Susceptibility of Leishmania to novel pentavalent organometallics: Investigating impact on DNA and membrane integrity in antimony(III)-sensitive and -resistant strains.

The aim the present study was to investigate the impact of novel pentavalent organobismuth and organoantimony complexes on membrane integrity and their interaction with DNA, activity against Sb(III)-sensitive and -resistant Leishmania strains and toxicity in mammalian peritoneal macrophages. Ph3M(L)2 type complexes were synthesized, where M = Sb(V) or Bi(V) and L = deprotonated 3-(dimethylamino)benzoic acid or 2-acetylbenzoic acid. Both organobismuth(V) and organoantimony(V) complexes exhibited efficacy at micromolar concentrations against Leishmania amazonensis and L. infantum but only the later ones demonstrated biocompatibility. Ph3Sb(L1)2 and Ph3Bi(L1)2 demonstrated distinct susceptibility profiles compared to inorganic Sb(III)-resistant strains of MRPA-overexpressing L. amazonensis and AQP1-mutated L. guyanensis. These complexes were able to permeate the cell membrane and interact with the Leishmania DNA, suggesting that this effect may contribute to the parasite growth inhibition via apoptosis. Taken altogether, our data substantiate the notion of a distinct mechanism of uptake pathway and action in Leishmania for these organometallic complexes, distinguishing them from the conventional inorganic antimonial drugs.

Phylogenetic analysis and antimony resistance of Leishmania major isolated from humans and rodents.

Cutaneous Leishmaniasis (CL) is one of the world's neglected diseases which is caused by Leishmania spp. The aim of this study was to assess molecular profile and antimony resistance of Leishmania isolated from human and rodent hosts. Samples were collected from suspected CL patients referred to health centres and wild rodent's traps in Gonbad-e-Qabus region, north-eastern Iran. Smears were subjected to PCR-RFLP to identify Leishmania species. In addition, ITS1-PCR products were sequenced for phylogenetic analysis. Clinical isolates and rodent samples were subjected to MTT assay to determine IC50 values and in vitro susceptibilities. Expression levels of antimony resistance-related genes were determined in CL isolates. Out of 1,949 suspected patients with CL and 148 rodents, 1,704 (87.4%) and 6 (4.05%) were positive with direct smear, respectively. Digestion patterns of BusRI (HaeIII) endonuclease enzyme were similar to what expected for Leishmania major. Phylogenetic analysis revealed that the highest interspecies similarity was found between current L. major sequences with L. major obtained from Russia and Uzbekistan. Out of 20 L. major samples tested, 13 (65%) were resistant to meglumine antimoniate (MA) treatment, with an activity index (AI) exceeding 4. The remaining 7 samples (35%) responded to MA treatment and were classified as sensitive isolates, with a confirmed sensitive phenotype based on their AI values. The comparison expression analysis of three major antimony resistance-associated genes in unresponsive clinical isolates demonstrated significant fold changes for TDR1 (4.78-fold), AQP1 (1.3-fold), and γ-GCS (1.17-fold) genes (P < 0.05). Herein, we demonstrate genetic diversity and antimony resistance of L. major isolated from human and reservoir hosts in north-eastern Iran, which could be the basis for planning future control strategies.

Insights into the trypanothione system in antimony-resistant and sensitive Leishmania tropica clinical isolates.

