Mona I. Shaaban

Suppressor Mutagenesis Identifies a Velvet Complex Remediator of Aspergillus nidulans Secondary Metabolism

ABSTRACT Fungal secondary metabolites (SM) are bioactive compounds that are important in fungal ecology and, moreover, both harmful and useful in human endeavors (e.g., as toxins and pharmaceuticals). Recently a nuclear heterocomplex termed the Velvet complex, characterized in the model ascomycete Aspergillus nidulans, was found to be critical for SM production. Deletion of two members of the Velvet complex, laeA and veA, results in near loss of SM and defective sexual spore production in A. nidulans and other species. Using a multicopy-suppressor genetics approach, we have isolated an Aspergillus nidulans gene named rsmA (remediation of secondary metabolism) based upon its ability to remediate secondary metabolism in ΔlaeA and ΔveA backgrounds. Overexpression of rsmA (OE::rsmA) restores production of sterigmatocystin (ST) (a carcinogenic SM) via transcriptional activation of ST biosynthetic genes. However, defects in sexual reproduction in either ΔlaeA or ΔveA strains cannot be overcome by OE::rsmA. An intact Velvet complex coupled with an OE::rsmA allele increases SM many fold over the wild-type level, but loss of rsmA does not decrease SM. RsmA encodes a putative bZIP basic leucine zipper-type transcription factor.

Involvement of transposon-like elements in penicillin gene cluster regulation.

Subtelomeric secondary metabolite (SM) gene clusters are frequently surrounded by DNA repeats and transposon-like elements. The Aspergillus nidulans penicillin cluster, 30kb from the telomere of chromosome VI, is bordered by such elements. Deletions of penicillin telomere proximal and distal border regions resulted in decreased penicillin production. A 3.7kb distal region called PbIa, consisting of the putative transposable element DNA-2, was examined further where its replacement by a pyrG marker presented a similar phenotype as loss of the global SM regulator LaeA, resulting in a decrease in penicillin gene expression and product formation. In contrast, placement of the pyrG marker on either side of PbIa had no effect on penicillin synthesis. A requirement for PbIa in penicillin production was also apparent in a histone deacetylase mutant, DeltahdaA, enhanced for penicillin production. Trans-complementation of the PbIa element near and within the terrequinone A cluster on chromosome V did not restore penicillin biosynthesis or increase production of terrequinone A. Taken together, this data provides evidence for transposon involvement in SM cluster regulation.

Mangostanaxanthones I and II, new xanthones from the pericarp of Garcinia mangostana.

Two new xanthones: mangostanaxanthones I (3) and II (5) were isolated from the pericarp of Garcinia mangostana, along with four known xanthones: 9-hydroxycalabaxanthone (1), parvifolixanthone C (2), α-mangostin (4), and rubraxanthone (6). Their structures were elucidated on the basis of IR, UV, 1D, 2D NMR, and MS spectroscopic data, in addition to comparison with literature data. The isolated compounds were evaluated for their antioxidant, antimicrobial, and quorum-sensing inhibitory activities. Compounds 3 and 5 displayed promising antioxidant activity with IC50 12.07 and 14.12 μM, respectively using DPPH assay. Compounds 4-6 had weak to moderate activity against Escherichia coli and Staphylococcus aureus, while demonstrated promising action against Bacillus cereus with MICs 0.25, 1.0, and 1.0mg/mL, respectively. The tested compounds were inactive against Candida albicans. However, they showed selective antifungal potential toward Aspergillus fumigatus. Compounds 3 and 4 possessed quorum-sensing inhibitory activity against Chromobacterium violaceum ATCC 12472.

Cryptic Aspergillus nidulans antimicrobials.

ABSTRACT Secondary metabolite (SM) production by fungi is hypothesized to provide some fitness attribute for the producing organisms. However, most SM clusters are “silent” when fungi are grown in traditional laboratory settings, and it is difficult to ascertain any function or activity of these SM cluster products. Recently, the creation of a chromatin remodeling mutant in Aspergillus nidulans induced activation of several cryptic SM gene clusters. Systematic testing of nine purified metabolites from this mutant identified an emodin derivate with efficacy against both human fungal pathogens (inhibiting both spore germination and hyphal growth) and several bacteria. The ability of catalase to diminish this antimicrobial activity implicates reactive oxygen species generation, specifically, the generation of hydrogen peroxide, as the mechanism of emodin hydroxyl activity.

