Bryan Troxell

Transcriptional regulation by Ferric Uptake Regulator (Fur) in pathogenic bacteria

In the ancient anaerobic environment, ferrous iron (Fe2+) was one of the first metal cofactors. Oxygenation of the ancient world challenged bacteria to acquire the insoluble ferric iron (Fe3+) and later to defend against reactive oxygen species (ROS) generated by the Fenton chemistry. To acquire Fe3+, bacteria produce low-molecular weight compounds, known as siderophores, which have extremely high affinity for Fe3+. However, during infection the host restricts iron from pathogens by producing iron- and siderophore-chelating proteins, by exporting iron from intracellular pathogen-containing compartments, and by limiting absorption of dietary iron. Ferric Uptake Regulator (Fur) is a transcription factor which utilizes Fe2+ as a corepressor and represses siderophore synthesis in pathogens. Fur, directly or indirectly, controls expression of enzymes that protect against ROS damage. Thus, the challenges of iron homeostasis and defense against ROS are addressed via Fur. Although the role of Fur as a repressor is well-documented, emerging evidence demonstrates that Fur can function as an activator. Fur activation can occur through three distinct mechanisms (1) indirectly via small RNAs, (2) binding at cis regulatory elements that enhance recruitment of the RNA polymerase holoenzyme (RNAP), and (3) functioning as an antirepressor by removing or blocking DNA binding of a repressor of transcription. In addition, Fur homologs control defense against peroxide stress (PerR) and control uptake of other metals such as zinc (Zur) and manganese (Mur) in pathogenic bacteria. Fur family members are important for virulence within bacterial pathogens since mutants of fur, perR, or zur exhibit reduced virulence within numerous animal and plant models of infection. This review focuses on the breadth of Fur regulation in pathogenic bacteria.

FNR Is a Global Regulator of Virulence and Anaerobic Metabolism in Salmonella enterica Serovar Typhimurium (ATCC 14028s)

Salmonella enterica serovar Typhimurium must successfully transition the broad fluctuations in oxygen concentrations encountered in the host. In Escherichia coli, FNR is one of the main regulatory proteins involved in O2 sensing. To assess the role of FNR in serovar Typhimurium, we constructed an isogenic fnr mutant in the virulent wild-type strain (ATCC 14028s) and compared their transcriptional profiles and pathogenicities in mice. Here, we report that, under anaerobic conditions, 311 genes (6.80% of the genome) are regulated directly or indirectly by FNR; of these, 87 genes (28%) are poorly characterized. Regulation by FNR in serovar Typhimurium is similar to, but distinct from, that in E. coli. Thus, genes/operons involved in aerobic metabolism, NO. detoxification, flagellar biosynthesis, motility, chemotaxis, and anaerobic carbon utilization are regulated by FNR in a fashion similar to that in E. coli. However, genes/operons existing in E. coli but regulated by FNR only in serovar Typhimurium include those coding for ethanolamine utilization, a universal stress protein, a ferritin-like protein, and a phosphotransacetylase. Interestingly, Salmonella-specific genes/operons regulated by FNR include numerous virulence genes within Salmonella pathogenicity island 1 (SPI-1), newly identified flagellar genes (mcpAC, cheV), and the virulence operon (srfABC). Furthermore, the role of FNR as a positive regulator of motility, flagellar biosynthesis, and pathogenesis was confirmed by showing that the mutant is nonmotile, lacks flagella, is attenuated in mice, and does not survive inside macrophages. The inability of the mutant to survive inside macrophages is likely due to its sensitivity to the reactive oxygen species generated by NADPH phagocyte oxidase.

Cyclic di-GMP is Essential for the Survival of the Lyme Disease Spirochete in Ticks

Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that modulates many biological processes. Although its role in bacterial pathogenesis during mammalian infection has been documented, the role of c-di-GMP in a pathogens life cycle within a vector host is less understood. The enzootic cycle of the Lyme disease pathogen Borrelia burgdorferi involves both a mammalian host and an Ixodes tick vector. The B. burgdorferi genome encodes a single copy of the diguanylate cyclase gene (rrp1), which is responsible for c-di-GMP synthesis. To determine the role of c-di-GMP in the life cycle of B. burgdorferi, an Rrp1-deficient B. burgdorferi strain was generated. The rrp1 mutant remains infectious in the mammalian host but cannot survive in the tick vector. Microarray analyses revealed that expression of a four-gene operon involved in glycerol transport and metabolism, bb0240-bb0243, was significantly downregulated by abrogation of Rrp1. In vitro, the rrp1 mutant is impaired in growth in the media containing glycerol as the carbon source (BSK-glycerol). To determine the contribution of the glycerol metabolic pathway to the rrp1 mutant phenotype, a glp mutant, in which the entire bb0240-bb0243 operon is not expressed, was generated. Similar to the rrp1 mutant, the glp mutant has a growth defect in BSK-glycerol medium. In vivo, the glp mutant is also infectious in mice but has reduced survival in ticks. Constitutive expression of the bb0240-bb0243 operon in the rrp1 mutant fully rescues the growth defect in BSK-glycerol medium and partially restores survival of the rrp1 mutant in ticks. Thus, c-di-GMP appears to govern a catabolic switch in B. burgdorferi and plays a vital role in the tick part of the spirochetal enzootic cycle. This work provides the first evidence that c-di-GMP is essential for a pathogens survival in its vector host.

Fur Negatively Regulates hns and Is Required for the Expression of HilA and Virulence in Salmonella enterica Serovar Typhimurium

Iron is an essential element for the survival of living cells. However, excess iron is toxic, and its uptake is exquisitely regulated by the ferric uptake regulator, Fur. In Salmonella, the Salmonella pathogenicity island 1 (SPI-1) encodes a type three secretion system, which is required for invasion of host epithelial cells in the small intestine. A major activator of SPI-1 is HilA, which is encoded within SPI-1. One known regulator of hilA is Fur. The mechanism of hilA regulation by Fur is unknown. We report here that Fur is required for virulence in Salmonella enterica serovar Typhimurium and that Fur is required for the activation of hilA, as well as of other HilA-dependent genes, invF and sipC. The Fur-dependent regulation of hilA was independent of PhoP, a known repressor of hilA. Instead, the expression of the gene coding for the histone-like protein, hns, was significantly derepressed in the fur mutant. Indeed, the activation of hilA by Fur was dependent on 28 nucleotides located upstream of hns. Moreover, we used chromatin immunoprecipitation to show that Fur bound, in vivo, to the upstream region of hns in a metal-dependent fashion. Finally, deletion of fur in an hns mutant resulted in Fur-independent activation of hilA. In conclusion, Fur activates hilA by repressing the expression of hns.

Transcriptional responses of Leptospira interrogans to host innate immunity: significant changes in metabolism, oxygen tolerance, and outer membrane.

Background Leptospira interrogans is the major causative agent of leptospirosis. Phagocytosis plays important roles in the innate immune responses to L. interrogans infection, and L. interrogans can evade the killing of phagocytes. However, little is known about the adaptation of L. interrogans during this process. Methodology/Principal Findings To better understand the interaction of pathogenic Leptospira and innate immunity, we employed microarray and comparative genomics analyzing the responses of L. interrogans to macrophage-derived cells. During this process, L. interrogans altered expressions of many genes involved in carbohydrate and lipid metabolism, energy production, signal transduction, transcription and translation, oxygen tolerance, and outer membrane proteins. Among them, the catalase gene expression was significantly up-regulated, suggesting it may contribute to resisting the oxidative pressure of the macrophages. The expressions of several major outer membrane protein (OMP) genes (e.g., ompL1, lipL32, lipL41, lipL48 and ompL47) were dramatically down-regulated (10–50 folds), consistent with previous observations that the major OMPs are differentially regulated in vivo. The persistent down-regulations of these major OMPs were validated by immunoblotting. Furthermore, to gain initial insight into the gene regulation mechanisms in L. interrogans, we re-defined the transcription factors (TFs) in the genome and identified the major OmpR TF gene (LB333) that is concurrently regulated with the major OMP genes, suggesting a potential role of LB333 in OMPs regulation. Conclusions/Significance This is the first report on global responses of pathogenic Leptospira to innate immunity, which revealed that the down-regulation of the major OMPs may be an immune evasion strategy of L. interrogans, and a putative TF may be involved in governing these down-regulations. Alterations of the leptospiral OMPs up interaction with host antigen-presenting cells (APCs) provide critical information for selection of vaccine candidates. In addition, genome-wide annotation and comparative analysis of TFs set a foundation for further studying regulatory networks in Leptospira spp.

