Effects of Neospora caninum infection on brain microvascular endothelial cells bioenergetics

Elsheikha, Hany M.; McKinlay, Charlotte L.; Elsaied, Nashwa A.; Smith, Paul A.

Abstract

Background: The brain is the most commonly affected organ during Neospora caninum infection but the mechanisms utilized by this protozoan parasite for traversal of the blood–brain barrier (BBB) are not yet understood. Herein, we investigate the cellular pathogenicity of N. caninum infection on bioenergetics of human brain microvascular endothelial cells (HBMECs), a fundamental component of the BBB.

Methods: We tracked the growth kinetics of N. caninum in HBMECs. Focusing on cell bioenergetics, oxygen consumption rate (OCR) was determined using Clark electrode system and mitochondrial membrane potential (ΔΨm) was evaluated using DePsipher staining by fluorescence microscopy in the presence and absence of infection.

Results: HBMECs provided a receptive environment for parasite proliferation. N. caninum tachyzoites were able to invade and replicate within HBMECs without significantly altering cell proliferation rate, as measured with the MTT assay, up to 24 hr post infection (pi). The oxygen consumption rate (OCR) was significantly inhibited (p < 0.001) by 10 mM glucose [from −2.26±0.23 to −0.6±0.21 nmol 106 cell min-1 and from −0.29±0.09 to −0.16±0.1 nmol 106 cell min-1 for uninfected HBMECs and free N. Caninum tachyzoites, respectively]. After normalization for DNA content the basal OCR did not differ between two host cell types: HBMECs and K562. The OCR of HBMECs was significantly elevated 24 hr pi in the absence of substrate, in 10 mM glucose and in the presence of a tetramethyl-p-phenylenediamine (TMPD)/ascorbate redox shuttle. Although quantitatively similar results were observed for uninfected K562 cells, there was no effect on their OCR 24 hr pi with N. caninum under any of the above substrate conditions. 6mM azide abolished OCR in all situations. Mitochondrial staining with DePsipher indicated no change in their membrane potential (Δψm) up to 24 hr pi.

Conclusions: N. caninum is able to grow in HBMECs without markedly disrupting their normal proliferation or mitochondrial integrity. However, it is associated with an increase in infected cell respiration. Whether this increase reflects numeric addition of the parasites own respiration or results from an additional energy demand upon the host cell remains to be elucidated.

Citation

Elsheikha, H. M., McKinlay, C. L., Elsaied, N. A., & Smith, P. A. (2013). Effects of Neospora caninum infection on brain microvascular endothelial cells bioenergetics. Parasites and Vectors, 6(24), https://doi.org/10.1186/1756-3305-6-24

• Journal Article Type: Article
• Publication Date: Jan 25, 2013
• Deposit Date: Apr 15, 2014
• Publicly Available Date: Apr 15, 2014
• Journal: Parasites and Vectors
• Electronic ISSN: 1756-3305
• Publisher: Springer Verlag
• Peer Reviewed: Peer Reviewed
• Volume: 6
• Issue: 24
• DOI: https://doi.org/10.1186/1756-3305-6-24
• Public URL: https://nottingham-repository.worktribe.com/output/712786
• Publisher URL: http://www.parasitesandvectors.com/content/6/1/24