Isolation and Propagation of Single Inclusion-Derived Chlamydia Using Laser Microdissection.

Other than its routine application for capturing pure cell populations from fixed tissue sections for diverse downstream molecular assays, laser microdissection enables isolation of single live cells. Here we describe a method for the isolation of single Chlamydia trachomatis-infected cells using a laser microdissection system, in which the dissected samples are captured via gravity. Cells infected by C. trachomatis at low multiplicity of infection are marked with the fluorescent Golgi-specific probe BODIPY® FL C5-ceramide, to facilitate identification of the cells with chlamydial inclusions under the microscope. Individual C. trachomatis-infected cells are harvested into separate wells with a pregrown host cell monolayer. Inclusions in harvested cells maturate, and the released elementary bodies infect the host cell monolayer, thus initiating propagation of single inclusion-derived Chlamydia. The method can be used for generation of microbiological clones of C. trachomatis and recovery of transformants and mutants. Isolated single Chlamydia-infected cells can also be examined by diverse downstream molecular assays to reveal unknown features of the Chlamydia replication at a single inclusion level.

Discovery of novel antimicrobial peptides: A transcriptomic study of the sea anemone Cnidopus japonicus.

As essential conservative component of the innate immune systems of living organisms, antimicrobial peptides (AMPs) could complement pharmaceuticals that increasingly fail to combat various pathogens exhibiting increased resistance to microbial antibiotics. Among the properties of AMPs that suggest their potential as therapeutic agents, diverse peptides in the venoms of various predators demonstrate antimicrobial activity and kill a wide range of microorganisms. To identify potent AMPs, the study reported here involved a transcriptomic profiling of the tentacle secretion of the sea anemone Cnidopus japonicus. An in silico search algorithm designed to discover toxin-like proteins containing AMPs was developed based on the evaluation of the properties and structural peculiarities of amino acid sequences. The algorithm revealed new proteins of the anemone containing antimicrobial candidate sequences, and 10 AMPs verified using high-throughput proteomics were synthesized. The antimicrobial activity of the candidate molecules was experimentally estimated against Gram-positive and -negative bacteria. Ultimately, three peptides exhibited antimicrobial activity against bacterial strains, which suggests that the method can be applied to reveal new AMPs in the venoms of other predators as well.

Isolation of single Chlamydia-infected cells using laser microdissection.

Chlamydia are obligate intracellular parasites of humans and animals that cause a wide range of acute and chronic infections. To elucidate the genetic basis of chlamydial parasitism, several approaches for making genetic modifications to Chlamydia have recently been reported. However, the lack of the available methods for the fast and effective selection of genetically modified bacteria restricts the application of genetic tools. We suggest the use of laser microdissection to isolate of single live Chlamydia-infected cells for the re-cultivation and whole-genome sequencing of single inclusion-derived Chlamydia. To visualise individual infected cells, we made use of the vital labelling of inclusions with the fluorescent Golgi-specific dye BODIPY® FL C5-ceramide. We demonstrated that single Chlamydia-infected cells isolated by laser microdissection and placed onto a host cell monolayer resulted in new cycles of infection. We also demonstrated the successful use of whole-genome sequencing to study the genomic variability of Chlamydia derived from a single inclusion. Our work provides the first evidence of the successful use of laser microdissection for the isolation of single live Chlamydia-infected cells, thus demonstrating that this method can help overcome the barriers to the fast and effective selection of Chlamydia.

Antimicrobial peptide from spider venom inhibits Chlamydia trachomatis infection at an early stage.

Antichlamydial activity of cyto-insectotoxin 1a (CIT 1a), representative of a unique class of antimicrobial peptides from the venom of the Central Asian spider Lachesana tarabaevi, was studied. A plasmid vector expressing the cit 1a gene controlled by a human cytomegalovirus tetracycline-dependent promoter was constructed. Impressive inhibition of Chlamydia trachomatis infection in HEK 293 cells transfected by the cit 1a-harboring vector was achieved. With the use of various schemes of cell infection and gene expression induction, it was shown for the first time that an antimicrobial peptide exerts its potent antichlamydial action at an early stage of the pathogen life cycle.

Cyto-Insectotoxin 1a from Lachesana tarabaevi Spider Venom Inhibits Chlamydia trachomatis Infection.

Venom of the ant spider Lachesana tarabaevi contains a wide variety of antimicrobial peptides. Among them, a special place belongs to cyto-insectotoxins, a class of cytolytic molecules showing equally potent antimicrobial and insecticidal effects. We tested one of them, CIT 1a, for ability to suppress Chlamydia trachomatis infection. HEK293 cells were transfected with plasmid vectors harboring the cit 1a gene. Controlled expression of the transgene led to a significant decrease in C. trachomatis viability inside the infected cells. Using proteomic and transcriptomic approaches, we found alterations in protein expression patterns and identified differentially expressed genes in transfected cells.

Spider venom peptides for gene therapy of Chlamydia infection.

Spider venoms are vast natural pharmacopoeias selected by evolution. The venom of the ant spider Lachesana tarabaevi contains a wide variety of antimicrobial peptides. We tested six of them (latarcins 1, 2a, 3a, 4b, 5, and cytoinsectotoxin 1a) for their ability to suppress Chlamydia trachomatis infection. HEK293 cells were transfected with plasmid vectors harboring the genes of the selected peptides. Controlled expression of the transgenes led to a significant decrease of C. trachomatis viability inside the infected cells.