Pentavalent antimonials are the mainstay treatment against different clinical forms of leishmaniasis. The emergence of resistant isolates in endemic areas has led to treatment failure. Unraveling the underlying resistance mechanism would assist in improving the treatment strategies against resistant isolates. This study aimed to investigate the RNA expression level of glutathione synthetase (GS), Spermidine synthetase (SpS), trypanothione synthetase (TryS) genes involved in trypanothione synthesis, and thiol-dependent reductase (TDR) implicated in drug reduction, in antimony-sensitive and -resistant Leishmania tropica isolates. We investigated 11 antimony-resistant and 11 antimony-sensitive L. tropica clinical isolates from ACL patients. Drug sensitivity of amastigotes was determined in mouse macrophage cell line J774A.1. The RNA expression level in the promastigote forms was analyzed by quantitative real-time PCR. The results revealed a significant increase in the average expression of GS, SpS, and TrpS genes by 2.19, 1.56, and 2.33-fold in resistant isolates compared to sensitive ones. The average expression of TDR was 1.24-fold higher in resistant isolates, which was insignificant. The highest correlation coefficient between inhibitory concentration (IC50) values and gene expression belonged to the TryS, GS, SpS, and TDR genes. Moreover, the intracellular thiol content was increased 2.17-fold in resistant isolates compared to sensitive ones and positively correlated with IC50 values. Our findings suggest that overexpression of trypanothione biosynthesis genes and increased thiol content might play a key role in the antimony resistance of L. tropica clinical isolates. In addition, the diversity of gene expression in the trypanothione system and thiol content among L. tropica clinical isolates highlighted the phenotypic heterogeneity of antimony resistance among the parasite population.

Potential selection of antimony and methotrexate cross-resistance in Leishmania infantum circulating strains.

BACKGROUND: Visceral leishmaniasis (VL) resolution depends on a wide range of factors, including the instauration of an effective treatment coupled to a functional host immune system. Patients with a depressed immune system, like the ones receiving methotrexate (MTX), are at higher risk of developing VL and refusing antileishmanial drugs. Moreover, the alarmingly growing levels of antimicrobial resistance, especially in endemic areas, contribute to the increasing the burden of this complex zoonotic disease. PRINCIPAL FINDINGS: To understand the potential links between immunosuppressants and antileishmanial drugs, we have studied the interaction of antimony (Sb) and MTX in a Leishmania infantum reference strain (LiWT) and in two L. infantum clinical strains (LiFS-A and LiFS-B) naturally circulating in non-treated VL dogs in Spain. The LiFS-A strain was isolated before Sb treatment in a case that responded positively to the treatment, while the LiFS-B strain was recovered from a dog before Sb treatment, with the dog later relapsing after the treatment. Our results show that, exposure to Sb or MTX leads to an increase in the production of reactive oxygen species (ROS) in LiWT which correlates with a sensitive phenotype against both drugs in promastigotes and intracellular amastigotes. LiFS-A was sensitive against Sb but resistant against MTX, displaying high levels of protection against ROS when exposed to MTX. LiFS-B was resistant to both drugs. Evaluation of the melting proteomes of the two LiFS, in the presence and absence of Sb and MTX, showed a differential enrichment of direct and indirect targets for both drugs, including common and unique pathways. CONCLUSION: Our results show the potential selection of Sb-MTX cross-resistant parasites in the field, pointing to the possibility to undermine antileishmanial treatment of those patients being treated with immunosuppressant drugs in Leishmania endemic areas.

Identification of bacterial dissimilatory antimonate reductase AnrA: genes and proteins involved in antimonate respiration and resistance in Geobacter sp. strain SVR.

