Iron-depleted Conditions Research Articles

Fur Represses Adhesion to, Invasion of, and Intracellular Bacterial Community Formation within Bladder Epithelial Cells and Motility in Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infections (UTIs). This bacterium adheres to and invades the host cells in the bladder, where it forms biofilm-like polymicrobial structures termed intracellular bacterial communities (IBCs) that protect UPEC from antimicrobial agents and the host immune systems. Using genetic screening, we found that deletion of the fur gene, which encodes an iron-binding transcriptional repressor for iron uptake systems, elevated the expression of type I fimbriae and motility when UPEC was grown under iron-rich conditions, and it led to an increased number of UPEC cells adhering to and internalized in bladder epithelial cells. Consequently, the IBC colonies that the fur mutant formed in host cells were denser and larger than those formed by the wild-type parent strain. Fur is inactivated under iron-restricted conditions. When iron was depleted from the bacterial cultures, wild-type UPEC adhesion, invasion, and motility increased, similar to the case with the fur mutant. The purified Fur protein bound to regions upstream of fimA and flhD, which encode type I fimbriae and an activator of flagellar expression that contributes to motility, respectively. These results suggest that Fur is a repressor of fimA and flhD and that its repression is abolished under iron-depleted conditions. Based on our in vitro experiments, we conclude that UPEC adhesion, invasion, IBC formation, and motility are suppressed by Fur under iron-rich conditions but derepressed under iron-restricted conditions, such as in patients with UTIs.

The genes that encode the gonococcal transferrin binding proteins, TbpB and TbpA, are differentially regulated by MisR under iron-replete and iron-depleted conditions.

Neisseria gonorrhoeae produces two transferrin binding proteins, TbpA and TbpB, which together enable efficient iron transport from human transferrin. We demonstrate that expression of the tbp genes is controlled by MisR, a response regulator in the two-component regulatory system that also includes the sensor kinase MisS. The tbp genes were up-regulated in the misR mutant under iron-replete conditions but were conversely down-regulated in the misR mutant under iron-depleted conditions. The misR mutant was capable of transferrin-iron uptake at only 50% of wild-type levels, consistent with decreased tbp expression. We demonstrate that phosphorylated MisR specifically binds to the tbpBA promoter and that MisR interacts with five regions upstream of the tbpB start codon. These analyses confirm that MisR directly regulates tbpBA expression. The MisR binding sites in the gonococcus are only partially conserved in Neisseria meningitidis, which may explain why tbpBA was not MisR-regulated in previous studies using this related pathogen. This is the first report of a trans-acting protein factor other than Fur that can directly contribute to gonococcal tbpBA regulation.

Iron-depletion promotes mitophagy to maintain mitochondrial integrity in pathogenic yeast Candida glabrata

ABSTRACTCandida glabrata, a haploid budding yeast, is the cause of severe systemic infections in immune-compromised hosts. The amount of free iron supplied to C. glabrata cells during systemic infections is severely limited by iron-chelating proteins such as transferrin. Thus, the iron-deficiency response in C. glabrata cells is thought to play important roles in their survival inside the host's body. In this study, we found that mitophagy was induced under iron-depleted conditions, and that the disruption of a gene homologous to ATG32, which is responsible for mitophagy in Saccharomyces cerevisiae, blocked mitophagy in C. glabrata. The mitophagic activity in C. glabrata cells was not detected on short-period exposure to nitrogen-starved conditions, which is a mitophagy-inducing condition used in S. cerevisiae. The mitophagy-deficient atg32Δ mutant of C. glabrata also exhibited decreased longevity under iron-deficient conditions. The mitochondrial membrane potential in Cgatg32Δ cells was significantly lower than that in wild-type cells under iron-depleted conditions. In a mouse model of disseminated infection, the Cgatg32Δ strain resulted in significantly decreased kidney and spleen fungal burdens compared with the wild-type strain. These results indicate that mitophagy in C. glabrata occurs in an iron-poor host tissue environment, and it may contribute to the longevity of cells, mitochondrial quality control, and pathogenesis.

