Deep-Sea Coral Garden Invertebrates and Their Associated Fungi Are Genetic Resources for Chronic Disease Drug Discovery.

Chronic diseases characterized by bone and cartilage loss are associated with a reduced ability of progenitor cells to regenerate new tissues in an inflammatory environment. A promising strategy to treat such diseases is based on tissue repair mediated by human mesenchymal stem cells (hMSCs), but therapeutic outcomes are hindered by the absence of small molecules to efficiently modulate cell behaviour. Here, we applied a high-throughput drug screening technology to bioprospect a large library of extracts from Irish deep-sea organisms to induce hMSC differentiation toward musculoskeletal lineages and reduce inflammation of activated macrophages. The library included extracts from deep-sea corals, sponges and filamentous fungi representing a novel source of compounds for the targeted bioactivity. A validated hit rate of 3.4% was recorded from the invertebrate library, with cold water sea pens (octocoral order Pennatulacea), such as Kophobelemnon sp. and Anthoptilum sp., showing the most promising results in influencing stem cell differentiation toward osteogenic and chondrogenic lineages. Extracts obtained from deep-sea fungi showed no effects on stem cell differentiation, but a 6.8% hit rate in reducing the inflammation of activated macrophages. Our results demonstrate the potential of deep-sea organisms to synthetize pro-differentiation and immunomodulatory compounds that may represent potential drug development candidates to treat chronic musculoskeletal diseases.

A Novel High-Throughput Screening Platform Identifies Itaconate Derivatives from Marine Penicillium antarcticum as Inhibitors of Mesenchymal Stem Cell Differentiation.

Worldwide diffused diseases such as osteoarthritis, atherosclerosis or chronic kidney disease are associated with a tissue calcification process which may involve unexpected local stem cell differentiation. Current pharmacological treatments for such musculoskeletal conditions are weakly effective, sometimes extremely expensive and often absent. The potential to develop new therapies is represented by the discovery of small molecules modulating resident progenitor cell differentiation to prevent aberrant tissue calcification. The marine environment is a rich reserve of compounds with pharmaceutical potential and many novel molecules are isolated from macro and microorganisms annually. The potential of small molecules synthetized by marine filamentous fungi to influence the osteogenic and chondrogenic differentiation of human mesenchymal stem/stromal cells (hMSCs) was investigated using a novel, high-throughput automated screening platform. Metabolites synthetized by the marine-derived fungus Penicillium antarcticum were evaluated on the platform. Itaconic acid derivatives were identified as inhibitors of calcium elaboration into the matrix of osteogenically differentiated hMSCs and also inhibited hMSC chondrogenic differentiation, highlighting their capacity to impair ectopic calcification. Bioactive small molecule discovery is critical to address ectopic tissue calcification and the use of biologically relevant assays to identify naturally occurring metabolites from marine sources represents a strategy that can contribute to this effort.

Effects of combined progesterone and 17ß-estradiol treatment on the transcriptome of cultured human myometrial smooth muscle cells.

A transcriptomic analysis of cultured human uterine smooth muscle cells (hUtSMCs) was performed to examine gene expression profiles in smooth muscle in an environment containing the two major steroid hormones that regulate the human myometrium in physiological states associated with estrous, pregnancy, labor, and pathophysiological states such as leiomyoma and endometrial cancer. hUtSMCs were treated with progesterone (P4) and 17ß-estradiol (E2) individually and in combination, in the presence and absence of RU486 (mifepristone). Transcription of many genes was modulated in the presence of P4 or E2 alone, but almost six times more genes were transcriptionally modulated in the presence of the P4/E2 hormone combination. In total 796 annotated genes were significantly differentially expressed in the presence of both P4 and E2 relative to their expression in untreated cells. Functional withdrawal of P4 by addition of RU486 effectively reversed almost all transcriptional changes caused by P4/E2 treatment. Gene ontology analysis of differentially expressed genes revealed a strong association between P4/E2 treatment and downregulated expression of genes involved in cell communication, signal transduction, channel activity, inflammatory response, and differentiation. Upregulated processes included cell survival, gene transcription, steroid hormone biosynthesis, muscle development, insulin receptor signaling, and cell growth.

Co-acting gene networks predict TRAIL responsiveness of tumour cells with high accuracy.

