Comprehensive overexpression analysis of cyclic-di-GMP signalling proteins in the phytopathogen Pectobacterium atrosepticum reveals diverse effects on motility and virulence phenotypes.

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous bacterial signalling molecule produced by diguanylate cyclases of the GGDEF-domain family. Elevated c-di-GMP levels or increased GGDEF protein expression is frequently associated with the onset of sessility and biofilm formation in numerous bacterial species. Conversely, phosphodiesterase-dependent diminution of c-di-GMP levels by EAL- and HD-GYP-domain proteins is often accompanied by increased motility and virulence. In this study, we individually overexpressed 23 predicted GGDEF, EAL or HD-GYP-domain proteins encoded by the phytopathogen Pectobacterium atrosepticum strain SCRI1043. MS-based detection of c-di-GMP and 5'-phosphoguanylyl-(3'-5')-guanosine in these strains revealed that overexpression of most genes promoted modest 1-10-fold changes in cellular levels of c-di-GMP, with the exception of the GGDEF-domain proteins ECA0659 and ECA3374, which induced 1290- and 7660-fold increases, respectively. Overexpression of most EAL domain proteins increased motility, while overexpression of most GGDEF domain proteins reduced motility and increased poly-ß-1,6-N-acetyl-glucosamine-dependent flocculation. In contrast to domain-based predictions, overexpression of the EAL protein ECA3549 or the HD-GYP protein ECA3548 increased c-di-GMP concentrations and reduced motility. Most overexpression constructs altered the levels of secreted cellulases, pectinases and proteases, confirming c-di-GMP regulation of virulence in Pe. atrosepticum. However, there was no apparent correlation between virulence-factor induction and the domain class expressed or cellular c-di-GMP levels, suggesting that regulation was in response to specific effectors within the network, rather than total c-di-GMP concentration. Finally, we demonstrated that the cellular localization patterns vary considerably for GGDEF/EAL/HD-GYP proteins, indicating it is a likely factor restricting specific interactions within the c-di-GMP network.

Permeability studies of Kavalactones using a Caco-2 cell monolayer model.

OBJECTIVE: To examine the bioavailability of kavalactones in vitro and the possible differences in their bioavailability because of variations in either chemical structure or the method of extraction used. RESEARCH DESIGN AND METHODS: Caco-2 cell monolayers were used to determine the potential bioavailability of kavalactones. Kavalactones were added to the apical layer and basolateral samples were taken over 150 min to examine the concentration diffusing across the cell monolayer. Kavalactone concentrations in these samples were determined by high pressure liquid chromatography. RESULTS: Kavalactones were found to be potentially bioavailable as they all readily crossed the Caco-2 monolayers with apparent permeabilities (P(app)) increasing from 42 x 10(-6) cm/s and most exhibiting more than 70% crossing within 90 min. Not all differences in their bioavailability can be related to kavalactone structural differences as it appears that bioavailability may also be affected by co-extracted compounds. For example, the P(app) for kawain from ethanol extracts was higher than the values obtained for the same compound from water extracts or for the kavalactone alone. CONCLUSIONS: While the extraction method used (ethanol or water) influences the total (but not the relative) concentrations of kavalactones, it does not markedly affect their bioavailability. Hence, any differences between an ethanolic or an aqueous extract in terms of the propensity of kava to cause liver damage is not because of differing kavalactone bioavailabilities.

Novel quorum-sensing-controlled genes in Erwinia carotovora subsp. carotovora: identification of a fungal elicitor homologue in a soft-rotting bacterium.

Seven new genes controlled by the quorum-sensing signal molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) have been identified in Erwinia carotovora subsp. carotovora. Using TnphoA as a mutagen, we enriched for mutants defective in proteins that could play a role in the interaction between E. carotovora subsp. carotovora and its plant hosts, and identified NipEcc and its counterpart in E. carotovora subsp. atroseptica. These are members of a growing family of proteins related to Nep1 from Fusarium oxysporum which can induce necrotic responses in a variety of dicotyledonous plants. NipEcc produced necrosis in tobacco, NipEca affected potato stem rot, and both affected virulence in potato tubers. In E. carotovora subsp. carotovora, nip was shown to be subject to weak repression by the LuxR family regulator, EccR, and may be regulated by the negative global regulator RsmA.

