Heterologous production in Wolinella succinogenes and characterization of the quinol:fumarate reductase enzymes from Helicobacter pylori and Campylobacter jejuni

Mileni, Mauro; MacMillan, Fraser; Tziatzios, Christos; Zwicker, Klaus; Haas, Alexander H.; Mäntele, Werner; Simon, Jörg; Lancaster, C. Roy D.

doi:10.1042/bj20051675

Cited by 38 publications

(25 citation statements)

References 60 publications

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“…Anthelmintics that have been used to cure helminthic infections in animals and humans have bacteriostatic and bactericidal effects against some bacteria by inhibiting the Frd enzyme complex (44,(46)(47)(48). In the present study, we demonstrated that P. gingivalis Frd was inhibited by oxantel in a dose-dependent manner with an IC 50 of 2.2 M, and at 5 M, oxantel effectively abolished Frd activity in a cytoplasmic extract (Table 2).…”

Section: Discussionsupporting

confidence: 52%

“…This is a much lower concentration than the previously determined MIC of 125 M, suggesting that entry into the cell is the limiting factor in growth inhibition (35). In addition, the P. gingivalis Frd was much more sensitive to oxantel inhibition than recombinantly expressed and purified C. jejuni and H. pylori Frd (48). Oxantel also inhibited the growth of the two other selected oral bacterial pathogens that are predicted to rely on Frd for catabolism.…”

Section: Discussionmentioning

confidence: 72%

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Oxantel Disrupts Polymicrobial Biofilm Development of Periodontal Pathogens

Dashper

O'Brien-Simpson

Liu

et al. 2014

Antimicrob Agents Chemother

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Bacterial pathogens commonly associated with chronic periodontitis are the spirochete Treponema denticola and the Gramnegative, proteolytic species Porphyromonas gingivalis and Tannerella forsythia. These species rely on complex anaerobic respiration of amino acids, and the anthelmintic drug oxantel has been shown to inhibit fumarate reductase (Frd) activity in some pathogenic bacteria and inhibit P. gingivalis homotypic biofilm formation. Here, we demonstrate that oxantel inhibited P. gingivalis Frd activity with a 50% inhibitory concentration (IC 50 ) of 2.2 M and planktonic growth of T. forsythia with a MIC of 295 M, but it had no effect on the growth of T. denticola. Oxantel treatment caused the downregulation of six P. gingivalis gene products and the upregulation of 22 gene products. All of these genes are part of a regulon controlled by heme availability. There was no large-scale change in the expression of genes encoding metabolic enzymes, indicating that P. gingivalis may be unable to overcome Frd inhibition. Oxantel disrupted the development of polymicrobial biofilms composed of P. gingivalis, T. forsythia, and T. denticola in a concentration-dependent manner. In these biofilms, all three species were inhibited to a similar degree, demonstrating the synergistic nature of biofilm formation by these species and the dependence of T. denticola on the other two species. In a murine alveolar bone loss model of periodontitis oxantel addition to the drinking water of P. gingivalis-infected mice reduced bone loss to the same level as the uninfected control.

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Section: Discussionsupporting

confidence: 52%

Section: Discussionmentioning

confidence: 72%

Oxantel Disrupts Polymicrobial Biofilm Development of Periodontal Pathogens

Dashper

O'Brien-Simpson

Liu

et al. 2014

Antimicrob Agents Chemother

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“…The bacterium can also generate energy aerobically directly from molecular hydrogen with the help of a hydrogenase (40). Moreover, H. pylori is able to use fumarate as an electron acceptor (17,33). No redox regulator Fnr homolog which might regulate the Cbb3 terminal oxidase has been identified, implying that its expression is constitutive.…”

Section: Discussionmentioning

confidence: 99%

Functional Characterization and Mutagenesis of the Proposed Behavioral Sensor TlpD of Helicobacter pylori

