Adaptive iron utilization compensates for the lack of an inducible uptake system in Naegleria fowleri and represents a potential target for therapeutic intervention

Abstract

Naegleria fowleri is a single-cell organism living in warm freshwater that can become a deadly human pathogen known as a brain-eating amoeba. The condition caused by N. fowleri, primary amoebic meningoencephalitis, is usually a fatal infection of the brain with rapid and severe onset. Iron is a common element on earth and a crucial cofactor for all living organisms. However, its bioavailable form can be scarce in certain niches, where it becomes a factor that limits growth. To obtain iron, many pathogens use different machineries to exploit an iron-withholding strategy that has evolved in mammals and is important to host-parasite interactions. The present study demonstrates the importance of iron in the biology of N. fowleri and explores the plausibility of exploiting iron as a potential target for therapeutic intervention. We used different biochemical and analytical methods to explore the effect of decreased iron availability on the cellular processes of the amoeba. We show that, under iron starvation, nonessential, iron-dependent, mostly cytosolic pathways in N. fowleri are downregulated, while the metal is utilized in the mitochondria to maintain vital respiratory processes. Surprisingly, N. fowleri fails to respond to acute shortages of iron by inducing the reductive iron uptake system that seems to be the main iron-obtaining strategy of the parasite. Our findings suggest that iron restriction may be used to slow the progression of infection, which may make the difference between life and death for patients.

Fig 1. Iron uptake in N. fowleri.

(A) Ferrous and ferric iron uptake by N. fowleri precultivated under iron-rich and iron-deficient conditions. Autoradiography of blue native electrophoresis gels of whole cell extracts from N. fowleri previously cultivated for 72 hours under iron-deficient conditions (25 μM BPS) or iron-rich conditions (25 μM Fe-NTA) and further incubated with ⁵⁵Fe(II) (ferrous ascorbate) or ⁵⁵Fe(III) (ferric citrate). Equal protein concentrations were loaded, and the loading control is shown in S1(A) Fig. The gel is representative of three independent replicates. Numbers indicate average relative densitometry values for the appropriate lines. Significant differences between band densitometry values are denoted by asterisk (*, p<0.05; **, p<0.01). (B) Ferric reductase activity under iron-rich and iron-deficient conditions. Relative values of N. fowleri ferric reductase activities in the iron-rich (Fe) and iron-deficient (BPS). The difference between the two conditions was not significant, p-value >0.05. Data are presented as the relative percentage ± SD from three independent replicates. (C) Growth restoration of N. fowleri in culture by hemin under iron-deficient conditions. Cells in regular growth medium (control) and with 50 μM hemin (hemin) exhibited similar levels of propagation, while the cells under iron-deficient conditions, achieved with 50 μM chelator bathophenanthroline disulfonic acid (BPS), showed stagnated growth in the first 24 hours. The addition of 50 μM hemin to the iron-deficient medium (BPS+hemin) partially restored culture growth. The boxed area indicates the time from which growth restoration was calculated. Data are presented as the means ± SD from four independent replicates.

Fig 2. Effect of iron deficiency on N. fowleri.

(A) Downregulation of N. fowleri hemerythrin under iron-deficient conditions. Results from the Western blot analysis of cells cultivated under iron-rich (Fe) and iron-deficient (BPS) conditions using an antibody against Naegleria gruberi hemerythrin. Equal protein concentrations were loaded, and the loading control is shown in S1(D) Fig. The gel represents one of three independent replicates. (B) Cellular content of selected amino acids in N. fowleri cells cultivated under iron-rich and iron-deficient conditions. Relative amounts of phenylalanine and tyrosine were significantly increased under the iron-deficient condition, while those of tryptophan and lysine remained unchanged. t-test p-values <0.01 are marked with a star. The total protein concentration was equal in all the samples, and the values shown are relative to those under the iron-rich conditions for each amino acid. Fe, cells cultivated under iron-rich conditions; BPS, cells cultivated under iron-deficient conditions. Data are presented as the means ± SD from three independent replicates. (C) Respiration of N. fowleri grown under iron-rich and iron-deficient conditions. Using selective inhibitors of complex IV (potassium cyanide) and of alternative oxidase (salicyl-hydroxamic acid), the contribution of alternative oxidase and complex IV activity was assessed with respect to total respiration levels. AOX, alternative oxidase; Fe, cells cultivated under iron-rich conditions; and BPS, cells cultivated under iron-deficient conditions. The t-test p-values <0.01 are marked with a star. Data are presented as the means ± SD from five independent replicates. (D) Activity of hydrogenase and NADH:ubiquinone dehydrogenase (complex I) under iron-rich and iron-deficient conditions. While hydrogenase was significantly downregulated, complex I was significantly upregulated as a result of the iron-deficient conditions. Relative values are shown. Fe, cells cultivated under iron-rich conditions; and BPS, cells cultivated under iron-deficient conditions. The t-test p-values <0.01 are marked with a star. Data are presented as the means ± SD from four independent replicates.

Fig 3. Illustration of the main effects of iron-deficient conditions on the selected cellular processes of N. fowleri.

The results of proteomic analysis for selected proteins are depicted in red, the results from measured metabolite levels are in green and the assessed enzyme activities are in purple. Upwards pointing arrows stand for increased in the iron-deficient condition, downwards pointing arrows represent decreased in the iron-deficient conditions and dashes mean no significant change in the different iron conditions. Respiration chain complexes are represented by appropriate numbers, Fe represents iron-containing/involving protein/process. C, cytochrome C; MTF, mitoferrin; MPCP, mitochondrial phosphate carrier protein; P, phosphate; Q, ubiquinol/ubiquinone.