Type 2 NADH dehydrogenase is the only point of entry for electrons into the Streptococcus agalactiae respiratory chain and is a potential drug target

The opportunistic pathogen Streptococcus agalactiae is the major cause of meningitis and sepsis in a newborn’s first week, as well as a considerable cause of pneumonia, urinary tract infections, and sepsis in immunocompromised adults. This pathogen respires aerobically if heme and quinone are available in the environment, and a functional respiratory chain is required for full virulence. Remarkably, it is shown here that the entire respiratory chain of S. agalactiae consists of only two enzymes, a type 2 NADH dehydrogenase (NDH-2) and a cytochrome bd oxygen reductase. There are no respiratory dehydrogenases other than NDH-2 to feed electrons into the respiratory chain, and there is only one respiratory oxygen reductase to reduce oxygen to water. Although S. agalactiae grows well in vitro by fermentative metabolism, it is shown here that the absence of NDH-2 results in attenuated virulence, as observed by reduced colonization in heart and kidney in a mouse model of systemic infection. The lack of NDH-2 in mammalian mitochondria and its important role for virulence suggest this enzyme may be a potential drug target. For this reason, in this study, S. agalactiae NDH-2 was purified and biochemically characterized, and the isolated enzyme was used to screen for inhibitors from libraries of FDA-approved drugs. Zafirlukast was identified to successfully inhibit both NDH-2 activity and aerobic respiration in intact cells. This compound may be useful as a laboratory tool to inhibit respiration in S. agalactiae and, since it has few side effects, it might be considered a lead compound for therapeutics development. IMPORTANCE S. agalactiae is part of the human intestinal microbiota and is present in the vagina of ~30% of healthy women. Although a commensal, it is also the leading cause of septicemia and meningitis in neonates and immunocompromised adults. This organism can aerobically respire, but only using external sources of heme and quinone, required to have a functional electron transport chain. Although bacteria usually have a branched respiratory chain with multiple dehydrogenases and terminal oxygen reductases, here we establish that S. agalactiae utilizes only one type 2 NADH dehydrogenase (NDH-2) and one cytochrome bd oxygen reductase to perform respiration. NADH-dependent respiration plays a critical role in the pathogen in maintaining NADH/NAD+ redox balance in the cell, optimizing ATP production, and tolerating oxygen. In summary, we demonstrate the essential role of NDH-2 in respiration and its contribution to S. agalactiae virulence and propose it as a potential drug target

The role of secretion systems and small molecules in soft-rot enterobacteriaceae pathogenicity

Soft-rot Enterobacteriaceae (SRE), which belong to the genera Pectobacterium and Dickeya, consist mainly of broad host-range pathogens that cause wilt, rot, and blackleg diseases on a wide range of plants. They are found in plants, insects, soil, and water in agricultural regions worldwide. SRE encode all six known protein secretion systems present in gram-negative bacteria, and these systems are involved in attacking host plants and competing bacteria. They also produce and detect multiple types of small molecules to coordinate pathogenesis, modify the plant environment, attack competing microbes, and perhaps to attract insect vectors. This review integrates new information about the role protein secretion and detection and production of ions and small molecules play in soft-rot pathogenicity

Caracterisation d'un nouvel operon intervenant dans le transport et la biosynthese de la chrysobactine, siderophore implique dans la virulence d'une enterobacterie phytopathogene Erwinia chrysanthemi

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 84829 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Role of iron homeostasis in the virulence of phytopathogenic bacteria: an "a la carte' menu

The interaction between pathogenic microbes and their hosts is determined by survival strategies on both sides. As a result of its redox properties, iron is vital for the growth and proliferation of nearly all organisms, including pathogenic bacteria. In bacteriavertebrate interactions, competition for this essential metal is critical for the outcome of the infection. The role of iron in the virulence of plant pathogenic bacteria has only been explored in a few pathosystems in the past. However, in the last 5years, intensive research has provided new insights into the mechanisms of iron homeostasis in phytopathogenic bacteria that are involved in virulence. This review, which includes important plant pathosystems, discusses the recent advances in the understanding of iron transport and homeostasis during plant pathogenesis. By summarizing the recent progress, we wish to provide an updated view clarifying the various roles played by this metal in the virulence of bacterial phytopathogens as a nutritional and regulatory element. The complex intertwining of iron metabolism and oxidative stress during infection is emphasized

