Maria Salomé Gomes

Evidence for a link between iron metabolism and Nramp1 gene function in innate resistance against Mycobacterium avium

In the mouse, the progression of the Mycobacterium avium infection is highly dependent on the Nramp1 gene. Strains of mice that express the Nramp1D169 allele are highly susceptible to M. avium infections, while Nramp1G169 strains of mice can control them. Recently, the Nramp1 gene has been cloned and characterized as coding a transmembrane protein, probably involved in divalent cation transport. One possible function of this protein could be the transport of iron out of the parasite‐harbouring phagosome. Previous work in our lab has shown both in vitro (in macrophage cultures) and in vivo, that the growth rate of M. avium is highly dependent on the amount of iron available in the system. To try to correlate this with the Nramp1 gene function, BALB/c (susceptible) and C.D2 (resistant) congenic mice were treated for 20 days with different doses of iron‐dextran, so as to induce different degrees of iron overload, and infected with M. avium 2447. Iron administration increased M. avium growth in infected organs in a dose‐dependent manner at the same time as it decreased the difference in mycobacterial growth between the two mouse strains. These results indicate that an excess of iron hampers Nramp1‐encoded function, strongly suggesting a direct involvement of the Nramp1‐encoded protein in the transport of this cation.

Heme Catabolism by Heme Oxygenase-1 Confers Host Resistance to Mycobacterium Infection

ABSTRACT Heme oxygenases (HO) catalyze the rate-limiting step of heme degradation. The cytoprotective action of the inducible HO-1 isoform, encoded by the Hmox1 gene, is required for host protection against systemic infections. Here we report that upregulation of HO-1 expression in macrophages (Mϕ) is strictly required for protection against mycobacterial infection in mice. HO-1-deficient (Hmox1 −/−) mice are more susceptible to intravenous Mycobacterium avium infection, failing to mount a protective granulomatous response and developing higher pathogen loads, than infected wild-type (Hmox1 +/+) controls. Furthermore, Hmox1 −/− mice also develop higher pathogen loads and ultimately succumb when challenged with a low-dose aerosol infection with Mycobacterium tuberculosis. The protective effect of HO-1 acts independently of adaptive immunity, as revealed in M. avium-infected Hmox1 −/− versus Hmox1 +/+ SCID mice lacking mature B and T cells. In the absence of HO-1, heme accumulation acts as a cytotoxic pro-oxidant in infected Mϕ, an effect mimicked by exogenous heme administration to M. avium-infected wild-type Mϕ in vitro or to mice in vivo. In conclusion, HO-1 prevents the cytotoxic effect of heme in Mϕ, contributing critically to host resistance to Mycobacterium infection.

Identification of a new hexadentate iron chelator capable of restricting the intramacrophagic growth of Mycobacterium avium

Iron accumulation has been suggested to contribute to an increase of the susceptibility to mycobacterial infections. In this study we tested the effect of an array of iron chelating ligands of the 3-hydroxy-4-pyridinone family, in the intramacrophagic growth of Mycobacterium avium. We found that bidentate chelators, namely N-alkyl-3-hydroxy-4-pyridinones and N-aryl-3-hydroxy-4-pyridinones, did not affect the growth of M. avium inside mouse macrophages. In the case of the hexadentate chelators, those synthesized using an alkylamine (CP262) or a benzene ring (CP252) to link the three bidentate units, did not have an inhibitory effect on intramacrophagic growth of M. avium while those synthesized from a tripodal structure to anchor the bidentate units were capable of inhibiting the intramacrophagic growth of M. avium. The molecule we designated CP777 had the strongest inhibitory activity. The growth-reducing activity of CP777 was abrogated when this molecule was saturated with iron. These results confirm that iron deprivation, by the use of iron chelating compounds, restricts M. avium growth and that new iron chelators offer an approach to controlling mycobacterial infections.

Increased susceptibility to Mycobacterium avium in hemochromatosis protein HFE-deficient mice

ABSTRACT Mycobacterium avium is an opportunistic infectious agent in immunocompromised patients, living inside macrophage phagosomes. As for other mycobacterial species, iron availability is a critical factor for M. avium survival and multiplication. Indeed, an association between host secondary iron overload and increased susceptibility to these mycobacteria is generally acknowledged. However, studies on the impact of primary iron overload on M. avium infection have not been performed. In this work, we used animal models of primary iron overload that mimic the human disease hereditary hemochromatosis. This pathology is characterized by increased serum transferrin saturation with iron deposition in parenchymal cells, mainly in the liver, and is most often associated with mutations in the gene encoding the molecule HFE. In this paper, we demonstrate that mice of two genetically determined primary iron overload phenotypes, Hfe−/− and β2m−/−, show an increased susceptibility to experimental infection with M. avium and that during infection these animals accumulate iron inside granuloma macrophages. β2m−/− mice were found to be more susceptible than Hfe−/− mice, but depleting Hfe−/− mice of CD8+ cells had no effect on resistance to infection. Overall, our results suggest that serum iron, rather than total liver iron, levels have a considerable impact on susceptibility to M. avium infection.