Geobacter sp. strain SVR uses antimonate [Sb(V)] as a terminal electron acceptor for anaerobic respiration. Here, we visualized a possible key enzyme, periplasmic Sb(V) reductase (Anr), via active staining and non-denaturing gel electrophoresis. Liquid chromatography-tandem mass spectrometry analysis revealed that a novel dimethyl sulfoxide (DMSO) reductase family protein, WP_173201954.1, is involved in Anr. This protein was closely related with AnrA, a protein suggested to be the catalytic subunit of a respiratory Sb(V) reductase in Desulfuribacillus stibiiarsenatis. The anr genes of strain SVR (anrXSRBAD) formed an operon-like structure, and their transcription was upregulated under Sb(V)-respiring conditions. The expression of anrA gene was induced by more than 1 µM of antimonite [Sb(III)]; however, arsenite [As(III)] did not induce the expression of anrA gene. Tandem mass tag-based proteomic analysis revealed that, in addition to Anr proteins, proteins in the following categories were upregulated under Sb(V)-respiring conditions: (i) Sb(III) efflux systems such as Ant and Ars; (ii) antioxidizing proteins such as ferritin, rubredoxin, and thioredoxin; (iii) protein quality control systems such as HspA, HslO, and DnaK; and (iv) DNA repair proteins such as UspA and UvrB. These results suggest that strain SVR copes with antimony stress by modulating pleiotropic processes to resist and actively metabolize antimony. To the best of our knowledge, this is the first report to demonstrate the involvement of AnrA in Sb(V) respiration at the protein level. Furthermore, this is the first example to show high expression of the Ant system proteins in the Sb(V)-respiring bacterium.IMPORTANCEAntimony (Sb) exists mainly as antimonite [Sb(III)] or antimonate [Sb(V)] in the environment, and Sb(III) is more toxic than Sb(V). Recently, microbial involvement in Sb redox reactions has received attention. Although more than 90 Sb(III)-oxidizing bacteria have been reported, information on Sb(V)-reducing bacteria is limited. Especially, the enzyme involved in dissimilatory Sb(V) reduction, or Sb(V) respiration, is unclear, despite this pathway being very important for the circulation of Sb in nature. In this study, we demonstrated that the Sb(V) reductase (Anr) of an Sb(V)-respiring bacterium (Geobacter sp. SVR) is a novel member of the dimethyl sulfoxide (DMSO) reductase family. In addition, we found that strain SVR copes with Sb stress by modulating pleiotropic processes, including the Ant and Ars systems, and upregulating the antioxidant and quality control protein levels. Considering the abundance and diversity of putative anr genes in the environment, Anr may play a significant role in global Sb cycling in both marine and terrestrial environments.

Deletion of the lipid droplet protein kinase gene affects lipid droplets biogenesis, parasite infectivity, and resistance to trivalent antimony in Leishmania infantum.

The Lipid Droplet Protein Kinase (LDK) facilitates lipid droplet (LD) biogenesis, organelles involved in various metabolic and signaling functions in trypanosomatids. As LDK's function has not been previously explored in Leishmania spp., we utilized CRISPR/Cas9 technology to generate LDK-knockout lines of Leishmania infantum to investigate its role in this parasite. Our findings demonstrate that LDK is not an essential gene for L. infantum, as its deletion did not impede parasite survival. Furthermore, removing LDK did not impact the growth of promastigote forms of L. infantum lacking LDK. However, a noticeable reduction in LDs occurred during the stationary phase of parasite growth following LDK deletion. In the presence of myriocin, a LD inducer, LDK-knockout parasites displayed reduced LD abundance during both logarithmic and stationary growth phases compared to control parasites. Moreover, an infection analysis involving THP-1 cells revealed that 72 h post-infection, LDK-knockout L. infantum lines exhibited fewer infected macrophages and intracellular amastigotes than control parasites. LDK-knockout L. infantum lines also displayed 1.7 to 1.8 -fold greater resistance to trivalent antimony than control parasites. There were no observed alterations in susceptibility to amphotericin B, miltefosine, or menadione in LDK-knockout L. infantum lines. Our results suggest that LDK plays a crucial role in the biogenesis and/or maintenance of LDs in L. infantum, as well as in parasite infectivity and resistance to trivalent antimony.

Cytotoxic auranofin analogues bearing phosphine, arsine and stibine ligands: A study on the possible role of the ligand on the biological activity.

Three gold(I) linear compounds, sharing the general formula [AuI(LPh3)], have been synthesized and characterized. The nature of the ligand has been modified by moving down among some of the elements of group 15, i.e. phosphorus, arsenic and antimony. The structures of derived compounds have been solved through XRD and the reactivity behaviour towards selected biomolecules has been investigated through a multi-technique approach involving NMR, high-resolution mass spectrometry and IR. Moreover, the biological activity of the investigated compounds has been comparatively analyzed through classical methodologies and the disclosed differences are discussed in detail.

Syntheses and characterization of novel antimony (III) and bismuth (III) derivatives containing ß-enamino ester along with antimicrobial evaluation, DFT calculation, and cytotoxic study.