Iron regulation and pathogenicity in Erwinia chrysanthemi 3937: Role of the fur repressor protein

International audienc

Contrôle de l'homéostasie du fer au cours du cycle infectieux d'Erwinia chrysantemi 3937

Erwinia chrysanthemi 3937 est une bactérie phytopathogène responsable de maladies de type pourriture molle sur une large gamme de plantes. Durant l infection, les bactéries se disséminent de manière extracellulaire, au niveau de l apoplasme des tissus aériens du végétal où elles doivent s adapter à des conditions de stress oxydant et une faible disponibilité en fer. Comme cet élément est essentiel et paradoxalement génère des radicaux hydroxyles hautement toxiques via la réaction de Fenton, une régulation fine des quantités intracellulaires en fer est primordiale pour la bactérie. L homéostasie du fer implique une classe de protéines dénommées ferritines qui séquestrent le fer sous forme non réactive et biodisponible notamment lorsque le métal devient limitant dans l environnement. Le génome d E. chrysanthemi 3937 comporte une centaine de gènes dédiés au métabolisme du fer dont 4 sont supposés être impliqués dans le stockage intracellulaire du fer : le gène ftnA codant une ferritine de type eucaryote, le gène bfr codant une bacterioferritine contenant des groupements hème et les gènes dps1 et dps2 codant deux protéines Dps (DNA-binding proteins from starved cells). L inactivation de ces gènes a montré que la ferritine FtnA contribue principalement au stockage intracellulaire du fer. Le rôle des ferritines ne se limite pas à servir de réserves de fer intracellulaire : ainsi la protéine FtnA participe à la résistance au stress oxydant et la protéine Dps1 pourrait jouer un rôle dans la détoxication du peroxyde d hydrogène. Conformément à leur rôle dans le stockage intracellulaire du fer, les gènes ftnA, bfr et dps1 sont exprimés en réponse à la biodisponibilité en fer par la protéine Fur (Ferric uptake repressor), mais de manière temporellement différentielle au cours de la croissance bactérienne et selon des mécanismes distincts. Seule l induction du gène ftnA par le fer et Fur est dépendante de l ARN anti-sens RyhB. Par ailleurs, les gènes bfr et dps1 sont induits en phase stationnaire de croissance par le facteur S. Les travaux réalisés au cours cette thèse ont permis de caractériser les intervenants de l homéostasie du fer chez E. chrysanthemi 3937, d acquérir une vue globale du trafic intracellulaire du fer et d en apprécier leur contribution respective dans la pathogénie.The pathogenic enterobacterium Erwinia chrysanthemi 3937 is able to cause soft-rot disease on a large set of plants. In the host, bacteria disseminate extracellularly and encounter an oxidative environment with low iron availability. Under these conditions, a tight control of the intracellular iron pool is important since excess of iron can exacerbate oxidative stress by generating the highly toxic radical OH through the Fenton s reaction. Iron homeostasis involves ferritins which sequester iron under a non reactive state. These iron stores can be used to enhance bacterial growth when external iron supplies are restricted. The genome of E. chrysanthemi 3937 presents a hundred of genes involved in iron metabolism including four loci that could be devoted to iron storage : the ftnA gene encoding an eukaryotic type haem-free ferritin, the bfr gene coding for a haem-containing bacterioferritin and, the dps1 and dps2 genes encoding two Dps proteins (DNA-binding proteins from starved cells). Our work is aimed at elucidating the role of these proteins in iron homeostasis and pathogenicity of E. chrysanthemi 3937. According to their differential role in iron storage, the ftnA, bfr and dps1 genes appeared to be controlled differently by iron and Fur (ferric uptake regulator). Only the iron and Fur-induction of ftnA gene is dependent of the small antisens RyhB RNA. Moreover, the dps1 and bfr genes are regulated by additional effectors including the S transcription factor. Disruption of the ftnA, bfr and dps1 genes showed that FtnA is the main cellular component involved in iron storage. However, these ferritins are not only involved in iron storage : FtnA contributes to oxidative stress resistance and Dps1 could be implicated in hydrogen peroxide detoxication through its ferroxidase center. The implication of these proteins to bacterial virulence is also discussed.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Role of the Dickeya dadantii Dps protein

