An anti-Aspergillus protein from Escherichia coli DH5α: putative inhibitor of siderophore biosynthesis in Aspergillus fumigatus.

An antifungal protein designated as anti-Aspergillus protein (AAP), produced by Escherichia coli DH5α, was purified and characterised. It exhibited a molecular weight of 60 kDa on Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis and depicted 99% purity on ultra performance liquid chromatography. The purified protein manifested antimycotic potential against pathogenic isolates of Aspergillus spp., depicting a minimum inhibitory concentration in the range 15.62-31.25 µg ml(-1) and 5.0-10.0 µg per disc, using microbroth dilution, spore germination inhibition and disc diffusion assays respectively. In vitro toxicity tests demonstrated that it showed no toxicity against human erythrocytes at doses up to 1000 µg ml(-1) . Matrix-assisted laser desorption ionisation-Time-of-flight analysis of trypsin-digested peptides of purified protein and subsequent Mascot search revealed that several peptides of AAP have identity with bacterial siderophore biosynthetic protein, i.e. non-ribosomal peptide synthetase enzyme, involved in critical step of fungal siderophore biosynthesis. Siderophore-based inhibition was further corroborated by Chrome azurol S assay. Hence, the antagonistic effect might be the result of impediment in siderophore-mediated iron uptake and transport process which may cause critical consequences on Aspergillus growth and virulence.

Differential expression of Aspergillus fumigatus protein in response to treatment with a novel antifungal compound, diethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate.

A dihydropyridine derivative, diethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate (2e), having potent antifungal activity against pathogenic species of Aspergillus was investigated for its possible molecular mechanism of action. The SDS-PAGE coupled with nano-high-performance liquid chromatography-tandem mass spectrometry was used directly to assess both absolute abundance and differential expression of proteins in the secretory phases of Aspergillus fumigatus under the influence of 2e. It was observed that the compound inhibited the expression of two proteins of 60.99 and 79.77 kDa. Both of these secretory proteins that were inhibited by 2e, were analysed further by matrix assisted laser desorption ionization time-of-flight mass spectrometry. The 60.99- and 79.77-kDa proteins were identified as probable retroelement pol polyprotein and elongation factor G respectively. These targeted proteins could be the products of potentially virulence-related genes of A. fumigatus which may unravel the mode of action of 2e and pathobiology of A. fumigatus.

An antifungal protein from Escherichia coli.

A cytosolic protein was purified from Escherichia coli BL21 that demonstrated potent antifungal activity against pathogenic strains of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Candida albicans. The MIC of purified protein from E. coli BL21 (PPEBL21) against Aspergillus species and C. albicans was 1.95-3.98 and 15.62 microg ml(-1), respectively. In vitro toxicity tests demonstrated no cytotoxicity of PPEBL21 to human erythrocytes up to the tested concentrations of 1250 microg ml(-1). Amphotericin B was lethal to 100 % of human erythrocytes at a concentration of 37.5 microg ml(-1). The N-terminal amino acid sequence of PPEBL21 was found to be DLAEVASR, which showed 75 % sequence similarity with alcohol dehydrogenase of yeast. Mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry also substantiated these observations. The results suggested that E. coli BL21 might be an important bioresource of lead molecules for developing new peptide-based therapies for treating fungal infections.

In vitro antifungal activity of 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate, a dihydropyrrole derivative.

A novel compound 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate was isolated from the plant Datura metel L. The in vitro activity of this dihydropyrrole derivative against Aspergillus and Candida species was evaluated by using standard methods approved by the National Committee for Clinical Laboratory Standards. The compound was found to be active against all the species tested, namely Candida albicans, Candida tropicalis, Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. The MIC at which more than 90% of growth was inhibited (MIC(90)) by the compound ranged from 21.87 to 43.75 microg ml(-1) against various fungal species by microbroth dilution assay. Since the compound 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate has antifungal activity it can be explored further to develop new antimycotic drugs.

A fraction from Escherichia coli with anti-Aspergillus properties.

The products of various strains of Escherichia coli (BL21, DH5alpha, HB101 and XL Blue) were investigated for antimycotic properties using pathogenic isolates of Aspergillus. Co-culture experiments revealed that E. coli strains exhibited variable activity against Aspergillus fumigatus. The lysates prepared from DH5alpha, HB101 and XL Blue strains of E. coli showed inhibitory activity against A. fumigatus in the protein concentration range of 62.50 to 250.00 microg ml(-1). The highest activity was seen in the lysate of BL21, which inhibited the growth of A. fumigatus and Aspergillus flavus completely at a concentration of 31.25 microg protein ml(-1). The MIC of BL21 lysate against Aspergillus niger was found to be 62.50 microg ml(-1). The in vitro toxicity of BL21 lysate was evaluated using a haemolytic assay. A BL21 lysate protein concentration of 1250.00 microg ml(-1) was found to be nontoxic to human erythrocytes. The standard drug amphotericin B lysed 100 % of erythrocytes at a concentration of 37.50 microg ml(-1). SDS-PAGE showed the presence of at least 15 major proteins in the lysate of BL21. Ion-exchange chromatography resolved the BL21 lysate into five fractions and fraction III was found to be endowed with anti-Aspergillus properties. The MIC of this fraction was found to be 3.90 microg ml(-1). Further work on the purification of the active molecule and its characterization is in progress.