Four novel antimony (III) and bismuth(III) complexes of the kind Cl-Sb-O-C(OR)-CH(CH3 )C-NH-(CH2 )2 -NH-C(CH3 )CH:C(OR)-O [where R = -CH3 , M = Sb (1a); R = -C2 H5 , M = Sb (1b); R = -CH3, M = Bi (1c); R = -C2 H5 , M = Bi (1d)] were successfully prepared by reacting antimony(III)chloride and bismuth(III)chloride with sodium salt of ß-enamino esters in 1:1 stoichiometry, which were further structurally characterized by physicochemical and IR, 1 H, 13 C NMR spectral and mass spectrometry. Structural analysis revealed that all four derivatives of both antimony and bismuth display octahedarl geometry which has been optimized through computational studies. These derivatives along with their parent ligands were subsequently assayed in vitro for antibacterial (Bacillus subtilis, Pseudomonas aeruginosa) and antifungal (Aspergillus niger and Candida albicans) activities. Synthesized complexes were more efficacious in terms of biological activities as compared to parent ligands Further synthesized compounds were evaluated for their in vitro cytotoxic activity against lung cancer cell line A549 using MTT method. IC50 value for all four complexes was determined and all of them are found active. Computational studies of the representative complexes have been done using B3LYP/631-G* basis sets to provide optimized geometry.

Antimony resistance and gene expression in Leishmania: spotlight on molecular and proteomic aspects.

Leishmaniasis is a vector-borne parasitic disease caused by Leishmania parasites with a spectrum of clinical manifestations, ranging from skin lesions to severe visceral complications. Treatment of this infection has been extremely challenging with the concurrent emergence of drug resistance. The differential gene expression and the discrepancies in protein functions contribute to the appearance of 2 distinct phenotypes: resistant and sensitive, but the current diagnostic tools fail to differentiate between them. The identification of gene expression patterns and molecular mechanisms coupled with antimony (Sb) resistance can be leveraged to prompt diagnosis and select the most effective treatment methods. The present study attempts to use comparative expression of Sb resistance-associated genes in resistant and sensitive Leishmania, to disclose their relative abundance in clinical or in vitro selected isolates to gain an understanding of the molecular mechanisms of Sb response/resistance. Data suggest that the analysis of resistance gene expression would verify the Sb resistance or susceptibility only to a certain extent; however, none of the individual expression patterns of the studied genes was diagnostic as a biomarker of Sb response of Leishmania. The findings highlighted will be useful in bridging the knowledge gap and discovering innovative diagnostic tools and novel therapeutic targets.

Selenite affected photosynthesis of Oryza sativa L. exposed to antimonite: Electron transfer, carbon fixation, pigment synthesis via a combined analysis of physiology and transcriptome.

Selenium (Se) is a microelement that can counteract (a)biotic stresses in plants. Excess antimony (Sb) will inhibit plant photosynthesis, which can be alleviated by appropriate doses of Se but the associated mechanisms at the molecular levels have not been fully explored. Here, a rice variety (Yongyou 9) was exposed to selenite [Se(IV), 0.2 and 0.8 mg L-1] alone or combined with antimonite [Sb(III), 5 and 10 mg L-1]. When compared to the 10 mg L-1 Sb treatment alone, addition of Se in a dose-dependent manner 1) reduced the heat dissipation efficiency resulting from the inhibited donors, Sb concentrations in shoots and roots, leaf concentrations of fructose, H2O2 and O2•-; 2) enhanced heat dissipation efficiency resulting from the inhibited accepters value, concentrations of Chl a, sucrose and starch, and the enzyme activity of adenosine diphosphate glucose pyrophosphorylase, sucrose phosphate synthase, and sucrose synthase; but 3) did not alter gas exchange parameters, concentrations of Chl b and total Chl, enzyme activity of soluble acid invertase, and values of maximum P700 signal, photochemical efficiency of PSI and electron transport rate of PSI. Se alleviated the damage caused by Sb to the oxygen-evolving complex and promoted the transfer of electrons from QA to QB. When compared to the 10 mg L-1 Sb treatment alone, addition of Se 1) up-regulated genes correlated to synthesis pathways of Chl, carotenoid, sucrose and glucose; 2) disturbed signal transduction pathway of abscisic acid; and 3) upregulated gene expression correlated to photosynthetic complexes (OsFd1, OsFER1 and OsFER2).

