FBXL5 Regulates IRP2 Stability in Iron Homeostasis via an Oxygen-Responsive [2Fe2S] Cluster.

Cellular iron homeostasis is dominated by FBXL5-mediated degradation of iron regulatory protein 2 (IRP2), which is dependent on both iron and oxygen. However, how the physical interaction between FBXL5 and IRP2 is regulated remains elusive. Here, we show that the C-terminal substrate-binding domain of FBXL5 harbors a [2Fe2S] cluster in the oxidized state. A cryoelectron microscopy (cryo-EM) structure of the IRP2-FBXL5-SKP1 complex reveals that the cluster organizes the FBXL5 C-terminal loop responsible for recruiting IRP2. Interestingly, IRP2 binding to FBXL5 hinges on the oxidized state of the [2Fe2S] cluster maintained by ambient oxygen, which could explain hypoxia-induced IRP2 stabilization. Steric incompatibility also allows FBXL5 to physically dislodge IRP2 from iron-responsive element RNA to facilitate its turnover. Taken together, our studies have identified an iron-sulfur cluster within FBXL5, which promotes IRP2 polyubiquitination and degradation in response to both iron and oxygen concentrations.

An Oxygen-Dependent Interaction between FBXL5 and the CIA-Targeting Complex Regulates Iron Homeostasis.

The iron-sensing protein FBXL5 is the substrate adaptor for a SKP1-CUL1-RBX1 E3 ubiquitin ligase complex that regulates the degradation of iron regulatory proteins (IRPs). Here, we describe a mechanism of FBXL5 regulation involving its interaction with the cytosolic Fe-S cluster assembly (CIA) targeting complex composed of MMS19, FAM96B, and CIAO1. We demonstrate that the CIA-targeting complex promotes the ability of FBXL5 to degrade IRPs. In addition, the FBXL5-CIA-targeting complex interaction is regulated by oxygen (O2) tension displaying a robust association in 21% O2 that is severely diminished in 1% O2 and contributes to O2-dependent regulation of IRP degradation. Together, these data identify a novel oxygen-dependent signaling axis that links IRP-dependent iron homeostasis with the Fe-S cluster assembly machinery.

Iron regulatory protein 2 contributes to antimicrobial immunity by preserving lysosomal function in macrophages.

Colorectal cancer and Crohn's disease patients develop pyogenic liver abscesses due to failures of immune cells to fight off bacterial infections. Here, we show that mice lacking iron regulatory protein 2 (Irp2), globally (Irp2-/-) or myeloid cell lineage (Lysozyme 2 promoter-driven, LysM)-specifically (Irp2ΔLysM), are highly susceptible to liver abscesses when the intestinal tissue was injured with dextran sodium sulfate treatment. Further studies demonstrated that Irp2 is required for lysosomal acidification and biogenesis, both of which are crucial for bacterial clearance. In Irp2-deficient liver tissue or macrophages, the nuclear location of transcription factor EB (Tfeb) was remarkably reduced, leading to the downregulation of Tfeb target genes that encode critical components for lysosomal biogenesis. Tfeb mislocalization was reversed by hypoxia-inducible factor 2 inhibitor PT2385 and, independently, through inhibition of lactic acid production. These experimental findings were confirmed clinically in patients with Crohn's disease and through bioinformatic searches in databases from Crohn's disease or ulcerative colitis biopsies showing loss of IRP2 and transcription factor EB (TFEB)-dependent lysosomal gene expression. Overall, our study highlights a mechanism whereby Irp2 supports nuclear translocation of Tfeb and lysosomal function, preserving macrophage antimicrobial activity and protecting the liver against invading bacteria during intestinal inflammation.

Iron regulatory protein two facilitates ferritinophagy and DNA damage/repair through guiding ATG9A trafficking.

