Identification of Polyketide Inhibitors Targeting 3-Dehydroquinate Dehydratase in the Shikimate Pathway of Enterococcus faecalis

Due to the emergence of resistance toward current antibiotics, there is a pressing need to develop the next generation of antibiotics as therapeutics against infectious and opportunistic diseases of microbial origins. The shikimate pathway is exclusive to microbes, plants and fungi, and hence is an attractive and logical target for development of antimicrobial therapeutics. The Gram-positive commensal microbe, Enterococcus faecalis, is a major human pathogen associated with nosocomial infections and resistance to vancomycin, the “drug of last resort”. Here, we report the identification of several polyketide-based inhibitors against the E. faecalis shikimate pathway enzyme, 3-dehydroquinate dehydratase (DHQase). In particular, marein, a flavonoid polyketide, both inhibited DHQase and retarded the growth of Enterococcus faecalis. The purification, crystallization and structural resolution of recombinant DHQase from E. faecalis (at 2.2 Å resolution) are also reported. This study provides a route in the development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the human pathogen E. faecalis.     

Blocking Phosphatidylcholine Utilization in Pseudomonas aeruginosa,via Mutagenesis of Fatty Acid,Glycerol and Choline Degradation Pathways,Confirms the Importance of This Nutrient Source In Vivo

Pseudomonas aeruginosa can grow to very high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of P. aeruginosa in vivo. The phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs) are released by enzymatic cleavage of PC by bacterial phospholipase C and lipases. Three different bacterial pathways, the choline, glycerol, and fatty acid degradation pathways, are then involved in the degradation of these PC components. Here, we identified five potential FA degradation (Fad) related fadBA-operons (fadBA1-5, each encoding 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase). Through mutagenesis and growth analyses, we showed that three (fadBA145) of the five fadBA-operons are dominant in medium-chain and long-chain Fad. The triple fadBA145 mutant also showed reduced ability to degrade PC in vitro. We have previously shown that by partially blocking Fad, via mutagenesis of fadBA5 and fadDs, we could significantly reduce the ability of P. aeruginosa to replicate on FA and PC in vitro, as well as in the mouse lung. However, no studies have assessed the ability of mutants, defective in choline and/or glycerol degradation in conjunction with Fad, to grow on PC or in vivo. Hence, we constructed additional mutants (ΔfadBA145ΔglpD, ΔfadBA145ΔbetAB, and ΔfadBA145ΔbetABΔglpD) significantly defective in the ability to degrade FA, choline, and glycerol and, therefore, PC. The analysis of these mutants in the BALB/c mouse lung infection model showed significant inability to utilize PC in vitro, resulted in decreased replication fitness and competitiveness in vivo compared to the complement strain, although there was little to no variation in typical virulence factor production (e.g., hemolysin, lipase, and protease levels). This further supports the hypothesis that lung surfactant PC serves as an important nutrient for P. aeruginosa during CF lung infection.     

Artificial and factitious foods support the development and reproduction of the predatory mite Amblyseius swirskii

The generalist predatory mite Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae) was reared on Ephestia kuehniella Zeller eggs (Lepidoptera: Pyralidae), decapsulated dry cysts of the brine shrimp Artemia franciscana Kellogg (Anostraca: Artemiidae), and on meridic artificial diets (composed of honey, sucrose, tryptone, yeast extract, and egg yolk) supplemented with pupal hemolymph of the Chinese oak silkworm Antheraea pernyi (Guérin-Méneville) (Lepidoptera: Saturniidae) (AD1), with E. kuehniella eggs (AD2) or with A. franciscana cysts (AD3). Development, reproduction and predation capacity of the predatory mites were assessed in the first (G1) and sixth generation (G6) of rearing on the different diets. Immature survival rates in G1 were similar on all diets (96.8–100 %). After six generations, however, survival of A. swirskii was significantly reduced on all diets except on A. franciscana cysts. Oviposition rates did not differ between generations when females were fed on E. kuehniella, AD2 or AD3. The total number of deposited eggs was similar among diets except in G6 where the females fed on A. franciscana cysts produced more eggs than those maintained on E. kuehniella eggs. On most diets the intrinsic rates of increase in G1 were superior to those in G6, except for predators supplied with A. franciscana cysts where no differences were observed among generations. Female mites did not lose their capacity to kill first instar Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) after six generations on the different diets, but predation rates in G6 on E. kuehniella were lower than in G1. In conclusion, the different factitious and artificial diets tested in the present study supported the development and reproduction of A. swirskii for a single generation but fitness losses occurred to a varying degree after several generations on E. kuehniella eggs or the artificial diets. Artificial diet enriched with A. franciscana cysts yielded better results than the other artificial diets. Amblyseius swirskii performed best on decapsulated Artemia cysts indicating their potential for use in the mass production of the predator or to sustain its populations in the crop after release.     