et al. 2008

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Helicobacter pylori requires flagellar motility and chemotaxis to establish and maintain chronic infection of the human stomach. The pH gradient in the stomach mucus is essential for bacterial orientation and guides the bacterium toward a narrow layer of the mucus, suggesting that H. pylori is capable of energy sensing or taxis. In the present study, H. pylori wild-type behavior in a temporal swimming assay could be altered by electron transport inhibitors, indicating that a connection between metabolism and behavior exists. In order to elucidate mechanisms of behavioral responses of H. pylori related to energy sensing, we investigated the phenotypes of single and multiple mutants of the four proposed chemotaxis sensor proteins. All sensor mutants were motile, but they diverged in their behavior in media supporting different energy yields. One proposed intracellular sensor, TlpD, was crucial for behavioral responses of H. pylori in defined media which did not permit growth and led to reduced bacterial energy levels. Suboptimal energetic conditions and inhibition of electron transport induced an increased frequency of stops and direction changes in the wild type but not in tlpD mutants. Loss of metabolism-dependent behavior in tlpD mutants could be reversed by complementation but not by electron donors bypassing the activity of the electron transport chain, in contrast to the case for the wild type. TlpD, which apparently lacks transmembrane domains, was detected both in the bacterial cytoplasm and at the bacterial periphery. The proposed energy sensor TlpD was found to mediate a repellent tactic response away from conditions of reduced electron transport.Helicobacter pylori has the ability to survive and multiply in the mucous layer of the human stomach, where it colonizes persistently for many years. Gastric Helicobacter species need complete flagella and full motility to colonize their hosts persistently (5,13,23,42). Both for the first entry of H. pylori into the human gastric mucus and for chronic colonization, H. pylori probably exploits horizontal and vertical substance gradients to reach and stay in its optimal habitat. Motile H. pylori mutants deficient in chemotaxis by knockout mutagenesis of the chemotaxis histidine kinase gene cheA and other chemotaxis proteins have been found to be unable to colonize in mouse and gerbil colonization models (16,26). Analysis of nanobiopsies taken from the stomach mucus of anesthetized, Helicobacterinfected mice and Mongolian gerbils has shown that Helicobacter actively accumulates in a very narrow zone of the proximal stomach mucus, at a distance from the epithelial surface of between 0 and 15 m (45, 46). It is guided there by a vertical proton gradient within the gastric mucus. Changes in other chemical gradients in the mucus in vivo did not alter the horizontal distribution of H. pylori, suggesting that pH taxis may be dominant over other chemotactic responses (45). pH taxis, a manifestation of energy-dependent taxis, was therefore shown to be essential for persiste...

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“…All these elements are predicted to be required for menaquinone-dependent succinate dehydrogenase activity (45). The fumarate reductase of W. succinogenes is capable of both specific enzymatic activity during succinate-dependent methylene blue reduction and benzyl viologen oxidation through fumarate reduction (26).…”

Section: Discussionmentioning

confidence: 99%

The Dual-Functioning Fumarate Reductase Is the Sole Succinate:Quinone Reductase in Campylobacter jejuni and Is Required for Full Host Colonization

2009

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Campylobacter jejuni encodes all the enzymes necessary for a complete oxidative tricarboxylic acid (TCA) cycle. Because of its inability to utilize glucose, C. jejuni relies exclusively on amino acids as the source of reduced carbon, and they are incorporated into central carbon metabolism. The oxidation of succinate to fumarate is a key step in the oxidative TCA cycle. C. jejuni encodes enzymes annotated as a fumarate reductase (Cj0408 to Cj0410) and a succinate dehydrogenase (Cj0437 to Cj0439). Null alleles in the genes encoding each enzyme were constructed. Both enzymes contributed to the total fumarate reductase activity in vitro. The frdA::cat ؉ strain was completely deficient in succinate dehydrogenase activity in vitro and was unable to perform whole-cell succinate-dependent respiration. The sdhA::cat ؉ strain exhibited wild-type levels of succinate dehydrogenase activity both in vivo and in vitro. These data indicate that Frd is the only succinate dehydrogenase in C. jejuni and that the protein annotated as a succinate dehydrogenase has been misannotated. The frdA::cat ؉ strain was also unable to grow with the characteristic wild-type biphasic growth pattern and exhibited only the first growth phase, which is marked by the consumption of aspartate, serine, and associated organic acids. Substrates consumed in the second growth phase (glutamate, proline, and associated organic acids) were not catabolized by the the frdA::cat ؉ strain, indicating that the oxidation of succinate is a crucial step in metabolism of these substrates. Chicken colonization trials confirmed the in vivo importance of succinate oxidation, as the frdA::cat ؉ strain colonized chickens at significantly lower levels than the wild type, while the sdhA::cat ؉ strain colonized chickens at wild-type levels.

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Heterologous production in Wolinella succinogenes and characterization of the quinol:fumarate reductase enzymes from Helicobacter pylori and Campylobacter jejuni

Cited by 38 publications

References 60 publications

Oxantel Disrupts Polymicrobial Biofilm Development of Periodontal Pathogens

Oxantel Disrupts Polymicrobial Biofilm Development of Periodontal Pathogens

Functional Characterization and Mutagenesis of the Proposed Behavioral Sensor TlpD of Helicobacter pylori

The Dual-Functioning Fumarate Reductase Is the Sole Succinate:Quinone Reductase in Campylobacter jejuni and Is Required for Full Host Colonization

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