Translational reprogramming as a driver of antimony-drug resistance in Leishmania.

Leishmania is a unicellular protozoan that has a limited transcriptional control and mostly uses post-transcriptional regulation of gene expression, although the molecular mechanisms of the process are still poorly understood. Treatments of leishmaniasis, pathologies associated with Leishmania infections, are limited due to drug resistance. Here, we report dramatic differences in mRNA translation in antimony drug-resistant and sensitive strains at the full translatome level. The major differences (2431 differentially translated transcripts) were demonstrated in the absence of the drug pressure supporting that complex preemptive adaptations are needed to efficiently compensate for the loss of biological fitness once they are exposed to the antimony. In contrast, drug-resistant parasites exposed to antimony activated a highly selective translation of only 156 transcripts. This selective mRNA translation is associated with surface protein rearrangement, optimized energy metabolism, amastins upregulation, and improved antioxidant response. We propose a novel model that establishes translational control as a major driver of antimony-resistant phenotypes in Leishmania.

Transcriptomic analysis reveals the molecular mechanisms of Boehmeria nivea L. in response to antimonite and antimonate stresses.

Soil antimony (Sb) pollution is a global concern that threatens food security and human health. Boehmeria nivea L. (ramie) is a promising phytoremediation plant exhibiting high tolerance and enrichment capacity for Sb. To reveal the molecular mechanisms and thus enhance the ramie uptake, transport, and detoxification of Sb with practical strategies, a hydroponic experiment was conducted to compare the physiological and transcriptomic responses of ramie towards antimonite (Sb(Ⅲ)) and antimonate (Sb(Ⅴ)). Phenotypic results showed that Sb(Ⅲ) had a stronger inhibitory effect on the growth of ramie. Root Sb content under Sb(Ⅲ) was 2.43 times higher than that in Sb(Ⅴ) treatment. Based on the ribonucleic acid sequencing (RNA-Seq) technique, 3915 and 999 significant differentially expressed genes (DEGs) were identified under Sb(Ⅲ) and Sb(Ⅴ), respectively. Transcriptomic analysis revealed that ramie showed different adaptation strategies to Sb(Ⅲ) and Sb(V). Key DEGs and their involved pathways such as catalytic activity, carbohydrate metabolisms, phenylpropanoid biosynthesis, and cell wall modification were identified to perform crucial roles in Sb tolerance and detoxification. Two heavy metal-associated domain-type genes, six heavy metal-associated isoprenylated plant proteins, and nine ABC transporters showed possible roles in the transport and detoxification of Sb. The significant upregulation of NRAMP5 and three NIPs suggested their roles in the transport of Sb(V). This study is the basis for future research to identify the exact genes and biological processes that can effectively enhance Sb accumulation or improve plant tolerance to Sb, thereby promoting the phytoremediation of Sb-polluted soils.

Novel Pyrido[2',1':2,3]imidazo[4,5-c]quinoline Derivative Selectively Poisons Leishmania donovani Bisubunit Topoisomerase 1 to Inhibit the Antimony-Resistant Leishmania Infection in Vivo.

The unique bisubunit structure of Leishmania donovani topoisomerase 1B (LdTop1) is a potential drug target in the parasites unlike the monomeric Top1 from its human host counterpart. Here, we report the design, synthesis, and validation of a chimeric pyrido[2',1':2,3]imidazo[4,5-c]quinoline derivative (C17) as a novel antileishmanial agent that poisons topoisomerase 1-DNA covalent complexes (LdTop1cc) inside the parasites and inhibits Top1 religation activity both in the drug sensitive and antimony-resistant L. donovani clinical isolates. Importantly, the human Top1 is not sensitive to C17. Further, C17 overcomes the chemical instability of camptothecin (CPT) by generating persistent LdTop1cc-induced DNA breaks inside the parasites even after 12 h of drug removal. Intraperitoneal administration of C17 results in marked reduction of the Leishmania amastigotes from the infected spleen and liver of BALB/c mice. C17 confers a host protective immune-response up-regulating the Th1 cytokines facilitating parasite clearance which can be exploited for treating drug-resistant leishmaniasis.

