Genome-Scale Screening of Vaccine Candidates Against Pseudomonas Aeruginosa

Abstract BackgroundInfections due to Pseudomonas aeruginosa (PA) are becoming a serious threat to patients in intensive care units. A PA vaccine is a practical and economical solution to solve the problems caused by PA infection successfully. In recent years, several antigen candidates have been tested in animal and human clinical trials, but none of them has been approved to date. An alternative strategy for antigen screening and protective antigens is in urgent demand.MethodsIn this study, we generated a genome-wide library of PA protein fragments tagged with maltose-binding protein (MBP). Using sera from patients who recovered after PA infection, we identified novel protective antigens and investigate the mechanism of these antigens induced protections.Resultswe identified a novel protective antigen, FlgE, which is the structural component of the flagella hook. Vaccination with recombinant FlgE (reFlgE) induced a Th2-predominant immune response and reduced bacterial load and inflammation in PA-infected mice. Anti-reFlgE antibodies recognized native FlgE on the bacterial membrane in vitro and conferred protection in mice, which may be due to the mediation of opsonophagocytic killing and inhibition of bacterial motility. In addition, the combination of reFlgE with rePcrVNH, an engineered antigen we reported previously, provided elevated protection against PA infection.ConclusionOur data demonstrate that FlgE is a promising vaccine candidate for PA and provide a new strategy for the efficient screening of antigens of other pathogens.

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A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast

BMC Genomics ◽

10.1186/s12864-021-07518-0 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Nicholas J. McGlincy ◽

Zuriah A. Meacham ◽

Kendra K. Reynaud ◽

Ryan Muller ◽

Rachel Baum ◽

...

Keyword(s):

In Vitro Transcription ◽

Essential Genes ◽

Competitive Growth ◽

Loss Of Function ◽

Design Rules ◽

Growth Defects ◽

Genome Wide ◽

A Genome ◽

Genome Scale

Abstract Background CRISPR/Cas9-mediated transcriptional interference (CRISPRi) enables programmable gene knock-down, yielding loss-of-function phenotypes for nearly any gene. Effective, inducible CRISPRi has been demonstrated in budding yeast, and genome-scale guide libraries enable systematic, genome-wide genetic analysis. Results We present a comprehensive yeast CRISPRi library, based on empirical design rules, containing 10 distinct guides for most genes. Competitive growth after pooled transformation revealed strong fitness defects for most essential genes, verifying that the library provides comprehensive genome coverage. We used the relative growth defects caused by different guides targeting essential genes to further refine yeast CRISPRi design rules. In order to obtain more accurate and robust guide abundance measurements in pooled screens, we link guides with random nucleotide barcodes and carry out linear amplification by in vitro transcription. Conclusions Taken together, we demonstrate a broadly useful platform for comprehensive, high-precision CRISPRi screening in yeast.

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Genome-Scale Metabolic Network Analysis of the Opportunistic Pathogen Pseudomonas aeruginosa PAO1

Journal of Bacteriology ◽

10.1128/jb.01583-07 ◽

2008 ◽

Vol 190 (8) ◽

pp. 2790-2803 ◽

Cited By ~ 175

Author(s):

Matthew A. Oberhardt ◽

Jacek Puchałka ◽

Kimberly E. Fryer ◽

Vítor A. P. Martins dos Santos ◽

Jason A. Papin

Keyword(s):

Pseudomonas Aeruginosa ◽

Metabolic Network ◽

Opportunistic Pathogen ◽

Scale Model ◽

Modeling Framework ◽

Major Life ◽

Metabolic Versatility ◽

A Genome ◽

Scale Properties ◽

Genome Scale

ABSTRACT Pseudomonas aeruginosa is a major life-threatening opportunistic pathogen that commonly infects immunocompromised patients. This bacterium owes its success as a pathogen largely to its metabolic versatility and flexibility. A thorough understanding of P. aeruginosa's metabolism is thus pivotal for the design of effective intervention strategies. Here we aim to provide, through systems analysis, a basis for the characterization of the genome-scale properties of this pathogen's versatile metabolic network. To this end, we reconstructed a genome-scale metabolic network of Pseudomonas aeruginosa PAO1. This reconstruction accounts for 1,056 genes (19% of the genome), 1,030 proteins, and 883 reactions. Flux balance analysis was used to identify key features of P. aeruginosa metabolism, such as growth yield, under defined conditions and with defined knowledge gaps within the network. BIOLOG substrate oxidation data were used in model expansion, and a genome-scale transposon knockout set was compared against in silico knockout predictions to validate the model. Ultimately, this genome-scale model provides a basic modeling framework with which to explore the metabolism of P. aeruginosa in the context of its environmental and genetic constraints, thereby contributing to a more thorough understanding of the genotype-phenotype relationships in this resourceful and dangerous pathogen.

