Generation, annotation, and analysis of ESTs from midgut tissue of adult female Anopheles stephensi mosquitoes

<p>Abstract</p> <p>Background</p> <p>Malaria is a tropical disease caused by protozoan parasite, <it>Plasmodium</it>, which is transmitted to humans by various species of female anopheline mosquitoes. <it>Anopheles stephensi </it>is one such major malaria vector in urban parts of the Indian subcontinent. Unlike <it>Anopheles gambiae</it>, an African malaria vector, transcriptome of <it>A. stephensi </it>midgut tissue is less explored. We have therefore carried out generation, annotation, and analysis of expressed sequence tags from sugar-fed and <it>Plasmodium yoelii </it>infected blood-fed (post 24 h) adult female <it>A. stephensi </it>midgut tissue.</p> <p>Results</p> <p>We obtained 7061 and 8306 ESTs from the sugar-fed and <it>P. yoelii </it>infected mosquito midgut tissue libraries, respectively. ESTs from the combined dataset formed 1319 contigs and 2627 singlets, totaling to 3946 unique transcripts. Putative functions were assigned to 1615 (40.9%) transcripts using BLASTX against UniProtKB database. Amongst unannotated transcripts, we identified 1513 putative novel transcripts and 818 potential untranslated regions (UTRs). Statistical comparison of annotated and unannotated ESTs from the two libraries identified 119 differentially regulated genes. Out of 3946 unique transcripts, only 1387 transcripts were mapped on the <it>A. gambiae </it>genome. These also included 189 novel transcripts, which were mapped to the unannotated regions of the genome. The EST data is available as ESTDB at <url>http://mycompdb.bioinfo-portal.cdac.in/cgi-bin/est/index.cgi</url>.</p> <p>Conclusion</p> <p>3946 unique transcripts were successfully identified from the adult female <it>A. stephensi </it>midgut tissue. These data can be used for microarray development for better understanding of vector-parasite relationship and to study differences or similarities with other malaria vectors. Mapping of putative novel transcripts from <it>A. stephensi </it>on the <it>A. gambiae </it>genome proved fruitful in identification and annotation of several genes. Failure of some novel transcripts to map on the <it>A. gambiae </it>genome indicates existence of substantial genomic dissimilarities between these two potent malaria vectors.</p

Comparison of Small Gut and Whole Gut Microbiota of First-Degree Relatives With Adult Celiac Disease Patients and Controls

Recent studies on celiac disease (CeD) have reported alterations in the gut microbiome. Whether this alteration in the microbial community is the cause or effect of the disease is not well understood, especially in adult onset of disease. The first-degree relatives (FDRs) of CeD patients may provide an opportunity to study gut microbiome in pre-disease state as FDRs are genetically susceptible to CeD. By using 16S rRNA gene sequencing, we observed that ecosystem level diversity measures were not significantly different between the disease condition (CeD), pre-disease (FDR) and control subjects. However, differences were observed at the level of amplicon sequence variant (ASV), suggesting alterations in specific ASVs between pre-disease and diseased condition. Duodenal biopsies showed higher differences in ASVs compared to fecal samples indicating larger disruption of the microbiota at the disease site. The duodenal microbiota of FDR was characterized by significant abundance of ASVs belonging to Parvimonas, Granulicatella, Gemella, Bifidobacterium, Anaerostipes, and Actinomyces genera. The duodenal microbiota of CeD was characterized by higher abundance of ASVs from genera Megasphaera and Helicobacter compared to the FDR microbiota. The CeD and FDR fecal microbiota had reduced abundance of ASVs classified as Akkermansia and Dorea when compared to control group microbiota. In addition, predicted functional metagenome showed reduced ability of gluten degradation by CeD fecal microbiota in comparison to FDRs and controls. The findings of the present study demonstrate differences in ASVs and predicts reduced ability of CeD fecal microbiota to degrade gluten compared to the FDR fecal microbiota. Further research is required to investigate the strain level and active functional profiles of FDR and CeD microbiota to better understand the role of gut microbiome in pathophysiology of CeD

Decoding the role of oxidative stress resistance and alternative carbon substrate assimilation in the mature biofilm growth mode of Candida glabrata

