternative. Genetic methods enable the DNA extraction from different biological samples, and both the presence of the bacteria and their resistance to one or more antibiotics can be determined from a single DNA sample. By studying the genes that induce antibiotic resistance and the plasmids that transfer such genes, the mechanism that leads to antibiotic resistance can be elucidated.

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A C. trachomatis Cloning Vector and the Generation of C. trachomatis Strains Expressing Fluorescent Proteins under the Control of a C. trachomatis Promoter

Cited by 109 publications

References 53 publications

Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

Using Fluorescent Proteins to Visualize and Quantitate <em>Chlamydia </em>Vacuole Growth Dynamics in Living Cells

Determining the Antibiotic Resistance of Bacterial Pathogens in Sexually Transmitted Diseases

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