Trace elemental iron is an essential nutrient that participates in diverse metabolic processes. Dysregulation of cellular iron homeostasis, both iron deficiency and iron overload, is detrimental and tightly associated with disease pathogenesis. IRPs-IREs system is located at the center for iron homeostasis regulation. Additionally, ferritinophagy, the autophagy-dependent ferritin catabolism for iron recycling, is emerging as a novel mechanism for iron homeostasis regulation. It is still unclear whether IRPs-IREs system and ferritinophagy are synergistic or redundant in determining iron homeostasis. Here we report that IRP2, but not IRP1, is indispensable for ferritinophagy in response to iron depletion. Mechanistically, IRP2 ablation results in compromised AMPK activation and defective ATG9A endosomal trafficking, leading to the decreased engulfment of NCOA4-ferritin complex by endosomes and the subsequent dysregulated endosomal microferritinophagy. Moreover, this defective endosomal microferritinophagy exacerbates DNA damage and reduces colony formation in IRP2-depleted cells. Collectively, this study expands the physiological function of IRP2 in endosomal microferritinophagy and highlights potential crosstalk between IRPs-IREs and ferritinophagy in manipulating iron homeostasis.

Ferredoxin reductase is critical for p53-dependent tumor suppression via iron regulatory protein 2.

Ferredoxin reductase (FDXR), a target of p53, modulates p53-dependent apoptosis and is necessary for steroidogenesis and biogenesis of iron-sulfur clusters. To determine the biological function of FDXR, we generated a Fdxr-deficient mouse model and found that loss of Fdxr led to embryonic lethality potentially due to iron overload in developing embryos. Interestingly, mice heterozygous in Fdxr had a short life span and were prone to spontaneous tumors and liver abnormalities, including steatosis, hepatitis, and hepatocellular carcinoma. We also found that FDXR was necessary for mitochondrial iron homeostasis and proper expression of several master regulators of iron metabolism, including iron regulatory protein 2 (IRP2). Surprisingly, we found that p53 mRNA translation was suppressed by FDXR deficiency via IRP2. Moreover, we found that the signal from FDXR to iron homeostasis and the p53 pathway was transduced by ferredoxin 2, a substrate of FDXR. Finally, we found that p53 played a role in iron homeostasis and was required for FDXR-mediated iron metabolism. Together, we conclude that FDXR and p53 are mutually regulated and that the FDXR-p53 loop is critical for tumor suppression via iron homeostasis.

Using Biotinylated Iron-Responsive Element to Analyze the Activity of Iron Regulatory Proteins.

Iron regulatory proteins (IRP1 and IRP2) are the master regulators of mammalian iron homeostasis. They bind to the iron-responsive elements (IREs) of the transcripts of iron-related genes to regulate their expression, thereby maintaining cellular iron availability. The primary method to measure the IRE-binding activity of IRPs is the electrophoresis mobility shift assay (EMSA). This method is particularly useful for evaluating IRP1 activity, since IRP1 is a bifunctional enzyme and its protein levels remain similar during conversion between the IRE-binding protein and cytosolic aconitase forms. Here, we exploited a method of using a biotinylated-IRE probe to separate IRE-binding IRPs followed by immunoblotting to analyze the IRE-binding activity. This method allows for the successful measurement of IRP activity in cultured cells and mouse tissues under various iron conditions. By separating IRE-binding IRPs from the rest of the lysates, this method increases the specificity of IRP antibodies and verifies whether a band represents an IRP, thereby revealing some previously unrecognized information about IRPs. With this method, we showed that the S711-phosphorylated IRP1 was found only in the IRE-binding form in PMA-treated Hep3B cells. Second, we found a truncated IRE-binding IRP2 isoform that is generated by proteolytic cleavage on sites in the 73aa insert region of the IRP2 protein. Third, we found that higher levels of SDS, compared to 1-2% SDS in regular loading buffer, could dramatically increase the band intensity of IRPs in immunoblots, especially in HL-60 cells. Fourth, we found that the addition of SDS or LDS to cell lysates activated protein degradation at 37 °C or room temperature, especially in HL-60 cell lysates. As this method is more practical, sensitive, and cost-effective, we believe that its application will enhance future research on iron regulation and metabolism.