HapX Mediates Iron Homeostasis in the Pathogenic Dermatophyte Arthroderma benhamiae but Is Dispensable for Virulence.

For many pathogenic fungi, siderophore-mediated iron acquisition is essential for virulence. The process of siderophore production and further mechanisms to adapt to iron limitation are strictly controlled in fungi to maintain iron homeostasis. Here we demonstrate that the human pathogenic dermatophyte Arthroderma benhamiae produces the hydroxamate siderophores ferricrocin and ferrichrome C. Additionally, we show that the iron regulator HapX is crucial for the adaptation to iron starvation and iron excess, but is dispensable for virulence of A. benhamiae. Deletion of hapX caused downregulation of siderophore biosynthesis genes leading to a decreased production of siderophores during iron starvation. Furthermore, HapX was required for transcriptional repression of genes involved in iron-dependent pathways during iron-depleted conditions. Additionally, the ΔhapX mutant of A. benhamiae was sensitive to high-iron concentrations indicating that HapX also contributes to iron detoxification. In contrast to other pathogenic fungi, HapX of A. benhamiae was redundant for virulence and a ΔhapX mutant was still able to infect keratinized host tissues in vitro. Our findings underline the highly conserved role of the transcription factor HapX for maintaining iron homeostasis in ascomycetous fungi but, unlike in many other human and plant pathogenic fungi, HapX of A. benhamiae is not a virulence determinant.

The iron-responsive microsomal proteome of Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen. Siderophore biosynthesis and iron acquisition are essential for virulence. Yet, limited data exist with respect to the adaptive nature of the fungal microsomal proteome under iron-limiting growth conditions, as encountered during host infection. Here, we demonstrate that under siderophore biosynthetic conditions — significantly elevated fusarinine C (FSC) and triacetylfusarinine C (TAFC) production (p<0.0001), extensive microsomal proteome remodelling occurs. Specifically, a four-fold enrichment of transmembrane-containing proteins was observed with respect to whole cell lysates following ultracentrifugation-based microsomal extraction. Comparative label-free proteomic analysis of microsomal extracts, isolated following iron-replete and -deplete growth, identified 710 unique proteins. Scatterplot analysis (MaxQuant) demonstrated high correlation amongst biological replicates from each growth condition (Pearson correlation >0.96 within groups; biological replicates (n=4)). Quantitative and qualitative comparison revealed 231 proteins with a significant change in abundance between the iron-replete and iron-deplete conditions (p<0.05, fold change ≥2), with 96 proteins showing increased abundance and 135 with decreased abundance following iron limitation, including predicted siderophore transporters. Fluorescently labelled FSC was only sequestered following A. fumigatus growth under iron-limiting conditions. Interestingly, human sera exhibited significantly increased reactivity (p<0.0001) against microsomal protein extracts obtained following iron-deplete growth. Biological significanceThe opportunistic fungal pathogen Aspergillus fumigatus must acquire iron to facilitate growth and pathogenicity. Iron-chelating non-ribosomal peptides, termed siderophores, mediate iron uptake via membrane-localised transporter proteins. Here we demonstrate for the first time that growth of A. fumigatus under iron-deplete conditions, concomitant with siderophore biosynthesis, leads to an extensive remodelling of the microsomal proteome which includes significantly altered levels of 231 constituent proteins (96 increased and 135 decreased in abundance), many of which have not previously been localised to the microsome. We also demonstrate the first synthesis of a fluorescent version of fusarinine C, an extracellular A. fumigatus siderophore, and its uptake and localization under iron-restricted conditions. This infers the use of an A. fumigatus siderophore as a ‘Trojan horse’ to potentiate the efficacy of anti-fungal drugs. Finally, in addition to revealing the Aspergillus-specific IgG reactivity in normal human sera against microsomal proteins, there appears to be a significantly increased reactivity against microsomal proteins obtained following iron-restricted growth. We hypothesise that iron-limiting environment in humans, which has evolved to nutritionally limit pathogen growth in vivo, may also alter the fungal microsomal proteome.

