Applied genomics for identification of virulent biothreats and for disease outbreak surveillance

Fortifying our preparedness to cope with biological threats by identifying and targeting virulence factors may be a preventative strategy for curtailing infectious disease outbreak. Virulence factors evoke successful pathogenic invasion, and the science and technology of genomics offers a way of identifying them, their agents and evolutionary ancestor. Genomics offers the possibility of deciphering if the release of a pathogen was intentional or natural by observing sequence and annotated data of the causative agent, and evidence of genetic engineering such as cloned vectors at restriction sites. However, to leverage and maximise the application of genomics to strengthen global interception system for real-time biothreat diagnostics, a complete genomic library of pathogenic and non-pathogenic agents will create a robust reference assembly that can be used to screen, characterise, track and trace new and existing strains. Encouraging ethical research sequencing pathogens found in animals and the environment, as well as creating a global space for collaboration will lead to effective global regulation and biosurveillance.

Phosphoinositide-Dependent Protein Kinases Regulate Cell Cycle Progression Through the SAD Kinase Cdr2 in Fission Yeast

Aberration in the control of cell cycle contributes to the development and progression of many diseases including cancers. Ksg1 is a Schizosaccharomyces pombe fission yeast homolog of mammalian phosphoinositide-dependent protein kinase 1 (PDK1) which is regarded as a signaling hub for human tumorigenesis. A previous study reported that Ksg1 plays an important role in cell cycle progression, however, the underlying mechanism remains elusive. Our genomic library screen for novel elements involved in Ksg1 function identified two serine/threonine kinases, namely SAD family kinase Cdr2 and another PDK1 homolog Ppk21, as multicopy suppressors of the thermosensitive phenotype of ksg1-208 mutant. We found that overexpression of Ppk21 or Cdr2 recovered the defective cell cycle transition of ksg1-208 mutant. In addition, ksg1-208 Δppk21 cells showed more marked defects in cell cycle transition than each single mutant. Moreover, overexpression of Ppk21 failed to recover the thermosensitive phenotype of the ksg1-208 mutant when Cdr2 was lacking. Notably, the ksg1-208 mutation resulted in abnormal subcellular localization and decreased abundance of Cdr2, and Ppk21 deletion exacerbated the decreased abundance of Cdr2 in the ksg1-208 mutant. Intriguingly, expression of a mitotic inducer Cdc25 was significantly decreased in ksg1-208, Δppk21, or Δcdr2 cells, and overexpression of Ppk21 or Cdr2 partially recovered the decreased protein level of Cdc25 in the ksg1-208 mutant. Altogether, our findings indicated that Cdr2 is a novel downstream effector of PDK1 homologs Ksg1 and Ppk21, both of which cooperatively participate in regulating cell cycle progression, and Cdc25 is involved in this process in fission yeast.

Construction of Genomic Library and Screening of Edwardsiella tarda Immunogenic Proteins for Their Protective Efficacy Against Edwardsiellosis

Edwardsiella tarda is a severe aquaculture pathogen that can infect many hosts including humans, animals, and fish. Timely diagnosis and treatment are crucial for the control of edwardsiellosis in the aqua industry. By using rabbit polyclonal antibody, an expression gene library of virulent Edwardsiella tarda strain ED-BDU 1 isolated in south India was constructed and screened. The identified immune expressive proteins were characterized, and the corresponding coding sequences were cloned, expressed, and the purified recombinant proteins were used as antigens. The identified immunoreactive proteins namely HflC, HflK, and YhcI were studied for their immune protective potential in vivo by challenge experiments. The protective efficacy of HflC, HflK, and YhcI showed that the clearance of Edwardsiella from the host with ~ 60% survivability. Further, the immunoreactive proteins induce a strong immune response upon infection and elicit the significant production of IL-10, IFN-γ, Th1, and Th2 mediated mRNA expression and were therefore effective in vaccine production for edwardsiellosis.

