Vaccination with DNA vector expressing chlamydial low calcium response protein E (LcrE) against Chlamydophila pneumoniae infection

Chlamydophila pneumoniae is an obligate intracellular human pathogen, which causes acute respiratory tract infections and can also cause chronic infections.C. pneumoniae possess type III secretion system (TTSS), which allows them to secrete effector molecules into the inclusion membrane and the host cell cytosol. Low calcium response protein E (LcrE) is a part of TTSS. The gene of LcrE in a 6His-tagged form was cloned from C. pneumoniae CWL029, expressed and purified from Escherichia coli using the HIS-select TALON CellThru Resin, this gene was also cloned into a eukaryotic expression vector (pΔRC). One group of BALB/c mice received an intramuscular pΔRC inoculation then the mice were immunized with purified LcrE protein; the second group of mice was immunized two times with the recombinant plasmid (pΔRCLcrE), and the third group was primed with pΔRCLcrE inoculation then boosted with LcrE protein. LcrE-specific antibody response was induced by DNA immunization with a shift towards Th1 isotype pattern compared to protein-immunization, this shifting pattern was observed in plasmid primed then protein-boosted animals. DNA immunization given as a priming and followed by a protein booster significantly reduced the number of viable bacteria in the lungs after challenge with C. pneumoniae. These results confirm that immunization with pΔRCLcrE can be an effective part of a vaccination schedule against C. pneumoniae

Production and purification of low calcium response protein h of chlamydophila pneumoniae

Chlamydophila pneumoniae possesses a type III secretion system (TTSS), which allows the bacteria to secrete effector molecules into the inclusion membrane and into the cytosol of the host cell. Low calcium response protein H (LcrH), as a part of the TTSS, is a chaperone protein expressed from the middle to late stages of the chlamydial developmental cycle. Gene of LcrH (CPn0811) in a 6His-tagged form was cloned from C. pneumoniae CWL029, expressed and purified from Escherichia coli using the HIS-select TALON CellThru Resin. The purity was checked with mass spectrometry. The samples were used for immunization of BALB/c mice. The inducible E. coli clone, which over-expresses the chlamydial LcrH, permits the study of the biological properties of this protein

Production and purification of low calcium response protein H of Chlamydophila pneumoniae

Chlamydophila pneumoniae possesses a type III secretion system (TTSS), which allows the bacteria to secrete effector molecules into the inclusion membrane and into the cytosol of the host cell. Low calcium response protein H (LcrH), as a part of the TTSS, is a chaperone protein expressed from the middle to late stages of the chlamydial developmental cycle. Gene of LcrH (CPn0811) in a 6His-tagged form was cloned from C. pneumoniae CWL029, expressed and purified from Escherichia coli using the HIS-select TALON CellThru Resin. The purity was checked with mass spectrometry. The samples were used for immunization of BALB/c mice. The inducible E. coli clone, which over-expresses the chlamydial LcrH, permits the study of the biological properties of this protein

Recombinant Mycobacterium smegmatis vaccine candidates

Mycobacterium smegmatis is a species of rapidly growing saprophytes with a number of properties that make it an effective vaccine vector. Recombinant M. smegmatis expressing protective antigens of different pathogens and molecules modulating the immune responses offers some potential for reduction of the burden of tuberculosis, HIV and hepatitis B infections. This paper discusses the molecular methods used to generate recombinant M. smegmatis and the results obtained with some of these recombinants. © 2011 Akadémiai Kiadó, Budapest

Recombinant Mycobacterium smegmatis vaccine candidates

Mycobacterium smegmatis is a species of rapidly growing saprophytes with a number of properties that make it an effective vaccine vector. Recombinant M. smegmatis expressing protective antigens of different pathogens and molecules modulating the immune responses offers some potential for reduction of the burden of tuberculosis, HIV and hepatitis B infections. This paper discusses the molecular methods used to generate recombinant M. smegmatis and the results obtained with some of these recombinants

Fluorinated Beta-diketo Phosphorus Ylides Are Novel Efflux Pump Inhibitors in Bacteria

