Investigations on the Interactions of λPhage-Derived Peptides Against the SrtA Mechanism in Bacillus anthracis

Bacillus anthracis is a well-known bioweapon pathogen, which coordinates the expression of its virulence factors in response to a specific environmental signal by its protein architecture. Absences of sortase signal functioning may fail to assemble the surface linked proteins and so B. anthracis cannot sustain an infection with host cells. Targeting the signaling mechanism of B. anthracis can be achieved by inhibition of SrtA enzyme through λphage-derived plyG. The lysin enzyme plyG is experimentally proven as bacteriolytic agent, specifically kill's B. anthracis by inhibiting the SrtA. Here, we have screened the peptides from λphage lysin, and these peptides are having the ability as LPXTG competitive inhibitors. In comparison to the activator peptide LPXTG binding motif, λphage lysin based inhibitor peptides are having much supremacy towards binding of SrtA. Finally, peptide structures extracted from PlyG are free from toxic, allergic abilities and also have the ability to terminate the signal transduction mechanism in B. anthracis.     

Targeted proteomics approach to species-level identification of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> spores by AP-MALDI-MS

Anthrax infections progress at a rapid pace, making rapid detection methods of utmost importance. MALDI-MS proteomics methods focused on Bacillus anthracis detection have targeted chromosomally encoded proteins, which are highly conserved between closely related species, hindering species identification. Presented here is an AP-MALDI-MS method targeting plasmid-borne proteins from Bacillus spores for species-level identification. A bioinformatics analysis revealed that 60.3% and 75.4% of tryptic peptides from plasmid-borne proteins of B. anthracis and B. thuringiensis were species-specific, respectively. Reported here is a method in which plasmid-borne Δ-endotoxins were extracted directly from B. thuringiensis spores in 100 mM KOH. The pH was then adjusted to 8 and a 5-min trypsin digestion was performed on the extracted proteins. The resulting tryptic peptides were analyzed by AP-MALDI-MS/MS, which produced a definitive identification the B. thuringiensis speciesspecific Cry1Ab protein with a MASCOT score of 278 and expect value of 7.5 × 10⁻²³. This method has demonstrated the detection and identification of B. thuringiensis spores at the species level following a 5-min trypsin digestion. The challenges in applying a similar approach to the detection of plasmid-borne protein toxins from B. anthracis are also discussed.     

Simultaneous identification and verification of Bacillus anthracis

Specific identification of Bacillus anthracis (B. anthracis) is vital for the accurate treatment of afflicted personnel during biological warfare situations and civilian terrorist attacks. In order to accomplish this, we have subjected the lysates from B. anthracis to affinity purification using monoclonal antibodies for the selected antigenic protein present in the bacteria. The bound antigenic protein was identified by multi-dimensional protein identification technology (MudPIT) to be a surface layer protein EA1. The same antigen was identified from the lysates from a few strains of B. anthracis demonstrating the observation to be common for B. anthracis strains. Hence, this presents an effective pathway for the identification of the bacteria present in unknown samples of various origins. Generation of a database containing the EA1 protein has been found to be useful in the database search of unknown samples.     

Bacillus anthracis: toxicology, epidemiology and current rapid-detection methods

B. anthracis, the causative agent for anthrax, has been well studied for over 150 years. Due to the genetic similarities among various Bacillus species, as well as its existence in both a spore form and a vegetative state, the detection and specific identification of B. anthracis have been proven to require complex techniques and/or laborious methods. With the heightened interest in the organism as a potential biological threat agent, a large number of interesting detection technologies have recently been developed, including methods involving immunological and nucleic acid-based assay formats. The technologies range from culture-based methods to portable Total Analysis Systems based on real-time PCR. This review with 170 references provides a brief background on the toxicology and epidemiology of B. anthracis, discusses challenges associated with its detection related to genetic similarities to other species, and reviews immunological and, with greater emphasis, nucleic acid-based detection systems. Harriet A. Clancy is currently on active duty with the U.S. Army’s 3rd COSCOM (Corps Support Command)     

Proteomic profiling of human urine using multi-dimensional protein identification technology

Human urine samples are ideal for proteomic profiling and have tremendous potential as sources of biomarkers. Multi-dimensional protein identification technology (MudPIT) is an effective approach to analyzing human urine or other fluids dominated by diverse metabolites. MudPIT analysis was used to identify 87 proteins in just 15 ml of human urine. A high throughput, reproducible, and sensitive technology, MudPIT may soon be used for more proteomic analyses of metabolites.     