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A genome-wide screen uncovers multiple roles for mitochondrial nucleoside diphosphate kinase D in inflammasome activation

Science Signaling ◽

10.1126/scisignal.abe0387 ◽

2021 ◽

Vol 14 (694) ◽

pp. eabe0387

Author(s):

Orna Ernst ◽

Jing Sun ◽

Bin Lin ◽

Balaji Banoth ◽

Michael G. Dorrington ◽

...

Keyword(s):

Nucleoside Diphosphate Kinase ◽

Multiple Roles ◽

Metabolic Reprogramming ◽

Inflammasome Activation ◽

Ros Production ◽

Genome Wide ◽

A Genome ◽

Mouse Macrophages ◽

Genome Scale ◽

Mitochondrial Dna Synthesis

Noncanonical inflammasome activation by cytosolic lipopolysaccharide (LPS) is a critical component of the host response to Gram-negative bacteria. Cytosolic LPS recognition in macrophages is preceded by a Toll-like receptor (TLR) priming signal required to induce transcription of inflammasome components and facilitate the metabolic reprograming that fuels the inflammatory response. Using a genome-scale arrayed siRNA screen to find inflammasome regulators in mouse macrophages, we identified the mitochondrial enzyme nucleoside diphosphate kinase D (NDPK-D) as a regulator of both noncanonical and canonical inflammasomes. NDPK-D was required for both mitochondrial DNA synthesis and cardiolipin exposure on the mitochondrial surface in response to inflammasome priming signals mediated by TLRs, and macrophages deficient in NDPK-D had multiple defects in LPS-induced inflammasome activation. In addition, NDPK-D was required for the recruitment of TNF receptor–associated factor 6 (TRAF6) to mitochondria, which was critical for reactive oxygen species (ROS) production and the metabolic reprogramming that supported the TLR-induced gene program. NDPK-D knockout mice were protected from LPS-induced shock, consistent with decreased ROS production and attenuated glycolytic commitment during priming. Our findings suggest that, in response to microbial challenge, NDPK-D–dependent TRAF6 mitochondrial recruitment triggers an energetic fitness checkpoint required to engage and maintain the transcriptional program necessary for inflammasome activation.

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Transcriptome analysis ofSchistosoma mansonilarval development using serial analysis of gene expression (SAGE)

Parasitology ◽

10.1017/s0031182009005733 ◽

2009 ◽

Vol 136 (5) ◽

pp. 469-485 ◽

Cited By ~ 22

Author(s):

A. S. TAFT ◽

J. J. VERMEIRE ◽

J. BERNIER ◽

S. R. BIRKELAND ◽

M. J. CIPRIANO ◽

...

Keyword(s):

Gene Expression ◽

Sequence Data ◽

Subsequent Development ◽

Differentially Expressed ◽

Cdna Libraries ◽

Genome Wide ◽

A Genome ◽

Genome Wide Expression ◽

Cell Conditioned Medium

SUMMARYInfection of the snail,Biomphalaria glabrata, by the free-swimming miracidial stage of the human blood fluke,Schistosoma mansoni, and its subsequent development to the parasitic sporocyst stage is critical to establishment of viable infections and continued human transmission. We performed a genome-wide expression analysis of theS. mansonimiracidia and developing sporocyst using Long Serial Analysis of Gene Expression (LongSAGE). Five cDNA libraries were constructed from miracidia andin vitrocultured 6- and 20-day-old sporocysts maintained in sporocyst medium (SM) or in SM conditioned by previous cultivation with cells of theB. glabrataembryonic (Bge) cell line. We generated 21 440 SAGE tags and mapped 13 381 to theS. mansonigene predictions (v4.0e) either by estimating theoretical 3′ UTR lengths or using existing 3′ EST sequence data. Overall, 432 transcripts were found to be differentially expressed amongst all 5 libraries. In total, 172 tags were differentially expressed between miracidia and 6-day conditioned sporocysts and 152 were differentially expressed between miracidia and 6-day unconditioned sporocysts. In addition, 53 and 45 tags, respectively, were differentially expressed in 6-day and 20-day cultured sporocysts, due to the effects of exposure to Bge cell-conditioned medium.