Sb-Phenyl-N-methyl-5,6,7,12-tetrahydrodibenz[c,f][1,5]azastibocine Induces Perlecan Core Protein Synthesis in Cultured Vascular Endothelial Cells.

Vascular endothelial cells synthesize and secrete perlecan, a large heparan sulfate proteoglycan that increases the anticoagulant activity of vascular endothelium by inducing antithrombin III and intensifying fibroblast growth factor (FGF)-2 activity to promote migration and proliferation in the repair process of damaged endothelium during the progression of atherosclerosis. However, the exact regulatory mechanisms of endothelial perlecan expression remain unclear. Since organic-inorganic hybrid molecules are being developed rapidly as tools to analyze biological systems, we searched for a molecular probe to analyze these mechanisms using a library of organoantimony compounds and found that the Sb-phenyl-N-methyl-5,6,7,12-tetrahydrodibenz[c,f][1,5]azastibocine (PMTAS) molecule promotes the expression of perlecan core protein gene without exhibiting cytotoxicity in vascular endothelial cells. In the present study, we characterized proteoglycans synthesized by cultured bovine aortic endothelial cells using biochemical techniques. The results indicated that PMTAS selectively induced perlecan core protein synthesis, without affecting the formation of its heparan sulfate chain, in vascular endothelial cells. The results also implied that this process is independent of the endothelial cell density, whereas in vascular smooth muscle cells, it occurred only at high cell density. Thus, PMTAS would be a useful tool for further studies on the mechanisms underlying perlecan core protein synthesis in vascular cells, which is critical in the progression of vascular lesions, such as those during atherosclerosis.

Assessing the effect of antimony pressure on trypanothione reductase activity in Leishmania (Viannia) braziliensis.

Leishmania parasites have an oxidative and chemical defense mechanism called trypanothione system (T[SH]2), the most abundant thiol system in trypanosomatids. This system has a central role in processing pentavalent antimony and resistance has been related to a better capacity to metabolize it through the activation of T[SH]2 enzymatic cascade. A biochemical approach was applied to assess the effect of trivalent (SbIII) and pentavalent antimony (SbV) on Trypanothione Reductase (TR) activity of two Leishmania (Viannia) braziliensis clinical isolates, which were labeled as responder (R) and non-responder (NR) after patient treatment with Glucantime®. Both isolates were characterized based on in vitro susceptibility to SbIII and SbV and trypanothione reductase (TR) activity. SbIII and SbV discriminated susceptibility profiles in all parasite forms, since isolate NR had significantly higher EC50 values than isolate R. Differences were observed in TR activity between promastigotes, axenic amastigotes and intracellular amastigotes: R (0.439 ± 0.009, 0.103 ± 0.01 and 0.185 ± 0.01AU.min-1.µg of protein-1) and NR (1.083 ± 0.04, 0.914 ± 0.04 and 0.343 ± 0.04 AU. min-1.µg of protein-1), respectively. Incubation with SbIII and SbV using each form EC50 value caused a time-dependent differential effect on TR activity suggesting that oxidative defense is related to the antimony susceptibility phenotype. Data gathered here shows a biochemical approach able to discriminate two L. (V.) braziliensis clinical isolates measurements TR activity of promastigotes, axenic amastigotes and intracellular amastigotes.

Antimony resistance mechanism in genetically different clinical isolates of Indian Kala-azar patients.