Association of the PIM3 allele of the alpha-1-antitrypsin gene with chronic obstructive pulmonary disease.

OBJECTIVE: The study investigated the association of genetic polymorphism of the alpha1AT gene with COPD. DESIGN AND METHODS: The mutations and polymorphism of alpha1AT gene were investigated by DNA sequence analysis using polymerase chain reaction. RESULTS: The frequency of the PIM3 allele in COPD patients was found to be significantly higher than the controls (P < 0.0001). Five SNPs, including a novel SNP (24_25insA), were observed near the junction of exon-intron I. The occurrence of these SNPs didn't show any association with COPD. However, the PIM3 allele of the alpha1AT gene was found to be associated with COPD. CONCLUSION: The PIM3 allele of the alpha1AT gene is found to have an association with the pathogenesis of COPD in the Indian population.

Antifungal potential of Indian medicinal plants.

Fourteen Indian plants, selected based on their use in respiratory and other disorders in traditional systems of medicine, were analyzed for their potential activity against fungi. The antifungal activity was investigated by disc diffusion, microbroth dilution and percent spore germination inhibition tests against pathogenic Aspergilli. Methanolic extracts of Solanum xanthocarpum and Datura metel inhibited the growth of Aspergillus fumigatus, A. flavus and A. niger and their in vitro MICs were found to be 1.25-2.50 mg/ml by both microbroth dilution and percent spore germination assays. In disc diffusion assay, a concentration of 0.062 mg/disc of methanol extract of D. metel showed significant activity against Aspergilli. S. xanthocarpum exhibited similar activity at 0.125 mg/disc.

Inhibition of conidiophore development in Aspergillus fumigatus by an Escherichia coli DH5α strain, a promising antifungal candidate against aspergillosis.

The opportunistic human pathogen Aspergillus fumigatus produces a massive number of asexual spores (conidia) as the primary means of dispersal, survival, genome protection and infection of hosts. In this report, we investigated secretory and cytosolic proteins of non-pathogenic bacterial species (mostly belonging to human microbiome) for antifungal potential against A. fumigatus, A. flavus and A. niger. Our preliminary results revealed that cytosolic proteins of E. coli DH5α were most active and the less toxic against various pathogenic isolates of A. fumigatus (the major pathogenic species), depicting a minimum inhibitory concentration (MIC) of 62.50 µg/mL, 62.50 µg/mL and 12.50 µg/disc using microbroth dilution assay (MDA), percentage spore germination inhibition assay (PSGI) and disc diffusion assay (DDA), respectively. E. coli protein was non-toxic against human erythrocytes at doses up to 1000 µg/mL as compared to standard drug, amphotericin B which lysed 100% of erythrocytes at a concentration of 37.50 µg/mL. Time kill analysis proved it to be fungicidal in a concentration and time-dependent manner. Scanning electron microscopic studies (SEM) were carried out to prevail what kind of damage it causes to A. fumigatus. SEM results reported that conidiophore (structures forming conidia) development was halted as a major consequence, reducing the number of conidiophores to insignificant values as well as alteration in their morphological attributes. This feature may contribute to the development of new prevention strategies against Aspergillus infections. Hyphal atrophy was also observed, evidenced by shrinking and flattening of hyphal walls and reduced, abrupt hyphal branching. Such actions may effectively reduce the invasive ability of Aspergillus as well as it can sterilize the fungal burden by obstructing the conidiation pathway of A. fumigatus. Hence, E. coli DH5α, being a commensal species, can lead to the development of antifungal molecule with novel targets in fungal metabolism, which will help in combating the antifungal resistance and toxicity associated with current therapy.

Advancement in infection control of opportunistic pathogen (Aspergillus spp.): adjunctive agents.

There is continuous emergence of resistant strains which leads to urgent need to discover new antifungal agents. The investigation of adjunctive agents for antifungal activity might help to optimize the therapy for Invasive Aspergillosis (IA). The chelating agents Ethylenediamine Tetraacetic Acid (EDTA) & Disodium salt of EDTA (DiEDTA) as adjunct to antifungal drugs have been investigated against 8 pathogenic isolates of Aspergillus spp. The MIC (Minimum Inhibitory Concentration) found by DDA (Disc Diffusion Assay) is 7.50-15.0 µg/disc; by MDA (Microbroth Dilution Assay) is 30.0-49.13 µg/ml & SGIA (Spore germination Inhibition Assay) is 30.0-49.13 µg/ml. Moreover, these agents did not show any toxicity up to a concentration of 312.5 µg/ml. The antifungal activity is also confirmed by another method i.e time kill curve analysis. While these agents were ten times less active than gold standard drug (Amphotericin B; AmpB) but eight times less toxic than AmpB. This leads to preliminary investigation of in vitro combination of chelating agents with antifungal drugs (Polyenes & Azoles) by DDA. These combinations showed a significant increase in zone of inhibition in contrast to single drug used. This preliminary work with chelating agents suggest that EDTA as an enhancing agent with antifungal properties in combination with antifungal drugs can be used in pharmaceutical preparations. Further investigation is in progress.