Exploring human plasma proteome strategies: high efficiency in-solution digestion protocol for multi-dimensional protein identification technology

Multi-dimensional protein identification technology (MudPIT) is becoming a prevalent proteomic approach due to its high-throughput separations and accurate mass detection. Prior to MudPIT analysis, complicated samples required in-solution digestion. Unlike in-gel digestion, in which enzymes work on just a few proteins, in-solution digestion involves simultaneous digestion of hundreds or thousands of proteins. In-solution digestion protocols must therefore be very efficient. Few investigations have evaluated the efficiency of in-solution digestion protocols. The present research compared three such protocols. Results suggest that a protocol utilizing trifluoroethanol (TFE) as denaturant is most efficient.     

Identification and discrimination of bacterial strains by laser induced breakdown spectroscopy and neural networks

A method based on laser induced breakdown spectroscopy (LIBS) and neural networks (NNs) has been developed and applied to the identification and discrimination of specific bacteria strains (Pseudomonas aeroginosa, Escherichia coli and Salmonella typhimurium). Instant identification of the samples is achieved using a spectral library, which was obtained by analysis using a single laser pulse of representative samples and treatment by neural networks. The samples used in this study were divided into three groups, which were prepared on three different days. The results obtained allow the identification of the bacteria tested with a certainty of over 95%, and show that only a difference between the bacteria can cause identification. Single-shot measurements were sufficient for clear identification of the bacterial strains studied. The method can be developed for automatic real time, fast, reliable and robust measurements and can be packaged in portable systems for non-specialist users.     

Rapid identification and classification of Mycobacterium spp. using whole-cell protein barcodes with matrix assisted laser desorption ionization time of flight mass spectrometry in comparison with multigene phylogenetic analysis

The need of quick diagnostics and increasing number of bacterial species isolated necessitate development of a rapid and effective phenotypic identification method. Mass spectrometry (MS) profiling of whole cell proteins has potential to satisfy the requirements. The genus Mycobacterium contains more than 154 species that are taxonomically very close and require use of multiple genes including 16S rDNA for phylogenetic identification and classification. Six strains of five Mycobacterium species were selected as model bacteria in the present study because of their 16S rDNA similarity (98.4–99.8%) and the high similarity of the concatenated 16S rDNA, rpoB and hsp65 gene sequences (95.9–99.9%), requiring high identification resolution. The classification of the six strains by MALDI TOF MS protein barcodes was consistent with, but at much higher resolution than, that of the multi-locus sequence analysis of using 16S rDNA, rpoB and hsp65. The species were well differentiated using MALDI TOF MS and MALDI BioTyper™ software after quick preparation of whole-cell proteins. Several proteins were selected as diagnostic markers for species confirmation. An integration of MALDI TOF MS, MALDI BioTyper™ software and diagnostic protein fragments provides a robust phenotypic approach for bacterial identification and classification.     

Characterization and in Vitro Inhibition Studies of Bacillus anthracis FtsZ: A Potential Antibacterial Target

FtsZ is an essential bacterial cell division protein that is an attractive target for the development of antibacterial agents. FtsZ is a homologue of eukaryotic tubulin, has GTPase activity, and forms a ring-type structure to initiate cell division. In this study, the FtsZ of Bacillus anthracis was cloned into a bacterial expression vector and overexpressed into Escherichia coli BL21 (DE3) cells. The overexpressed B. anthracis FtsZ was soluble and purified to homogeneity using Ni-His-tag affinity chromatography. Like other known FtsZs, the recombinant B. anthracis FtsZ also showed GTP-dependent polymerization, which was analyzed using both spectrophotometric and Transmission Electronic Microscopic (TEM) analysis. Using the purified FtsZ, we screened a naturally extracted chemical library to identify potent and novel inhibitors. The screening yielded three chemicals, SA-011, SA-059, and SA-069, that inhibited the in vitro polymerization activity of FtsZ in the micromolar range (IC₅₀ of 55–168 μM). The inhibition potency was significantly comparable with that of berberine, a known potential inhibitor of FtsZ. Understanding the biochemical basis of the effect of these inhibitors on B. anthracis growth would provide a promising path for the development of new antianthracis drugs.     