The central theme of this enterprise is to find common features, if any, displayed by genetically different antimony (Sb)-resistant viscerotropic Leishmania parasites to impart Sb resistance. In a limited number of clinical isolates (n = 3), we studied the breadth of variation in the following dimensions: (a) intracellular thiol content, (b) cell surface expression of glycan having N-acetyl-D-galactosaminyl residue as the terminal sugar, and (c) gene expression of thiol-synthesizing enzymes (CBS, MST, gamma-GCS, ODC, and TR), antimony-reducing enzymes (TDR and ACR2), and antimonial transporter genes (AQP1, MRPA, and PRP1). One of the isolates, T5, that was genotypically characterized as Leishmania tropica, caused Indian Kala-azar and was phenotypically Sb resistant (T5-LT-SSG-R), while the other two were Leishmania donovani, out of which one isolate, AG83, is antimony sensitive (AG83-LD-SSG-S) and the other isolate, T8, is Sb resistant (T8-LD-SSG-R). Our study showed that the Sb-resistant parasites, regardless of their genotype, showed significantly higher intracellular thiol compared with Sb-sensitive AG83-LD-SSG-S. Seemingly, T5-LT-SSG-R showed about 1.9-fold higher thiol content compared with T8-LD-SSG-R which essentially mirrored cell surface N-acetyl-D-galactosaminyl expression. Except TR, the expression of the remaining thiol-synthesizing genes was significantly higher in T8-LD-SSG-R and T5-LT-SSG-R than the sensitive one, and between the Sb-resistant parasites, the latter showed a significantly higher expression. Furthermore, the genes for Sb-reducing enzymes increased significantly in resistant parasites regardless of genotype compared with the sensitive one, and between two resistant parasites, there was hardly any difference in expression. Out of three antimony transporters, AQP1 was decreased with the concurrent increase in MRPA and PRP1 in resistant isolates when compared with the sensitive counterpart. Interestingly, no difference in expression of the above-mentioned transporters was noted between two Sb-resistant isolates. The enduring image that resonated from our study is that the genetically diverse Sb-resistant parasites showed enhanced thiol-synthesizing and antimony transporter gene expression than the sensitive counterpart to confer a resistant phenotype.

Non-Antibiotic Antimony-Based Antimicrobials.

A series of the eight novel organoantimony(V) cyanoximates of Sb(C6H5)4L composition was synthesized using the high-yield heterogeneous metathesis reaction between solid AgL (or TlL) and Sb(C6H5)4Br in CH3CN at room temperature. Cyanoximes L were specially selected from a large group of 48 known compounds of this subclass of oximes on the basis of their water solubility and history of prior biological activity. The synthesized compounds are well soluble in organic solvents and were studied using a variety of conventional spectroscopic and physical methods. The crystal structures of all reported organometallic compounds were determined and revealed the formation of the distorted trigonal bipyramidal environment of the Sb atom and monodentate axial binding of acido-ligands via the O atom of the oxime group. The compounds are thermally stable in the solid state and in solution molecular compounds. For the first time, this specially designed series of organoantimony(V) compounds is investigated as potential non-antibiotic antimicrobial agents against three bacterial and two fungal human pathogens known for their increasing antimicrobial resistance. Bacterial pathogens included Gram-negative Escherichia coli and Pseudomonas aeruginosa, and Gram-positive Staphylococcus aureus. Fungal pathogens included Cryptococcus neoformans and Candida albicans. The cyanoximates alone showed no antimicrobial impact, and the incorporation of the SbPh4 group enabled the antimicrobial effect. Overall, the new antimony compounds showed a strong potential as both broad- and narrow-spectrum antimicrobials against selected bacterial and fundal pathogens and provide insights for further synthetic modifications of the compounds to increase their activities.

Effect of nitrate and sulfate coexistence on hydrogen autotrophic reduction of antimonate (Sb(V)) and microbial community structures.

