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Iron is a transition metal used as a cofactor in many biochemical reactions. In bacteria, iron homeostasis involves Fur-mediated de-repression of iron uptake systems, such as the iron-chelating compounds siderophores. In this work, we identified and characterized novel regulatory systems that control siderophores in the environmental opportunistic pathogen Chromobacterium violaceum. Screening of a 10,000-transposon mutant library for siderophore halos identified seven possible regulatory systems involved in siderophore-mediated iron homeostasis in C. violaceum. Further characterization revealed a regulatory cascade that controls siderophores involving the transcription factor VitR acting upstream of the quorum-sensing (QS) system CviIR. Mutation of the regulator VitR led to an increase in siderophore halos, and a decrease in biofilm, violacein, and protease production. We determined that these effects occurred due to VitR-dependent de-repression of vioS. Increased VioS leads to direct inhibition of the CviR regulator by protein-protein interaction. Indeed, insertion mutations in cviR and null mutations of cviI and cviR led to an increase of siderophore halos. RNA-seq of the cviI and cviR mutants revealed that CviR regulates CviI-dependent and CviI-independent regulons. Classical QS-dependent processes (violacein, proteases, and antibiotics) were activated at high cell density by both CviI and CviR. However, genes related to iron homeostasis and many other processes were regulated by CviR but not CviI, suggesting that CviR acts without its canonical CviI autoinducer. Our data revealed a complex regulatory cascade involving QS that controls siderophore-mediated iron homeostasis in C. violaceum.IMPORTANCEThe iron-chelating compounds siderophores play a major role in bacterial iron acquisition. Here, we employed a genetic screen to identify novel siderophore regulatory systems in Chromobacterium violaceum, an opportunistic human pathogen. Many mutants with increased siderophore halos had transposon insertions in genes encoding transcription factors, including a novel regulator called VitR, and CviR, the regulator of the quorum-sensing (QS) system CviIR. We found that VitR is upstream in the pathway and acts as a dedicated repressor of vioS, which encodes a direct CviR-inhibitory protein. Indeed, all QS-related phenotypes of a vitR mutant were rescued in a vitRvioS mutant. At high cell density, CviIR activated classical QS-dependent processes (violacein, proteases, and antibiotics production). However, genes related to iron homeostasis and type-III and type-VI secretion systems were regulated by CviR in a CviI- or cell density-independent manner. Our data unveil a complex regulatory cascade integrating QS and siderophores in C. violaceum.

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Thyroid hormones (THs) are crucial in bodily functions, while iron is essential for processes like oxygen transport. Specialized proteins maintain iron balance, including ferritin, transferrin, ferroportin, and hepcidin. Research suggests that THs can influence iron homeostasis by affecting mRNA and protein expression, such as ferritin and transferrin. Our study focused on male rats to assess mRNA expression of iron homeostasis-related proteins and metabolomics in thyroid dysfunction. We found altered gene expression across various tissues (liver, duodenum, spleen, and kidney) and identified disrupted metabolite patterns in thyroid dysfunction. These findings highlight tissue-specific effects of thyroid dysfunction on essential iron homeostasis proteins and provide insights into associated metabolic changes. Our research contributes to understanding the intricate interplay between thyroid hormones and iron balance. By unveiling tissue-specific gene expression alterations and metabolic disruptions caused by thyroid dysfunction, our work lays a foundation for future investigations to explore underlying mechanisms and develop targeted strategies for managing iron-related complications in thyroid disorders.

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Serious concerns have recently been raised regarding the association of Fe excess with neurodegenerative diseases in mammals and nutritional and oxidative disorders in plants. Therefore, the current study aimed to understand the physiological changes induced by Fe excess in Pistia stratiotes, a species often employed in phytoremediation studies. P. stratiotes were subjected to five concentrations of Fe: 0.038 (control), 1.0, 3.0, 5.0 and 7.0 mM. Visual symptoms of Fe-toxicity such as bronzing of leaf edges in 5.0 and 7.0 mM-grown plants were observed after 5 days. Nevertheless, no major changes were observed in photosynthesis-related parameters at this time-point. In contrast, plants growing for 10 days in high Fe concentrations showed decreased chlorophyll concentrations and lower net CO2 assimilation rate. Notwithstanding, P. stratiotes accumulated high amounts of Fe, especially in roots (maximum of 10,000 µg g-1 DW) and displayed a robust induction of the enzymatic antioxidant system. In conclusion, we demonstrated that P. stratiotes can be applied to clean up Fe-contaminated water, as the species displays high Fe bioaccumulation, mostly in root apoplasts, and can maintain physiological processes under Fe excess. Our results further revealed that by monitoring visual symptoms, P. stratiotes could be applied for bioindication purposes.