Sodium ascorbate as a quorum sensing inhibitor of Pseudomonas aeruginosa

Quorum sensing circuits regulate virulence factors in Pseudomonas aeruginosa and coordinate bacterial pathogenicity. We are interested in exploring available medications for their antiquorum sensing activity.

Synthesis, Antimicrobial, Antiquorum-Sensing, and Cytotoxic Activities of New Series of Isoindoline-1,3-dione, Pyrazolo[5,1-a]isoindole, and Pyridine Derivatives

New series of isoindoline‐1,3‐diones 2–9, pyrazolo[5,1‐a]isoindoles 10–14, and pyridines 16–18 were synthesized. Twenty of the synthesized compounds were screened for their antibacterial activity against S. aureus, B. cereus, and E. coli. Compound 5 was proved to be the most active member in this study, showing the highest antibacterial activity against the three selected microorganisms. The antifungal activity of these compounds was also tested against C. albicans and A. flavus 3375. Compounds 4, 5, 8, and 17a exhibited the best antifungal activity against A. flavus 3375. The same compounds were examined for their antiquorum‐sensing activity against Ch. violacium ATCC 12472, whereas compound 5 displayed strong antiquorum‐sensing activity. The in vitro cytotoxicity testing of compounds 4–9 and 17a against human normal lung fibroblast (W138) cell line revealed that compounds 4, 5, and 8 are the least cytotoxic analogs in this study. In vivo acute toxicity testing of compounds 4, 5, and 8 was performed. The DNA‐binding affinity of compounds 4–9 and 17a was also tested and the obtained results showed that all tested compounds have moderate DNA‐binding affinity.

Quorum Sensing Inhibiting Activity of Streptomyces coelicoflavus Isolated from Soil

Quorum sensing (QS) systems communicate bacterial population and stimulate microbial pathogenesis through signaling molecules. Inhibition of QS signals potentially suppresses microbial infections. Antimicrobial properties of Streptomyces have been extensively studied, however, less is known about quorum sensing inhibitory (QSI) activities of Streptomyces. This study explored the QSI potential of Streptomyces isolated from soil. Sixty-five bacterial isolates were purified from soil samples with morphological characteristics of Streptomyces. The three isolates: S6, S12, and S17, exhibited QSI effect by screening with the reporter, Chromobacterium violaceum. Isolate S17 was identified as Streptomyces coelicoflavus by sequencing of the hypervariable regions (V1–V6) of 16S rRNA and was assigned gene bank number KJ855087. The QSI effect of the cell-free supernatant of isolate S17 was not abolished by proteinase K indicating the non-enzymatic activity of QSI components of S17. Three major compounds were isolated and identified, using spectroscopic techniques (1D, 2D NMR, and Mass spectrometry), as behenic acid (docosanoic acid), borrelidin, and 1H-pyrrole-2-carboxylic acid. 1H-pyrrole-2-carboxylic acid inhibited QS and related virulence factors of Pseudomonas aeruginosa PAO1 including; elastase, protease, and pyocyanin without affecting Pseudomonas viability. At the molecular level, 1H-pyrrole-2-carboxylic acid suppressed the expression of QS genes (lasI, lasR, lasA, lasB, rhlI, rhlR, pqsA, and pqsR). Moreover, QSI activity of S17 was assessed under different growth conditions and ISP2 medium supplemented with glucose 0.4% w/v and adjusted at pH 7, showed the highest QSI action. In conclusion, 1H-pyrrole-2-carboxylic acid, one of the major metabolites of Streptomyces isolate S17, inhibited QS and virulence determinants of P. aeruginosa PAO1. The findings of the study open the scope to exploit the in vivo efficacy of this active molecule as anti-pathogenic and anti-virulence of P. aeruginosa.

Formulation of carbapenems loaded gold nanoparticles to combat multi-antibiotic bacterial resistance: In vitro antibacterial study