ß-catenin-IRP2-primed iron availability to mitochondrial metabolism is druggable for active ß-catenin-mediated cancer.

BACKGROUND: Although ß-catenin signaling cascade is frequently altered in human cancers, targeting this pathway has not been approved for cancer treatment. METHODS: High-throughput screening of an FDA-approved drug library was conducted to identify therapeutics that selectively inhibited the cells with activated ß-catenin. Efficacy of iron chelator and mitochondrial inhibitor was evaluated for suppression of cell proliferation and tumorigenesis. Cellular chelatable iron levels were measured to gain insight into the potential vulnerability of ß-catenin-activated cells to iron deprivation. Extracellular flux analysis of mitochondrial function was conducted to evaluate the downstream events of iron deprivation. Chromatin immunoprecipitation, real-time quantitative PCR and immunoblotting were performed to identify ß-catenin targets. Depletion of iron-regulatory protein 2 (IRP2), a key regulator of cellular iron homeostasis, was carried out to elucidate its significance in ß-catenin-activated cells. Online databases were analyzed for correlation between ß-catenin activity and IRP2-TfR1 axis in human cancers. RESULTS: Iron chelators were identified as selective inhibitors against ß-catenin-activated cells. Deferoxamine mesylate, an iron chelator, preferentially repressed ß-catenin-activated cell proliferation and tumor formation in mice. Mechanically, ß-catenin stimulated the transcription of IRP2 to increase labile iron level. Depletion of IRP2-sequered iron impaired ß-catenin-invigorated mitochondrial function. Moreover, mitochondrial inhibitor S-Gboxin selectively reduced ß-catenin-associated cell viability and tumor formation. CONCLUSIONS: ß-catenin/IRP2/iron stimulation of mitochondrial energetics is targetable vulnerability of ß-catenin-potentiated cancer.

Optimization of Biotinylated RNA or DNA Pull-Down Assays for Detection of Binding Proteins: Examples of IRP1, IRP2, HuR, AUF1, and Nrf2.

Investigation of RNA- and DNA-binding proteins to a defined regulatory sequence, such as an AU-rich RNA and a DNA enhancer element, is important for understanding gene regulation through their interactions. For in vitro binding studies, an electrophoretic mobility shift assay (EMSA) was widely used in the past. In line with the trend toward using non-radioactive materials in various bioassays, end-labeled biotinylated RNA and DNA oligonucleotides can be more practical probes to study protein-RNA and protein-DNA interactions; thereby, the binding complexes can be pulled down with streptavidin-conjugated resins and identified by Western blotting. However, setting up RNA and DNA pull-down assays with biotinylated probes in optimum protein binding conditions remains challenging. Here, we demonstrate the step-by step optimization of pull-down for IRP (iron-responsive-element-binding protein) with a 5'-biotinylated stem-loop IRE (iron-responsive element) RNA, HuR, and AUF1 with an AU-rich RNA element and Nrf2 binding to an antioxidant-responsive element (ARE) enhancer in the human ferritin H gene. This study was designed to address key technical questions in RNA and DNA pull-down assays: (1) how much RNA and DNA probes we should use; (2) what binding buffer and cell lysis buffer we can use; (3) how to verify the specific interaction; (4) what streptavidin resin (agarose or magnetic beads) works; and (5) what Western blotting results we can expect from varying to optimum conditions. We anticipate that our optimized pull-down conditions can be applicable to other RNA- and DNA-binding proteins along with emerging non-coding small RNA-binding proteins for their in vitro characterization.

Managing the Dual Nature of Iron to Preserve Health.