Abstract 4243: Iron control is a novel therapeutic target of cancer stem cells

Abstract Background: Iron overload is known to cause cancer in animal models. Several studies have shown that iron deprivation treatment has a strong anti-cancer effect. However, it is unclear whether iron deprivation treatment suppresses cancer stem cells or not. A cancer stem cell model, miPS-LLCcm, was recently epigenetically established from murine induced pluripotent stem cells (miPS cells) in Okayama University. In this model, the green fluorescent protein (GFP) and the puromycin resistant gene were inserted into the 59-bp untranslated region of the Nanog gene of miPS cells. By this procedure, undifferentiated cancer progenitor cells are recognized as GFP positive cells. We then examined the iron dependency of these cancer stem cells both in in vitro and in vivo studies and we used this model to examine the possibility of cancer stem cells by iron deprivation. Materials and Methods: In in vitro studies, the miPS-LLCcm cells were used as cancer stem cells and colon26 and 4T1 cells were used as differentiated murine cancer cells. Puromycin was used to purify the cancer stem cells before seeding of the miPS-LLCcm cells. The dependency on iron for cell proliferation was examined following transferrin addition. Transferrin (Holo) was used to simulate an iron rich condition. Iron free medium and the iron chelators, Deferasirox and Deferoxamine, plus 1% fetal bovine serum (FBS) were used to simulate iron depletion conditions. Cell proliferation assays and flow cytometric analyses were performed 48 hours after adjustment of the iron concentration level. In in vivo studies, an iron depleted diet was used to simulate iron depleted conditions. Nude mice were divided into normal diet and iron depleted diet groups. The mice were fed with these diets for three weeks and then a suspension of miPS-LLCcm cells was injected into the backs of the nude mice. Tumor size was measured and the tumors were immunohistologically examined. Results: In the in vitro studies, transferrin strongly promoted the proliferation of cancer stem cells under iron depletion conditions compared to no transferrin(p&amp;lt;0.001). However, transferrin did not promote the proliferation of the differentiated 4T1 and colon26 cancer cells. The number of GFP-expressing cancer stem cells decreased as the iron concentration was decreased. In the vivo studies, iron depletion significantly suppressed the tumor growth of the cancer stem cells (p&amp;lt;0.05). Immunohistological analysis indicated that Nanog expression was also decreased in the tumors of the iron depletion diet group. Conclusions: Iron is a key element for the proliferation and differentiation of cancer stem cells. Iron controlling therapy including iron chelators is a novel therapeutic target of cancer stem cells. Citation Format: Takayuki Ninomiya, Toshiaki Ohara, Hajime Kashima, Ryoichi Katsube, Kazuhiro Noma, Yasuko Tomono, Akifumi Mizutani, Tomonari Kasai, Masaharu Seno, Shinji Kuroda, Hiroyuki Kishimoto, Hiroshi Tazawa, Yasuhiro Shirakawa, Shunsuke Kagawa, Toshiyoshi Fujiwara. Iron control is a novel therapeutic target of cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4243. doi:10.1158/1538-7445.AM2015-4243

IscR of Rhodobacter sphaeroides functions as repressor of genes for iron-sulfur metabolism and represents a new type of iron-sulfur-binding protein.

IscR proteins are known as transcriptional regulators for Fe–S biogenesis. In the facultatively phototrophic bacterium, Rhodobacter sphaeroides IscR is the product of the first gene in the isc-suf operon. A major role of IscR in R. sphaeroides iron-dependent regulation was suggested in a bioinformatic study (Rodionov et al., PLoS Comput Biol 2:e163, 2006), which predicted a binding site in the upstream regions of several iron uptake genes, named Iron-Rhodo-box. Most known IscR proteins have Fe–S clusters featuring (Cys)3(His)1 ligation. However, IscR proteins from Rhodobacteraceae harbor only a single-Cys residue and it was considered unlikely that they can ligate an Fe–S cluster. In this study, the role of R. sphaeroides IscR as transcriptional regulator and sensor of the Fe–S cluster status of the cell was analyzed. A mutant lacking IscR is more impaired in growth under iron limitation than the wild-type and exhibits significantly increased ROS levels in iron-replete and iron-deplete conditions. Expression studies reveal that R. sphaeroides IscR in its cluster-bound form functions as transcriptional repressor of genes involved in iron metabolism by direct binding to the promoter region of genes preceded by the motif. A total of 110 genes are directly or indirectly affected by IscR. Furthermore, IscR possesses a unique Fe–S cluster ligation scheme with only a single cysteine involved.

