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Identification of universally applicable and species-specific marker peptides for Bacillus anthracis

ABSTRACT: Anthrax is a zoonotic infection caused by the bacterium Bacillus anthracis (BA), a gram-positive, aerobic, spore-forming bacterium that can be misused as a biowarfare agent. The major patho-genicity factors of BA are encoded by genes located on two extrachromosomal plasmids, which are often targeted for specific identification of this pathogen. However, more recent findings show that these plasmids are not a unique feature of BA but can also occur in other Bacillus species. Furthermore, BA is a member of the Bacillus cereus group, a subgroup of closely related Bacilli. Due to the high genetic similarity within this group, it is a challenge to distinguish BA from other members of this group. In this study, we investigated if it is possible to identify species-specific and universally applicable marker peptides for BA. For this purpose, we applied a high-resolution mass spectrometry-based approach for 42 BA isolates. Together with the genomic sequencing data and by developing a bioinformatics data evaluation pipeline, which uses a database containing most of the publicly available protein sequences worldwide (UniParc), we were able to identify eleven universal marker peptides unique to BA, which are located on the chromosome and there-fore might overcome known problems like observable loss of plasmids in environmental species, plasmid loss during cultivation in the lab and that the virulence plasmids are not necessarily a unique feature of BA. The identified chromosomally encoded markers in this study could extend the small panel of already existing chromosomal targets and together with targets for the viru-lence plasmids may pave the way to an even more reliable identification of BA using genomics- as well as proteomics-based techniques.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Bacillus Anthracis Bacteria

DISEASE(S): Anthrax Disease

SUBMITTER: David Meierhofer

LAB HEAD: David Meierhofer

PROVIDER: PXD036243 | Pride | 2023-01-05

REPOSITORIES: Pride

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Project description:To reveal the comparative pharmacological mechanism of differential compounds and its combination, we used a custom cNDA microarray to screen gene expression on ischemia mouse hippocampus. Cerebral ischemia was induced in anesthetized mice by urethane (1.5g/kg, i.p) to 38-48g male or female mice by occlusion of the middle cerebral artery (MCA) for 15 minutes twice with microvascular clips, followed by reperfusion for 10 minutes after the first occlusion. Experimental animal were divided into groups: Sham-operated, vehicle (M3,ischemic mice 3 hour;12 hour;24 hour),Baicalin(BA) (5mg/ml), Jasminoidin(JA) (25mg/ml),Jasminoidin(H) (50mg/ml),Cholic acid(CA) (7mg/ml),Concha margaritifera(CM) (50mg/ml), Nimodipine (NI) (50mg/ml),and JA+BA, JA+CA, JA+BA+CA, JA+BA+CA+CM. The prescription of combination therapies was composed of the same ratio on every compound. Treated compound dosage according to 0.2ml physic liquor/100g mouse or rat body weight treated at 1.5h post operation given through tail vein. Sham-operated mice underwent identical procedures except therapy were vehicle (2ml/kg; 100% DMSO). Infarct Volume and Neurological Score were tested to display variation on comeout change. Hippocampus (subfields CA1-4 of Ammon's horn) were carefully dissected out from re-anesthetized mice under RNAase-free conditions, flash frozen and stored at -75 C. Total RNA of each sample was extracted with Trizol reagent (Invitrogen, procedure according to the instruction manual). Three animals per group were pooled and homogenized in denaturing solution with a Polytron. Three pooled per group and nine times repeted microarray experiments of each pooled were preformed. Each microarray contained 374 cDNA derived from cDNA library of mouse brain (Invitrogen, Cat.1065-025). Plasmid magnified using Invitrogen provided library. Design and synthesize Primer according the information provided by selected genes.Total gene segment is 416, two of them is control(lambda-A and pUC19). The criterion of qualified PCR extending is: the total gained volume of the purpose strap DNA is greater or equal to 7?g;the purpose strap DNA gained volume/total DNA gained volume is greater or equal to 80%.Finally, the DNA concentration is adjusted to 0.5 ?g/ ?l.After refinded PCR_for every gene, 2?l DNA solution was taken to proceeding electrophoresis on the electrophoresis condition of 2%Gel.Then fix quantify analysis according electrophoresis photo have taken.Choosing 5 PCR refined products sequencing from two direction after amplification.These 5 genes all chosen from UniGene sample which marked by ?,one of them is used Dalian ? as cyclostyle to amplification.The five sequence genes was certified as aimed gene segment by Blast(Completed in TaKaRa(Dalian))

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Identification of universally applicable and species-specific marker peptides for Bacillus anthracis

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