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Gluconacetobacter diazotrophicus has been the focus of several studies aiming to understand the mechanisms behind this endophytic diazotrophic bacterium. The present study is the first global analysis of the early transcriptional response of exponentially growing G. diazotrophicus to iron, an essential cofactor for many enzymes involved in various metabolic pathways. RNA-seq, targeted gene mutagenesis and computational motif discovery tools were used to define the G. diazotrophicusfur regulon. The data analysis showed that genes encoding functions related to iron homeostasis were significantly upregulated in response to iron limitations. Certain genes involved in secondary metabolism were overexpressed under iron-limited conditions. In contrast, it was observed that the expression of genes involved in Fe-S cluster biosynthesis, flagellar biosynthesis and type IV secretion systems were downregulated in an iron-depleted culture medium. Our results support a model that controls transcription in G. diazotrophicus by fur function. The G. diazotrophicusfur protein was able to complement an E. colifur mutant. These results provide new insights into the effects of iron on the metabolism of G. diazotrophicus, as well as demonstrate the essentiality of this micronutrient for the main characteristics of plant growth promotion by G. diazotrophicus.

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Chromobacterium violaceum is an environmental Gram-negative beta-proteobacterium that causes systemic infections in humans. C. violaceum uses siderophore-based iron acquisition systems to overcome the host-imposed iron limitation, but its capacity to use other iron sources is unknown. In this work, we characterized ChuPRSTUV as a heme utilization system employed by C. violaceum to explore an important iron reservoir in mammalian hosts, free heme and hemoproteins. We demonstrate that the chuPRSTUV genes comprise a Fur-repressed operon that is expressed under iron limitation. The chu operon potentially encodes a small regulatory protein (ChuP), an outer membrane TonB-dependent receptor (ChuR), a heme degradation enzyme (ChuS), and an inner membrane ABC transporter (ChuTUV). Our nutrition growth experiments using C. violaceum chu deletion mutants revealed that, with the exception of chuS, all genes of the chu operon are required for heme and hemoglobin utilization in C. violaceum. The mutant strains without chuP displayed increased siderophore halos on CAS plate assays. Significantly, we demonstrate that ChuP connects heme and siderophore utilization by acting as a positive regulator of chuR and vbuA, which encode the TonB-dependent receptors for the uptake of heme (ChuR) and the siderophore viobactin (VbuA). Our data favor a model of ChuP as a heme-binding post-transcriptional regulator. Moreover, our virulence data in a mice model of acute infection demonstrate that C. violaceum uses both heme and siderophore for iron acquisition during infection, with a preference for siderophores over the Chu heme utilization system.

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It is known that iron transporter proteins and their regulation can modulate the fish's immune system, suggesting these proteins as a potential candidate for fish vaccines. Previous studies have evidenced the effects of Atlantic salmon immunized with the chimeric iron-related protein named IPath® against bacterial and ectoparasitic infections. The present study aimed to explore the transcriptome modulation and the morphology of the sea louse Caligus rogercresseyi in response to Atlantic salmon injected with IPath®. Herein, Atlantic salmon were injected with IPath® and challenged to sea lice in controlled laboratory conditions. Then, female adults were collected after 25 days post-infection for molecular and morphological evaluation. Transcriptome analysis conducted in lice collected from immunized fish revealed high modulation of transcripts compared with the control groups. Notably, the low number of up/downregulated transcripts was mainly found in lice exposed to the IPath® fish group. Among the top-25 differentially expressed genes, Vitellogenin, Cytochrome oxidases, and proteases genes were strongly downregulated, suggesting that IPath® can alter lipid transport, hydrogen ion transmembrane transport, and proteolysis. The morphological analysis in lice collected from IPath® fish revealed abnormal embryogenesis and inflammatory processes of the genital segment. Furthermore, head kidney, spleen, and skin were also analyzed in immunized fish to evaluate the transcription expression of immune and iron homeostasis-related genes. The results showed downregulation of TLR22, MCHII, IL-1ß, ALAs, HO, BLVr, GSHPx, and Ferritin genes in head kidney and skin tissues; meanwhile, those genes did not show significant differences in spleen tissue. Overall, our findings suggest that IPath® can be used to enhance the fish immune response, showing a promissory commercial application against lice infections.

