The growing demand to fulfill the needs of present-day medicine in terms of novel effective molecules has lead to reexamining some of the old and known bacterial secondary metabolites. Bacterial ...prodigiosins (prodiginines) have a long history of being re markable multipurpose compounds, best examined for their anticancer and antimalarial activities. Production of prodigiosin in the most common producer strain Serratia marcescens has been described in great detail. However, few reports have discussed the ecophysiological roles of these molecules in the producing strains, as well as their antibiotic and UV-protective properties. This review describes recent advances in the production process, biosynthesis, properties, and applications of bacterial prodigiosins. Special emphasis is put on undecylprodigiosin which has generally been a less studied member of the prodigiosin family. In addition, it has been suggested that proteins involved in undecylprodigiosin synthesis, RedG and RedH, could be a useful addition to the biocatalytic toolbox being able to mediate regio- and stereoselective oxidative cyclization. Judging by the number of recent references (216 for the 2007–2013 period), it has become clear that undecylprodigiosin and other bacterial prodigiosins still hold surprises in terms of valuable properties and applicative potential to medical and other industrial fields and that they still deserve continuing research curiosity.
Bacterial infections have become increasingly difficult to treat due to the occurrence of antibiotic-resistant strains. A promising strategy to increase the efficacy of therapy is to combine ...antibacterials with agents that decrease pathogen virulence
via
the modulation of the quorum sensing (QS). Lactonases inhibit acylated homoserine lactone (AHL)-mediated QS in Gram-negative bacteria, including the leading nosocomial pathogen
Pseudomonas aeruginosa
. Here we describe the characteristics of heterologously expressed YtnP lactonase from
Bacillus paralicheniformis
ZP1 (YtnP-ZP1) isolated from agricultural soil using the culture enrichment method. Purified YtnP-ZP1 hydrolyzed different AHLs with preference to substrates with long acyl residues as evaluated in assays with biosensors and HPLC. The enzyme showed good thermostability and activity in a wide temperature range. YtnP-ZP1 in 50 μg mL
–1
concentration reduced the amount of
P. aeruginosa
-produced long-chain AHLs by 85%, while it hydrolyzed 50% of short-chain AHLs. Incubation of
P. aeruginosa
PAO1 with YtnP-ZP1 reduced its swarming motility and elastolytic activity without bactericidal effect. YtnP-ZP1 caused the inhibition of biofilm formation and disintegration of mature biofilms in
P. aeruginosa
PAO1 and multiresistant clinical strain BR5H that was visualized by crystal violet staining. The treatment with YtnP-ZP1 in concentrations higher than 25 μg mL
–1
improved the survival of
P. aeruginosa
PAO1-infected zebrafish (
Danio rerio
), rescuing 80% of embryos, while in combination with tobramycin or gentamicin survival rate increased to 100%. The treatment of
P. aeruginosa
PAO1 biofilms on infected zebrafish tail wounds with 50 μg mL
–1
YtnP-ZP1 and 2 × MIC tobramycin led to infection clearing in 2 days. The extensive toxicity studies proved YtnP-ZP1 was non-toxic to human cells and zebrafish. In conclusion, novel YtnP-ZP1 lactonase with its effective anti-virulence activity could be used to increase the efficacy of clinically approved antibiotics in clearing both systemic and biofilm-associated
P. aeruginosa
infections.
Candidiasis caused by Candida albicans is one of the most common fungal infections in modern society. The limited arsenal of clinical drugs, their side effects and emerging resistance, largely ...contribute to the low efficacy of current antifungal therapies. The morphogenetic yeast-to-hyphae transition is the key virulence feature for the establishment of local and systemic C. albicans infections. We show that a delfinidin-rich fruit extract from Aristotelia chilensis (Molina, Stuntz) (maqui berry) inhibits filamentation of C. albicans, in both laboratory and clinical strains. The extract acts synergistically with nystatin, with filament formation completely prevented by the combination of ¼×MIC of nystatin and 0.125 mg/ml of the extract. The combination treatment results in increased survival of C. albicans-infected zebrafish embryos compared to treatment with nystatin. Neither the extract nor its combination with nystatin was toxic at effective doses. These results warrant further investigation of maqui berry extract as adjuvant antifungal treatment.