Validation of a Reference Gene (BdFIM) for Quantifying Transgene Copy Numbers in Brachypodium distachyon by Real-Time PCR

Brachypodium distachyon has been proposed as a new model system for gramineous plants with a sequenced genome and an efficient transformation system. Many transgenic B. distachyon plants have been generated in recent years. To develop a reliable fast method for detecting transgenic B. distachyon and quantifying its transgene copy numbers, a species-specific reference gene is of great priority to be validated both in qualitative PCR and quantitative real-time PCR detection. In this study, we first proved that the BdFIM (B. distachyon fimbrin-like protein) gene is a suitable reference gene in qualitative PCR and quantitative real-time PCR for B. distachyon. Fourteen different B. distachyon varieties were tested by both qualitative and quantitative PCRs, and identical amplification products of BdFIM were obtained with all of them, while no amplification products were observed with samples from 14 other plant species, suggesting that BdFIM gene was specific to B. distachyon. The results of Southern blot analysis revealed that the BdFIM gene was low copy number in seven tested B. distachyon varieties. In conclusion, the BdFIM gene can be used as a reference gene, since it had species specificity, low heterogeneity, and low copy number among the tested B. distachyon varieties. Furthermore, the copy number of inserted sequences from transgenic B. distachyon obtained by real-time PCR methods and Southern blot confirmed that the BdFIM gene was an applicable reference gene in B. distachyon.     

One-Component Styrene Monooxygenases: An Evolutionary View on a Rare Class of Flavoproteins

Styrene monooxygenases (SMOs) are catalysts for the enantioselective epoxidation of terminal alkenes. Most representatives comprise a reductase and a monooxygenase which are encoded by separate genes (styA, styB). Only six presumed self-sufficient one-component SMOs (styA2B) have previously been submitted to databases, and one has so far been characterized. StyA2B can be supported by another epoxidase (StyA1) encoded by styA1, a gene in direct neighborhood of styA2B. The present report describes the identification of a further styA1/styA2B-like SMO, which was detected in Rhodococcus opacus MR11. Based on the initially available sequences of styA2B-type SMOs, primers directed at conserved sequences were designed and a 7,012-bp genomic fragment from strain MR11 was obtained after PCRs and subsequent genome walking. Six open reading frames (ORFs) were detected and compared to genomic fragments of strains comprising either two- or one-component SMOs. Among the proteins encoded by the ORFs, the monooxygenase StyA1/StyA2B showed the highest divergence on amino acid level when comparing proteins from different sources. That finding, a rare distribution of styA2B genes among bacteria, and the general observation of evolution from simple to complex systems indicate that one-component SMOs evolved from two-component ancestors. Analysis of gene products from styA/styB- and styA1/styA2B-like SMOs revealed that a fusion of styA/styB to styA2B might have happened at least twice among microorganisms. This points to a convergent evolution of one-component SMOs.     

A rapid biosensor for viable <Emphasis Type="Italic">B. anthracis</Emphasis> spores

A simple membrane-strip-based biosensor assay has been combined with a nucleic acid sequence-based amplification (NASBA) reaction for rapid (4 h) detection of a small number (ten) of viable B. anthracis spores. The biosensor is based on identification of a unique mRNA sequence from one of the anthrax toxin genes, the protective antigen (pag), encoded on the toxin plasmid, pXO1, and thus provides high specificity toward B. anthracis. Previously, the anthrax toxins activator (atxA) mRNA had been used in our laboratory for the development of a biosensor for the detection of a single B. anthracis spore within 12 h. Changing the target sequence to the pag mRNA provided the ability to shorten the overall assay time significantly. The vaccine strain of B. anthracis (Sterne strain) was used in all experiments. A 500-L sample containing as few as ten spores was mixed with 500 L growth medium and incubated for 30 min for spore germination and mRNA production. Thus, only spores that are viable were detected. Subsequently, RNA was extracted from lysed cells, selectively amplified using NASBA, and rapidly identified by the biosensor. While the biosensor assay requires only 15 min assay time, the overall process takes 4 h for detection of ten viable B. anthracis spores, and is shortened significantly if more spores are present. The biosensor is based on an oligonucleotide sandwich-hybridization assay format. It uses a membrane flow-through system with an immobilized DNA probe that hybridizes with the target sequence. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye sulforhodamine B. The amount of liposomes captured in the detection zone can be read visually or quantified with a hand-held reflectometer. The biosensor can detect as little as 1 fmol target mRNA (1 nmol L^–1). Specificity analysis revealed no cross-reactivity with 11 organisms tested, among them closely related species such as B. cereus, B. megaterium, B. subtilis, B. thuringiensis, Lactococcus lactis, Lactobacillus plantarum, and Chlostridium butyricum. Also, no false positive signals were obtained from nonviable spores. We suggest that this inexpensive biosensor is a viable option for rapid, on-site analysis providing highly specific data on the presence of viable B. anthracis spores.     

