Liposomal Encapsulation Increases the Efficacy of Azithromycin against Chlamydia trachomatis

Chlamydia trachomatis (C. trachomatis) is an obligate intracellular bacterium linked to ocular and urogenital infections with potentially serious sequelae, including blindness and infertility. First-line antibiotics, such as azithromycin (AZT) and doxycycline, are effective, but treatment failures have also been reported. Encapsulation of antibiotics in liposomes is considered an effective approach for improving their local effects, bioavailability, biocompatibility and antimicrobial activity. To test whether liposomes could enhance the antichlamydial action of AZT, we encapsulated AZT in different surface-charged elastic liposomes (neutral, cationic and anionic elastic liposomes) and assessed their antibacterial potential against the C. trachomatis serovar D laboratory strain as well as the clinical isolate C. trachomatis serovar F. A direct quantitative polymerase chain reaction (qPCR) method was used to measure chlamydial genome content 48 h post infection and to determine the recoverable chlamydial growth. All the liposomes efficiently delivered AZT to HeLa 229 cells infected with the laboratory Chlamydia strain, exhibiting the minimal inhibitory concentrations (MIC) and the minimal bactericidal concentrations (MBC) of AZT even 4–8-fold lower than those achieved with the free AZT. The tested AZT-liposomes were also effective against the clinical Chlamydia strain by decreasing MIC values by 2-fold relative to the free AZT. Interestingly, the neutral AZT-liposomes had no effect on the MBC against the clinical strain, while cationic and anionic AZT-liposomes decreased the MBC 2-fold, hence proving the potential of the surface-charged elastic liposomes to improve the effectiveness of AZT against C. trachomatis.

Chlamydia Genomic MinD Protein Has not the Function of Regulating Plasmid-dependent Genes as Pgp5

Chlamydia has a unique intracellular developmental cycle which has hindered the function study of Chlamydia. Transformation system of Chlamydia developed recently has greatly advanced the chlamydial function research and has been used to find chlamydial plasmid-encoded pgp5 can down-regulate plasmid-dependent genes. It is predicted that pgp5 has similar function with MinD protein encoded by chlamydial genome. However，it is unknown whether MinD has the function of regulating these plasmid-dependent genes. The pgp5 gene in the shuttle vector pGFP::CM was replaced with MinD gene of Chlamydia trachomatis (CT0582) or Chlamydia muridarum(TC0871). The recombinant plasmid was transformed into plasmid-free organisms-CMUT3.Real time PCR was used to detect the genes transcription level in these pgp5 replacement organisms. GlgA, one of the plasmid-regulated gene products was detected by the immunofluorescence assay. After recombination, transformation and plaque purification, the stable transformants CMUT3-pGFP::CM CT0582Rpgp5 and CMUT3-pGFP::CM TC0871Rpgp5 were generated. In these transformants, the plasmid-dependent genes were up-regulated, similar with those in the pgp5 premature stop mutant and pgp5 replacement with mCherry mutant. GlgA protein was also increased in all pgp5 mutants including CT0582Rpgp5 and TC0871Rpgp5. Thus, the transformation system has allowed us to identify the function of MinD that is useful for further understanding the chlamydiae.

The Chlamydial Plasmid-Encoded Protein pgp3 Is Secreted into the Cytosol of Chlamydia-Infected Cells

ABSTRACT The chlamydial cryptic plasmid encodes eight putative open reading frames (ORFs), designated pORF1 to -8. Antibodies raised against these ORF proteins were used to localize the endogenous proteins during chlamydial infection. We found that the pORF5 protein (also known as pgp3) was detected mainly in the cytosol of Chlamydia-infected cells, while the remaining seven proteins were found inside the chlamydial inclusions only. The pgp3 distribution pattern in the host cell cytosol is similar to but not overlapping with that of chlamydial protease/proteasome-like activity factor (CPAF), a chlamydial genome-encoded protein known to be secreted from chlamydial inclusions into the host cell cytosol. The anti-pgp3 labeling was removed by preabsorption with pgp3 but not CPAF fusion proteins and vice versa, demonstrating that pgp3 is a unique secretion protein. This conclusion is further supported by the observation that pgp3 was highly enriched in cytosolic fractions and had a minimal presence in the inclusion-containing nuclear fractions prepared from Chlamydia-infected cells. The pgp3 protein was detected as early as 12 h after infection and was secreted by all chlamydial species that carry the cryptic plasmid, suggesting that there is a selection pressure for maintaining pgp3 secretion during chlamydial infection. Although expression of pgp3 in the host cell cytosol via a transgene did not alter the susceptibility of the transfected cells to the subsequent chlamydial infection, purified pgp3 protein stimulated macrophages to release inflammatory cytokines, suggesting that pgp3 may contribute to chlamydial pathogenesis.

Profiling of Human Antibody Responses to Chlamydia trachomatis Urogenital Tract Infection Using Microplates Arrayed with 156 Chlamydial Fusion Proteins

ABSTRACT The available chlamydial genome sequences have made it possible to comprehensively analyze host responses to all chlamydial proteins, which is essential for further understanding of chlamydial pathogenesis and development of effective chlamydial vaccines. Microplates arrayed with 156 Chlamydia trachomatis fusion proteins were used to evaluate antibody responses in women urogenitally infected with C. trachomatis. Based on both the antibody recognition frequency and titer, seven chlamydial antigens encoded by open reading frames (ORFs) CT089, CT147, CT226, CT681, CT694, CT795, and CT858, respectively, were identified as relatively immunodominant; six of these are encoded by hypothetical ORFs. Antibody binding to these chlamydial fusion proteins was blocked by C. trachomatis-infected but not by normal HeLa cell lysates or irrelevant bacterial lysates. These results have revealed novel immune-reactive chlamydial antigens, not only indicating that the hypothetical ORF-encoded proteins are expressed during chlamydial infection in humans but also providing the proof of principle that the fusion protein-based approach can be used to profile human immune responses to chlamydial infection at the whole-genome scale.