Towards the Creation of a Transgenic Possum Parasite

<p>The brushtail possum. Trichosurus vulpecula, is New Zealand's most serious vertebrate pest; possums destroy native flora and fauna and are vectors of bovine Tb. Conventional control is considered to be unsustainable and, in the long term, biological control is seen as the only solution to reducing possum numbers. The aim of this project is to contribute to the development of a self-disseminating vector that will spread a control molecule throughout the possum population reducing fecundity or increasing mortality. The possum-specific parasite Parastrongyloides trichosuri has considerable potential a-s such a vector. A protein from P. trichosuri specifically, was found to be antigenic in possums. The antibodies to this protein were purified from positive possum serum and used to detect the antigen on the surface of infective larvae but not in the excretory/secretory products of either larvae or adults. The protein was isolated from crude infective larvae and found to show homology to the heat-shock 70 family of proteins. Genomic DNA was extracted, an oligonucleotide probe made and a genomic library screened for the Hsp70 gene. Several positive clones were found and DNA isolated and sequenced from one such clone. Five kilo bases of unambiguous sequence was obtained in which was an open reading frame of 2 kb. Theoretical translation of this gave a protein of 64 amino acids with 80% homology to the Hsp70A protein of C. elegans. The region upstream of the ATG initiator codon was amplified and 1.3 kb of the putative promoter region was cloned into a vector containing the gfp:lacZ reporter genes. This construct was microinjected, first into C. elegans to demonstrate promoter function, and then into both tree-living and parasitic adults of P. trichosuri. Reporter gene expression was shown in the progeny of microinjected parasitic adults. RNA was made from infective P. trichosuri larvae, reverse transcribed and the coding sequence for the PtHsp70 protein cloned into an expression vector and expressed in E. coli, The recombinant protein pattern had a similar pattern of trypsin digestion products as the native protein, as shown by MALDI-TOF mass spectrometry, but it was immunologically distinct from the native protein. The culmination of this project was the generation of a transgenic P trichosuri, the first vertebrate endoparasitic nematode to be heritably transformed. This is a necessary step in the development of a self-disseminating vector to be used in the biocontrol of possums.</p>

Towards the Creation of a Transgenic Possum Parasite

<p>The brushtail possum. Trichosurus vulpecula, is New Zealand's most serious vertebrate pest; possums destroy native flora and fauna and are vectors of bovine Tb. Conventional control is considered to be unsustainable and, in the long term, biological control is seen as the only solution to reducing possum numbers. The aim of this project is to contribute to the development of a self-disseminating vector that will spread a control molecule throughout the possum population reducing fecundity or increasing mortality. The possum-specific parasite Parastrongyloides trichosuri has considerable potential a-s such a vector. A protein from P. trichosuri specifically, was found to be antigenic in possums. The antibodies to this protein were purified from positive possum serum and used to detect the antigen on the surface of infective larvae but not in the excretory/secretory products of either larvae or adults. The protein was isolated from crude infective larvae and found to show homology to the heat-shock 70 family of proteins. Genomic DNA was extracted, an oligonucleotide probe made and a genomic library screened for the Hsp70 gene. Several positive clones were found and DNA isolated and sequenced from one such clone. Five kilo bases of unambiguous sequence was obtained in which was an open reading frame of 2 kb. Theoretical translation of this gave a protein of 64 amino acids with 80% homology to the Hsp70A protein of C. elegans. The region upstream of the ATG initiator codon was amplified and 1.3 kb of the putative promoter region was cloned into a vector containing the gfp:lacZ reporter genes. This construct was microinjected, first into C. elegans to demonstrate promoter function, and then into both tree-living and parasitic adults of P. trichosuri. Reporter gene expression was shown in the progeny of microinjected parasitic adults. RNA was made from infective P. trichosuri larvae, reverse transcribed and the coding sequence for the PtHsp70 protein cloned into an expression vector and expressed in E. coli, The recombinant protein pattern had a similar pattern of trypsin digestion products as the native protein, as shown by MALDI-TOF mass spectrometry, but it was immunologically distinct from the native protein. The culmination of this project was the generation of a transgenic P trichosuri, the first vertebrate endoparasitic nematode to be heritably transformed. This is a necessary step in the development of a self-disseminating vector to be used in the biocontrol of possums.</p>

Unraveling Protein Interactions between the Temperate Virus Bam35 and Its Bacillus Host Using an Integrative Yeast Two Hybrid–High Throughput Sequencing Approach

Bacillus virus Bam35 is the model Betatectivirus and member of the family Tectiviridae, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. Interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, which are known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein–protein interactions (PPIs) network for a tectivirus–host system by studying the Bam35–Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and high-throughput sequencing (Y2H-HTS). We generated and thoroughly analyzed a genomic library of Bam35′s host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait–prey couples. Initial analysis of the raw data enabled the identification of over 4000 candidate interactions, which were sequentially filtered to produce 182 high-confidence interactions that were defined as part of the core virus–host interactome. Overall, host metabolism proteins and peptidases were particularly enriched within the detected interactions, distinguishing this host–phage system from the other reported host–phage PPIs. Our approach also suggested biological roles for several Bam35 proteins of unknown function, including the membrane structural protein P25, which may be a viral hub with a role in host membrane modification during viral particle morphogenesis. This work resulted in a better understanding of the Bam35–B. thuringiensis interaction at the molecular level and holds great potential for the generalization of the Y2H-HTS approach for other virus–host models.