BACKGROUND: Identification of differentially expressed genes from transcriptomic studies is one of the most common mechanisms to identify tumor biomarkers. This approach however is not well suited to identify interaction between genes whose protein products potentially influence each other, which limits its power to identify molecular wiring of tumour cells dictating response to a drug. Due to the fact that signal transduction pathways are not linear and highly interlinked, the biological response they drive may be better described by the relative amount of their components and their functional relationships than by their individual, absolute expression. RESULTS: Gene expression microarray data for 109 tumor cell lines with known sensitivity to the death ligand cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was used to identify genes with potential functional relationships determining responsiveness to TRAIL-induced apoptosis. The machine learning technique Random Forest in the statistical environment "R" with backward elimination was used to identify the key predictors of TRAIL sensitivity and differentially expressed genes were identified using the software GeneSpring. Gene co-regulation and statistical interaction was assessed with q-order partial correlation analysis and non-rejection rate. Biological (functional) interactions amongst the co-acting genes were studied with Ingenuity network analysis. Prediction accuracy was assessed by calculating the area under the receiver operator curve using an independent dataset. We show that the gene panel identified could predict TRAIL-sensitivity with a very high degree of sensitivity and specificity (AUC=0·84). The genes in the panel are co-regulated and at least 40% of them functionally interact in signal transduction pathways that regulate cell death and cell survival, cellular differentiation and morphogenesis. Importantly, only 12% of the TRAIL-predictor genes were differentially expressed highlighting the importance of functional interactions in predicting the biological response. CONCLUSIONS: The advantage of co-acting gene clusters is that this analysis does not depend on differential expression and is able to incorporate direct- and indirect gene interactions as well as tissue- and cell-specific characteristics. This approach (1) identified a descriptor of TRAIL sensitivity which performs significantly better as a predictor of TRAIL sensitivity than any previously reported gene signatures, (2) identified potential novel regulators of TRAIL-responsiveness and (3) provided a systematic view highlighting fundamental differences between the molecular wiring of sensitive and resistant cell types.

Inhibition of protein synthesis and JNK activation are not required for cell death induced by anisomycin and anisomycin analogues.

Anisomycin was identified in a screen of clinical compounds as a drug that kills breast cancer cells (MDA16 cells, derived from the triple negative breast cancer cell line, MDA-MB-468) that express high levels of an efflux pump, ABCB1. We show the MDA16 cells died by a caspase-independent mechanism, while MDA-MB-468 cells died by apoptosis. There was no correlation between cell death and either protein synthesis or JNK activation, which had previously been implicated in anisomycin-induced cell death. In addition, anisomycin analogues that did not inhibit protein synthesis or activate JNK retained the ability to induce cell death. These data suggest that either a ribosome-ANS complex is a death signal in the absence of JNK activation or ANS kills cells by binding to an as yet unidentified target.

The vapA co-expressed virulence plasmid gene vcgB (orf10) of the intracellular actinomycete Rhodococcus equi.

The virulence plasmid of the pathogenic actinomycete Rhodococcus equi is essential for proliferation of this pathogen in macrophages and the development of disease. The pathogenicity island of this plasmid encodes a family of virulence-associated proteins (Vap), one of which (VapA) is a virulence factor. This paper describes the vcgAB operon (vapA co-expressed gene), located upstream of the vapA operon. Transcription of the vcgAB operon gave rise to transcripts with a half-life similar to those determined for other virulence plasmid genes (1.8 min). Transcription started at a promoter similar to the vapA promoter, and proceeded through an inefficient terminator into the downstream vcgC gene. In addition, vcgC is also transcribed from a promoter downstream of vcgB. The vcgAB and vapA operons were coordinately regulated by temperature and pH in a synergistic manner. The latter parameter only affected transcription at higher growth temperatures, indicating that temperature is the dominant regulatory signal. Transcription of the vcgAB operon increased 10-fold during the late exponential and stationary growth phases. Transcription was also upregulated during the initial hours following phagocytosis by phagocytic cells. In contrast to vcgA and vcgC, the vcgB gene is conserved in the porcine VapB-encoding plasmid, as well as in pathogenic mycobacteria. The coordinated regulation of vcgB and vapA, transcription of vcgB following phagocytosis and conservation of vcgB in pathogenic mycobacteria indicate a role for vcgB and the vcg genes in the virulence of R. equi.

A robust platform for high-throughput screening of therapeutic strategies for acute and chronic spinal cord injury.