Abstract Background Biofilm formation is viewed as a vital mechanism in C. glabrata pathogenesis. Although, it plays a significant role in virulence but transcriptomic architecture and metabolic pathways governing the biofilm growth mode of C. glabrata remain elusive. The present study intended to investigate the genes implicated in biofilm growth phase of C. glabrata through global transcriptomic approach. Results Functional analysis of Differentially expressed genes (DEGs) using gene ontology and pathways analysis revealed that upregulated genes are involved in the glyoxylate cycle, carbon-carbon lyase activity, pre-autophagosomal structure membrane and vacuolar parts whereas, down- regulated genes appear to be associated with glycolysis, ribonucleoside biosynthetic process, ribosomal and translation process in the biofilm growth condition. The RNA-Seq expression of eight selected DEGs (CgICL1, CgMLS1, CgPEP1, and CgNTH1, CgERG9, CgERG11, CgTEF3, and CgCOF1) was performed with quantitative real-time PCR (RT-qPCR). The gene expression profile of selected DEGs with RT-qPCR displayed a similar pattern of expression as observed in RNA-Seq. Phenotype screening of mutant strains generated for genes CgPCK1 and CgPEP1, showed that Cgpck1∆ failed to grow on alternative carbon substrate (Glycerol, Ethanol, Oleic acid) and similarly, Cgpep1∆ unable to grow on YPD medium supplemented with hydrogen peroxide. Our results suggest that in the absence of glucose, C. glabrata assimilate glycerol, oleic acid and generate acetyl coenzyme-A (acetyl-CoA) which is a central and connecting metabolite between catabolic and anabolic pathways (glyoxylate and gluconeogenesis) to produce glucose and fulfil energy requirements. Conclusions The study was executed using various approaches (transcriptomics, functional genomics and gene deletion) and it revealed that metabolic plasticity of C. glabrata (NCCPF-100,037) in biofilm stage modulates its virulence and survival ability to counter the stress and may promote its transition from commensal to opportunistic pathogen. The observations deduced from the present study along with future work on characterization of the proteins involved in this intricate process may prove to be beneficial for designing novel antifungal strategies

Characterization of Bordetella pertussis Strains Isolated from India

Despite high level vaccination and the availability of two different types of vaccines, whole cell (wP) and acellular vaccines (aP), the resurgence of pertussis has been reported in many countries. Antigenic variation within circulating and vaccine strains is the most documented reason reported for the resurgence of pertussis. Research on genetic divergence among circulating and vaccine strains has largely been reported in countries using aP vaccines. There are inadequate data available for antigenic variation in B. pertussis from wP-using countries. India has used wP for more than 40 years in their primary immunization program. The present study reports five clinical isolates of B. pertussis from samples of pediatric patients with pertussis symptoms observed in India. Genotypic and phenotypic characterization of clinical isolates were performed by serotyping, genotyping, whole genome analyses and comparative genomics. All clinical isolates showed serotype 1, 2 and 3 based on the presence of fimbriae 2 and 3. Genotyping showed genetic similarities in allele types for five aP genes within vaccine strains and clinical isolates reported from India. The presence of the ptxP3 genotype was observed in two out of five clinical isolates. Whole-genome sequencing was performed for clinical isolates using the hybrid strategy of combining Illumina (short reads) and oxford nanopore (long reads) sequencing strategies. Clinical isolates (n = 5) and vaccine strains (n = 7) genomes of B. pertussis from India were compared with 744 B. pertussis closed genomes available in the public databases. The phylogenomic comparison of B. pertussis genomes reported from India will be advantageous in better understanding pertussis resurgence reported globally with respect to pathogen adaptation

Contrasting Composition, Diversity and Predictive Metabolic Potential of the Rhizobacterial Microbiomes Associated with Native and Invasive Prosopis Congeners

Invasive plants are known to alter the soil microbial communities; however, the effects of co-occurring native and invasive congeners on the soil bacterial diversity and their predictive metabolic profiles are not known. Here, we compared the rhizosphere bacterial communities of invasive Prosopis juliflora and its native congener Prosopis cineraria using high-throughput sequencing of the 16S rRNA gene. Unweighted Pair Group Method with Arithmetic mean (UPGMA) based dendrogram revealed significant variation in the communities of these co-occurring Prosopis species. Additionally, Canonical Correspondence Analysis (CCA) based on microbial communities in addition to the soil physiochemical parameters viz. soil pH, electrical conductivity, moisture content and sampling depth showed ~ 80% of the variation in bacterial communities of the rhizosphere and control soil. We observed that Proteobacteria was the predominant phylum of P. juliflora rhizosphere and the control soil, while P. cineraria rhizosphere was dominated by Cyanobacteria. Notably, the invasive P. juliflora rhizosphere showed an enhanced abundance of bacterial phyla like Actinobacteria, Chloroflexi, Firmicutes and Acidobacteria compared to the native P. cineraria as well as the control soil. Predictive metagenomics revealed that the bacterial communities of the P. juliflora rhizosphere had a higher abundance of pathways involved in antimicrobial biosynthesis and degradation, suggesting probable exposure to enemy attack and an active response mechanism to counter it as compared to native P. cineraria. Interestingly, the higher antimicrobial biosynthesis predicted in the invasive rhizosphere microbiome is further corroborated by the fact that the bacterial isolates purified from the rhizosphere of P. juliflora belonged to genera like Streptomyces, Isoptericola and Brevibacterium from the phylum Actinobacteria, which are widely reported for their antibiotic production ability. In conclusion, our results demonstrate that the co-occurring native and invasive Prosopis species have significantly different rhizosphere bacterial communities in terms of composition, diversity and their predictive metabolic potentials. In addition, the rhizosphere microbiome of invasive Prosopis proffers it a fitness advantage and influences invasion success of the species