Hydrogen autotrophic bioreduction of antimonate (Sb(V)) to antimonite (Sb(III)) is an alternative approach for removing antimony (Sb) from water. This study investigated Sb(V) reduction kinetics and the effects of various parameters on the Sb(V) removal performance in a hydrogen autotrophic reaction system (HARS). Sb(V) reduction in the HARS was well fitted to the Michaelis-Menten model, showing a positive correlation between the reaction rate and biomass. The maximum specific substrate removal rates were 0.29-4.86 and 6.82-15.87 mg Sb(V)/(g·VSS·h) at initial Sb(V) concentrations of 500 µg/L and 10 mg/L, respectively. Coexisting nitrate significantly inhibited Sb(V) reduction, and the inhibition intensified with increasing nitrate concentration. However, coexisting sulfate had a positive effect on Sb(V) reduction, and the sulfate effectively enhanced total antimony (TSb) removal performance by generating sulfide from sulfate reduction. Illumina high-throughput sequencing technology was used to determine the changes in microbial community structure during different periods in the HARS, revealing the effects of co-existing ions on the dominant Sb(V) reducing bacteria. In the HARS, Longilinea and Terrimonas were the dominant genera in the presence of nitrate, and Longilinea was the dominant genus in the presence of sulfate, at initial Sb(V) concentration of 500 µg/L. When the concentration of Sb(V) was 10 mg/L, Longilinea and Thauera were the dominant genus in the HARS for treating water co-polluted with nitrate and sulfate, respectively. These results provide a theoretical basis of the application of HARS for the bio-remediation of Sb(V) contaminated water.

Exploring direct and indirect targets of current antileishmanial drugs using a novel thermal proteomics profiling approach.

Visceral leishmaniasis (VL), caused by Leishmania infantum, is an oft-fatal neglected tropical disease. In the absence of an effective vaccine, the control of leishmaniasis relies exclusively on chemotherapy. Due to the lack of established molecular/genetic markers denoting parasite resistance, clinical treatment failure is often used as an indicator. Antimony-based drugs have been the standard antileishmanial treatment for more than seven decades, leading to major drug resistance in certain regions. Likewise, drug resistance to miltefosine and amphotericin B continues to spread at alarming rates. In consequence, innovative approaches are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. To this end, we have implemented a novel approach based on thermal proteome profiling (TPP) to further characterize the mode of action of antileishmanials antimony, miltefosine and amphotericin B, as well as to better understand the mechanisms of drug resistance deployed by Leishmania. Proteins become more resistant to heat-induced denaturation when complexed with a ligand. In this way, we used multiplexed quantitative mass spectrometry-based proteomics to monitor the melting profile of thousands of expressed soluble proteins in WT, antimony-resistant, miltefosine-resistant, and amphotericin B-resistant L. infantum parasites, in the presence (or absence) of the above-mentioned drugs. Bioinformatics analyses were performed, including data normalization, melting profile fitting, and identification of proteins that underwent changes (fold change > 4) caused by complexation with a drug. With this unique approach, we were able to narrow down the regions of the L. infantum proteome that interact with antimony, miltefosine, and amphotericin B; validating previously-identified and unveiling novel drug targets. Moreover, analyses revealed candidate proteins potentially involved in drug resistance. Interestingly, we detected thermal proximity coaggregation for several proteins belonging to the same metabolic pathway (i.e., tryparedoxin peroxidase and aspartate aminotransferase in proteins exposed to antimony), highlighting the importance of these pathways. Collectively, our results could serve as a jumping-off point for the future development of innovative diagnostic tools for the detection and evaluation of antimicrobial-resistant Leishmania populations, as well as open the door for new on-target therapies.

A Water-Soluble Antimony-Rich Polyoxometalate with Broad-Spectrum Antitumor Activities.

Herein, a giant Sb-rich polyoxometalate (POM) {Sb21 Tb7 W56 } is reported, which contains the largest number of Sb atoms in a POM so far. The Sb-rich POM has many interesting structural features and is a rare example of a soluble and water-stable giant POM. Biomedical studies indicate that the Sb-rich POM exhibits broad-spectrum antitumor activity against various cancer cell lines by reactivating the P53-dependent apoptotic processes and disrupting the mitochondrial membrane. In addition, this Sb-rich POM was capable of suppressing the growth and metastasis of a breast cancer in vivo. This work demonstrates that Sb-rich POMs are promising candidates for the development of new anticancer drugs.