Because of its peculiar redox properties, iron is an essential element in living organisms, being involved in crucial biochemical processes such as oxygen transport, energy production, DNA metabolism, and many others. However, its propensity to accept or donate electrons makes it potentially highly toxic when present in excess and inadequately buffered, as it can generate reactive oxygen species. For this reason, several mechanisms evolved to prevent both iron overload and iron deficiency. At the cellular level, iron regulatory proteins, sensors of intracellular iron levels, and post-transcriptional modifications regulate the expression and translation of genes encoding proteins that modulate the uptake, storage, utilization, and export of iron. At the systemic level, the liver controls body iron levels by producing hepcidin, a peptide hormone that reduces the amount of iron entering the bloodstream by blocking the function of ferroportin, the sole iron exporter in mammals. The regulation of hepcidin occurs through the integration of multiple signals, primarily iron, inflammation and infection, and erythropoiesis. These signals modulate hepcidin levels by accessory proteins such as the hemochromatosis proteins hemojuvelin, HFE, and transferrin receptor 2, the serine protease TMPRSS6, the proinflammatory cytokine IL6, and the erythroid regulator Erythroferrone. The deregulation of the hepcidin/ferroportin axis is the central pathogenic mechanism of diseases characterized by iron overload, such as hemochromatosis and iron-loading anemias, or by iron deficiency, such as IRIDA and anemia of inflammation. Understanding the basic mechanisms involved in the regulation of hepcidin will help in identifying new therapeutic targets to treat these disorders.

Mdm2 is a target and mediator of IRP2 in cell growth control.

Iron is an essential element to all living organisms and plays a vital role in many cellular processes, such as DNA synthesis and energy production. The Mdm2 oncogene is an E3 ligase and known to promote tumor growth. However, the role of Mdm2 in iron homeostasis is not certain. Here, we showed that Mdm2 expression was increased by iron depletion but decreased by iron repletion. We also showed that Iron Regulatory Protein 2 (IRP2) mediated iron-regulated Mdm2 expression. Specifically, Mdm2 expression was increased by ectopic IRP2 but decreased by knockdown or knockout of IRP2 in human cancer cells as well as in mouse embryonic fibroblasts. In addition, we showed that IRP2-regulated Mdm2 expression was independent of tumor suppressor p53. Mechanistically, we found that IRP2 stabilized Mdm2 transcript via binding to an iron response element (IRE) in the 3'UTR of Mdm2 mRNA. Finally, we showed that Mdm2 is required for IRP2-mediated cell proliferation and Mdm2 expression is highly associated with IRP2 in both the normal and cancerous liver tissues. Together, we uncover a novel regulation of Mdm2 by IRP2 via mRNA stability and that the IRP2-Mdm2 axis may play a critical role in cell growth.

Regulatory Connections between Iron and Glucose Metabolism.

Iron is essential for energy metabolism, and states of iron deficiency or excess are detrimental for organisms and cells. Therefore, iron and carbohydrate metabolism are tightly regulated. Serum iron and glucose levels are subjected to hormonal regulation by hepcidin and insulin, respectively. Hepcidin is a liver-derived peptide hormone that inactivates the iron exporter ferroportin in target cells, thereby limiting iron efflux to the bloodstream. Insulin is a protein hormone secreted from pancreatic ß-cells that stimulates glucose uptake and metabolism via insulin receptor signaling. There is increasing evidence that systemic, but also cellular iron and glucose metabolic pathways are interconnected. This review article presents relevant data derived primarily from mouse models and biochemical studies. In addition, it discusses iron and glucose metabolism in the context of human disease.

Iron regulatory protein 2 deficiency may correlate with insulin resistance.

Iron is known to be a crucial regulator of glucose, and several studies have demonstrated that iron overload is one of the risk factors for insulin resistance and diabetes; however, the mechanism has not yet been clarified. To investigate the effect of iron overload on glucose metabolism and the underlying mechanism, Irp2 knockout (Irp2-/-) mice (endogenous iron overload model) were used. We found that Irp2-/- mice exhibited hyperglycemia and iron overload in the liver and skeletal muscle. Increased MDA, decreased SOD levels, and increased cell apoptosis were also found in the liver and muscle of Irp2-/- mice. Glucose concentrations were significantly higher in Irp2-/- mice in insulin tolerance tests. However, early-phase insulin secretion was not altered in Irp2-/- mice. The expression of hepatic IRS2 and muscle GLUT4 was declined in Irp2-/- mice at both mRNA and protein levels when compared with those of wild-type control. In conclusions, Irp2-/- mice showed hyperglycemia, which might due to insulin resistance rather than due to impaired insulin secretion.