The adaptability of halophytes to increased soil salinity is related to complex rhizosphere interactions. In this study, an integrative approach, combining culture-independent and culture-dependent ...techniques was used to analyze the bacterial communities in the endorizosphere of indigenous succulent halophytes Salicornia europaea, Suaeda maritima, and Camphorosma annua from the natural salt marshes of Slano Kopovo (Serbia). The 16 S rDNA analyses gave, for the first time, an insight into the composition of the endophytic bacterial communities of S. maritima and C. annua. We have found that the composition of endophyte microbiomes in the same habitat is to some extent influenced by plant species. A cultivable portion of the halophyte microbiota was tested at different NaCl concentrations for the set of plant growth promoting (PGP) traits. Through the mining of indigenous halotolerant endophytes, we obtained a collection representing a core endophyte microbiome conferring desirable PGP traits. The majority (65%) of the selected strains belonged to the common halotolerant/halophilic genera Halomonas, Kushneria, and Halobacillus, with representatives exhibiting multiple PGP traits, and retaining beneficial traits in conditions of the increased salinity. The results suggest that the root endosphere of halophytes is a valuable source of PGP bacteria supporting plant growth and fitness in salt-affected soils.
sp. NP10 was previously shown to synthesize large amounts of free fatty acids (FFAs). In this work, we report the first insights into the biosynthesis of these fatty acids (FAs) gained after genome ...sequencing and identification of the genes involved. Analysis of the
sp. NP10 draft genome revealed that it is closely related to several strains of
. Comparative analyses of secondary metabolite biosynthetic gene clusters, as well as those presumably involved in FA biosynthesis, allowed identification of an unusual cluster C12-2, which could be identified in only one other
-related streptomycete. To prove the involvement of identified cluster in FFA biosynthesis, one of its three ketosynthase genes was insertionally inactivated to generate mutant strain mNP10. Accumulation of FFAs in mNP10 was almost completely abolished, reaching less than 0.01% compared to the wild-type strain. Cloning and transfer of the C12-2 cluster to the mNP10 mutant partially restored FFA production, albeit to a low level. The discovery of this rare FFA biosynthesis cluster opens possibilities for detailed characterization of the roles of individual genes and their products in the biosynthesis of FFAs in NP10.
Glycosylation and pH stability of penicillin G acylase from Providencia rettgeri produced in Pichia pastoris Senerovic, L., Max Plank Institute for Infectious Biology, Berlin (Germany); Stankovic, N., Institute of Molecular Genetics and Genetic Engineering, Belgrade (Serbia); Ljubijankic, G., Institute of Molecular Genetics and Genetic Engineering, Belgrade (Serbia) ...
Archives of biological sciences,
(2009), Letnik:
61, Številka:
4
Journal Article
Odprti dostop
Penicillin G acylase (PAC) is one of the most widely used enzymes in industrail synthesis of semi-synthetic antibiotics. The Providencia rettgeri pac gene was expressed to a level of 2.7 U/ml using ...the Pichia pastoris expression system. The recombinant enzyme was purified and its glycosylation status was determined. It was found that both subunits (alfa and beta) of the enzyme were N-glycosylated, while the beta-subunit also contained O-glycans. It was also observed that rPACP.rett. was stable in a wide range of pH, which, in addition to the previously proved high thermostability, makes it an attractive biocatalyst from an industrial point of view.
Actinomycetes are Gram-positive soil bacteria known as producers of secondary
bioactive metabolites. In the search for new bioactive compounds collection
of actinomycetes from the Laboratory for ...Microbial Molecular Genetics and
Ecology (Institute of Molecular Genetic and Genetic Engineering, University
of Belgrade) was screened according to several criteria - bioactivity, the
presence of the genes for polyketide synthase (PKS), pigmentation, and crude
culture extracts absorption profile in ultraviolet and visible (UV/Vis)
spectra. Based on these we have isolated three strains NP10, JS520, and
Streptomyces durmitotensis, previously renewed for didehydroroflamicoin
(DDHR) production. Isolates NP10 and JS520 were identified to belong to the
genus Streptomyces. Streptomyces sp. NP10 accumulated branched chain fatty
acids, predominately isopalmitic, and cyclic dipeptides consisting of proline
in combination with valine, isoleucine or alanine, that have a stimulating
effect on the trophoblast cell line in culture at low concentrations (1 ng/ml
and 1 mg/ml) and cytotoxic effect at high concentrations (1 mg/ml).