Transcriptional response of Atlantic salmon families to Piscirickettsia salmonis infection highlights the relevance of the iron-deprivation defence system

BackgroundPiscirickettsiosis or Salmonid Rickettsial Septicaemia (SRS) is a bacterial disease that has a major economic impact on the Chilean salmon farming industry. Despite the fact that Piscirickettsia salmonis has been recognized as a major fish pathogen for over 20 years, the molecular strategies underlying the fish response to infection and the bacterial mechanisms of pathogenesis are poorly understood. We analysed and compared the head kidney transcriptional response of Atlantic salmon (Salmo salar) families with different levels of susceptibility to P. salmonis infection in order to reveal mechanisms that might confer infection resistance.ResultsWe ranked forty full-sibling Atlantic salmon families according to accumulated mortality after a challenge with P. salmonis and selected the families with the lowest and highest cumulative mortalities for microarray gene expression analysis. A comparison of the response to P. salmonis infection between low and high susceptibility groups identified biological processes presumably involved in natural resistance to the pathogen. In particular, expression changes of genes linked to cellular iron depletion, as well as low iron content and bacterial load in the head kidney of fish from low susceptibility families, suggest that iron-deprivation is an innate immunity defence mechanism against P. salmonis. To complement these results, we predicted a set of iron acquisition genes from the P. salmonis genome. Identification of putative Fur boxes and expression of the genes under iron-depleted conditions revealed that most of these genes form part of the Fur regulon of P. salmonis.ConclusionsThis study revealed, for the first time, differences in the transcriptional response to P. salmonis infection among Atlantic salmon families with varied levels of susceptibility to the infection. These differences correlated with changes in the abundance of transcripts encoding proteins directly and indirectly involved in the immune response; changes that highlighted the role of nutritional immunity through iron deprivation in host defence mechanisms against P. salmonis. Additionally, we found that P. salmonis has several mechanisms for iron acquisition, suggesting that this bacterium can obtain iron from different sources, including ferric iron through capturing endogenous and exogenous siderophores and ferrous iron. Our results contribute to determining the underlying resistance mechanisms of Atlantic salmon to P. salmonis infection and to identifying future treatment strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1716-9) contains supplementary material, which is available to authorized users.

RGS19 converts iron deprivation stress into a growth-inhibitory signal

Iron chelation is a promising therapeutic strategy for cancer that works, in part, by inducing overexpression of N-myc downstream-regulated gene 1 protein (NDRG1), a known growth inhibitor and metastasis suppressor. However, details of the signaling cascades that convert physical stress into a biological response remain elusive. We investigated the role of RGS19, a regulator of G-protein signaling, in iron chelator-induced NDRG1 overexpression in HeLa cells. Knockdown of RGS19 diminished the expression of genes involved in desferrioxamine (DFO)-induced growth inhibition. Conversely, overexpression of RGS19 enhanced the expression of these genes. Moreover, overexpression of RGS19 reduced cell viability. Overexpression of G-protein alpha subunit i3 (Gαi3) repressed the induction of NDRG1 expression. Selective inhibition of downstream targets of Gαi3 abrogated DFO-induced overexpression of NDRG1. DFO protected RGS19 from proteolysis induced by GAIP interacting protein N terminus (GIPN); moreover, an iron-deficient RGS19 mutant was stable in the presence of GIPN and retained GTPase-activating protein activity. RGS19 was co-purified with iron and showed unique UV-absorption characteristics frequently observed in iron-binding proteins. This study demonstrates that RGS19 senses cellular iron availability and is stabilized under iron-depleted conditions, resulting in the induction of a growth-inhibitory signal.

Shotgun proteome analysis of Bordetella pertussis reveals a distinct influence of iron availability on the bacterial metabolism, virulence, and defense response.