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Genome-wide association study of signature genetic alterations among pseudomonas aeruginosa cystic fibrosis isolates

PLoS Pathogens ◽

10.1371/journal.ppat.1009681 ◽

2021 ◽

Vol 17 (6) ◽

pp. e1009681

Author(s):

Wontae Hwang ◽

Ji Hyun Yong ◽

Kyung Bae Min ◽

Kang-Mu Lee ◽

Ben Pascoe ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Association Study ◽

Genome Wide Association Study ◽

Genetic Alterations ◽

Genome Wide Association ◽

Small Scale ◽

Outer Membrane Receptor ◽

Genome Wide ◽

A Genome

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes diverse human infections including chronic airway infection in patients with cystic fibrosis (CF). Comparing the genomes of CF and non-CF PA isolates has great potential to identify the genetic basis of pathogenicity. To gain a deeper understanding of PA adaptation in CF airways, we performed a genome-wide association study (GWAS) on 1,001 PA genomes. Genetic variations identified among CF isolates were categorized into (i) alterations in protein-coding regions, either large- or small-scale, and (ii) polymorphic variation in intergenic regions. We introduced each CF-associated genetic alteration into the genome of PAO1, a prototype PA strain, and validated the outcomes experimentally. Loci readily mutated among CF isolates included genes encoding a probable sulfatase, a probable TonB-dependent receptor (PA2332~PA2336), L-cystine transporter (YecS, PA0313), and a probable transcriptional regulator (PA5438). A promoter region of a heme/hemoglobin uptake outer membrane receptor (PhuR, PA4710) was also different between the CF and non-CF isolate groups. Our analysis highlights ways in which the PA genome evolves to survive and persist within the context of chronic CF infection.

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The FAM171A2 gene is a key regulator of progranulin expression and modifies the risk of multiple neurodegenerative diseases

Science Advances ◽

10.1126/sciadv.abb3063 ◽

2020 ◽

Vol 6 (43) ◽

pp. eabb3063

Author(s):

Wei Xu ◽

Si-Da Han ◽

Can Zhang ◽

Jie-Qiong Li ◽

Yan-Jiang Wang ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Genome Wide Association Study ◽

In Vitro Study ◽

Genome Wide ◽

A Genome ◽

Increased Risk ◽

Pathophysiological Role ◽

Independent Cohort

Progranulin (PGRN) is a secreted pleiotropic glycoprotein associated with the development of common neurodegenerative diseases. Understanding the pathophysiological role of PGRN may help uncover biological underpinnings. We performed a genome-wide association study to determine the genetic regulators of cerebrospinal fluid (CSF) PGRN levels. Common variants in region of FAM171A2 were associated with lower CSF PGRN levels (rs708384, P = 3.95 × 10−12). This was replicated in another independent cohort. The rs708384 was associated with increased risk of Alzheimer’s disease, Parkinson’s disease, and frontotemporal dementia and could modify the expression of the FAM171A2 gene. FAM171A2 was considerably expressed in the vascular endothelium and microglia, which are rich in PGRN. The in vitro study further confirmed that the rs708384 mutation up-regulated the expression of FAM171A2, which caused a decrease in the PGRN level. Collectively, genetic, molecular, and bioinformatic findings suggested that FAM171A2 is a key player in regulating PGRN production.

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Complete biosynthetic pathways of ascofuranone and ascochlorin inAcremonium egyptiacum

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1819254116 ◽

2019 ◽

Vol 116 (17) ◽

pp. 8269-8274 ◽

Cited By ~ 18

Author(s):

Yasuko Araki ◽

Takayoshi Awakawa ◽

Motomichi Matsuzaki ◽

Rihe Cho ◽

Yudai Matsuda ◽

...

Keyword(s):

Differential Expression Analysis ◽

Cost Effective ◽

Drug Candidate ◽

P450 Monooxygenase ◽

Genome Wide ◽

Common Precursor ◽

A Genome ◽

Membrane Bound ◽

The Cost

Ascofuranone (AF) and ascochlorin (AC) are meroterpenoids produced by various filamentous fungi, includingAcremonium egyptiacum(synonym:Acremonium sclerotigenum), and exhibit diverse physiological activities. In particular, AF is a promising drug candidate against African trypanosomiasis and a potential anticancer lead compound. These compounds are supposedly biosynthesized through farnesylation of orsellinic acid, but the details have not been established. In this study, we present all of the reactions and responsible genes for AF and AC biosyntheses inA. egyptiacum, identified by heterologous expression, in vitro reconstruction, and gene deletion experiments with the aid of a genome-wide differential expression analysis. Both pathways share the common precursor, ilicicolin A epoxide, which is processed by the membrane-bound terpene cyclase (TPC) AscF in AC biosynthesis. AF biosynthesis branches from the precursor by hydroxylation at C-16 by the P450 monooxygenase AscH, followed by cyclization by a membrane-bound TPC AscI. All genes required for AC biosynthesis (ascABCDEFG) and a transcriptional factor (ascR) form a functional gene cluster, whereas those involved in the late steps of AF biosynthesis (ascHIJ) are present in another distantly located cluster. AF is therefore a rare example of fungal secondary metabolites requiring multilocus biosynthetic clusters, which are likely to be controlled by the single regulator, AscR. Finally, we achieved the selective production of AF inA. egyptiacumby genetically blocking the AC biosynthetic pathway; further manipulation of the strain will lead to the cost-effective mass production required for the clinical use of AF.

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