Astrocytes and microglia are critical regulators of inflammatory cascade after spinal cord injury (SCI). Existing glial in vitro studies do not replicate inflammatory phases associated with SCI. Here, we report an in vitro model of mixed glial culture where inflammation is induced by the administration of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1ß, and interleukin-6) to promote pathologically relevant "acute" and "chronic" inflammatory phases. We observed SCI relevant differential modulation of inflammatory pathways, cytokines, chemokines, and growth factors over 21 days. Mitochondrial dysfunction was associated with a cytokine combination treatment. Highly expressed cytokine induced neutrophil chemoattractant (CINC-3) chemokine was used as a biomarker to establish an enzyme-linked immunosorbent assay-based high-throughput screening (HTS) platform. We screened a 786-compound drug library to demonstrate the efficacy of the HTS platform. The developed model is robust and will facilitate in vitro screening of anti-reactive glial therapeutics for the treatment of SCI.

Gene expression analysis of the microvascular compartment in multiple sclerosis using laser microdissected blood vessels.

The blood brain barrier (BBB) is formed by capillary endothelial cells with inter-endothelial cell tight junctions and other cells such as pericytes and astrocytes present. Previous studies have shown a role for tight junction abnormalities in BBB leakage in multiple sclerosis (MS) brain. This marks a key stage in the development of inflammatory demyelination in MS. The aim of this study was to identify aberrantly expressed genes involved in BBB changes in MS lesions. A focused endothelial cell biology microarray, capable of detecting changes in expression of 113 endothelial cell-specific genes, was employed to analyse endothelial cell mRNA extracted from post-mortem control white matter, MS normal appearing white matter (NAWM), chronic active or inactive lesions by laser capture microdissection. Microarray analysis found 52 genes out of 113 analysed, predominantly in the activation functional group, to be differentially expressed in lesions compared to control or NAWM (p < 0.01). The majority of the differentially expressed genes were validated by quantitative real time PCR. In addition, the protein expression profiles of ICAM2, MMP2, and VEGFR1 were examined by immunofluorescent staining of selected tissue blocks. ICAM-2 was expressed at a higher level in chronic inactive lesions than control or NAWM, corresponding with the increased mRNA measured by microarray and real time PCR. The data shown, presenting a number of differentially expressed genes in the microvascular compartment of MS lesions, may shed light on the molecular mechanisms that are involved in the breakdown of the BBB. This moves us a step closer to the identification of potential therapeutic targets for repair of the compromised BBB.

Apoptosome-dependent myotube formation involves activation of caspase-3 in differentiating myoblasts.

Caspase-2, -9, and -3 are reported to control myoblast differentiation into myotubes. This had been previously explained by phosphatidylserine exposure on apoptotic myoblasts inducing differentiation in neighboring cells. Here we show for the first time that caspase-3 is activated in the myoblasts undergoing differentiation. Using RNAi, we also demonstrate that differentiation requires both cytochrome c and Apaf-1, and by using a new pharmacological approach, we show that apoptosome formation is required. We also show that Bid, whose cleavage links caspase-2 to the mitochondrial death pathway, was required for differentiation, and that the caspase cleavage product, tBid, was generated during differentiation. Taken together, these data suggest that myoblast differentiation requires caspase-2 activation of the mitochondrial death pathway, and that this occurs in the cells that differentiate. Our data also reveal a hierarchy of caspases in differentiation with caspase-2 upstream of apoptosome activation, and exerting a more profound control of differentiation, while caspases downstream of the apoptosome primarily control cell fusion.

Droplet Combinations: A Scalable Microfluidic Platform for Biochemical Assays.

Droplet-based microfluidics holds enormous potential for transforming high-throughput drug screening. Miniaturization through droplets in combination with automation contributes to reduce reagent use and analysis time as well as minimizing or eliminating labor-intensive steps leading to associated reductions in cost. In this paper, we demonstrate the potential of automated and cost-effective microfluidic droplet-generating technology in the context of an enzymatic activity assay for screening collagenase inhibitors. Experimental results show reproducible and accurate creation and mixing of droplet combinations resulting in biochemical data comparable to data produced by an industry standard instrument. This microfluidic platform that can generate and combine multiple droplets represents a promising tool for high-throughput drug screening.

A new split-luciferase complementation assay identifies pentachlorophenol as an inhibitor of apoptosome formation.