Iron in intracellular infection: to provide or to deprive?

Due to their chemical versatility, transition metals were incorporated as cofactors for several basic metabolic pathways in living organisms. This same characteristic makes them potentially harmful, since they can be engaged in deleterious reactions like Fenton chemistry. As such, organisms have evolved highly specialized mechanisms to supply their own metal needs while keeping their toxic potential in check. This dual character comes into play in host-pathogen interactions, given that the host can either deprive the pathogen of these key nutrients or exploit them to induce toxicity toward the invading agent. Iron stands as the prototypic example of how a metal can be used to limit the growth of pathogens by nutrient deprivation, a mechanism widely studied in Mycobacterium infections. However, the host can also take advantage of iron-induced toxicity to control pathogen proliferation, as observed in infections caused by Leishmania. Whether we may harness either of the two pathways for therapeutical purposes is still ill-defined. In this review, we discuss how modulation of the host iron availability impacts the course of infections, focusing on those caused by two relevant intracellular pathogens, Mycobacterium and Leishmania.

Iron overload favors the elimination of Leishmania infantum from mouse tissues through interaction with reactive oxygen and nitrogen species.

Iron plays a central role in host-parasite interactions, since both intervenients need iron for survival and growth, but are sensitive to iron-mediated toxicity. The hosts iron overload is often associated with susceptibility to infection. However, it has been previously reported that iron overload prevented the growth of Leishmania major, an agent of cutaneous leishmaniasis, in BALB/c mice. In order to further clarify the impact of iron modulation on the growth of Leishmania in vivo, we studied the effects of iron supplementation or deprivation on the growth of L. infantum, the causative agent of Mediterranean visceral leishmaniasis, in the mouse model. We found that dietary iron deficiency did not affect the protozoan growth, whereas iron overload decreased its replication in the liver and spleen of a susceptible mouse strain. The fact that the iron-induced inhibitory effect could not be seen in mice deficient in NADPH dependent oxidase or nitric oxide synthase 2 suggests that iron eliminates L. infantum in vivo through the interaction with reactive oxygen and nitrogen species. Iron overload did not significantly alter the mouse adaptive immune response against L. infantum. Furthermore, the inhibitory action of iron towards L. infantum was also observed, in a dose dependent manner, in axenic cultures of promastigotes and amastigotes. Importantly, high iron concentrations were needed to achieve such effects. In conclusion, externally added iron synergizes with the hosts oxidative mechanisms of defense in eliminating L. infantum from mouse tissues. Additionally, the direct toxicity of iron against Leishmania suggests a potential use of this metal as a therapeutic tool or the further exploration of iron anti-parasitic mechanisms for the design of new drugs.

Rhodamine labeling of 3-hydroxy-4-pyridinone iron chelators is an important contribution to target Mycobacterium avium infection.

We have recently demonstrated that tripodal hexadentate chelators, based on 3-hydroxy-4-pyridinone units, can limit the access of iron to bacteria and have a significant inhibitory effect in the intramacrophagic growth of Mycobacterium avium. The results showed that the chelation of iron is a determinant although not sufficient property for antimicrobial activity. The rhodamine B isothiocyanate labelled chelator (MRH7) exhibited the strongest inhibitory activity and was identified as a lead compound since a dose response effect was observed. Significant inhibition of M. avium growth was achieved at a concentration as low as 1 μM. To identify key molecular features essential for the biological activity we designed parent hexadentate and bidentate chelators, in which different structural groups are introduced in the molecular framework. Herein, we report the work concerning three novel fluorescent chelators: a hexadentate ligand labelled with 5(6)-carboxytetramethylrhodamine (MRH8) and two 3-hydroxy-4-pyridinone fluorescent bidentate ligands labelled with rhodamine B isothiocyanate (MRB7) and 5(6)-carboxytetramethylrhodamine (MRB8). The results show that all fluorescent chelators are capable of restricting the intramacrophagic growth of M. avium and that the inhibitory effect is dependent on the fluorophore. In fact, for compounds bearing the same fluorophore the results obtained with the hexadentate or bidentate chelator (MRH7/MRB7 or MRH8/MRB8) are identical as long as the appropriate stoichiometric amount of chelator is used. The inhibitory effect of the rhodamine B isothiocyanate labelled compounds (MRH7 and MRB7) is significantly greater than that observed for the other two chelators, thus pointing out the significance of the rhodamine B isothiocyanate molecular fragment.

