Evolutionary Diversity in the Intracellular Microsporidian Parasite Nosema sp. Infecting Wild Silkworm Revealed by IGS Nucleotide Sequence Diversity.

Intracellular microsporidian Nosema mylitta infects Indian wild silkworm Antheraea mylitta causing pebrine disease. Genetic structure and phylogeny of N. mylitta are analysed using nucleotide variability in 5S ribosomal DNA and intergenic spacer (IGS) sequence from 20 isolates collected from Southern, Northern and Central regions of Jharkhand State. Nucleotide diversity (π) and genetic differentiation Gst were highest in the Central isolates whereas lowest in the North. Among the isolates, absence of nucleotides, transitions and transversions were observed. Haplotyping showed nucleotide variability at 83 positions in IGS and 13 positions in 5S rDNA. Haplotype-based genetic differentiation was 0.96 to 0.97 whereas nucleotide sequence-based genetic differentiation was higher (Ks = 22.29) between Southern and Central isolates. Bottleneck analysis showed negative value for Tajima's D and other summary statistics revealing induction of loss of rare alleles and population explosion. From IGS, 17 ancestral sequences were inferred by Network algorithm. Core of nine closely related nodes having ancient nucleotides and peripheral nodes with highly divergent nucleotides were derived. Most diverged peripheral haplotype was Bero (H11) from the Central region whereas Deoghar (H3) of the Northern region diverged early. Phylogeny of N. mylitta grouped Southern and Northern isolates together revealed weak phylogenetic signal for these locations. Phylogeny of N. mylitta with Nosema sp. infecting other lepidopterans clustered N. mylitta isolates with N. antheraea and N. philosamiae of China indicating genetic similarity whereas other species were dissimilar showing diversity irrespective of country of origin.

In-depth 2-DE reference map of Aspergillus fumigatus and its proteomic profiling on exposure to itraconazole.

Aspergillus fumigatus (A. fumigatus) is a medically important opportunistic fungus that may lead to invasive aspergillosis in humans with weak immune system. Proteomic profiling of this fungus on exposure to itraconazole (ITC), an azole antifungal drug, may lead to identification of its molecular targets and better understanding on the development of drug resistance against ITC in A. fumigatus. Here, proteome analysis was performed using 2-DE followed by mass spectrometric analysis which resulted in identification of a total of 259 unique proteins. Further, proteome profiling of A. fumigatus was carried out on exposure to ITC, 0.154 µg/ml, the minimum inhibitory concentration (MIC50). Image analysis showed altered levels of 175 proteins (66 upregulated and 109 downregulated) of A. fumigatus treated with ITC as compared to the untreated control. Peptide mass fingerprinting led to the identification of 54 proteins (12 up-regulated and 42 down-regulated). The differentially expressed proteins include proteins related to cell stress, carbohydrate metabolism and amino acid metabolism. We also observed four proteins, including nucleotide phosphate kinase (NDK), that are reported to interact with calcineurin, a protein involved in regulation of cell morphology and fungal virulence. Comparison of differentially expressed proteins on exposure to ITC with artemisinin (ART), an antimalarial drug with antifungal activity(1), revealed a total of 26 proteins to be common among them suggesting that common proteins and pathways are targeted by these two antifungal agents. The proteins targeted by ITC may serve as important leads for development of new antifungal drugs.

Genetic characterisation of microsporidia infecting Indian tasar silkworm, Antheraea mylitta, using morphology and molecular tools.

The utility of inter simple sequence repeat-PCR (ISSR-PCR) assay in the genetic characterisation and elucidation of the phylogenetic relationship of different microsporidian isolates infecting tropical tasar silkworm, Antheraea mylitta Drury, is demonstrated. A total of 22 different microsporidians collected from the diseased tasar silkworms from Jharkhand state of India were analysed using morphological characters and ISSR-PCR. Observations spores under phase contrast microscope revealed oval to elongate in shape with length ranging from 3.8 µm to 5.1 µm and width from 2.6 µm to 3.3 µm. All the microsporidian isolates except MIJ-1gC showed gonadal infection and transovarial transmission in infected tasar silkworms. Fourteen out of 20 ISSR primers tested generated reproducible profiles and yielded a total of 281 fragments, of which 273 were polymorphic (97%). The degree of banding pattern was used to evaluate genetic distances and for phylogenetic analysis. The results demonstrated that ISSR analysis may be a useful and efficient tool for taxonomical grouping and phylogenetic classification of different microsporidians in general.

Transcriptomic and proteomic profile of Aspergillus fumigatus on exposure to artemisinin.

Artemisinin, an antimalarial drug, and its derivatives are reported to have antifungal activity against some fungi. We report its antifungal activity against Aspergillus fumigatus (A. fumigatus), a pathogenic filamentous fungus responsible for allergic and invasive aspergillosis in humans, and its synergistic effect in combination with itraconazole (ITC), an available antifungal drug. In order to identify its molecular targets, we further analyzed transcript and proteomic profiles of the fungus on exposure to the artemisinin. In transcriptomic analysis, a total of 745 genes were observed to be modulated on exposure to artemisinin, and some of them were confirmed by real-time polymerase chain reaction analysis. Proteomic profiles of A. fumigatus treated with artemisinin showed modulation of 175 proteins (66 upregulated and 109 downregulated) as compared to the control. Peptide mass fingerprinting led to the identification of 85 proteins-29 upregulated and 56 downregulated, 65 of which were unique proteins. Consistent with earlier reports of molecular mechanisms of artemisinin and that of other antifungal drugs, we believe that oxidative phosphorylation pathway (64 kDa mitochondrial NADH dehydrogenase), cell wall-associated proteins and enzymes (conidial hydrophobin B protein, cell wall phiA protein, extracellular thaumatin domain protein, 1,3-beta-glucanosyltransferase Gel2) and genes involved in ergosterol biosynthesis (ERG6 and coproporphyrinogen III oxidase, HEM13) are potential targets of artemisinin for further investigations.