One of the mechanisms involved in host immunity is the limitation of iron accessibility to pathogens, which in turn provokes the corresponding physiological adaptation of pathogens. This study reports a gel-free nanoLC-MS/MS-based comparative proteome analysis of Bordetella pertussis grown under iron-excess and iron-depleted conditions. Out of the 926 proteins covered 98 displayed a shift in their abundance in response to low iron availability. Forty-seven of them were found to be increased in level while 58 were found with decreased protein levels under iron starvation. In addition to proteins previously reported to be influenced by iron in B. pertussis, we observed changes in metabolic proteins involved in fatty acid utilization and poly-hydroxybutyrate production. Additionally, many bacterial virulence factors regulated by the BvgAS two-component system were found at decreased levels in response to iron limitation. These results, together with the increased production of proteins potentially involved in oxidative stress resistance, seem to indicate that iron starvation provokes changes in B. pertussis phenotype that might shape host-pathogen interaction.

Mitogen-activated protein kinases are associated with the regulation of physiological traits and virulence in Fusarium oxysporum f. sp. cubense.

Fusarium oxysporum f. sp. cubense (FOC) is an important soil-borne fungal pathogen causing devastating vascular wilt disease of banana plants and has become a great concern threatening banana production worldwide. However, little information is known about the molecular mechanisms that govern the expression of virulence determinants of this important fungal pathogen. In this study, we showed that null mutation of three mitogen-activated protein (MAP) kinase genes, designated as FoSlt2, FoMkk2 and FoBck1, respectively, led to substantial attenuation in fungal virulence on banana plants. Transcriptional analysis revealed that the MAP kinase signaling pathway plays a key role in regulation of the genes encoding production of chitin, peroxidase, beauvericin and fusaric acid. Biochemical analysis further confirmed the essential role of MAP kinases in modulating the production of fusaric acid, which was a crucial phytotoxin in accelerating development of Fusarium wilt symptoms in banana plants. Additionally, we found that the MAP kinase FoSlt2 was required for siderophore biosynthesis under iron-depletion conditions. Moreover, disruption of the MAP kinase genes resulted in abnormal hypha and increased sensitivity to Congo Red, Calcofluor White and H2O2. Taken together, these results depict the critical roles of MAP kinases in regulation of FOC physiology and virulence.

Pseudomonas fluorescens: Iron-responsive proteins and their involvement in host infection

For pathogenic bacteria, the ability to acquire iron is vital to survival in the host. In consequence, many genes involved in iron acquisition are associated with bacterial virulence. Pseudomonas fluorescens is a bacterial pathogen to a variety of farmed fish. However, the global regulatory function of iron in pathogenic P. fluorescens is essentially unknown. In this study, in order to identify proteins affected by iron condition at the expression level, we performed proteomic analysis to compare the global protein profiles of P. fluorescens strain TSS, a fish pathogen, cultured under iron-replete and iron-deplete conditions. Twenty-two differentially expressed proteins were identified, most of which were confirmed to be regulated by iron at the mRNA level. To investigate their potential involvement in virulence, the genes encoding four of the 22 proteins, i.e. HemO (heme oxygenase), PspB (serine protease), Sod (superoxide dismutase), and TfeR (TonB-dependent outermembrane ferric enterobactin receptor), were knocked out, and the pathogenicity of the mutants was examined in a model of turbot (Scophthalmus maximus). The results showed that compared to the wild type, the hemO, pspB, and tfeR knockouts were significantly impaired in the ability to survive in host serum, to invade host tissues, and to cause host mortality. Immunization of turbot with recombinant TfeR (rTfeR) and PspB induced production of specific serum antibodies and significant protections against lethal TSS challenge. Further analysis showed that rTfeR antibodies recognized and bound to TSS, and that treatment of TSS with rTfeR antibodies significantly impaired the infectivity of TSS to fish cells. Taken together, these results indicate for the first time that in pathogenic P. fluorescens, iron affects the expression of a large number of proteins including those that are involved in host infection.

Regulation of Helicobacter pylori Virulence Within the Context of Iron Deficiency.