Peptidomimetic and organometallic derivatives of primaquine active against Leishmania infantum

ABSTRACT The current treatment of visceral leishmaniasis is made difficult by the low efficacy, elevated costs, low bioavailability, and high toxicity of many of the available drugs. Primaquine, an antimalarial 8-aminoquinoline, displays activity against Leishmania spp., and several of its derivatives have been developed as potential antileishmanial drugs. However, primaquine exhibits low oral bioavailability due to oxidative deamination of its aliphatic chain. We previously developed peptidomimetic and organometallic derivatives of primaquine, with higher resistance to proteolytic degradation and oxidative deamination, which presented significant activity against primaquine-sensitive pathogens such as Plasmodium or Pneumocystis. In light of these relevant findings, we decided to evaluate these compounds against both the promastigote and intramacrophagic amastigote forms of Leishmania infantum, the agent of Mediterranean visceral leishmaniasis. We found that several of these compounds had significant activity against L. infantum. One of the peptidomimetic (3c) and one of the organometallic (7a) derivatives of primaquine were active against the clinically relevant intramacrophagic amastigote form of the parasite, causing >96% reductions in the number of amastigotes per 100 macrophages at 60 and 40 μM, respectively, while being less cytotoxic for host cells than the reference drugs sitamaquine and miltefosine. Hence, compounds 3c and 7a represent new entries toward the development of new antileishmanial leads.

Killing of Mycobacterium avium by Lactoferricin Peptides: Improved Activity of Arginine- and d-Amino-Acid-Containing Molecules

ABSTRACT Mycobacterium avium causes respiratory disease in susceptible individuals, as well as disseminated infections in immunocompromised hosts, being an important cause of morbidity and mortality among these populations. Current therapies consist of a combination of antibiotics taken for at least 6 months, with no more than 60% overall clinical success. Furthermore, mycobacterial antibiotic resistance is increasing worldwide, urging the need to develop novel classes of antimicrobial drugs. One potential and interesting alternative strategy is the use of antimicrobial peptides (AMP). These are present in almost all living organisms as part of their immune system, acting as a first barrier against invading pathogens. In this context, we investigated the effect of several lactoferrin-derived AMP against M. avium. Short peptide sequences from both human and bovine lactoferricins, namely, hLFcin1-11 and LFcin17-30, as well as variants obtained by specific amino acid substitutions, were evaluated. All tested peptides significantly inhibited the axenic growth of M. avium, the bovine peptides being more active than the human. Arginine residues were found to be crucial for the display of antimycobacterial activity, whereas the all-d-amino-acid analogue of the bovine sequence displayed the highest mycobactericidal activity. These findings reveal the promising potential of lactoferricins against mycobacteria, thus opening the way for further research on their development and use as a new weapon against mycobacterial infections.

Antimycobacterial activity of rhodamine 3,4-HPO iron chelators against Mycobacterium avium: analysis of the contribution of functional groups and of chelator's combination with ethambutol

Rhodamine-labelled 3-hydroxy-4-pyridinone (3,4-HPO) chelators exhibit antimycobacterial activity, related but not limited to their iron binding capacity. We previously found that bacterial growth inhibition observed for chelators with ethyl substituents on the amino groups of the xanthene ring of rhodamine and a thiourea linkage between rhodamine and the chelating unit (MRH7 and MRB7) was different from that of compounds with methyl substituents and an amide linkage (MRH8 and MRB8). In this work we evaluated the antimycobacterial activity of two new chelators (MRH10 and MRB9) expressly designed to allow: (a) the direct comparison of the influence of the functional groups per se and (b) identification of the finest combination to achieve a higher biological activity. The activity of the chelators was assessed, as previously, by measuring their effect against M. avium. In this study we also report the antimycobacterial effect of MRH7, which proved to be the best performer of all four chelators, in combination with ethambutol, which is one of the antibiotics currently in use to treat mycobacterial infections. The results are indicative that a combination of 3,4-HPO iron chelators with an antibiotic is a promising strategy to fight M. avium infections. The current results are relevant for the choice of the best chelator in our set of compounds and also for the design of novel molecular architectures to target cellular membranes.