Safety and efficacy of green foliage in the treatment of fungal infections and associated clinical conditions.

Forty five crude extracts of nine selected medicinal plants, based on their use in respiratory and other disorders in traditional systems of medicine were analyzed for their potential activity against three pathogenic species of Aspergillus. The presence of phenols, tannins, flavanoids, terpenoid, steroids, alkaloids and saponins in the different extracts was established. The crude extracts were examined for antifungal activity in concentration ranging from 5000.0 to 19.53 µg/ml using microbroth dilution assay in which twenty two extracts exhibited the anti-Aspergilli activity. The petroleum ether extract of Justicia adhatoda and water extract of Commelina bengalensis exhibited the maximum activity against Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. Their in vitro minimum inhibitory concentrations (MICs) were found to be 156.0-312.0 µg/ml by microbroth dilution and spore germination inhibition assays. In disc diffusion assay, at concentration of 10 µg/disc of some crude extracts showed significant activity against Aspergilli. The toxicity (in vitro and in vivo) of bio-active fractions was evaluated, in which the extracts isolated from J. adhatoda were found to be non-toxic. Gas chromatography-mass spectrometry (GCMS) studies were performed for various extracts (petroleum ether, chloroform and acetone) of J. adhatoda which resulted in the identification of several bioactive compounds. The antifungal activity along with acute toxicity, cyto-toxicity as well as genotoxicity of extract fractions from J. adhatoda justifies the use of such screening in the expedition for new drugs.

Invasive aspergillosis: adjunctive combination therapy.

Invasive aspergillosis remains a serious opportunistic fungal infection particularly in patients with a reduced immune defense such as those with hematological malignancies or transplant recipients. The mortality of invasive infections due to Aspergillus spp. is still high. The main reasons for this are the difficulty in diagnosing of these infections and the limited efficacy of antifungal agents. There is no optimal therapy for invasive aspergillosis, and therefore many clinicians have attempted to utilize a combination approach to improve outcomes. The current antifungal classes of drugs targeting the cell wall and cell membrane may need adjunctive agents focused on separate cellular pathways that can be used in combination therapy to maximize the efficacy, a valuable alternative to the monotherapy. The endeavor of this article is to review the literature on combination therapy by using adjunctive agents against Aspergillus spp and assess its eventual usability in the treatment of invasive aspergillosis.

Investigations on anti-Aspergillus properties of bacterial products.

AIMS: To investigate the anti-Aspergillus properties of bacterial products. METHODS AND RESULTS: In the present study, 12 bacterial strains were screened for antifungal activity against Aspergilli. The culture supernatant and lysates of Pseudomonas aeruginosa, Bacillus cereus, Escherichia coli (BL21, DH5alpha, HB101, XL Blue), Klebsiella pneumoniae, Streptomyces thermonitrificans, Streptococcus pneumoniae, Enterobacter aerogenes, Staphylococcus aureus and Salmonella typhi were examined for antifungal activity in protein concentration ranging from 1000.0 to 7.8 microg ml-1 using microbroth dilution assay. The lysate of Salm. typhi and E. coli BL21 exhibited the maximum activity against Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. Their in vitro minimum inhibitory concentrations (MICs) were found to be 15.6-31.2 microg ml-1 by microbroth dilution and spore germination inhibition assays. In disc diffusion assay, a concentration of 3.1 microg disc-1 of Salm. typhi lysate showed significant activity against Aspergilli. Escherichia coli BL21 exhibited similar activity at 6.2 microg disc-1. The work on identification of molecule endowed with antimycotic properties is in progress. CONCLUSION: The products of Salm. typhi and E. coli demonstrated significant activity against Aspergillus species. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that E. coli has been reported for anti-Aspergillus activity. It could be an important source of biologically active compounds useful for developing better new antifungal drugs/or probiotics.

Cytogenetic damage in plastic industry workers exposed to polyvinyl chloride.

Cytogenetic investigations on peripheral blood lymphocytes of 40 workers exposed to polyvinyl chloride in the plastic industry were undertaken. These were compared with an equal number of occupationally unexposed and matched controls in relation to age, sex and smoking habits. The mitotic index (MI), chromosomal aberrations (CA), sister chromatid exchanges (SCE) and satellite associations (SA) were analysed. All the parameters showed a significant increase (p <0.01) in the exposed sample compared with the controls, viz MI, 3.64-6.30, CA 1.02-3.77, SCE 3.40-7.83 and SA 5.57-12.05. The occurrence of the DG type of satellite association B was highest and that of 3D type lowest. The frequencies of all the parameters increased with the duration of exposure, but MI declined after 15 years of exposure.