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Electroporation enhances the ability of lactobacilli to remove cholesterol

Lye HS, Karim AA, Rusul G, Liong MT

J Dairy Sci, 2011 Oct;94(10):4820-30.
PMID: 21943733 DOI: 10.3168/jds.2011-4426

The objective of the present study was to evaluate the effect of electroporation on the membrane properties of lactobacilli and their ability to remove cholesterol in vitro. The growth of lactobacilli cells treated at 7.5 kV/cm for 4 ms was increased by 0.89 to 1.96 log(10) cfu/mL upon fermentation at 37 °C for 20 h, the increase being attributed to the reversible and transient formation of pores and defragmentation of clumped cells. In addition, an increase of cholesterol assimilation as high as 127.2% was observed for most cells electroporated at a field strength of 7.5 kV/cm for 3.5 ms compared with a lower field strength of 2.5 kV/cm. Electroporation also increased the incorporation of cholesterol into the cellular membrane, as shown by an increased cholesterol:phospholipids ratio (50.0-59.6%) upon treatment at 7.5 kV/cm compared with treatment at 2.5 kV/cm. Saturation of cholesterol was observed in different regions of the membrane bilayer such as upper phospholipids, apolar tail, and polar heads, as indicated by fluorescence anisotropy using 3 fluorescent probes. Electroporation could be a useful technique to increase the ability of lactobacilli to remove cholesterol for possible use as cholesterol-lowering adjuncts in the future.

Matched MeSH terms: Cell Membrane Permeability/physiology
Envelope proteome changes driven by RamA overproduction in Klebsiella pneumoniae that enhance acquired β-lactam resistance

Jiménez-Castellanos JC, Wan Nur Ismah WAK, Takebayashi Y, Findlay J, Schneiders T, Heesom KJ, et al.

J Antimicrob Chemother, 2018 Jan 01;73(1):88-94.
PMID: 29029194 DOI: 10.1093/jac/dkx345

Objectives: In Klebsiella pneumoniae, overproduction of RamA results in reduced envelope permeability and reduced antimicrobial susceptibility but clinically relevant resistance is rarely observed. Here we have tested whether RamA overproduction can enhance acquired β-lactam resistance mechanisms in K. pneumoniae and have defined the envelope protein abundance changes upon RamA overproduction during growth in low and high osmolarity media.
Methods: Envelope permeability was estimated using a fluorescent dye accumulation assay. β-Lactam susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and transcript levels were quantified using real-time RT-PCR.
Results: RamA overproduction enhanced β-lactamase-mediated β-lactam resistance, in some cases dramatically, without altering β-lactamase production. It increased production of efflux pumps and decreased OmpK35 porin production, though micF overexpression showed that OmpK35 reduction has little impact on envelope permeability. A survey of K. pneumoniae bloodstream isolates revealed ramA hyperexpression in 3 of 4 carbapenemase producers, 1 of 21 CTX-M producers and 2 of 19 strains not carrying CTX-M or carbapenemases.
Conclusions: Whilst RamA is not a key mediator of antibiotic resistance in K. pneumoniae on its own, it is potentially important for enhancing the spectrum of acquired β-lactamase-mediated β-lactam resistance. LC-MS/MS proteomics analysis has revealed that this enhancement is achieved predominantly through activation of efflux pump production.

Matched MeSH terms: Cell Membrane Permeability/physiology*
Design and tuning of a cell-penetrating albumin derivative as a versatile nanovehicle for intracellular drug delivery

Ichimizu S, Watanabe H, Maeda H, Hamasaki K, Nakamura Y, Chuang VTG, et al.

J Control Release, 2018 05 10;277:23-34.
PMID: 29530390 DOI: 10.1016/j.jconrel.2018.02.037

Human serum albumin (HSA) is a superior carrier for delivering extracellular drugs. However, the development of a cell-penetrating HSA remains a great challenge due to its low membrane permeability. We report herein on the design of a series of palmitoyl-poly-arginine peptides (CPPs) and an evaluation of their cell-penetrating effects after forming a complex with HSA for use in intracellular drug delivery. The palmitoyl CPPs forms a stable complex with HSA by anchoring itself to the high affinity palmitate binding sites of HSA. Among the CPPs evaluated, a cyclic polypeptide composed of D-dodecaarginines, palmitoyl-cyclic-(D-Arg)12 was the most effective for facilitating the cellular uptake of HSA by HeLa cells. Such a superior cell-penetrating capability is primarily mediated by macropinocytosis. The effect of the CPP on pharmacological activity was examined using three drugs loaded in HSA via three different methods: a) an HSA-paclitaxel complex, b) an HSA-doxorubicin covalent conjugate and c) an HSA-thioredoxin fusion protein. The results showed that cell-penetrating efficiency was increased with a corresponding and significant enhancement in pharmacological activity. In conclusion, palmitoyl-cyclic-(D-Arg)12/HSA is a versatile cell-penetrating drug delivery system with great potential for use as a nano-carrier for a wide diversity of pharmaceutical applications.

Matched MeSH terms: Cell Membrane Permeability/physiology

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