The Fur regulon in anaerobically grown Salmonella enterica sv . Typhimurium: identification of new Fur targets

BackgroundThe Ferric uptake regulator (Fur) is a transcriptional regulator that controls iron homeostasis in bacteria. Although the regulatory role of Fur in Escherichia coli is well characterized, most of the studies were conducted under routine culture conditions, i.e., in ambient oxygen concentration. To reveal potentially novel aspects of the Fur regulon in Salmonella enterica serovar Typhimurium under oxygen conditions similar to that encountered in the host, we compared the transcriptional profiles of the virulent wild-type strain (ATCC 14028s) and its isogenic Δfur strain under anaerobic conditions.ResultsMicroarray analysis of anaerobically grown Δfur S. Typhimurium identified 298 differentially expressed genes. Expression of several genes controlled by Fnr and NsrR appeared to be also dependent on Fur. Furthermore, Fur was required for the activity of the cytoplasmic superoxide disumutases (MnSOD and FeSOD). The regulation of FeSOD gene, sodB, occurred via small RNAs (i.e., the ryhB homologs, rfrA and rfrB) with the aid of the RNA chaperone Hfq. The transcription of sodA was increased in Δfur; however, the enzyme was inactive due to the incorporation of iron instead of manganese in SodA. Additionally, in Δfur, the expression of the gene coding for the ferritin-like protein (ftnB) was down-regulated, while the transcription of the gene coding for the nitric oxide (NO·) detoxifying flavohemoglobin (hmpA) was up-regulated. The promoters of ftnB and hmpA do not contain recognized Fur binding motifs, which indicated their probable indirect regulation by Fur. However, Fur activation of ftnB was independent of Fnr. In addition, the expression of the gene coding for the histone-like protein, H-NS (hns) was increased in Δfur. This may explain the observed down-regulation of the tdc operon, responsible for the anaerobic degradation of threonine, and ftnB in Δfur.ConclusionsThis study determined that Fur is a positive factor in ftnB regulation, while serving to repress the expression of hmpA. Furthermore, Fur is required for the proper expression and activation of the antioxidant enzymes, FeSOD and MnSOD. Finally, this work identified twenty-six new targets of Fur regulation, and demonstrates that H-NS repressed genes are down-regulated in Δfur.

Borrelia burgdorferi, a Pathogen That Lacks Iron, Encodes Manganese-dependent Superoxide Dismutase Essential for Resistance to Streptonigrin

Background: Borrelia burgdorferi contains a single superoxide dismutase (SOD). Results: This is a Mn-SOD that is manganese-induced, zinc-repressed, and required for resistance to the metal-dependent redox compound streptonigrin. Conclusion: Manganese is a key aspect in the defense against oxidative stress for B. burgdorferi. Significance: Our work provides insight into mechanism of streptonigrin toxicity and metal-dependent gene regulation within an iron-lacking bacterial species. Borrelia burgdorferi, the causative agent of Lyme disease, exists in nature through a complex life cycle involving ticks of the Ixodes genus and mammalian hosts. During its life cycle, B. burgdorferi experiences fluctuations in oxygen tension and may encounter reactive oxygen species (ROS). The key metalloenzyme to degrade ROS in B. burgdorferi is SodA. Although previous work suggests that B. burgdorferi SodA is an iron-dependent superoxide dismutase (SOD), later work demonstrates that B. burgdorferi is unable to transport iron and contains an extremely low intracellular concentration of iron. Consequently, the metal cofactor for SodA has been postulated to be manganese. However, experimental evidence to support this hypothesis remains lacking. In this study, we provide biochemical and genetic data showing that SodA is a manganese-dependent enzyme. First, B. burgdorferi contained SOD activity that is resistant to H2O2 and NaCN, characteristics associated with Mn-SODs. Second, the addition of manganese to the Chelex-treated BSK-II enhanced SodA expression. Third, disruption of the manganese transporter gene bmtA, which significantly lowers the intracellular manganese, greatly reduced SOD activity and SodA expression, suggesting that manganese regulates the level of SodA. In addition, we show that B. burgdorferi is resistant to streptonigrin, a metal-dependent redox cycling compound that produces ROS, and that SodA plays a protective role against the streptonigrin. Taken together, our data demonstrate the Lyme disease spirochete encodes a manganese-dependent SOD that contributes to B. burgdorferi defense against intracellular superoxide.

Pyruvate protects pathogenic spirochetes from H2O2 killing.