Leishmania donovani inhibits ferroportin translation by modulating FBXL5-IRP2 axis for its growth within host macrophages.

Hepcidin mediated ferroportin (Fpn) degradation in macrophages is a well adopted strategy to limit iron availability towards invading pathogens. Leishmania donovani (LD), a protozoan parasite, resides within macrophage and competes with host for availing iron. Using in vitro and in vivo model of infection, we reveal that LD decreases Fpn abundance in host macrophages by hepcidin independent mechanism. Unaffected level of Fpn-FLAG in LD infected J774 macrophage confirms that Fpn down-regulation is not due its degradation. While increased Fpn mRNA but decreased protein expression in macrophages suggests blocking of Fpn translation by LD infection that is confirmed by 35 S-methionine labelling assay. We further reveal that LD blocks Fpn translation by induced binding of iron regulatory proteins (IRPs) to the iron responsive element present in its 5'UTR. Supershift analysis provides evidence of involvement of IRP2 particularly during in vivo infection. Accordingly, a significant increase in IRP2 protein expression with simultaneous decrease in its stability regulator F-box and leucine-rich repeat Protein 5 (FBXL5) is detected in splenocytes of LD-infected mice. Increased intracellular growth due to compromised expressions of Fpn and FBXL5 by specific siRNAs reveals that LD uses a novel strategy of manipulating IRP2-FBXL5 axis to inhibit host Fpn expression.

The indispensable role of mammalian iron sulfur proteins in function and regulation of multiple diverse metabolic pathways.

In recent years, iron sulfur (Fe-S) proteins have been identified as key players in mammalian metabolism, ranging from long-known roles in the respiratory complexes and the citric acid cycle, to more recently recognized roles in RNA and DNA metabolism. Fe-S cofactors have often been missed because of their intrinsic lability and oxygen sensitivity. More Fe-S proteins have now been identified owing to detection of their direct interactions with components of the Fe-S biogenesis machinery, and through use of informatics to detect a motif that binds the co-chaperone responsible for transferring nascent Fe-S clusters to domains of recipient proteins. Dissection of the molecular steps involved in Fe-S transfer to Fe-S proteins has revealed that direct and shielded transfer occurs through highly conserved pathways that operate in parallel in the mitochondrial matrix and in the cytosolic/nuclear compartments of eukaryotic cells. Because Fe-S clusters have the unusual ability to accept or donate single electrons in chemical reactions, their presence renders complex chemical reactions possible. In addition, Fe-S clusters may function as sensors that interconnect activity of metabolic pathways with cellular redox status. Presence in pathways that control growth and division may enable cells to regulate their growth according to sufficiency of energy stores represented by redox capacity, and oxidation of such proteins could diminish anabolic activities to give cells an opportunity to restore energy supplies. This review will discuss mechanisms of Fe-S biogenesis and delivery, and methods that will likely reveal important roles of Fe-S proteins in proteins not yet recognized as Fe-S proteins.

A synergistic role of IRP1 and FBXL5 proteins in coordinating iron metabolism during cell proliferation.