Helicobacter pylori strains that harbor the oncoprotein CagA increase gastric cancer risk, and this risk is augmented under iron-deficient conditions. We demonstrate here that iron depletion induces coccoid morphology in strains lacking cagA. To evaluate the stability of augmented H. pylori virulence phenotypes stimulated by low-iron conditions, H. pylori isolated from iron-depleted conditions in vivo were serially passaged in vitro. Long-term passage decreased the ability of hypervirulent strains to translocate CagA or induce interleukin 8, indicating that hypervirulent phenotypes stimulated by low-level iron conditions are reversible. Therefore, rectifying iron deficiency may attenuate disease among H. pylori-infected persons with no response to antibiotics.

Protein-based biofilm matrices in Staphylococci.

Staphylococcus aureus and Staphylococcus epidermidis are the most important etiological agents of biofilm associated-infections on indwelling medical devices. Biofilm infections may also develop independently of indwelling devices, e.g., in native valve endocarditis, bone tissue, and open wounds. After attachment to tissue or indwelling medical devices that have been conditioned with host plasma proteins, staphylococcal biofilms grow, and produce a specific environment which provides the conditions for cell–cell interaction and formation of multicellular communities. Bacteria living in biofilms express a variety of macromolecules, including exopolysaccharides, proteins, extracellular eDNA, and other polymers. The S. aureus surface protein C and G (SasC and SasG), clumping factor B (ClfB), serine aspartate repeat protein (SdrC), the biofilm-associated protein (Bap), and the fibronectin/fibrinogen-binding proteins (FnBPA and FnBPB) are individually implicated in biofilm matrix formation. In S. epidermidis, a protein named accumulation-associated protein (Aap) contributes to both the primary attachment phase and the establishment of intercellular connections by forming fibrils on the cell surface. In S. epidermidis, proteinaceous biofilm formation can also be mediated by the extracellular matrix binding protein (Embp) and S. epidermidis surface protein C (SesC). Additionally, multifunctional proteins such as extracellular adherence protein (Eap) and extracellular matrix protein binding protein (Emp) of S. aureus and the iron-regulated surface determinant protein C (IsdC) of S. lugdunensis can promote biofilm formation in iron-depleted conditions. This multitude of proteins intervene at different stages of biofilm formation with certain proteins contributing to biofilm accumulation and others mediating primary attachment to surfaces. This review examines the contribution of proteins to biofilm formation in Staphylococci. The potential to develop vaccines to prevent protein-dependent biofilm formation during staphylococcal infection is discussed.

IscR regulation of capsular polysaccharide biosynthesis and iron-acquisition systems in Klebsiella pneumoniae CG43.

IscR, an Fe–S cluster-containing transcriptional factor, regulates genes involved in various cellular processes. In response to environmental stimuli such as oxidative stress and iron levels, IscR switches between its holo and apo forms to regulate various targets. IscR binding sequences are classified into two types: the type 1 IscR box that is specific for holo-IscR binding, and the type 2 IscR box that binds holo- and apo-IscR. Studying Klebsiella pneumoniae CG43S3, we have previously shown that iron availability regulates capsular polysaccharide (CPS) biosynthesis and iron-acquisition systems. The present study investigated whether IscR is involved in this regulation. Compared with that in CG43S3, the amount of CPS was decreased in AP001 (ΔiscR) or AP002 (iscR 3CA), a CG43S3-derived strain expressing mutated IscR mimicked apo-IscR, suggesting that only holo-IscR activates CPS biosynthesis. Furthermore, a promoter-reporter assay verified that the transcription of cps genes was reduced in AP001 and AP002. Purified IscR::His6, but not IscR3CA::His6, was also found to bind the predicted type 1 IscR box specifically in the cps promoter. Furthermore, reduced siderophore production was observed in AP004 (Δfur-ΔiscR) but not in AP005 (Δfur-iscR 3CA), implying that apo-IscR activates iron acquisition. Compared with those in AP004, mRNA levels of three putative iron acquisition systems (fhu, iuc, and sit) were increased in AP005, and both purified IscR::His6 and IscR3CA::His6 bound the predicted type 2 IscR box in the fhuA, iucA, and sitA promoters, whereas IscR3CA::His6 displayed a lower affinity. Finally, we analyzed the effect of external iron levels on iscR expression. The transcription of iscR was increased under iron-depleted conditions as well as in AP001 and AP002, suggesting an auto-repression exerted by apo-IscR. Our results show that in K. pneumoniae, IscR plays a dual role in the regulation of CPS biosynthesis and iron-acquisition systems in response to environmental iron availability.