Potato plants genetically modified to produce N-acylhomoserine lactones increase susceptibility to soft rot erwiniae.

Many gram-negative bacteria employ N-acylhomoserine lactones (AHL) to regulate diverse physiological processes in concert with cell population density (quorum sensing [QS]). In the plant pathogen Erwinia carotovora, the AHL synthesized via the carI/expI genes are responsible for regulating the production of secreted plant cell wall-degrading exoenzymes and the antibiotic carbapen-3-em carboxylic acid. We have previously shown that targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in planta of the cognate AHL signaling molecules N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL), which in turn, were able to complement a carI-QS mutant. In the present study, we demonstrate that transgenic potato plants containing the yenI gene are also able to express AHL and that the presence and level of these AHL in the plant increases susceptibility to infection by E. carotovora. Susceptibility is further affected by both the bacterial level and the plant tissue under investigation.

Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors.

The bacterial family Enterobacteriaceae is notable for its well studied human pathogens, including Salmonella, Yersinia, Shigella, and Escherichia spp. However, it also contains several plant pathogens. We report the genome sequence of a plant pathogenic enterobacterium, Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043, the causative agent of soft rot and blackleg potato diseases. Approximately 33% of Eca genes are not shared with sequenced enterobacterial human pathogens, including some predicted to facilitate unexpected metabolic traits, such as nitrogen fixation and opine catabolism. This proportion of genes also contains an overrepresentation of pathogenicity determinants, including possible horizontally acquired gene clusters for putative type IV secretion and polyketide phytotoxin synthesis. To investigate whether these gene clusters play a role in the disease process, an arrayed set of insertional mutants was generated, and mutations were identified. Plant bioassays showed that these mutants were significantly reduced in virulence, demonstrating both the presence of novel pathogenicity determinants in Eca, and the impact of functional genomics in expanding our understanding of phytopathogenicity in the Enterobacteriaceae.

Permeability studies of alkylamides and caffeic acid conjugates from echinacea using a Caco-2 cell monolayer model.

BACKGROUND: Echinacea is composed of three major groups of compounds that are thought to be responsible for stimulation of the immune system--the caffeic acid conjugates, alkylamides and polysaccharides. This study has focussed on the former two classes, as these are the constituents found in ethanolic liquid extracts. OBJECTIVE: To investigate the absorption of these two groups of compounds using Caco-2 monolayers, which are a model of the intestinal epithelial barrier. RESULTS: The caffeic acid conjugates (caftaric acid, echinacoside and cichoric acid) permeated poorly through the Caco-2 monolayers although one potential metabolite, cinnamic acid, diffused readily with an apparent permeability (Papp) of 1 x 10(-4) cm/s. Alkylamides were found to diffuse through Caco-2 monolayers with Papp ranging from 3 x 10(-6) to 3 x 10(-4) cm/s. This diversity in Papp for the different alkylamides correlates to structural variations, with saturation and N-terminal methylation contributing to decreases in Papp. The transport of the alkylamides is not affected by the presence of other constituents and the results for synthetic alkylamides were in line with those for the alkylamides in the echinacea preparation. CONCLUSION: Alkylamides but not caffeic acid conjugates are likely to cross the intestinal barrier.

A potato gene encoding a WRKY-like transcription factor is induced in interactions with Erwinia carotovora subsp. atroseptica and Phytophthora infestans and is coregulated with class I endochitinase expression.

A potato gene encoding a putative WRKY protein was isolated from a cDNA library enriched by suppression subtractive hybridization for sequences upregulated 1 h postinoculation with Erwinia carotovora subsp. atroseptica. The cDNA encodes a putative polypeptide of 172 amino acids, containing a single WRKY domain with a zinc finger motif and preceded by a potential nuclear localization site. St-WRKY1 was strongly upregulated in compatible, but only weakly in incompatible, interactions with Phytophthora infestans where, in all cases, it was coregulated with class I endochitinase, associating its expression with a known defense response. Whereas St-WRKY1 was strongly induced by E. carotovora culture filtrate (CF), confirming it to be an elicitor-induced gene, no such induction was detected after treatment with salicylic acid, methyl jasmonate, ethylene, or wounding. St-WRKY1 was upregulated by treatment of potato leaves with CFs from recombinant Escherichia coli containing plasmids expressing E. carotovora pectate lyase genes pelB and pelD, suggesting that either proteins encoded by these genes, or oligogalacturonides generated by their activity, elicit a potato defense pathway associated with St-WRKY1.