Differential survival of Escherichia coli uvrA,uvrB, and uvrC mutants to psoralen plus UV-A (PUVA): Evidence for uncoupled action of nucleotide excision repair to process DNA adducts

The nucleotide excision repair mechanism (NER) of Escherichia coli is responsible for the recognition and elimination of more than twenty different DNA lesions. Herein, we evaluated the in vivo role of NER in the repair of DNA adducts generated by psoralens (mono- or bi-functional) and UV-A light (PUVA) in E. coli. Cultures of wild-type E. coli K12 and mutants for uvrA, uvrB, uvrC or uvrAC genes were treated with PUVA and cell survival was determined. In parallel, kinetics of DNA repair was also evaluated by the comparison of DNA sedimentation profiles in all the strains after PUVA treatment. The uvrB mutant was more sensitive to PUVA treatment than all the other uvr mutant strains. Wild-type strain, and uvrA and uvrC mutants were able to repair PUVA-induced lesions, as seen by DNA sedimentation profiles, while the uvrB mutant was unable to repair the lesions. In addition, a quadruple fpg nth xth nfo mutant was unable to nick PUVA-treated DNA when the crude cell-free extract was used to perform plasmid nicking. These data suggest that DNA repair of PUVA-induced lesions may require base excision repair functions, despite proficient UvrABC activity. These results point to a specific role for UvrB protein in the repair of psoralen adducts, which appear to be independent of UvrA or UvrC proteins, as described for the classical UvrABC endonuclease mechanism.     

Discovering protein-binding RNA motifs with a generative model of RNA sequences

Recent advances in high-throughput experimental technologies have generated a huge amount of data on interactions between proteins and nucleic acids. Motivated by the big experimental data, several computational methods have been developed either to predict binding sites in a sequence or to determine if an interaction exists between protein and nucleic acid sequences. However, most of the methods cannot be used to discover new nucleic acid sequences that bind to a target protein because they are classifiers rather than generators. In this paper we propose a generative model for constructing protein-binding RNA sequences and motifs using a long short-term memory (LSTM) neural network. Testing the model for several target proteins showed that RNA sequences generated by the model have high binding affinity and specificity for their target proteins and that the protein-binding motifs derived from the generated RNA sequences are comparable to the motifs from experimentally validated protein-binding RNA sequences. The results are promising and we believe this approach will help design more efficient in vitro or in vivo experiments by suggesting potential RNA aptamers for a target protein.     

Repeatability and pattern recognition of bacterial fatty acid profiles generated by direct mass spectrometric analysis of in situ thermal hydrolysis/methylation of whole cells

Direct CI mass spectrometry profiling of fatty acid methyl esters (FAMEs) from in situ thermal hydrolysis/methylation (THM) of whole bacterial cells with tetramethylammonium hydroxide (TMAH) has been demonstrated as a potential method for real time and fieldable detection/identification of microorganisms. Bacillus anthracis (Ames), Yersinia pestis (Nair. Kenya), Vibrio cholerae (E1 Tor), Brucella melitensis (Abortus wild) and Francisella tularensis (LVS vaccine) were profiled by this method during a 10-month period. Repeatability of the in situ FAME data was calculated using one-way analysis of variance (ANOVA) and a t-test. Artificial neural network (ANN) and multivariate statistics of the FAME profiles were also compared for bacterial identification/classification. Equivalent results were obtained with a multivariate rule building expert system (MuRES) and the ANN. However, the ANN analysis required much less computer time and was deemed the best choice for this application. In situ THM FAME profiles of the bacterial samples provided comparable results with those obtained from the Microbial Identification System (MIDI) (Newark, DE) wet chemistry-gas chromatographic based system.     

Shotgun proteomic analysis of Arabidopsis thaliana leaves

Two shotgun tandem MS proteomics approaches, multidimensional protein identification technology (MudPIT) and 1-D gel-LC-MS/MS, were used to identify Arabidopsis thaliana leaf proteins. These methods utilize different protein/peptide separation strategies. Detergents not compatible with MudPIT were used with 1-D gel-LC-MS/MS to help enrich for the detection of membrane-spanning and hydrophobic proteins. By combining the data from all MudPIT and 1-D gel-LC-MS/MS experiments, 2342 nonredundant proteins spanning a broad range of molecular weights and pI values were detected. With the exception of unknown proteins, the distribution of gene ontology (GO) classifications for the detected proteins was similar to that encoded by the genome, which shows that these extraction and separation procedures are useful for a broad proteomic survey of plant cells. Unknown proteins will likely have to be targeted by using additional methods, some of which should be compatible with separation strategies taken here.     