During infection, the phytopathogenic enterobacterium Dickeya dadantii has to cope with iron-limiting conditions and the production of reactive oxygen species by plant cells. A tight control of the bacterial intracellular iron content is necessary for full virulence of D. dadantii: previous studies have shown that the ferritin FtnA and the bacterioferrtin Bfr, devoted to iron storage, contribute differentially to the virulence of this species. In this work, we investigated the role of the Dps miniferritin in iron homeostasis in D. dadantii. We constructed a Dps-deficient mutant by reverse genetics. This mutant grew like the wild-type stain under iron starvation and showed no decreased iron content. However, the dps mutant displayed an increased sensitivity to hydrogen peroxide in comparison to the wild-type strain. This hydrogen peroxide susceptibility only occurs when bacteria are in the stationary phase. Unlike the bfr and the ftnA mutants, the dps mutant is not affected in its pathogenicity on host plants. The dps gene expression is induced at the stationary phase of growth. The Sigma S transcriptional factor is necessary for this control. Furthermore, dps expression is positively regulated by the oxidative stress response regulator OxyR during the exponential growth phase, after hydrogen peroxide treatment. These results indicate that the Dps miniferritin from D. dadantii has a minor role in iron homeostasis, but is important in conferring tolerance to hydrogen peroxide and for survival of cells that enter the stationary phase of growth

Erwinia chrysanthemi requires a second iron transport route dependent of the siderophore achromobactin for extracellular growth and plant infection

International audienceFull virulence of the pectinolytic enterobacterium Erwinia chrysanthemi strain 3937 depends on the production in planta of the catechol-type siderophore chrysobactin. Under iron-limited conditions, E. chrysanthemi synthesizes a second siderophore called achromobactin belonging to the hydroxy/carboxylate class of siderophore. In this study, we cloned and functionally characterized a 13 kb long operon comprising seven genes required for the biosynthesis (acs) and extracellular release (yhcA) of achromobactin, as well as the gene encoding the specific outer membrane receptor for its ferric complex (acr). The promoter of this operon was negatively regulated by iron. In a fur null mutant, transcriptional fusions to the acsD and acsA genes were constitutively expressed. Band shift assays showed that the purified E. chrysanthemi Fur repressor protein specifically binds in vitro to the promoter region of the acsF gene confirming that the metalloregulation of the achromobactin operon is achieved directly by Fur. The temporal production of achromobactin in iron-depleted bacterial cultures was determined: achromobactin is produced before chrysobactin and its production decreases as that of chrysobactin increases. Pathogenicity tests performed on African violets showed that achromobactin production contributes to the virulence of E. chrysanthemi. Thus, during infection, synthesis of these two different siderophores allows E. chrysanthemi cells to cope with the fluctuations of iron availability encountered within plant tissues. Interestingly, iron transport mediated by achromobactin or a closely related siderophore probably exists in other phytopathogenic bacterial species such as Pseudomonas syringae

Iron regulation and pathogenicity in Erwinia chrysanthemi 3937: Role of the fur repressor protein

International audienc

Genetic analysis of the Erwinia chrysanthemi 3937 chrysobactin iron-transport system: Characterization of a gene cluster involved in uptake and biosynthetic pathways

Twenty of the twenty-two Mu dII1734 insertions impairing the chrysobactin iron-assimilation system of Erwinia chrysanthemi 3937 were localized to a 50kbp genomic insert contained in the R-prime plasmid, R′4 (Enard et al. 1988). Using the conjugative plasmid pULB110 (RP4:mini-Mu) and the generalized transducing phage ΦEC2, we located this iron-transport region and the two unlinked mutations on the chromosome linkage map. Chrysobactin is a catechol-type siderophore and, as we have previously observed with the entA locus of Escherichia coli, the E. chrysanthemi-derived R′4 was found to complement E. coli entB and entE mutations. A 2.9kb Eco Ri and a 4.8kb BamHI fragment in the R′4 sharing homology with the E. coli entCEBAP15 operon DNA were subcloned. These fragments were used as DNA/DNA hybridization probes to screen a wild-type gene library, yielding a recombinant cosmid (pEC7) able to complement mutations disrupting the 2,3-dihydroxybenzoic acid biosynthetic pathway in both Erwinia and Escherichia spp. as well as the E. coli entE mutation. Physical mapping of the genomic Mu dII1734 insertions corresponding to these mutations led to the identification of a cluster of genes confined to a DNA sequence of about 10 kb required for both biosynthetic and receptor functions.SCOPUS: ar.jFLWNAinfo:eu-repo/semantics/publishe