Characterization of bacterial community shift in human Ulcerative Colitis patients revealed by Illumina based 16S rRNA gene amplicon sequencing

BACKGROUND: The healthy human intestine is represented by the presence of bacterial communities predominantly belonging to obligate anaerobes; however disparity and dysanaerobiosis in intestinal microflora may lead to the progression of ulcerative colitis (UC). The foremost aim of this study is to consider and compare the gut microbiota composition in patients suffering from different stages of UC. METHODS: This study represents data from the biopsy samples of six individuals suffering from UC. The samples were collected by colonoscopy and were processed immediately for isolation of DNA. Mucosal microbiota was analyzed by means of 16S rRNA gene-based Illumina high throughput sequencing. Quantitative real-time PCR (qPCR) was performed to determine total bacterial abundances. RESULTS: Analysis of 23,927 OTUs demonstrated a significant reduction of bacterial diversity consistently from phylum to species level (p < 0.05) for individuals suffering from severe stage of UC. Significant increase in abundance of unusual aerobes and facultative anaerobes, including members from the phylum Proteobacteria (p- = 0.031) was also observed. A 10 fold increase in the total bacterial count was detected in patients suffering from severe inflammatory stage (2.98 +/-0.49 E + 09/ml) when compared with patients with moderate (1.03+/-0.29 E + 08/ml) and mild (1.76 +/-0.34 E + 08/ml) stages of inflammation. CONCLUSION: The reduction of bacterial diversity with an increase in the total bacterial count indicates a shift of bacterial communities which signifies dysbiosis and dysanaerobiosis at the mucosal level for patients suffering from UC

Insights into Diversity and Imputed Metabolic Potential of Bacterial Communities in the Continental Shelf of Agatti Island.

Marine microbes play a key role and contribute largely to the global biogeochemical cycles. This study aims to explore microbial diversity from one such ecological hotspot, the continental shelf of Agatti Island. Sediment samples from various depths of the continental shelf were analyzed for bacterial diversity using deep sequencing technology along with the culturable approach. Additionally, imputed metagenomic approach was carried out to understand the functional aspects of microbial community especially for microbial genes important in nutrient uptake, survival and biogeochemical cycling in the marine environment. Using culturable approach, 28 bacterial strains representing 9 genera were isolated from various depths of continental shelf. The microbial community structure throughout the samples was dominated by phylum Proteobacteria and harbored various bacterioplanktons as well. Significant differences were observed in bacterial diversity within a short region of the continental shelf (1-40 meters) i.e. between upper continental shelf samples (UCS) with lesser depths (i.e. 1-20 meters) and lower continental shelf samples (LCS) with greater depths (i.e. 25-40 meters). By using imputed metagenomic approach, this study also discusses several adaptive mechanisms which enable microbes to survive in nutritionally deprived conditions, and also help to understand the influence of nutrition availability on bacterial diversity

Genomic and functional features of the biosurfactant producing Bacillus sp. AM13

Genomic studies provide deeper insights into secondary metabolites produced by diverse bacterial communities, residing in various environmental niches. This study aims to understand the potential of a biosurfactant producing Bacillussp. AM13, isolated from soil. An integrated approach of genomic and chemical analysis was employed to characterize the antibacterial lipopeptide produced by the strain AM13. Genome analysis revealed that strain AM13 harbors a nonribosomal peptide synthetase (NRPS) cluster; highly similar with known biosynthetic gene clusters from surfactin family: lichenysin (85 %) and surfactin (78 %). These findings were substantiated with supplementary experiments of oil displacement assay and surface tension measurements, confirming the biosurfactant production. Further investigation using LCMS approach exhibited similarity of the biomolecule with biosurfactants of the surfactin family. Our consolidated effort of functional genomics provided chemical as well as genetic leads for understanding the biochemical characteristics of the bioactive compound