Abstract
C. trachomatis is the most common sexually transmitted bacterial infection in the world. Although the infection can be easily controlled by the use of antibiotics, several reports of clinical isolates that are resistant to antibiotics have prompted us to search for alternative strategies to manage this disease. In this paper, we developed a nanoparticle formulation (PDGFR-β siRNA-PEI-PLGA-PEG NP) that can simultaneously induce autophagy in human cells and knock down PDGFR-β gene expression, an important surface binding protein for C. trachomatis, as a strategy to reduce vaginal infection of C. trachomatis. PDGFR-β siRNA-PEI-PLGA-PEG NP significantly induced autophagy in human vaginal epithelial cells (VK2/E6E7) 48 hr post treatment by improving autophagic degradation activity without causing inflammation, apoptosis or any decrease in cell viability. Beclin-1, VPS34 (markers for initiation stage of autophagy), UVRAG, TECPR-1 (markers for degradation stage of autophagy) were found to be significantly upregulated after treatment with PDGFR-β siRNA-PEI-PLGA-PEG NP. Furthermore, PDGFR-β siRNA-PEI-PLGA-PEG NP decreased PDGFR-β mRNA expression by 50% and protein expression by 43% in VK2/E6E7 cells 48 hr post treatment. Treatment of cells with PDGFR-β siRNA-PEI-PLGA-PEG NP significantly decreased the intracellular C. trachomatis and extracellular release of C. trachomatis by approximately 65% and 67%, respectively, in vitro through augmenting autophagic degradation pathways and reducing bacterial binding simultaneously.
Highlights
Chlamydia trachomatis (C. trachomatis) is a gram-negative bacterium that preferentially infects epithelial cells of the genital tract and causes the most common sexually transmitted bacterial infection in the world[1]
Our results indicated that compared to naïve control, nonsilencing small interfering RNA (siRNA)-PLGA-PEG NP significantly increased the intracellular level of light chain 3B (LC3B) by 43.1 ± 11.9% (p = 0.0029), while nonsilencing siRNA-PEI-PLGA-PEG NP and platelet derived growth factor receptor-β (PDGFR-β) siRNA-PEI-PLGA-PEG NP did not increase the intracellular level of LC3B at all
This does not mean that nonsilencing siRNA-PEI-PLGA-PEG NP and PDGFR-β siRNA-PEI-PLGA-PEG NP did not exert an effect on the formation and conversion of autophagosomes, because when we compared the LC3B levels in these two groups to that in nonsilencing siRNA PLGA-PEG NP-treated group, we found that the intracellular levels of LC3B were significantly decreased by 23.8 ± 4.5% (p = 0.0119) and 38.7 ± 5.5% (p = 0.0006) respectively, and no significant change in LC3B was detected between nonsilencing siRNA-PEI-PLGA-PEG NP-treated group and PDGFR-β siRNA-PEI-PLGA-PEG NP-treated group (Fig. 1D)
Summary
Chlamydia trachomatis (C. trachomatis) is a gram-negative bacterium that preferentially infects epithelial cells of the genital tract and causes the most common sexually transmitted bacterial infection in the world[1]. A membrane called phagophore is invaginated from cell membranes and elongated to sequester components targeted for degradation, forming an enclosed double-membrane vesicle called autophagosome This stage is subsequently followed by the degradation stage, during which the autophagosomes will fuse with the acidic, enzyme-enriched lysosome, forming the degradative vesicle, autolysosome. C. trachomatis primarily targets epithelial cells as the first step to establish genital infection and forms inclusions to escape the endo-lysosomal degradation pathway. These two conditions predispose the genital epithelial cells with higher vulnerability to C. trachomatis compared to other immune cells in the genital tract. A therapy that can reduce bacterial binding to epithelial cells and induce autophagy in infected epithelial cells for the elimination of intracellular bacteria would be beneficial for combating the infection
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