Iron- and hemin-dependent gene expression of Porphyromonas gingivalis.

Although iron under anaerobic conditions is more accessible and highly reactive because of its reduced form, iron-dependent regulation is not well known in anaerobic bacteria. Here, we investigated iron- and hemin-dependent gene regulation in Porphyromonas gingivalis, an established periodontopathogen that primarily inhabits anaerobic pockets. Whole-genome microarrays of P.gingivalis genes were used to compare the levels of gene expression under iron-replete and iron-depleted conditions as well as under hemin-replete and hemin-depleted conditions. Under iron-depleted conditions, the expression of genes encoding proteins that participate in iron uptake and adhesion/invasion of host cells was increased, while that of genes encoding proteins involved in iron storage, energy metabolism, and electron transport was decreased. Interestingly, many of the genes with altered expression had no known function. Limiting the amount of hemin also resulted in a reduced expression of the genes encoding proteins involved in energy metabolism and electron transport. However, hemin also had a significant effect on many other biological processes such as oxidative stress protection and lipopolysaccharide synthesis. Overall, comparison of the data from iron-depleted conditions to those from hemin-depleted ones showed that although some regulation is through the iron derived from hemin, there also is significant distinct regulation through hemin only. Furthermore, our data showed that the molecular mechanisms of iron-dependent regulation are novel as the deletion of the putative Fur protein had no effect on the expression of iron-regulated genes. Finally, our functional studies demonstrated greater survivability of host cells in the presence of the iron-stressed bacterium than the iron-replete P.gingivalis cells. The major iron-regulated proteins encoded by PG1019-20 may play a role in this process as deletion of these sequences also resulted in reduced survival of the bacterium when grown with eukaryotic cells. Taken together, the results of this study demonstrated the utility of whole-genome microarray analysis for the identification of genes with altered expression profiles during varying growth conditions and provided a framework for the detailed analysis of the molecular mechanisms of iron and hemin acquisition, metabolism and virulence of P.gingivalis.

Alcanivorax borkumensis produces an extracellular siderophore in iron-limitation condition maintaining the hydrocarbon-degradation efficiency

Obligate marine hydrocarbonoclastic bacteria possess genetic and physiological features to use hydrocarbons as sole source of carbon and to compete for the uptake of nutrients in usually nutrient-depleted marine habitats. In the present work we have studied the siderophore-based iron uptake systems in Alcanivorax borkumensis SK2 and their functioning during biodegradation of an aliphatic hydrocarbon, tetradecane, under iron limitation conditions. The antiSMASH analysis of SK2 genome revealed the presence of two different putative operons of siderophore synthetases. Search for the predicted core structures indicated that one siderophore is clearly affiliated to the family of complex oligopeptidic siderophores possessing an Orn-Ser-Orn carboxyl motif whereas the second one is likely to belong to the family of SA (salicylic acid)–based siderophores.Analyzing the supernatant of SK2 culture, an extracellular siderophore was identified and its structure was resolved. Thus, along with the recently described membrane-associated amphiphilic tetrapeptidic siderophore amphibactin, strain SK2 additionally produces an extracellular type of iron-chelating molecule with structural similarity to pseudomonins. Comparative Q-PCR analysis of siderophore synthetases demonstrated their significant up-regulation in iron-depleted medium. Different expression patterns were recorded for two operons during the early and late exponential phases of growth, suggesting a different function of these two siderophores under iron-depleted conditions.

Metachelins, mannosylated and N-oxidized coprogen-type siderophores from Metarhizium robertsii.

