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J Antimicrob Chemother. 2019 Jan 1;74(1):48-57. doi: 10.1093/jac/dky409.

Intracellular localization of polymyxins in human alveolar epithelial cells.

Author information

1
Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
2
Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University, Victoria, Australia.
3
Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, West Lafayette, IN, USA.
4
Monash Micro Imaging, Monash University, Victoria, Australia.
5
School of Pharmacy, The University of Sydney, Camperdown, NSW, Australia.

Abstract

Background:

Current inhaled polymyxin therapy is empirical and often large doses are administered, which can lead to pulmonary adverse effects. There is a dearth of information on the mechanisms of polymyxin-induced lung toxicity and their intracellular localization in lung epithelial cells.

Objectives:

To investigate the intracellular localization of polymyxins in human lung epithelial A549 cells.

Methods:

A549 cells were treated with polymyxin B and intracellular organelles (early and late endosomes, endoplasmic reticulum, mitochondria, lysosomes and autophagosomes), ubiquitin protein and polymyxin B were visualized using immunostaining and confocal microscopy. Fluorescence intensities of the organelles and polymyxin B were quantified and correlated for co-localization using ImageJ and Imaris platforms.

Results:

Polymyxin B co-localized with early endosomes, lysosomes and ubiquitin at 24 h. Significantly increased lysosomal activity and the autophagic protein LC3A were observed after 0.5 and 1.0 mM polymyxin B treatment at 24 h. Polymyxin B also significantly co-localized with mitochondria (Pearson's R = 0.45) and led to the alteration of mitochondrial morphology from filamentous to fragmented form (n = 3, P < 0.001). These results are in line with the polymyxin-induced activation of the mitochondrial apoptotic pathway observed in A549 cells.

Conclusions:

Accumulation of polymyxins on mitochondria probably caused mitochondrial toxicity, resulting in increased oxidative stress and cell death. The formation of autophagosomes and lysosomes was likely a cellular response to the polymyxin-induced stress and played a defensive role by disassembling dysfunctional organelles and proteins. Our study provides new mechanistic information on polymyxin-induced lung toxicity, which is vital for optimizing inhaled polymyxins in the clinic.

PMID:
30357331
PMCID:
PMC6293086
[Available on 2019-10-23]
DOI:
10.1093/jac/dky409

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