Detection of tetrodotoxin-producing Providencia rettgeri T892 in Lagocephalus pufferfish

Tetrodotoxin (TTX) is a potent toxin but it could be used in pharmaceutical field. Identification of TTX producing bacteria in pufferfish is necessary for TTX yield and the pufferfish conservation. In this study, Lagocephalus was collected from Cam Ranh Sea, a central part of Vietnam during spring season. The liver and intestine were incubated in 0.9 % NaCl for TTX detection in pufferfish. To be benefited from the isolation of new TTX producing bacteria, the liver and intestine were incubated in 6.5 % NaCl. The cultures were used to test the toxin and to isolate the bacterial community that could yield TTX. Surprisingly, Providencia rettgeri T892 in intestine could produce TTX identified by biochemical test and 16S rRNA sequencing. This strain was used to test the production of TTX, based on thin layer chromatography (TLC), mouse bioassay and high performance liquid chromatography (HPLC) analysis. The bacterium was optimized for TTX production in media prepared from the meat-washing water of Auxis thazard, Megalaspis cordyla and Decapterus maruadsi. Interestingly, the TTX obtained 0.106 mg/mL and 0.055 mg/mL in medium prepared from A. thazard and M. cordyla, respectively while there was no TTX production detected in medium prepared from D. maruadsi. This paper could contribute to warn to the human health care system about a possible TTX poisoning in some cases related to eating fishes.     

Porcine lactoferrin as feedstuff additive elevates avian immunity and potentiates vaccination

In this study, recombinant porcine lactoferrin (PLF) was used as feedstuff additive to investigate the effects of peripheral lymphocyte proliferation and serum antibody titers in chickens vaccinated against the infectious bursal disease (IBD) virus. Treatment groups were fed three doses of PLF powder in their diet (0.5, 1.0, and 2.0% w/w), and the IBD vaccine was administrated at 1 and 3 weeks of age. At 8, 12, and 16 weeks after vaccination, serum IBD antibody titers were measured via the micro-method and T cell proliferation rates were evaluated. The results revealed that a high dose of PLF led to significant increases in serum IgA, IgG and IBD-specific antibody titers (P < 0.05). PLF administration, at either low or high doses, enhanced the expression of IFN-γ and IL-12 in chicken T lymphocytes. These results suggest that PLF enhances cell-mediated immunity and augment the ability of IBD vaccination to strengthen subsequent anti-viral responses.     

The p-type ATPase superfamily

P-type ATPases function to provide homeostasis in higher eukaryotes, but they are essentially ubiquitous, being found in all domains of life. Thever and Saier [J Memb Biol 2009;229:115-130] recently reported analyses of eukaryotic P-type ATPases, dividing them into nine functionally characterized and 13 functionally uncharacterized (FUPA) families. In this report, we analyze P-type ATPases in all major prokaryotic phyla for which complete genome sequence data are available, and we compare the results with those for eukaryotic P-type ATPases. Topological type I (heavy metal) P-type ATPases predominate in prokaryotes (approx. tenfold) while type II ATPases (specific for Na(+),K(+), H(+) Ca(2+), Mg(2+) and phospholipids) predominate in eukaryotes (approx. twofold). Many P-type ATPase families are found exclusively in prokaryotes (e.g. Kdp-type K(+) uptake ATPases (type III) and all ten prokaryotic FUPA familes), while others are restricted to eukaryotes (e.g. phospholipid flippases and all 13 eukaryotic FUPA families). Horizontal gene transfer has occurred frequently among bacteria and archaea, which have similar distributions of these enzymes, but rarely between most eukaryotic kingdoms, and even more rarely between eukaryotes and prokaryotes. In some bacterial phyla (e.g. Bacteroidetes, Flavobacteria and Fusobacteria), ATPase gene gain and loss as well as horizontal transfer occurred seldom in contrast to most other bacterial phyla. Some families (i.e. Kdp-type ATPases) underwent far less horizontal gene transfer than other prokaryotic families, possibly due to their multisubunit characteristics. Functional motifs are better conserved across family lines than across organismal lines, and these motifs can be family specific, facilitating functional predictions. In some cases, gene fusion events created P-type ATPases covalently linked to regulatory catalytic enzymes. In one family (FUPA Family 24), a type I ATPase gene (N-terminal) is fused to a type II ATPase gene (C-terminal) with retention of function only for the latter. Several pseudogene-encoded nonfunctional ATPases were identified. Genome minimalization led to preferential loss of P-type ATPase genes. We suggest that in prokaryotes and some unicellular eukaryotes, the primary function of P-type ATPases is protection from extreme environmental stress conditions. The classification of P-type ATPases of unknown function into phylogenetic families provides guides for future molecular biological studies.     

Importance of replication in analyzing time-series gene expression data: Corticosteroid dynamics and circadian patterns in rat liver

Background  

Comprehensively understanding corticosteroid pharmacogenomic effects is an essential step towards an insight into the underlying molecular mechanisms for both beneficial and detrimental clinical effects. Nevertheless, even in a single tissue different methods of corticosteroid administration can induce different patterns of expression and regulatory control structures. Therefore, rich in vivo datasets of pharmacological time-series with two dosing regimens sampled from rat liver are examined for temporal patterns of changes in gene expression and their regulatory commonalities.