Iron-regulatory protein 1 (IRP1) belongs to a family of RNA-binding proteins that modulate metazoan iron metabolism. Multiple mechanisms are employed to control the action of IRP1 in dictating changes in the uptake and metabolic fate of iron. Inactivation of IRP1 RNA binding by iron primarily involves insertion of a [4Fe-4S] cluster by the cytosolic iron-sulfur cluster assembly (CIA) system, converting it into cytosolic aconitase (c-acon), but can also involve iron-mediated degradation of IRP1 by the E3 ligase FBXL5 that also targets IRP2. How CIA and FBXL5 collaborate to maintain cellular iron homeostasis through IRP1 and other pathways is poorly understood. Because impaired Fe-S cluster biogenesis associates with human disease, we determined the importance of FBXL5 for regulating IRP1 when CIA is impaired. Suppression of FBXL5 expression coupled with induction of an IRP1 mutant (IRP13C>3S) that cannot insert the Fe-S cluster, or along with knockdown of the CIA factors NUBP2 or FAM96A, reduced cell viability. Iron supplementation reversed this growth defect and was associated with FBXL5-dependent polyubiquitination of IRP1. Phosphorylation of IRP1 at Ser-138 increased when CIA was inhibited and was required for iron rescue. Impaired CIA activity, as noted by reduced c-acon activity, was associated with enhanced FBXL5 expression and a concomitant reduction in IRP1 and IRP2 protein level and RNA-binding activity. Conversely, expression of either IRP induced FBXL5 protein level, demonstrating a negative feedback loop limiting excessive accumulation of iron-response element RNA-binding activity, whose disruption reduces cell growth. We conclude that a regulatory circuit involving FBXL5 and CIA acts through both IRPs to control iron metabolism and promote optimal cell growth.

Hypobaric Hypoxia Regulates Brain Iron Homeostasis in Rats.

Disruption of iron homeostasis in brain has been found to be closely involved in several neurodegenerative diseases. Recent studies have reported that appropriate intermittent hypobaric hypoxia played a protective role in brain injury caused by acute hypoxia. However, the mechanisms of this protective effect have not been fully understood. In this study, Sprague-Dawley (SD) rat models were developed by hypobaric hypoxia treatment in an altitude chamber, and the iron level and iron related protein levels were determined in rat brain after 4 weeks of treatment. We found that the iron levels significantly decreased in the cortex and hippocampus of rat brain as compared to that of the control rats without hypobaric hypoxia treatment. The expression levels of iron storage protein L-ferritin and iron transport proteins, including transferrin receptor-1 (TfR1), divalent metal transporter 1 (DMT1), and ferroportin1 (FPN1), were also altered. Further studies found that the iron regulatory protein 2 (IRP2) played a dominant regulatory role in the changes of iron hemostasis, whereas iron regulatory protein 1 (IRP1) mainly acted as cis-aconitase. These results, for the first time, showed the alteration of iron metabolism during hypobaric hypoxia in rat models, which link the potential neuroprotective role of hypobaric hypoxia treatment to the decreased iron level in brain. This may provide insight into the treatment of iron-overloaded neurodegenerative diseases. J. Cell. Biochem. 118: 1596-1605, 2017. © 2016 Wiley Periodicals, Inc.

Iron regulatory protein 2 in ovarian endometrial cysts.

Ovarian endometrial cysts cause some kinds of ovarian cancer, and iron is considered as one factor of carcinogenesis. In contrast, hypoxia is associated with progression, angiogenesis, metastasis, and resistance to therapy in cancer. We investigated hypoxia-induced perturbation of iron homeostasis in terms of labile iron, iron deposition, and iron regulatory protein (IRP) in ovarian endometrial cysts. Iron deposition, expression of IRPs, and a protein marker of hypoxia in human ovarian endometrial cysts were analyzed histologically. The concentration of free iron and the pO2 level of the cyst fluid of human ovarian cysts (n = 9) were measured. The expression of IRP2 under hypoxia was investigated in vitro by using Ishikawa cells as a model of endometrial cells. Iron deposition and the expression of IRP2 and Carbonic anhydrase 9 (CA9) were strong in endometrial stromal cells in the human ovarian endometrial cysts. The average concentration of free iron in the cyst fluid was 8.1 ± 2.9 mg/L, and the pO2 was 22.4 ± 5.2 mmHg. A cell-based study using Ishikawa cells revealed that IRP2 expression was decreased by an overload of Fe(II) under normoxia but remained unchanged under hypoxia even in the presence of excess Fe(II). An increase in the expression of IRP2 caused upregulation of intracellular iron as a result of the response to iron deficiency, whereas the protein was degraded under iron-rich conditions. We found that iron-rich regions existed in ovarian endometrial cysts concomitantly with the high level of IRP2 expression, which should generally be decomposed upon an overload of iron. We revealed that an insufficient level of oxygen in the cysts is the main factor for the unusual stabilization of IRP2 against iron-mediated degradation, which provides aberrant uptake of iron in ovarian endometrial stromal cells and can potentially lead to carcinogenesis.