Proteomic Analysis of Sarcoplasmic Peptides of Two Related Fish Species for Food Authentication

Detection of species-specific sarcoplasmic peptides can be used as proteomic markers for fish food authentication and identification of species of origin in processed products. In the present study, proteomics technology was employed for differential characterization of sarcoplasmic peptides of two closely related fish species, Sperata seenghala and Sperata aor. Species-specific peptides were searched in white muscle extracts of the two species for identification of unique peptides that might aid in differentiation of the species, under two-dimensional gel electrophoresis platform. A total of 19 proteins were identified by combined matrix-assisted laser desorption ionization time-of-flight mass spectrometry and liquid chromatography-tandem mass spectrometry, of which nine and two proteins were found to be unique to S. seenghala and S. aor, respectively. One of the proteins, triosephosphate isomerase (TPI) was found to have three isoforms, out of which two were specific to S. aor, and one was specific to S. seenghala. All the three isoforms of TPI were present in the mixed samples of raw protein extracts of S. seenghala and S. aor, an observation that can be exploited to differentiate between the species and detection of deceptive practices of fraudulent substitution of commercially valuable fish species with inferior ones and differential characterization between closely related fish species.     

Genome-wide analysis of Excretory/Secretory proteins in root-knot nematode,Meloidogyne incognita provides potential targets for parasite control

The root-knot nematode, Meloidogyne incognita causes significant damage to various economically important crops. Infection is associated with secretion of effector proteins into host cytoplasm and interference with host innate immunity. To combat this infection, the identification and functional annotations of Excretory/Secretory (ES) proteins serve as a key to produce durable control measures. The identification of ES proteins through experimental methods are expensive and time consuming while bioinformatics approaches are cost-effective by prioritizing the experimental analysis of potential drug targets for parasitic diseases. In this study, we predicted and functionally annotated the 1889 ES proteins in M. incognita genome using integration of several bioinformatics tools. Of these 1889 ES proteins, 473 (25%) had orthologues in free living nematode Caenorhabditis elegans, 825(67.8%) in parasitic nematodes whereas 561 (29.7%) appeared to be novel and M. incognita specific molecules. Of the C. elegans homologues, 17 ES proteins had “loss of function phenotype” by RNA interference and could represent potential drug targets for parasite intervention and control. We could functionally annotate 429 (22.7%) ES proteins using Gene Ontology (GO) terms, 672 (35.5%) proteins to protein domains and established pathway associations for 223 (11.8%) sequences using Kyoto Encyclopaedia of Genes and Genomes (KEGG). The 162 (8.5%) ES proteins were also mapped to several important plant cell-wall degrading CAZyme families including chitinase, cellulase, xylanase, pectate lyase and endo-β-1,4-xylanase. Our comprehensive analysis of M. incognita secretome provides functional information for further experimental study.     

High-throughput profiling of the mitochondrial proteome using affinity fractionation and automation

Recent studies have demonstrated the need for complementing cellular genomic information with specific information on expressed proteins, or proteomics, since the correlation between the two is poor. Typically, proteomic information is gathered by analyzing samples on two-dimensional gels with the subsequent identification of specific proteins of interest by using trypsin digestion and mass spectrometry in a process termed peptide mass fingerprinting. These procedures have, as a rule, been labor-intensive and manual, and therefore of low throughput. The development of automated proteomic technology for processing large numbers of samples simultaneously has made the concept of profiling entire proteomes feasible at last. In this study, we report the initiation of the (eventual) complete profile of the rat mitochondrial proteome by using high-throughput automated equipment in combination with a novel fractionation technique using minispin affinity columns. Using these technologies, approximately one hundred proteins could be identified in several days. In addition, separate profiles of calcium binding proteins, glycoproteins, and hydrophobic or membrane proteins could be generated. Because mitochondrial dysfunction has been implicated in numerous diseases, such as cancer, Alzheimer's disease and diabetes, it is probable that the identification of the majority of mitochondrial proteins will be a beneficial tool for developing drug and diagnostic targets for associated diseases.