Streptomyces sp. JS520 was determined to be exceptionally good producer of
pigment undecylprodigiosine (UP) with antibacterial, antifungal, antioxidant
and UV protective activities showing cytotoxic effects on trophoblast cell
line in culture at high concentrations (1 ng/ml). Cytotoxicity of polyene
macrolide antibiotic DDHR from S. durmitotensis was confirmed and it’s
antifungal effects were shown. PKS cluster responsible for DDHR synthesis was
subcloned in the cosmid library and partially sequenced. Optimization of
culture conditions resulted in 2.12 times increased production of UP, and 1.1
times increased production of DDHR.
Aktinomicete su Gram-pozitivne zemljišne bakterije poznate kao proizvođači
bioaktivnih sekundarnih metabolita. U cilju pronalaženja novih bioaktivnih
jedinjenja pretraživana je kolekcija aktinomiceta Laboratorije za molekularnu
gentiku i ekologiju mikroorganizama Instituta za molekularnu gentiku i
genetičko inženjerstvo Univerziteta u Beogradu po više kriterijuma –
bioaktivnosti, prisustvu gena za poliketid sintaze (PKS), pigmentaciji i
profilu apsorpcije ukupnih ekstrakata kultura u ultraljubičastom i vidljivom
(UV/Vis) delu spektra. Na osnovu ovoga izdvojena su tri izolata NP10, JS520 i
Streptomyces durmitorensis, od ranije poznati proizvođač
didehidroroflamikoina (DDHR). Izolati NP10 i JS520 identifikovani su kao
pripadnici roda Streptomyces. Streptomyces sp. NP10 akumulirao je masne
kiseline račvastog niza, pre svega izopalmitinsku kiselinu, i ciklične
dipeptide sastavljene od prolina u kombinaciji sa valinom, izoleucinom ili
alaninom koji su imali stimulativno dejstvo na trofoblastne ćelije u kulturi
u niskim koncentracijama (1 ng/ml i 1 μg/ml) i citotoksično dejstvo u visokim
(1 mg/ml) koncentracijama. Pokazano je da Streptomyces sp. JS520 ima
sposobnost proizvodnje velike količine pigmenta undecilprodigiozina (UP) sa
antibakterijskim, antimikotičkim, antioksidativnim i UV protektivnim
osobinama koji je imao citotoksično dejstvo na trofoblastne ćelije u kulturi
u visokim koncentracijama (1 ng/ml). Citotoksičnost polienskog makrolidnog
antibiotika DDHR iz S. durmitorensis je potvrđena, a pokazan je i njegov
antimikotički efekat. PKS klaster zadužen za sintezu DDHR je subkloniran u
kozmidnu biblioteku i delimično sekvenciran. Optimizacijom uslova gajenja
količina proizvedenog UP povećana je 2,12 puta, a DDHR 1,1 puta.
A Gram-positive, red-pigment-producing bacterial strain, designated JS520 was isolated from the pristine sediment from the cave on mountain Miroc in Serbia. Strain was confirmed to belong to
...Streptomyces
genus based on phenotypic and genetic analysis.