BACKGROUND: One of the most important resistance mechanisms in bacteria is the increased expression of multidrug efflux pumps. To combat efflux-related resistance, the development of new efflux pump inhibitors is essential. MATERIALS AND METHODS: Ten phosphorus ylides were compared based on their MDR-reverting activity in multidrug efflux pump system consisting of the subunits acridine-resistance proteins A and B (AcrA and AcrB) and the multidrug efflux pump outer membrane factor TolC (TolC) of Escherichia coli K-12 AG100 strain and its AcrAB-TolC-deleted strain. Efflux inhibition was assessed by real-time fluorimetry and the inhibition of quorum sensing (QS) was also investigated. The relative gene expression of efflux QS genes was determined by real-time reverse transcriptase quantitative polymerase chain reaction. RESULTS: The most potent derivative was Ph3P=C(COC2F5)CHO and its effect was more pronounced on the AcrAB-TolC-expressing E. coli strain, furthermore the most active compounds, Ph3P=C(COCF3)OMe, Ph3P=C(COC2F5)CHO and Ph3P=C(COCF3)COMe, reduced the expression of efflux pump and QS genes. CONCLUSION: Phosphorus ylides might be valuable EPI compounds to reverse efflux related MDR in bacteria

Efflux Pump Inhibiting Properties of Racemic Phenothiazine Derivatives and their Enantiomers on the Bacterial AcrAB-TolC System

Background/Aim: Bacterial resistance to antibiotics has become a serious problem in antibacterial chemotherapy and resistance of bacteria to chemicallyunrelated anti-microbial agents can be associated with the over-expression of efflux pumps. The simultaneous therapy with efflux pump inhibitors (EPIs) could be a solution to improve the effectiveness of antibiotics. The response of an organism to an EPI often depends on how that molecule fits a particular site of a protein. Because enantiomers of a given compound rotate plane-polarized light in a solution by the same angle but in opposite directions, the rational drug design should take the chirality into account if there is a difference between the racemic compound and its enantiomers. Materials and Methods: The main goal of the present study was to elucidate the role of chirality of Nhydroxyalkyl- 2-aminophenothiazines as effective EPIs by an automated method that uses the general efflux pump substrate ethidium bromide (EB) for the assessment of AcrAB-TolC system of wild-type Escherichia coli K-12 AG100. It has been shown that the most active EPIs among the N-hydroxyalkyl-2-aminophenothiazines were the compounds rac-3i, (+)-3i, and (–)-3i by modulating the AcrAB-TolC efflux pump. Conclusion: Comparison of effects of enantiomeric pairs revealed that their activities were similar to that of racemic derivatives. Moreover, there was no significant difference between the racemic compounds and their enantiomers related to their antibacterial and efflux pump inhibiting effects

Protection promoted by pGP3 or pGP4 against Chlamydia muridarum is mediated by CD4 cells in C57BL/6N mice

Urogenital tract infection with Chlamydia trachomatis is a leading cause of sexually transmitted infections. There is currently no commercially available vaccine against C. trachomatis. The highly conserved plasmid of chlamydiae has been considered to be a virulence factor and the plasmid proteins have important roles in the Chlamydia-specific immune response. This study was designed to evaluate the efficacy of vaccination with plasmid proteins in the prevention of C. muridarum lung infection in a mouse model. C57BL/6N mice were immunised 3 times subcutaneously with recombinant pGP3 or pGP4 and infected with C. muridarum. Immunisation of the mice with recombinant pGP3 or pGP4 protein caused a significantly lower chlamydial burden in the lungs of the infected mice; the lower IFN-gamma level indicated a reduced extent of inflammation. In vitro or in vivo neutralisation of C. muridarum with sera obtained from immunised mice did not reduce the number of viable C. muridarum in the lungs of mice. However, adoptive transfer of the CD4+ spleen cells isolated from the immunised mice resulted in a significantly reduced bacterial burden. Our results indicate that it is not the pGP3- and pGP4-specific antibodies, but the CD4+ cells that are responsible for the protective effect of the immune response to plasmid proteins