Function and Characterization of Fungal Communities in Chestnut Soils (Castanea crenata) of Kansai Region, Japan

Chestnut (Castanea crenata) is an important fruit crop in Japan, grown under three cultivation systems in Kansai region, which succumb to fungal root disease pathogens. The fungal community in soils of chestnut in these cultivation systems were characterized along with the potential of soil bacterial species as biological control agent against these root-invading fungi. Bacteria from the chestnut soil rhizosphere were identified and their ability to suppress diseases in vitro was evaluated. Bacteria DAC17225011 and DAC17225014 showed 99% similarity to Bacillus aryabhattai and Pseudomonas frederiksbergensis, respectively, which could suppress the growth of Armilaria mellea and Phytophtora cambivora, respectively, in in vitro conditions. The assay in vivo indicated the positive effect of these bacteria on the reduction of disease infection spots in chestnut roots; however, no visible symptoms were detected aboveground. For microbial community analysis, chestnut soil was sampled from four locations (Wachi, Ayabe, Fukuchiyama and Sasayama) considering three management systems, conventional, organic and wild. The amplicon from the ITS region (The genomic library of the fungal detection in soils) was sequenced by Illumina MiSeq 250bp and used to analyze the fungal community in the sampled soil. Nectriaceae, which contains pathogenic fungi, was very common in all samples, but lower in wild areas. Ceratobasidiacea was also higher in conventional areas. For the symbiotic families, Hypocraceae and Russulaceae were typical in wild soils, whereas Amanitaceae was found in organic soils. The fungal community was clearly distinct in the wild system, differing from conventional and organic systems.

Metabolomics integrated with transcriptomics reveals the distribution of iridoid and crocin metabolic flux in Gardenia jasminoides Ellis

Gardenia jasminoides Ellis (G. jasminoides) fruits are used as a resource for obtaining natural colorants and in traditional Chinese herbal medicine. However, G. jasminoides presents a relatively long flowering period and different ripening periods, so there are significant differences in the accumulation of metabolites in fruits of different colors. In addition, the complete metabolic pathways of iridoidsand crocins, which are used as medicinal composition of G. jasminoides, are poorly understood at present. In this research, we comprehensively compared the transcriptome and metabolites profiles of the developmental stages and locations of iridoid and crocin biosynthesis. A large number of differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were detected in four groups of samples, and clear variation in the pattern of metabolite abundance and gene expression were observed among different fruit colors and parts. Geniposide and gardenoside mainly accumulated in the sarcocarp of green fruit (GFS) and the sarcocarp of red fruit (FS), respectively. Crocin mainly accumulated in the peel and sarcocarp of red fruits. In the iridoid pathway, we hypothesized that there was a transport mechanism from the sarcocarp to the peel of G. jasminoides because of the inconsistent expression of G8O, 10-HGO and IS associated with differences in fruit ripening. UGTs play an important role in the biosynthesis of the active components of G. jasminoides. Combined transcriptome and metabonomics analysis showed a negative correlation between the biosynthesis of geniposide and crocin. The redirection of the metabolic flux and the regulation of key enzymes may be the main reasons for the changes in the biosynthesis of iridoid and crocin in G. jasminoides fruit. Our study expended valuable information for functional genomic library and provided new insights for metabolic engineering of secondary metabolite in G. Jasminoides.

Draft De Novo Genome Assembly of Acinetobacter pittii Strain VKPM B-3780, a Prospective Multifunctional Bioremediation Agent

Acinetobacter pittii strain B-3780 is a prospective degrader of oil and methanol, isolated from industrial wastewater. Here, we present the draft genome sequence of strain B-3780, obtained using Illumina sequencing of the fragment genomic library.

Yeast Nst1 is a Novel Component of P-bodies and is a Specific Suppressor of Proteasome Base Assembly Defects

Proteasome assembly utilizes multiple dedicated assembly chaperones and is regulated by signaling pathways that respond to diverse stress conditions. To discover new factors influencing proteasome base assembly, we screened a tiled high-copy yeast genomic library to identify dosage suppressors of a temperature-sensitive proteasome regulatory particle (RP) base mutant. The screen identified Nst1, a protein that when overexpressed, specifically suppressed the temperature sensitivity and proteasome-assembly defects of multiple base mutants. Nst1 overexpression reduced cytosolic RP ATPase (Rpt) aggregates in nas6Δ rpn14Δ cells, which lack two RP assembly chaperones. Nst1 is highly polar and predicted to have numerous intrinsically disordered regions, characteristics commonly found in proteins that can segregate into membraneless condensates. In agreement with this, both endogenous and overexpressed Nst1 could form cytosolic puncta that co-localized with processing body (P-body) components. Consistent with the accumulation of translationally inactive mRNAs in P-bodies, Nst1 overexpression inhibited global protein translation in nas6Δ rpn14Δ cells. Translational inhibition is known to suppress aggregation and proteasome assembly defects in base mutants under heat stress . Our data indicate that Nst1 is a previously overlooked P-body component that when expressed at elevated levels inhibits translation, prevents Rpt subunit aggregation, and rescues proteasome assembly under stress conditions.