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Glutaredoxin, Grx, is a small protein containing an active site cysteine pair and was discovered in 1976 by Arne Holmgren. The Grx system, comprised of Grx, glutathione, glutathione reductase, and NADPH, was first described as an electron donor for Ribonucleotide Reductase but, from the first discovery in E.coli, the Grx family has impressively grown, particularly in the last two decades. Several isoforms have been described in different organisms (from bacteria to humans) and with different functions. The unique characteristic of Grxs is their ability to catalyse glutathione-dependent redox regulation via glutathionylation, the conjugation of glutathione to a substrate, and its reverse reaction, deglutathionylation. Grxs have also recently been enrolled in iron sulphur cluster formation. These functions have been implied in various physiological and pathological conditions, from immune defense to neurodegeneration and cancer development thus making Grx a possible drug target. This review aims to give an overview on Grxs, starting by a phylogenetic analysis of vertebrate Grxs, followed by an analysis of the mechanisms of action, the specific characteristics of the different human isoforms and a discussion on aspects related to human physiology and diseases.

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Iron is a highly reactive metal that participates in several processes in prokaryotic and eukaryotic cells. Hosts and pathogens compete for iron in the context of infection. Chromobacterium violaceum, an environmental Gram-negative bacterial pathogen, relies on siderophores to overcome iron limitation in the host. In this work, we studied the role of the ferric uptake regulator Fur in the physiology and virulence of C. violaceum A Δfur mutant strain showed decreased growth and fitness under regular in vitro growth conditions and presented high sensitivity to iron and oxidative stresses. Furthermore, the absence of fur caused derepression of siderophore production and reduction in swimming motility and biofilm formation. Consistent with these results, the C. violaceum Δfur mutant was highly attenuated for virulence and liver colonization in mice. In contrast, a manganese-selected spontaneous fur mutant showed only siderophore overproduction and sensitivity to oxidative stress, indicating that Fur remained partially functional in this strain. We found that mutations in genes related to siderophore biosynthesis and a putative CRISPR-Cas locus rescued the Δfur mutant growth defects, indicating that multiple Fur-regulated processes contribute to maintaining bacterial cell fitness. Overall, our data indicated that Fur is conditionally essential in C. violaceum mainly by protecting cells from iron overload and oxidative damage. The requirement of Fur for virulence highlights the importance of iron in the pathogenesis of C. violaceumIMPORTANCE Maintenance of iron homeostasis, i.e., avoiding both deficiency and toxicity of this metal, is vital to bacteria and their hosts. Iron sequestration by host proteins is a crucial strategy to combat bacterial infections. In bacteria, the ferric uptake regulator Fur coordinates the expression of several iron-related genes. Sometimes, Fur can also regulate several other processes. In this work, we performed an in-depth phenotypic characterization of fur mutants in the human opportunistic pathogen Chromobacterium violaceum We determined that fur is a conditionally essential gene necessary for proper growth under regular conditions and is fully required for survival under iron and oxidative stresses. Fur also controlled several virulence-associated traits, such as swimming motility, biofilm formation, and siderophore production. Consistent with these results, a C. violaceumfur null mutant showed attenuation of virulence. Therefore, our data established Fur as a major player required for C. violaceum to manage iron, including during infection in the host.

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AIMS: The importance of bacterioferritin in the virulence and pathogenicity of the genus Mycobacterium is still unclear. The aim of this study was to analyse if the expression of a recombinant bacterioferritin from M. tuberculosis (Mtb) by Mycma could improve the capacity of this bacillus to resist the host defence mechanisms. METHODS AND RESULTS: Recombinant Mycma, expressing bacterioferritin (Rv1876) from Mtb, was developed by transformation with pMIP12_Rv1876. To determine bacterioferritin influence on Mycma physiology and virulence, the mycobacteria growth was analysed in vitro and in vivo. It was observed that the expression of bacterioferritin improved the growth rate of recombinant Mycma_BfrA under iron excess and oxidative stress, as compared to the wild type. Furthermore, in the murine model of infection, it was observed that Mycma_BfrA-infected mice had higher bacillary load and a more pronounced lesion in the lungs when compared with the wild type. CONCLUSION: This study showed that bacterioferritin confers additional resistance to stress conditions, resulting in increased pathogenicity of Mycma during mice infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new insights about the importance of bacterioferritin in the virulence and pathogenicity of the Mycobacterium genus.

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