The expense and time required for in vivo reproductive and developmental toxicity studies have driven the development of in vitro alternatives. Here, we used a new in vitro split luciferase-based assay to screen a library of 177 toxicants for inhibitors of apoptosome formation. The apoptosome contains seven Apoptotic Protease-Activating Factor-1 (Apaf-1) molecules and induces cell death by activating caspase-9. Apaf-1-dependent caspase activation also plays an important role in CNS development and spermatogenesis. In the in vitro assay, Apaf-1 fused to an N-terminal fragment of luciferase binds to Apaf-1 fused to a C-terminal fragment of luciferase and reconstitutes luciferase activity. Our assay indicated that pentachlorophenol (PCP) inhibits apoptosome formation, and further investigation revealed that PCP binds to cytochrome c. PCP is a wood preservative that reduces male fertility by ill-defined mechanisms. Although the data show that PCP inhibited apoptosome formation, the concentration required suggests that other mechanisms may be more important for PCP's effects on spermatogenesis. Nonetheless, the data demonstrate the utility of the new assay in identifying apoptosome inhibitors, and we suggest that the assay may be useful in screening for reproductive and developmental toxicants.

Differential mRNA stability of the vapAICD operon of the facultative intracellular pathogen Rhodococcus equi.

The gene encoding virulence associated protein A (VapA) is clustered with three vapA homologues (vapICD) within the pathogenicity island of the virulence plasmid of Rhodococcus equi. Northern blot analysis showed a vapA transcript of c. 700 nucleotides (nt) suggesting that vapA is a monocistronic transcript. However, using the more sensitive RT-PCR, it was shown that vapA is cotranscribed with the downstream vapICD genes forming a 2.3-kb operon. This initial transcript is subsequently processed to give rise to a 700 nt vapA transcript with a half-life of 7.5 min. In contrast, the vapI, vapC and vapD transcripts have an average half-life of 1.8 min, identical to that of the five cistronic virR operon located upstream of the vapA operon. It is speculated that the need for differential gene expression arises from the different localisation of the Vap proteins. VapA is tethered to the surface of the cell wall, whereas VapC and VapD are secreted, diffusable proteins. The intercistronic region between vapC and vapD harbours two short ORFs (OrfA, OrfB). These ORFs are translationally coupled to vapC and vapD in which the start codon overlaps the stop codon of the preceding gene.

A restatement of the natural science evidence concerning catchment-based 'natural' flood management in the UK.

Flooding is a very costly natural hazard in the UK and is expected to increase further under future climate change scenarios. Flood defences are commonly deployed to protect communities and property from flooding, but in recent years flood management policy has looked towards solutions that seek to mitigate flood risk at flood-prone sites through targeted interventions throughout the catchment, sometimes using techniques which involve working with natural processes. This paper describes a project to provide a succinct summary of the natural science evidence base concerning the effectiveness of catchment-based 'natural' flood management in the UK. The evidence summary is designed to be read by an informed but not technically specialist audience. Each evidence statement is placed into one of four categories describing the nature of the underlying information. The evidence summary forms the appendix to this paper and an annotated bibliography is provided in the electronic supplementary material.

Sensitivity Profiles of Human Prostate Cancer Cell Lines to an 80 Kinase Inhibitor Panel.

BACKGROUND: Taxanes and anti-androgen therapies are routinely used for the treatment of metastatic prostate cancer, however the majority of patients eventually develop resistance. MATERIALS AND METHODS: Eighty kinase inhibitors were screened regarding their ability to inhibit cell viability in CWR22, 22Rv1, PC-3 and DU145 prostate cancer cells using automated toxicity assays. Four kinase inhibitors were selected for further investigation. RESULTS: No significant difference in sensitivity patterns was found between the androgen receptor wild-type CWR22 and its androgen receptor mutant variant 22Rv1, indicating that androgen receptor mutation did not impact on kinase inhibitor sensitivity in this model. Metastatic PC-3 and DU145 prostate cancer cell lines were less sensitive to kinase inhibitors than the non-metastatic CWR22 and 22Rv1. All four cell lines responded to GSK-3 inhibitor BIO, and MEK inhibitor PD198306. DU145 cells were resistant to p75NTR/TrkA and CHK4 inhibitors, RO-082750 and Ryuvidine. CONCLUSION: Kinase inhibition may be an appropriate strategy for the treatment of prostate cancer.