Systemic iron homeostasis and erythropoiesis.

Iron is an essential nutrient that is potentially toxic due to its redox reactivity. Insufficient iron supply to erythroid cells, the major iron consumers in the body, leads to various forms of anemia. On the other hand, iron overload (hemochromatosis) is associated with tissue damage and diseases of liver, pancreas, and heart. Physiological iron balance is tightly controlled at the cellular and systemic level by iron regulatory proteins (IRP1, IRP2) and the iron regulatory hormone hepcidin, respectively. Underlying mechanisms often intersect to achieve optimal iron utilization, to control immune responses, and to prevent iron toxicity. This review focuses on systemic iron homeostasis in the context of erythropoiesis, a highly iron-demanding process. We discuss the function and regulation of hepcidin by various stimuli, and highlight hepcidin-dependent and -independent mechanisms that link iron utilization with maturation of erythroid progenitor cells. © 2017 IUBMB Life, 69(6):399-413, 2017.

Frequency of iss and irp2 genes by PCR method in Escherichia coli isolated from poultry with colibacillosis in comparison with healthy chicken in poultry farms of Zabol, South East of Iran.

There is no special trait for differentiation of Avian Pathogenic Escherichia coli from Avian Fecal Escherichia coli. This investigation is aimed, as a case control study, to evaluate and compare the frequency of iss and irp2 in 43 AFEC strains and also 40 and 56 E. coli strains isolated from the liver and kidney of chickens with colibacillosis, respectively, farmed in Zabol, as a border region of Iran, by PCR. 86.9% and 37.2% of isolates collected from chickens with colibacillosis and feces samples obtained from healthy chickens were positive for iss gene, respectively (P<0.05). On average, 59.3% of E. coli strains isolated from colibacillosis have irp2 gene while 27.9% of isolates from the feces of healthy birds were positive (P<0.05). 52.15% of isolates from colibacillosis and 19.62% of isolates from healthy chicken feces were positive for both genes, with statistical significant difference (p<0.05). This marked difference in the distribution of iss and irp2 genes makes these two genes good markers to differentiate AFEC and APEC strains especially in Sistan region to improve colibacillosis control measurements.

Occurrence and analysis of irp2 virulence gene in isolates of Klebsiella pneumoniae and Enterobacter spp. from microbiota and hospital and community-acquired infections.

Eighty-five isolates of Klebsiella pneumoniae and Enterobacter spp., originating from hospital- and community-acquired infections and from oropharyngeal and faecal microbiota from patients in Recife-PE, Brazil, were analyzed regarding the presence of irp2 gene. This is a Yersinia typical gene involved in the synthesis of siderophore yersiniabactin. DNA sequencing confirmed the identity of irp2 gene in five K. pneumoniae, five Enterobacter aerogenes and one Enterobacter amnigenus isolates. To our knowledge in the current literature, this is the first report of the irp2 gene in E. amnigenus, a species considered an unusual human pathogen, and in K. pneumoniae and E. aerogenes isolates from the normal microbiota and from community infections, respectively. Additionally, the analyses of nucleotide and amino acid sequences suggest the irp2 genes derived from isolates used in this study are more closely related to that of Yersinia pestis P.CE882 than to that of Yersinia enterocolitica 8081. These data demonstrated that K. pneumoniae and Enterobacter spp. from normal microbiota and from community- and hospital-acquired infections possess virulence factors important for the establishment of extra-intestinal infections.