Under iron-depleted culture conditions, the entomopathogenic fungus Metarhizium robertsii (Bischoff, Humber, and Rehner) (= M. anisopliae) produces a complex of extracellular siderophores including novel O-glycosylated and N-oxidized coprogen-type compounds as well as the known fungal siderophores N(α)-dimethylcoprogen (NADC) and dimerumic acid (DA). Metachelin A (1), the most abundant component in the M. robertsii siderophore mixture, was characterized as a 1094 Da analogue of NADC that is O-glycosylated by β-mannose at both terminal hydroxyl groups and N-oxidized at the dimethylated α-nitrogen. The mixture also contained a 1078 Da analogue, metachelin B (2), which lacks the N-oxide modification. Also characterized were the aglycone of 1, i.e., the N-oxide of NADC (3), and the monomannoside of DA (6). N-Oxide and O-glycosyl substituents are unprecedented among microbial siderophores. At high ESIMS source energy and at room temperature in DMSO, 1 underwent Cope elimination, resulting in loss of the N(α)-dimethyl group and dehydration of the α-β bond. High-resolution ESIMS data confirmed that all tri- and dihydroxamate siderophores (1-6) complex with trivalent Fe, Al, and Ga. In a chrome azurol S assay, all of the M. robertsii siderophores showed iron-binding activity roughly equivalent to that of desferrioxamine B.

Mycobacterium tuberculosis Rv3402c enhances mycobacterial survival within macrophages and modulates the host pro-inflammatory cytokines production via NF-kappa B/ERK/p38 signaling.

Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis, a process which depends on an array of virulence factors to colonize and replicate within the host. The M. tuberculosis iron regulated open reading frame (ORF) rv3402c, encoding a conserved hypothetical protein, was shown to be up-regulated upon infection in both human and mice macrophages. To explore the function of this ORF, we heterologously expressed the rv3402c gene in the non-pathogenic fast-growing Mycobacterium smegmatis strain, and demonstrated that Rv3402c, a cell envelope-associated protein, was able to enhance the intracellular survival of recombinant M. smegmatis. Enhanced growth was not found to be the result of an increased resistance to intracellular stresses, as growth of the Rv3402c expressing strain was unaffected by iron depletion, H2O2 exposure, or acidic conditions. Colonization of macrophages by M. smegmatis expressing Rv3402c was associated with substantial cell death and significantly greater amount of TNF-α and IL-1β compared with controls. Rv3402c-induced TNF-α and IL-1β production was found to be mediated by NF-κB, ERK and p38 pathway in macrophages. In summary, our study suggests that Rv3402c delivered in a live M. smegmatis vehicle can modify the cytokines profile of macrophage, promote host cell death and enhance the persistence of mycobacterium within host cells.

Unravelling the cross-talk between iron starvation and oxidative stress responses highlights the key role of PerR (alr0957) in peroxide signalling in the cyanobacterium Nostoc PCC 7120.

The cyanobacterial phylum includes oxygenic photosynthetic prokaryotes of a wide variety of morphologies, metabolisms and ecologies. Their adaptation to their various ecological niches is mainly achieved by sophisticated regulatory mechanisms and depends on a fine cross-talk between them. We assessed the global transcriptomic response of the filamentous cyanobacterium Nostoc PCC 7120 to iron starvation and oxidative stress. More than 20% of the differentially expressed genes in response to iron stress were also responsive to oxidative stress. These transcripts include antioxidant proteins-encoding genes that confirms that iron depletion leads to reactive oxygen accumulation. The activity of the Fe-superoxide dismutase was not significantly decreased under iron starvation, indicating that the oxidative stress generated under iron deficiency is not a consequence of (SOD) deficiency. The transcriptional data indicate that the adaptation of Nostoc to iron-depleted conditions displays important differences with what has been shown in unicellular cyanobacteria. While the FurA protein that regulates the response to iron deprivation has been well characterized in Nostoc, the regulators in charge of the oxidative stress response are unknown. Our study indicates that the alr0957 (perR) gene encodes the master regulator of the peroxide stress. PerR is a peroxide-sensor repressor that senses peroxide by metal-catalysed oxidation.