A high through-put screen for small molecules modulating MCM2 phosphorylation identifies Ryuvidine as an inducer of the DNA damage response.

DNA replication is an essential process for cell division and as such it is a process that is directly targeted by several anticancer drugs. CDC7 plays an essential role in the activation of replication origins and has recently been proposed as a novel target for drug discovery. The MCM DNA helicase complex (MCM2-7) is a key target of the CDC7 kinase, and MCM phosphorylation status at specific sites is a reliable biomarker of CDC7 cellular activity. In this work we describe a cell-based assay that utilizes the "In Cell Western Technique" (ICW) to identify compounds that affect cellular CDC7 activity. By screening a library of approved drugs and kinase inhibitors we found several compounds that can affect CDC7-dependent phosphorylation of MCM2 in HeLa cells. Among these, Mitoxantrone, a topoisomerase inhibitor, and Ryuvidine, previously described as a CDK4 inhibitor, cause a reduction in phosphorylated MCM2 levels and a sudden blockade of DNA synthesis that is accompanied by an ATM-dependent checkpoint response. This study sheds light on the previously observed cytotoxity of Ryuvidine, strongly suggesting that it is related to its effect of causing DNA damage.

A comparison of the efficacy of transplantation of bone marrow-derived mesenchymal stem cells and unrestricted somatic stem cells on outcome after acute myocardial infarction.

INTRODUCTION: A number of questions remain unanswered in the field of cell therapy for acute myocardial infarction, including what is the optimal cell type, and can therapeutic efficacy be enhanced by conditioning regimens. In this study, we sought to address these questions by directly comparing the effect of bone marrow-derived mesenchymal stem cells and unrestricted somatic stem cells delivered 24 hours post-myocardial infarction and by determining if the therapeutic efficacy of unrestricted somatic stem cells could be enhanced by exposing the cells to guiding factors before cell transplantation. METHODS: Unrestricted somatic stem cells were guided by exposure to 50 ng/mL basic fibroblast growth factor, 20 ng/mL hepatocyte growth factor and 20 ng/mL bone morphogenetic protein-2 for 24 hours. Using a Sprague-Dawley rat model of acute myocardial infarction, we transplanted cells by intramyocardial injection 24 hours post-myocardial infarction. Cardiac function was serially measured using echocardiography, and histological analyses of infarct morphology, angiogenesis and apoptosis were obtained. Transcriptomic and proteomic changes were assessed using microarray and real-time quantitative PCR. RESULTS: When assessed 28 days after the myocardial infarction, the delivery of mesenchymal stem cells 24 hours post-myocardial infarction did not improve ejection fraction (P = 0.19), and did not prevent the decline in ejection fraction observed in the absence of cell therapy (P = 0.17). The administration of unrestricted somatic stem cells also did not improve ejection fraction (P = 0.11), but did prevent a further decline in ejection fraction (P = 0.001). Delivery of guided unrestricted somatic stem cells significantly improved ejection fraction (P = 0.03). Guided unrestricted somatic stem cells restored function to a greater extent than mesenchymal stem cells (P = 0.03). The infarct area (P = 0.2), apoptosis (P = 0.07) and angiogenesis (P = 0.09) did not differ between groups. Microarray analysis revealed that, following pre-implantation guiding, the gene groupings of mitosis, signalling and angiogenesis were highly overrepresented, mediators of apoptosis were overrepresented, and cardiomyocyte-associated genes were not differentially expressed. CONCLUSIONS: These results suggest that guided unrestricted somatic stem cells have a moderate capacity to repair cardiac damage and that they are more effective than mesenchymal stem cells in restoring cardiac function after a myocardial infarction. The mechanism of the benefit was not fully elucidated in this study, but these observations may be mediated by favorable dysregulation of angiogenic and apoptotic gene groupings.

Evaluating the potential of quantum dots for in vitro biological studies: effects on gene expression using microarray analysis.

Quantum dots have potential applications in the biomedical field and especially in bioimaging owing to their tunable fluorescent properties. Although many phenotypic studies have been carried out using QDs on different cell lines, only very few of them involved the analysis of the effect of QDs on gene expression. Here, we describe the application of microarray gene expression analysis for studying the differential expression of genes in the cells treated with QDs.