A competitive PCR-based method for the detection and quantification of Erwinia carotovora subsp. atroseptica On potato tubers.

A PCR-based method was developed for the simultaneous detection and quantification of the potato pathogen Erwinia carotovora subsp. atroseptica (Eca) on potato tubers. The method incorporates a competitor PCR template cloned into Escherichia coli in vector pGEM-T (E. coli 4R l/l). Predetermined numbers of E. coli 4R were added to potato peel extract, either pre-inoculated with Eca or from naturally contaminated tubers, and Eca numbers estimated by comparing the ratio of products generated from Eca target DNA and competitor template DNA following PCR. Estimates of Eca numbers were consistent with counts obtained on crystal violet pectate medium and immunofluorescence colony staining. Unlike these methods, however, the PCR-based method is not affected by the presence of other erwinias and saprophytes and is able to detect all serogroups of Eca. Based on this method, a key was produced relating product ratios, obtained following PCR from contaminated tuber stocks, to the likelihood of blackleg disease incidence. This is the first quantitative PCR-based detection method described for Eca and is the first for any bacterial plant pathogen to incorporate a DNA extraction control.

In vitro inhibition of testicular delta 5-3 beta-hydroxysteroid dehydrogenase and prostatic 5 alpha-reductase activities in rats and humans by strogen forte extract.

Clinical findings indicate that Strogen forte (a standardized extract of the plant Sabalis serrulata) can be use successfully in the medical treatment of benign prostatic hyperplasia. The aim of the present study was to investigate the possible inhibitory effects of the Strogen forte extract on rat and human testicular delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta-HSD) and prostatic 5 alpha-reductase (5 alpha-R). Strogen forte proved to be a direct inhibitor of medium effectivity, with similar IC50 values for rat testicular delta 5-3 beta-HSD (400 +/- 23 micrograms/ml) and human testicular delta 5-3 beta-HSD (212 +/- 8.6 micrograms/ml). 5 alpha-R activities were analysed by in vitro incubation of rat and human prostatic tissue homogenates with 14C-labelled testosterone as substrate in KRPG-DTT medium (pH = 7.4) with NADPH coenzyme, in air, at 37 degrees C, in the presence of Strogen forte extract. The results clearly demonstrate that Strogen forte is a potent inhibitor of prostatic 5 alpha-R, with IC50 values of 385 +/- 35.6 micrograms/ml for rat and 245 +/- 64.6 micrograms/ml for human prostatic 5 alpha-R. The present study has revealed that this plant extract inhibits not only prostatic 5 alpha-R, but also testicular delta 5-3 beta-HSD.

Ascorbic acid enhances iron-induced ferritin translation in human leukemia and hepatoma cells.

Ascorbate is an important cofactor in many cellular metabolic reactions and is intimately linked to iron homeostasis. Continuously cultured cells are ascorbate deficient due to the lability of the vitamin in solution and to the fact that daily supplementation of media with ascorbate is unusual. We found that ascorbate repletion alone did not alter ferritin synthesis. However, ascorbate-replete human hepatoma cells, Hep3B and HepG2, as well as K562 human leukemia cells achieved a substantially higher cellular ferritin content in response to a challenge with iron than did their ascorbate-deficient counterparts grown under standard culture conditions. Most of the elevation in ferritin content was due to an increase in de novo ferritin synthesis of greater than 50-fold, as shown by in vivo labeling with [35S]methionine and immunoprecipitation. RNA-blot analysis showed only minor changes in steady state levels of ferritin mRNA, suggesting that ascorbate enhances iron-induced ferritin synthesis primarily by post-transcriptional events. Transient gene expression experiments using chloramphenicol acetyltransferase reporter gene constructs showed that the ascorbate effect on ferritin translation is not mediated through the stem-loop near the translational start site that transduces ferritin synthesis in response to cytokines. The data suggest that ascorbate possibly modifies the action of the iron-responsive element on ferritin translation, although more precise structure-function studies are needed to clarify this issue. These data demonstrate a novel role of ascorbate as a signaling molecule in post-transcriptional gene regulation. The mechanism by which ascorbate modulates cellular iron metabolism is complex and requires additional detailed investigation.

