Single-channel characterization of the chitooligosaccharide transporter chitoporin (SmChiP) from the opportunistic pathogen Serratia marcescens.

Serratia marcescens is an opportunistic pathogen that can utilize chitin as a carbon source, through its ability to produce chitin-degrading enzymes to digest chitin and membrane transporters to transport the degradation products (chitooligosaccharides) into the cells. Further characterization of these proteins is important to understand details of chitin metabolism. Here, we investigate the properties and function of the S. marcescens chitoporin, namely SmChiP, a chitooligosaccharide transporter. We show that SmChiP is a monomeric porin that forms a stable channel in artificial phospholipid membranes, with an average single-channel conductance of 0.5 ± 0.02 nS in 1 M KCl electrolyte. Additionally, we demonstrated that SmChiP allowed the passage of small molecules with a size exclusion limit of <300 Da and exhibited substrate specificity toward chitooligosaccharides, both in membrane and detergent-solubilized forms. We found that SmChiP interacted strongly with chitopentaose (Kd = 23 ± 2.0 µM) and chitohexaose (Kd = 17 ± 0.6 µM) but did not recognize nonchitose oligosaccharides (maltohexaose and cellohexaose). Given that S. marcescens can use chitin as a primary energy source, SmChiP may serve as a target for further development of nutrient-based antimicrobial therapies directed against multidrug antibiotic-resistant S. marcescens infections.

In silico and in vitro design of cordycepin encapsulation in liposomes for colon cancer treatment.

Cordycepin or 3'-deoxyadenosine is an interesting anti-cancer drug candidate that is found in abundance in the fungus Cordyceps militaris. It inhibits cellular growth of many cancers including lung carcinoma, melanoma, bladder cancer, and colon cancer by inducing apoptosis, anti-proliferation, anti-metastasis and by arresting the cell cycle. Cordycepin has, however, poor stability and low solubility in water, resulting in loss of its bioactivity. Liposomes can be used to overcome these obstacles. Our aim is to improve cordycepin's anti-colon cancer activity by liposome encapsulation. Cordycepin-encapsulated liposomes were designed and fabricated based on a combination of theoretical and experimental studies. Molecular dynamics (MD) simulations and free energy calculations suggest that phosphatidylcholine (PC) lipid environment is favorable for cordycepin adsorption. Cordycepin passively permeates into PC lipid bilayers without membrane damage and strongly binds to the lipids' polar groups by flipping its deoxyribose sugar toward the bilayer center. Our fabricated liposomes containing 10 : 1 molar ratio of egg yolk PC : cholesterol showed encapsulation efficiency (%EE) of 99% using microfluidic hydrodynamic focusing (MHF) methods. In our in vitro study using the HT-29 colon cancer cell line, cordycepin was able to inhibit growth by induction of apoptosis. Cell viability was significantly decreased below 50% at 125 µg mL-1 dosage after 48 h treatment with non-encapsulated and encapsulated cordycepin. Importantly, encapsulation provided (1) a 2-fold improvement in the inhibition of cancer cell growth at 125 µg mL-1 dosage and (2) 4-fold increase in release time. These in silico and in vitro studies indicate that cordycepin-encapsulated liposomes could be a potent drug candidate for colon cancer therapy.

Chitoporin from Serratia marcescens: recombinant expression, purification and crystallization.

Serratia marcescens is an opportunistic pathogen that commonly causes hospital-acquired infections and can utilize chitin-enriched nutrients as an alternative energy source. This study reports the identification of a chitoporin (ChiP), termed SmChiP, from the outer membrane of S. marcescens. Sequence alignment with genetically characterized ChiPs suggests that SmChiP is more closely related to the monomeric EcChiP from Escherichia coli than to the trimeric VhChiP from Vibrio campbellii. A single crystal of SmChiP grown under the condition 22%(w/v) PEG 8000, 0.1 M calcium acetate, 0.1 M MES pH 6.0 diffracted X-ray synchrotron radiation to 1.85 Šresolution. SmChiP co-crystallized with chitohexaose under the condition 19%(w/v) PEG 1500, 2 M ammonium phosphate monobasic, 0.1 M HEPES pH 7.0 diffracted X-rays to 2.70 Šresolution. Preliminary crystallographic analysis shows that both SmChiP crystal forms contain one molecule per asymmetric unit and that they belong to the tetragonal space groups P42212 and P41212, respectively. The SmChiP crystal has unit-cell parameters a = 82.97, b = 82.97, c = 189.53 Å, α = ß = γ = 90°, while the crystal of SmChiP in complex with chitohexaose has unit-cell parameters a = 73.24, b = 73.24, c = 213.46 Å, α = ß = γ = 90°. Initial assessment of the complex structure clearly revealed electron density for the sugar ligand. Structure determination of SmChiP in the absence and presence of chitohexaose should reveal the molecular basis of chitin utilization by S. marcescens.