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Cell wall as a target for bacteria inactivation by pulsed electric fields.

ABSTRACT: The integrity and morphology of bacteria is sustained by the cell wall, the target of the main microbial inactivation processes. One promising approach to inactivation is based on the use of pulsed electric fields (PEF). The current dogma is that irreversible cell membrane electro-permeabilisation causes the death of the bacteria. However, the actual effect on the cell-wall architecture has been poorly explored. Here we combine atomic force microscopy and electron microscopy to study the cell-wall organization of living Bacillus pumilus bacteria at the nanoscale. For vegetative bacteria, exposure to PEF led to structural disorganization correlated with morphological and mechanical alterations of the cell wall. For spores, PEF exposure led to the partial destruction of coat protein nanostructures, associated with internal alterations of cortex and core. Our findings reveal for the first time that the cell wall and coat architecture are directly involved in the electro-eradication of bacteria.

SUBMITTER: Pillet F

PROVIDER: S-EPMC4735277 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Cell wall as a target for bacteria inactivation by pulsed electric fields.

Pillet Flavien F Formosa-Dague Cécile C Baaziz Houda H Dague Etienne E Rols Marie-Pierre MP

Scientific reports 20160201

The integrity and morphology of bacteria is sustained by the cell wall, the target of the main microbial inactivation processes. One promising approach to inactivation is based on the use of pulsed electric fields (PEF). The current dogma is that irreversible cell membrane electro-permeabilisation causes the death of the bacteria. However, the actual effect on the cell-wall architecture has been poorly explored. Here we combine atomic force microscopy and electron microscopy to study the cell-wa ...[more]

PMID: 26830154

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Project description:Glioblastoma multiforme (GBM), is the most common form of adult malignant brain tumor, highly heterogeneous and relatively resistant to therapy with a poor prognosis. As other cancers, GBM starts from a relatively small fraction of poorly differentiated and particularly aggressive cancer stem cells (CSCs), responsible for aberrant proliferation and invasion, rapidly spreading the tumor growth in the other parts of the central nervous system. Due to the extreme tumor heterogeneity, standard therapies (i.e., surgery, chemotherapy and radiotherapy) provide poor positive outcomes and cancers usually recur. Therefore, alternative approaches, possibly targeting CSCs, are necessary for searching effective therapeutic strategies against GBM. Among many emerging new therapeutic strategies, ultra-short pulsed electric fields (PEF) are considered extremely promising in cancer therapy and our previous results demonstrated the capability of a specific electric pulse protocol, characterized by a high pulse amplitude and a short duration, to selectively affect medulloblastoma CSCs preserving normal cells. Here, we tested the same exposure protocol to investigate the response of U87 GBM cells, and of U87-derived neurospheres. By analyzing different in vitro biological endpoints and taking advantage of transcriptomic and bioinformatics analyses, we found that, independently of CSCs content, PEF exposure affected cell proliferation and differentially regulated hypoxia, inflammation and P53/cell cycle checkpoints. PEF exposure also significantly reduced the clonogenic potential, reducing the ability to form new neurospheres, and inhibited the invasion potential. Importantly, exclusively in U87 neurospheres, PEF exposure changed the expression of stemness/differentiation genes. Our results, confirm this physical stimulus as a promising treatment to destabilize the extreme intra-tumoral GBM heterogeneity opening up the possibility to develop future therapeutic strategies mediated by PEF exposure.

Project description:Pulsed electric fields with microsecond pulse width (?sPEFs) are used clinically; namely, irreversible electroporation/Nanoknife is used for soft tissue tumor ablation. The ?sPEF pulse parameters used in irreversible electroporation (0.5-1 kV/cm, 80-100 pulses, ?100 ?s each, 1 Hz frequency) may cause an internal field to develop within the cell because of the disruption of the outer cell membrane and subsequent penetration of the electric field. An internal field may disrupt voltage-sensitive mitochondria, although the research literature has been relatively unclear regarding whether such disruptions occur with ?sPEFs. This investigation reports the influence of clinically used ?sPEF parameters on mitochondrial respiration in live cells. Using a high-throughput Agilent Seahorse machine, it was observed that ?sPEF exposure comprising 80 pulses with amplitudes of 600 or 700 V/cm did not alter mitochondrial respiration in 4T1 cells measured after overnight postexposure recovery. To record alterations in mitochondrial function immediately after ?sPEF exposure, high-resolution respirometry was used to measure the electron transport chain state via responses to glutamate-malate and ADP and mitochondrial membrane potential via response to carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. In addition to measuring immediate mitochondrial responses to ?sPEF exposure, measurements were also made on cells permeabilized using digitonin and those with compromised cytoskeleton due to actin depolymerization via treatment with the drug latrunculin B. The former treatment was used as a control to tease out the effects of plasma membrane permeabilization, whereas the latter was used to investigate indirect effects on the mitochondria that may occur if ?sPEFs impact the cytoskeleton on which the mitochondria are anchored. Based on the results, it was concluded that within the pulse parameters tested, ?sPEFs alone do not hinder mitochondrial physiology but can be used to impact the mitochondria upon compromising the actin. Mitochondrial susceptibility to ?sPEF after actin depolymerization provides, to our knowledge, a novel avenue for cancer therapeutics.

Dataset Information

Cell wall as a target for bacteria inactivation by pulsed electric fields.

Publications

Cell wall as a target for bacteria inactivation by pulsed electric fields.

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