Chelation of Mitochondrial Iron as an Antiparasitic Strategy.

Iron, as an essential micronutrient, plays a crucial role in host-pathogen interactions. In order to limit the growth of the pathogen, a common strategy of innate immunity includes withdrawing available iron to interfere with the cellular processes of the microorganism. Against that, unicellular parasites have developed powerful strategies to scavenge iron, despite the effort of the host. Iron-sequestering compounds, such as the approved and potent chelator deferoxamine (DFO), are considered a viable option for therapeutic intervention. Since iron is heavily utilized in the mitochondrion, targeting iron chelators in this organelle could constitute an effective therapeutic strategy. This work presents mitochondrially targeted DFO, mitoDFO, as a candidate against a range of unicellular parasites with promising in vitro efficiency. Intracellular Leishmania infection can be cleared by this compound, and experimentation with Trypanosoma brucei 427 elucidates its possible mode of action. The compound not only affects iron homeostasis but also alters the physiochemical properties of the inner mitochondrial membrane, resulting in a loss of function. Furthermore, investigating the virulence factors of pathogenic yeasts confirms that mitoDFO is a viable candidate for therapeutic intervention against a wide spectrum of microbe-associated diseases.

The 4-Aminomethylphenoxy-Benzoxaborole AN3057 as a Potential Treatment Option for Primary Amoebic Meningoencephalitis.

Primary amoebic meningoencephalitis is a rare but fatal central nervous system (CNS) disease caused by the "brain-eating amoeba" Naegleria fowleri. A major obstacle is the requirement for drugs with the ability to cross the blood-brain barrier, which are used in extremely high doses, cause severe side effects, and are usually ineffective. We discovered that the 4-aminomethylphenoxy-benzoxaborole AN3057 exhibits nanomolar potency against N. fowleri, and experimental treatment of infected mice significantly prolonged survival and demonstrated a 28% relapse-free cure rate.

Repurposing of MitoTam: Novel Anti-Cancer Drug Candidate Exhibits Potent Activity against Major Protozoan and Fungal Pathogens.

Many of the currently available anti-parasitic and anti-fungal frontline drugs have severe limitations, including adverse side effects, complex administration, and increasing occurrence of resistance. The discovery and development of new therapeutic agents is a costly and lengthy process. Therefore, repurposing drugs with already established clinical application offers an attractive, fast-track approach for novel treatment options. In this study, we show that the anti-cancer drug candidate MitoTam, a mitochondria-targeted analog of tamoxifen, efficiently eliminates a wide range of evolutionarily distinct pathogens in vitro, including pathogenic fungi, Plasmodium falciparum, and several species of trypanosomatid parasites, causative agents of debilitating neglected tropical diseases. MitoTam treatment was also effective in vivo and significantly reduced parasitemia of two medically important parasites, Leishmania mexicana and Trypanosoma brucei, in their respective animal infection models. Functional analysis in the bloodstream form of T. brucei showed that MitoTam rapidly altered mitochondrial functions, particularly affecting cellular respiration, lowering ATP levels, and dissipating mitochondrial membrane potential. Our data suggest that the mode of action of MitoTam involves disruption of the inner mitochondrial membrane, leading to rapid organelle depolarization and cell death. Altogether, MitoTam is an excellent candidate drug against several important pathogens, for which there are no efficient therapies and for which drug development is not a priority.

Elucidation of iron homeostasis in Acanthamoeba castellanii.

Acanthamoeba castellanii is a ubiquitously distributed amoeba that can be found in soil, dust, natural and tap water, air conditioners, hospitals, contact lenses and other environments. It is an amphizoic organism that can cause granulomatous amoebic encephalitis, an infrequent fatal disease of the central nervous system, and amoebic keratitis, a severe corneal infection that can lead to blindness. These diseases are extremely hard to treat; therefore, a more comprehensive understanding of this pathogen's metabolism is essential for revealing potential therapeutic targets. To propagate successfully in human tissues, the parasites must resist the iron depletion caused by nutritional immunity. The aim of our study is to elucidate the mechanisms underlying iron homeostasis in A. castellanii. Using a comparative whole-cell proteomic analysis of cells grown under different degrees of iron availability, we identified the primary proteins involved in Acanthamoeba iron acquisition. Our results suggest a two-step reductive mechanism of iron acquisition by a ferric reductase from the STEAP family and a divalent metal transporter from the NRAMP family. Both proteins are localized to the membranes of acidified digestive vacuoles where endocytosed medium and bacteria are trafficked. The expression levels of these proteins are significantly higher under iron-limited conditions, which allows Acanthamoeba to increase the efficiency of iron uptake despite the observed reduced pinocytosis rate. We propose that excessive iron gained while grown under iron-rich conditions is removed from the cytosol into the vacuoles by an iron transporter homologous to VIT/Ccc1 proteins. Additionally, we identified a novel protein that may participate in iron uptake regulation, the overexpression of which leads to increased iron acquisition.

The response of Naegleria gruberi to oxidative stress.

Aerobic organisms require oxygen for respiration but must simultaneously cope with oxidative damages inherently linked with this molecule. Unicellular amoeboflagellates of the genus Naegleria, containing both free-living species and opportunistic parasites, thrive in aerobic environments. However, they are also known to maintain typical features of anaerobic organisms. Here, we describe the mechanisms of oxidative damage mitigation in Naegleria gruberi and focus on the molecular characteristics of three noncanonical proteins interacting with oxygen and its derived reactive forms. We show that this protist expresses hemerythrin, protoglobin, and an aerobic-type rubrerythrin, with spectral properties characteristic of the cofactors they bind. We provide evidence that protoglobin and hemerythrin interact with oxygen in vitro and confirm the mitochondrial localization of rubrerythrin by immunolabeling. Our proteomic analysis and immunoblotting following heavy metal treatment revealed upregulation of hemerythrin, while rotenone treatment resulted in an increase in rubrerythrin protein levels together with a vast upregulation of alternative oxidase. Our study provided new insights into the mechanisms employed by N. gruberi to cope with different types of oxidative stress and allowed us to propose specific roles for three unique and understudied proteins: hemerythrin, protoglobin, and rubrerythrin.

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

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.

Iron economy in Naegleria gruberi reflects its metabolic flexibility.

Naegleria gruberi is a free-living amoeba, closely related to the human pathogen Naegleria fowleri, the causative agent of the deadly human disease primary amoebic meningoencephalitis. Herein, we investigated the effect of iron limitation on different aspects of N. gruberi metabolism. Iron metabolism is among the most conserved pathways found in all eukaryotes. It includes the delivery, storage and utilisation of iron in many cell processes. Nevertheless, most of the iron metabolism pathways of N. gruberi are still not characterised, even though iron balance within the cell is crucial. We found a single homolog of ferritin in the N. gruberi genome and showed its localisation in the mitochondrion. Using comparative mass spectrometry, we identified 229 upregulated and 184 down-regulated proteins under iron-limited conditions. The most down-regulated protein under iron-limited conditions was hemerythrin, and a similar effect on the expression of hemerythrin was found in N. fowleri. Among the other down-regulated proteins were [FeFe]-hydrogenase and its maturase HydG and several heme-containing proteins. The activities of [FeFe]-hydrogenase, as well as alcohol dehydrogenase, were also decreased by iron deficiency. Our results indicate that N. gruberi is able to rearrange its metabolism according to iron availability, prioritising mitochondrial pathways. We hypothesise that the mitochondrion is the center for iron homeostasis in N. gruberi, with mitochondrially localised ferritin as a potential key component of this process.