A novel chemical approach to drug delivery: lipidic amino acid conjugates.

A novel infinity-amino acid based oligopeptide system has been designed, which combines structural features of lipids with those of amino acids and peptides. Because of their bifunctional nature, the fatty amino acids and peptides have the capacity to be chemically conjugated to drugs and peptides with a wide variety of functional groups. The linkage between drug and lipidic unit may either be biologically stable (ie. a new drug is formed) or exhibit biological or chemical instability (ie. the conjugate is a pro-drug). In either case, the resulting conjugates would be expected to possess a high degree of membrane-like character, which may be sufficient to facilitate their passage across membranes. The long alkyl side chains may also have the additional effect of protecting a labile parent drug from enzymatic attack. The lipidic system has been conjugated to a wide variety of different compounds, including (i) alkaloids (ii) beta-lactam antibiotics, (iii) anticancer compounds (iv) CNS drugs and (v) peptides. The biological examination of the conjugates showed that an increase in lipophilicity caused an increase in the in vitro cellular and in vivo oral uptake, as well as passage through the blood-brain-barrier, suggesting that conjugation to lipidic amino acids and peptides is a useful approach to improve the absorption of poorly-absorbed drugs. Lipidic conjugates of peptides (TRH, LHRH) resulted in higher enzymatic stability of the conjugates, proving that the long alkyl side chains also have the additional effect of protecting a labile parent drug or peptide in a biological environment. A novel Lipid-Core-Peptide (LCP) system has also been synthesised by incorporating lipidic amino acids to a lysine based polyamino acid system to enhance lipophilicity and membrane binding effects and the metabolic stability of the compound. The LCP system as a combined adjuvant-carrier-vaccine greatly increased the immunogenicity of synthetic peptides.

A pleiotropic reduced virulence (Rvi-) mutant of Erwinia carotovora subspecies atroseptica is defective in flagella assembly proteins that are conserved in plant and animal bacterial pathogens.

Erwinia carotovora subsp. atroseptica was mutagenized and assayed for virulence in planta. Those mutants which exhibited reduced virulence (Rvi-) were assayed for growth rate, auxotrophy and extracellular enzyme secretion and seven mutants were found to be wild type for all of these phenotypes. When screened for other phenotypes, two were found to be non-motile. One mutant was complemented for motility by a heterologous gene library. A 2.7kb XmaIII-ClaI complementing fragment was sequenced and the gene products were found to have similarity to flagella biosynthesis gene products from several bacteria. Further similarity was found to a pathogenicity protein from the plant pathogen Xanthomonas campestris pv. glycines and to the Spa pathogenicity proteins of the human pathogen Shigella flexneri, which are involved in the surface presentation of antigens. These studies highlight the emergence of common themes in the molecular strategies employed by both plant and animal bacterial pathogens for the targeting of proteins involved in the elaboration of disease.

Antibiotic resistance and plasmid profiles of Escherichia coli and Klebsiella isolated from in-patients receiving prolonged antibiotic therapy.

Distribution by serogroup, phage type, colicin production, colicin type, sensitivity to antibiotics and plasmid characteristics of 74 Escherichia coli and 11 Klebsiella strains isolated from hospitalized patients receiving prolonged antibiotic therapy indicated that the infections were not associated with the hospital environment. Resistance was tested to 26 antibiotics, some of them being not generally used in therapy; 30 strains were resistant to 4 to 17 antibiotics. There was a significant difference in the antibiotic resistance of strains derived from patients with urinary-tract infections (UTI) and with leukaemia (LP). As compared to the UTI group, among E. coli strains in the LP group the frequency of multiple resistance was significantly higher, the MIC values were higher and R-plasmids were more frequent. Out of 30 multiple resistant E. coli strains 27 were R-plasmid carriers. Three different kinds of plasmid profile were shown in more than one strain (2 out of 10 UTI strains and 3 and 2 out of 10 LP strains). The rest of the isolates differed in plasmid profile from these and from one another; the presence of "epidemic plasmid" was not demonstrated. Plasmid epidemiological examinations may forecast the efficacy of an antibiotic or of a group of antibiotics.