Localization and functional characterization of the alternative oxidase in Naegleria

Abstract

The alternative oxidase (AOX) is a protein involved in supporting enzymatic reactions of the Krebs cycle in instances when the canonical (cytochrome-mediated) respiratory chain has been inhibited, while allowing for the maintenance of cell growth and necessary metabolic processes for survival. Among eukaryotes, alternative oxidases have dispersed distribution and are found in plants, fungi, and protists, including Naegleria ssp. Naegleria species are free-living unicellular amoeboflagellates and include the pathogenic species of N. fowleri, the so-called "brain-eating amoeba." Using a multidisciplinary approach, we aimed to understand the evolution, localization, and function of AOX and the role that plays in Naegleria's biology. Our analyses suggest that AOX was present in last common ancestor of the genus and structure prediction showed that all functional residues are also present in Naegleria species. Using cellular and biochemical techniques, we also functionally characterize N. gruberi's AOX in its mitochondria, and we demonstrate that its inactivation affects its proliferation. Consequently, we discuss the benefits of the presence of this protein in Naegleria species, along with its potential pathogenicity role in N. fowleri. We predict that our findings will spearhead new explorations to understand the cell biology, metabolism, and evolution of Naegleria and other free-living relatives.

Keywords: Naegleria; adaptation; confocal microscopy; evolution; mitochondria; respiration.

FIGURE 1

Phylogenetic trees demonstrating the origin of alternative oxidases in heterobolosea. Maximum likelihood tree of AOX homologs in eukaryotes and bacteria (323 sequences, 148 amino acid positions). The tree was inferred with IQTREE using the LG + I + G4 model selected under the BIC criterion. Gray dots correspond to supports higher than 80%. The scale bar corresponds to the average number of substitutions per site. Inset focus on the heterobolsean section of the tree, demonstrating the evolution and duplications events through the Naegleria genus. Full phylogenetic tree can be found in Figure S1

FIGURE 2

Structural modeling of Naegleria gruberi AOX reveals canonical features. Using Phyre2 we modeled the structure of ngAOX. We observed the presence of the di‐iron domain and alpha‐helical bundles, as viewed from the side (A) and top‐down (B). Rendering the structure by hydrophobicity shows the typical hydrophobic patch required for membrane anchoring (C). Close up view of the di‐iron domain model derived by Phyre2 (D)

FIGURE 3

Confocal microscopy and western blot reveal mitochondrial localization of AOX. (A and B) Naegleria gruberi cells were treated with mitotracker red prior to fixation and then probed with AOX antibodies (green). Nuclear marker and mtDNA staining is shown in blue (DAPI staining) Our confocal imaging reveals a high degree of colocalization between the mitotracker red signal and the green signal derived from immunoprobing AOX. These results provide visual confirmation of the expression of AOX and their localization in the mitochondria. Localization figures of more N. gruberi cells can be found in Figure S3. For western blotting lysates were fractionated by centrifugation to yield samples containing pelleted mitochondrial fraction and a clarified cytosolic fraction. Successful fractionation from the whole cell lysate was confirmed by immunoblotting (left‐hand side) for hydrogenase maturase E (HydE) that localizes in the cytosol, and succinate dehydrogenase B (SdhB), which localizes exclusively in the mitochondria (C). Immunostaining against AOX revealed a band around ~28 kDa in the whole cell lysate and mitochondrial fraction only. A coomassie stain was carried out to the parallel SDS‐PAGE gel parallel to assess equal loading between samples (right‐hand side)

FIGURE 4

Immunoelectron microscopy reveals AOX localization within the inner mitochondrial membrane. Fixed Naegleria gruberi samples were probed for IEM to assess their localization in the mitochondria (A). The AOX signal derived by the immunogold secondary antibodies bound against AOX primary antibodies localizes predominantly inside the mitochondria (B, C). The gold particles were then quantified based on their localisation (D).

FIGURE 5

High resolution real‐time respirometry reveals cyanide resistant respiration. Naegleria gruberi cells were subjected to real‐time respirometry in the presence of metabolic inhibitors to assess respiration using either M7 (A, C) or PYNFH (B, D) culture media without glucose. Cells were added to the chambers without the presence of inhibitors to assess normal respiration, followed by the addition of a mitochondrial complex inhibitor. The order of inhibitors was KCN (complex IV inhibitor) followed by Antimycin A (complex III inhibitor) and SHAM (AOX inhibitor) (A, B). The experiments were then completed in reverse order (C, D). KCN did not display decreases in respiration, whereas SHAM significantly reduced respiration. Results consist of three independent biological repetitions. Student's t tests were carried out between each drug condition to the routine respiration. p values; *<0.05, **<0.01 ***<0.001

FIGURE 6

Growth of Naegleria gruberi is reduced in increasing concentrations of SHAM. Using the JuLI™Stage Live cell monitoring system, we were able to monitor the growth rates of N. gruberi cells in varying concentrations of SHAM. Cells were seeded in wells of a 96‐well plate and left to grow for 48 h prior to the addition of SHAM at 1, 0.1, 0.01 and 0.001 mM concentration. By counting cells, we noticed a significant decrease in cell numbers when challenged with 1 and 0.1 mM SHAM. In the presence of 0.01 mM SHAM we saw a decrease in proliferation rate, peaking at 96 h. We observed no effect using SHAM at 0.001 mM. Error bars are standard error of the mean