Despite the fact that carbapenems (powerful β-lactams antibiotics) were able to fight serious infectious diseases, nowadays the spread of carbapenems-resistant bacteria is considered the main challenge in antibacterial therapy. In this study, we focused on evaluating the surface conjugation of carbapenems (imipenem and meropenem) with gold nanoparticles as a delivering strategy to specifically and safely maximize their therapeutic efficacy while destroying the developing resistance of the pathogens. Different particle size formulae (35, 70 and 200nm) were prepared by citrate reduction method. The prepared nanoparticles were functionalized with imipenem (Ipm) or meropenem (Mem) and physico-chemically characterized for loading efficiency, particle size, morphology, and in-vitro release. The antibacterial efficacy was also evaluated against carbapenems resistant Gram-negative bacteria isolated from infected human, through measuring the minimum inhibitory concentration and antibiotic kill test. All the obtained gold nanoparticles showed a distinct nano-size with loading efficiency up to 72% and 74% for Ipm and Mem, respectively. The conjugation and physico-chemical stability of the formulated carbapenems were confirmed by FTIR and X-RPD. Diffusion driven release behavior was observed for both Ipm and Mem from all of the loaded gold nanoparticles. For both Ipm and Mem, formula with 35nm diameter showed the most significant enhancement in antibacterial activity against all the selected isolates including Klebsiella pneumoniae, Proteus mirabilis and Acinteobacter baumanii. Ipm loaded Gold nanoparticles demonstrated decrease in the MIC of Ipm down to four folds, whereas, Mem loaded gold nanoparticles showed decrease in the MIC of Mem down to three folds on the tested bacterial isolates. Based on these results, the formulation of carbapenems-loaded gold nanoparticles demonstrated to be a promising nano-size delivery vehicle for improving the therapeutic activity and destroying the bacterial resistance for carbapenems.

Antimicrobial and Antiquorum-Sensing Studies. Part 3: Synthesis and Biological Evaluation of New Series of [1,3,4]Thiadiazoles and Fused [1,3,4]Thiadiazoles

New series of [1,3,4]thiadiazoles and fused [1,3,4]thiadiazoles were synthesized. The newly synthesized compounds were screened for their antibacterial activity against Staphylococcus aureus, Bacillus cereus, and Escherichia coli. Compounds 3b and 10a displayed the highest activity against E. coli with MIC value of 78.125 μg/mL. In addition, compound 10a exhibited the highest activity against B. cereus with MIC value of 156.25 μg/mL. The antifungal activity of these compounds was also tested against Candida albicans and Aspergillus flavus 3375. Compounds 3b, 5a, 10a, and 12b showed the best activity against A. flavus 3375 with MIC value of 19.531 μg/mL. The same compounds were examined for their antiquorum‐sensing activity against Chromobacterium violaceum ATCC 12472, whereas compounds 3b, 5a, and 12b exhibited moderate activity. In vitro cytotoxicity testing of compounds 3b,c, 5a, 6a, 10a, and 12a,b against human normal lung fibroblast (W138) cell line was performed. The in vivo acute toxicity of the same compounds was also tested and the obtained results indicated that compound 10a is the least toxic analog. The same compounds were studied for their DNA‐binding affinity and the obtained results showed that compounds 3b, 10a, and 12a,b have moderate DNA‐binding affinity.

Synthesis of silver nanoparticles with antimicrobial and anti-adherence activities against multidrug-resistant isolates from Acinetobacter baumannii

Objectives The spread of multidrug-resistant pathogens poses a major health threat. Silver nanoparticles represent a new-class of antimicrobial agents. The aim of this study is the microbial synthesis of silver nanoparticles and the evaluation of their antimicrobial and antibiofilm activities. Methods Silver nanoparticles were synthesized using cell free supernatants of Acinetobacter baumannii. Silver nanoparticles were characterized by particle size analysis and transmission electron microscopy (TEM), and the antimicrobial and antibiofilm activities of the synthesized silver nanoparticles were assessed. Results The silver nanoparticle synthesis was monitored primarily by the conversion of the pale yellow colour of the bacteria free supernatants into a dark brown colour. Silver nanoparticles had uniform spherical shape, with particle sizes ranging from 37 to 168 nm and a zeta potential of −11.7 mV. Acinetobacter silver nanoparticles were effective against multidrug-resistant Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae with minimal inhibitory concentrations of 3.1, 1.56 and 3.1 μg/ml, respectively. Moreover, acinetobacter silver nanoparticles significantly reduced the attachment activities of E. coli, P. aeruginosa and K. pneumoniae by 66.6%, 86.5% and 75%, respectively. Conclusion Silver nanoparticles, synthesized from Acinetobacter baumannii, inhibited microbial growth and eradicated biofilm assembly by multidrug-resistant isolates that were derived from uropathogenic infection. These results suggested the possibility of using silver nanoparticles as effective antimicrobial and antibiofilm agents against infections caused by resistant isolates.