Streptomyces
sp. JS520 has the ability to produce exceptionally high amounts of deep red pigment into both solid and liquid media. Liquid chromatography and mass spectroscopy of the purified pigments revealed the major component to be undecylprodigiosin (93 %) with minor component being oxidatively cyclized derivative. The pigment production was affected by medium composition, temperature, pH, and the aeration rate. By medium optimization, yields of undecylprodigiosin of 138 mg l
−1
were achieved, what is the highest level of undecylprodigiosin production reported for the members of Gram-positive
Streptomyces
genus. Purified pigment had antimicrobial properties against bacterial
Bacillus
and
Micrococcus
species (50 μg ml
−1
) and against
Candida albicans
species (100–200 μg ml
−1
range). The ability to affect auto-oxidation of the linoleic acid was demonstrated for the purified undecylprodigiosin, suggesting antioxidative properties of this pigment. Multiple ecophysiological roles of the pigment were revealed by comparing cultures grown under pigment-producing and pigment-nonproducing conditions. Cells grown under undecylprodigiosin-producing conditions could tolerate presence of hydrogen peroxide exhibiting three times smaller zones of inhibition at 100 mM H
2
O
2
. Undecylprodigiosin-producing cells were also less susceptible to tetracycline, kanamycin, chloramphenicol, and 8-hydroxyquinoline. While the growth of the cells not producing pigment was completely inhibited by 15 min of exposure to ultraviolet light (254 nm), cells producing undecylprodigiosin and cells supplied with purified pigment in vitro showed survival rates at 22 and 8 %, respectively.
Candida albicans remains the main causal agent of candidiasis, the most common fungal infection with disturbingly high mortality rates worldwide. The limited diversity and efficacy of clinical ...antifungal drugs, exacerbated by emerging drug resistance, have resulted in the failure of current antifungal therapies. This imposes an urgent demand for the development of innovative strategies for effective eradication of candidal infections. While the existing clinical drugs display fungicidal or fungistatic activity, the strategy specifically targeting C. albicans filamentation, as the most important virulence trait, represents an attractive approach for overcoming the drawbacks related to clinical antifungals. The results acquired in this study revealed the significant potential of 5-aminotetrazoles as a new class of effective and safe anti-virulence agents. Moreover, these novel agents were active when applied both alone and in combination with clinically approved polyenes. Complete prevention of C. albicans morphogenetic yeast-to-hyphae transition was achieved at doses as low as 1.3 μM under conditions mimicking various filamentation-responsive stimuli in the human body, while no cardio- or hepatotoxicity was observed at doses as high as 200 μM. The treatment of C. albicans-infected zebrafish embryos with nystatin alone had low efficacy, while the combination of nystatin and selected 5-aminotetrazoles prevented fungal filamentation, successfully eliminating the infection and rescuing the infected embryos from lethal disseminated candidiasis. In addition, the most potent anti-virulence 5-aminotetrazole prevented C. albicans in developing the resistance to nystatin when applied in combination, keeping the fungus sensitive to the antifungal drug.
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•5-Aminotetrazoles as novel antivirulence agents showed in vitro and in vivo efficacy.•Inhibition of C. albicans filamentation was achieved under various stimuli.•5-Aminotetrazoles were non-toxic at a high dose of 200 μM in the zebrafish model.•Ethyladamantyl-bearing derivatives were the most potent being active at 1.25 μM.•Nystatin + 5d combination therapy was effective against disseminated candidiasis.
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•MBE has anti-virulence properties and inhibits Candida albicans filamentation.•MBE has synergistic activity with nystatin.•MBE is safe in vivo at the filamentation inhibitory ...doses.•Combination treatment with nystatin and MBE is more effective than treatments alone.
Candidiasis caused by Candida albicans is one of the most common fungal infections in modern society. The limited arsenal of clinical drugs, their side effects and emerging resistance, largely contribute to the low efficacy of current antifungal therapies. The morphogenetic yeast-to-hyphae transition is the key virulence feature for the establishment of local and systemic C. albicans infections. We show that a delfinidin-rich fruit extract from Aristotelia chilensis (Molina, Stuntz) (maqui berry) inhibits filamentation of C. albicans, in both laboratory and clinical strains. The extract acts synergistically with nystatin, with filament formation completely prevented by the combination of ¼×MIC of nystatin and 0.125 mg/ml of the extract. The combination treatment results in increased survival of C. albicans-infected zebrafish embryos compared to treatment with nystatin. Neither the extract nor its combination with nystatin was toxic at effective doses. These results warrant further investigation of maqui berry extract as adjuvant antifungal treatment.
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