Regulation of the DNA damage response and gene expression by the Dot1L histone methyltransferase and the 53Bp1 tumour suppressor.

BACKGROUND: Dot1L, a histone methyltransferase that targets histone H3 lysine 79 (H3K79), has been implicated in gene regulation and the DNA damage response although its functions in these processes remain poorly defined. METHODOLOGY/PRINCIPAL FINDINGS: Using the chicken DT40 model system, we generated cells in which the Dot1L gene is disrupted to examine the function and focal recruitment of the 53Bp1 DNA damage response protein. Detailed kinetic and dose response assays demonstrate that, despite the absence of H3K79 methylation demonstrated by mass spectrometry, 53Bp1 focal recruitment is not compromised in these cells. We also describe, for the first time, the phenotypes of a cell line lacking both Dot1L and 53Bp1. Dot1L⁻/⁻ and wild type cells are equally resistant to ionising radiation, whereas 53Bp1⁻/⁻/Dot1L⁻/⁻ cells display a striking DNA damage resistance phenotype. Dot1L and 53Bp1 also affect the expression of many genes. Loss of Dot1L activity dramatically alters the mRNA levels of over 1200 genes involved in diverse biological functions. These results, combined with the previously reported list of differentially expressed genes in mouse ES cells knocked down for Dot1L, demonstrates surprising cell type and species conservation of Dot1L-dependent gene expression. In 53Bp1⁻/⁻ cells, over 300 genes, many with functions in immune responses and apoptosis, were differentially expressed. To date, this is the first global analysis of gene expression in a 53Bp1-deficient cell line. CONCLUSIONS/SIGNIFICANCE: Taken together, our results uncover a negative role for Dot1L and H3K79 methylation in the DNA damage response in the absence of 53Bp1. They also enlighten the roles of Dot1L and 53Bp1 in gene expression and the control of DNA double-strand repair pathways in the context of chromatin.

Human coronary artery smooth muscle cell response to a novel PLA textile/fibrin gel composite scaffold.

Previous studies have demonstrated the potential of fibrin as a cell carrier for cardiovascular tissue engineering applications. Unfortunately, fibrin exhibits poor mechanical properties. One method of addressing this issue is to incorporate a textile in fibrin to provide structural support. However, it is first necessary to develop a deeper understanding of the effect of the textile on cell response. In this study, the cytotoxicity of a polylactic acid (PLA) warp-knit textile was assessed with human coronary artery smooth muscle cells (HCASMC). Subsequently, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was employed to examine the gene expression of HCASMC embedded in fibrin with and without the textile. Five genes were examined over a 3-week period: smooth muscle alpha-actin (SMalphaA), myosin heavy chain 11 smooth muscle (SM1/SM2), calponin, myosin heavy chain 10 non-muscle (SMemb) and collagen. Additionally, a microarray analysis was performed to examine a wider range of genes. The knitting process did not adversely affect the cell response; there was no dramatic change in cell number or metabolic rate compared to the negative control. After 3 weeks, there was no significant difference in gene expression, except for a slight decrease of 10% in SMemb in the fibrin with textile. After 3 weeks, there were no obvious cytotoxic effects observed as a result of the knitting process and the gene expression profile did not appear to be altered in the presence of the mesh in the fibrin gel.

The iron-regulated iupABC operon is required for saprophytic growth of the intracellular pathogen Rhodococcus equi at low iron concentrations.

Rhodococcus equi is a facultative intracellular pathogen which proliferates rapidly in both manure-enriched soil and alveolar macrophages. Although both environments are characterized by extremely low concentrations of free iron, very little is known regarding the strategies employed by R. equi to thrive under these conditions. This paper reports the characterization of an R. equi transposome mutant that fails to grow at low iron concentrations. The transposome was shown to be inserted into iupA, the first gene of the iupABC operon encoding an ABC transport system highly similar to siderophore uptake systems. Disruption of the iupA gene also resulted in a failure of R. equi to utilize heme and hemoglobin as a source of iron. Introduction of the iupABC operon in trans restored the wild-type phenotype of the mutant strain. iupABC transcripts were 180-fold more abundant in R. equi grown in iron-depleted medium than in organisms grown in iron-replete medium. Proliferation of the iupABC mutant strain in macrophages was comparable to that of the wild-type strain. Furthermore, the iupABC mutant was not attenuated in mice, showing that the iupABC operon is not required for virulence.