Pathogenic spirochetes cause clinically relevant diseases in humans and animals, such as Lyme disease and leptospirosis. The causative agent of Lyme disease, Borrelia burgdorferi, and the causative agent of leptospirosis, Leptospria interrogans, encounter reactive oxygen species (ROS) during their enzootic cycles. This report demonstrated that physiologically relevant concentrations of pyruvate, a potent H2O2 scavenger, and provided passive protection to B. burgdorferi and L. interrogans against H2O2. When extracellular pyruvate was absent, both spirochetes were sensitive to a low dose of H2O2 (≈0.6 µM per h) generated by glucose oxidase (GOX). Despite encoding a functional catalase, L. interrogans was more sensitive than B. burgdorferi to H2O2 generated by GOX, which may be due to the inherent resistance of B. burgdorferi because of the virtual absence of intracellular iron. In B. burgdorferi, the nucleotide excision repair (NER) and the DNA mismatch repair (MMR) pathways were important for survival during H2O2 challenge since deletion of the uvrB or the mutS genes enhanced its sensitivity to H2O2 killing; however, the presence of pyruvate fully protected ΔuvrB and ΔmutS from H2O2 killing further demonstrating the importance of pyruvate in protection. These findings demonstrated that pyruvate, in addition to its classical role in central carbon metabolism, serves as an important H2O2 scavenger for pathogenic spirochetes. Furthermore, pyruvate reduced ROS generated by human neutrophils in response to the Toll-like receptor 2 (TLR2) agonist zymosan. In addition, pyruvate reduced neutrophil-derived ROS in response to B. burgdorferi, which also activates host expression through TLR2 signaling. Thus, pathogenic spirochetes may exploit the metabolite pyruvate, present in blood and tissues, to survive H2O2 generated by the host antibacterial response generated during infection.

Manganese and zinc regulate virulence determinants in borrelia burgdorferi

ABSTRACT Borrelia burgdorferi, the causative agent of Lyme disease, must adapt to two diverse niches, an arthropod vector and a mammalian host. RpoS, an alternative sigma factor, plays a central role in spirochetal adaptation to the mammalian host by governing expression of many genes important for mammalian infection. B. burgdorferi is known to be unique in metal utilization, and little is known of the role of biologically available metals in B. burgdorferi. Here, we identified two transition metal ions, manganese (Mn2+) and zinc (Zn2+), that influenced regulation of RpoS. The intracellular Mn2+ level fluctuated approximately 20-fold under different conditions and inversely correlated with levels of RpoS and the major virulence factor OspC. Furthermore, an increase in intracellular Mn2+ repressed temperature-dependent induction of RpoS and OspC; this repression was overcome by an excess of Zn2+. Conversely, a decrease of intracellular Mn2+ by deletion of the Mn2+ transporter gene, bmtA, resulted in elevated levels of RpoS and OspC. Mn2+ affected RpoS through BosR, a Fur family homolog that is required for rpoS expression: elevated intracellular Mn2+ levels greatly reduced the level of BosR protein but not the level of bosR mRNA. Thus, Mn2+ and Zn2+ appeared to be important in modulation of the RpoS pathway that is essential to the life cycle of the Lyme disease spirochete. This finding supports the emerging notion that transition metals such as Mn2+ and Zn2+ play a critical role in regulation of virulence in bacteria.

Ferric Uptake Regulator-Dependent Antinitrosative Defenses in Salmonella enterica Serovar Typhimurium Pathogenesis

ABSTRACT Herein we report an important role for the ferric uptake regulator (Fur) in the resistance of Salmonella enterica serovar Typhimurium to the reactive nitrogen species produced by inducible nitric oxide (NO) synthase in an NRAMP1r murine model of acute systemic infection. The expression of fur protected Salmonella grown under normoxic and hypoxic conditions against the bacteriostatic activity of NO. The hypersusceptibility of fur-deficient Salmonella to the cytotoxic actions of NO coincides with a marked repression of respiratory activity and the reduced ability of the bacteria to detoxify NO. A fur mutant Salmonella strain contained reduced levels of the terminal quinol oxidases of the electron transport chain. Addition of the heme precursor δ-aminolevulinic acid restored the cytochrome content, respiratory activity, NO consumption, and wild-type growth in bacteria undergoing nitrosative stress. The innate antinitrosative defenses regulated by Fur added to the adaptive response associated with the NO-detoxifying activity of the flavohemoprotein Hmp. Our investigations indicate that, in addition to playing a critical role in iron homeostasis, Fur is an important antinitrosative determinant of Salmonella pathogenesis.