A novel anti-virulence gene revealed by proteomic analysis in Shigella flexneri 2a

<p>Abstract</p> <p>Background</p> <p><it>Shigella flexneri </it>is a gram-negative, facultative pathogen that causes the majority of communicable bacterial dysenteries in developing countries. The virulence factors of <it>S. flexneri </it>have been shown to be produced at 37 degrees C but not at 30 degrees C. To discover potential, novel virulence-related proteins of <it>S. flexneri</it>, we performed differential in-gel electrophoresis (DIGE) analysis to measure changes in the expression profile that are induced by a temperature increase.</p> <p>Results</p> <p>The ArgT protein was dramatically down-regulated at 37 degrees C. In contrast, the ArgT from the non-pathogenic <it>E. coli </it>did not show this differential expression as in <it>S. flexneri</it>, which suggested that <it>argT </it>might be a potential anti-virulence gene. Competitive invasion assays in HeLa cells and in BALB/c mice with <it>argT </it>mutants were performed, and the results indicated that the over-expression of ArgT_Y225Dwould attenuate the virulence of <it>S. flexneri</it>. A comparative proteomic analysis was subsequently performed to investigate the effects of ArgT in <it>S. flexneri </it>at the molecular level. We show that HtrA is differentially expressed among different derivative strains.</p> <p>Conclusion</p> <p>Gene <it>argT </it>is a novel anti-virulence gene that may interfere with the virulence of <it>S. flexneri </it>via the transport of specific amino acids or by affecting the expression of the virulence factor, HtrA.</p

Application of CRISPR/Cas9 System for Plasmid Elimination and Bacterial Killing of Bacillus cereus Group Strains

The CRISPR-Cas system has been widely applied in prokaryotic genome editing with its high efficiency and easy operation. We constructed some “scissors plasmids” via using the temperature-sensitive pJOE8999 shuttle plasmid, which carry the different 20nt (N20) guiding the Cas9 nuclease as a scissors to break the target DNA. We successfully used scissors plasmids to eliminate native plasmids from Bacillus anthracis and Bacillus cereus, and specifically killed B. anthracis. When curing pXO1 and pXO2 virulence plasmids from B. anthracis A16PI2 and A16Q1, respectively, we found that the plasmid elimination percentage was slightly higher when the sgRNA targeted the replication initiation region (96–100%), rather than the non-replication initiation region (88–92%). We also tried using a mixture of two scissors plasmids to simultaneously eliminate pXO1 and pXO2 plasmids from B. anthracis, and the single and double plasmid-cured rates were 29 and 14%, respectively. To our surprise, when we used the scissor plasmid containing two tandem sgRNAs to cure the target plasmids pXO1 and pXO2 from wild strain B. anthracis A16 simultaneously, only the second sgRNA could guide Cas9 to cleave the target plasmid with high efficiency, while the first sgRNA didn't work in all the experiments we designed. When we used the CRISPR/cas9 system to eliminate the pCE1 mega-virulence plasmid from B. cereus BC307 by simply changing the sgRNA, we also obtained a plasmid-cured isogenic strain at a very high elimination rate (69%). The sterilization efficiency of B. anthracis was about 93%, which is similar to the efficiency of plasmid curing, and there was no significant difference in the efficiency of among the scissors plasmids containing single sgRNA, targeting multi-sites, or single-site targeting and the two tandem sgRNA. This simple and effective curing method, which is applicable to B. cereus group strains, provides a new way to study these bacteria and their virulence profiles

A Reference Proteomic Database of Lactobacillus plantarum CMCC-P0002

Lactobacillus plantarum is a widespread probiotic bacteria found in many fermented food products. In this study, the whole-cell proteins and secretory proteins of L. plantarum were separated by two-dimensional electrophoresis method. A total of 434 proteins were identified by tandem mass spectrometry, including a plasmid-encoded hypothetical protein pLP9000_05. The information of first 20 highest abundance proteins was listed for the further genetic manipulation of L. plantarum, such as construction of high-level expressions system. Furthermore, the first interaction map of L. plantarum was established by Blue-Native/SDS-PAGE technique. A heterodimeric complex composed of maltose phosphorylase Map3 and Map2, and two homodimeric complexes composed of Map3 and Map2 respectively, were identified at the same time, indicating the important roles of these proteins. These findings provided valuable information for the further proteomic researches of L. plantarum

Curing of Plasmid pXO1 from Bacillus anthracis Using Plasmid Incompatibility

The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures) can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome

Cluster Headache Genomewide Association Study and Meta-Analysis Identifies Eight Loci and Implicates Smoking as Causal Risk Factor

Objective: The objective of this study was to aggregate data for the first genomewide association study meta-analysis of cluster headache, to identify genetic risk variants, and gain biological insights. Methods: A total of 4,777 cases (3,348 men and 1,429 women) with clinically diagnosed cluster headache were recruited from 10 European and 1 East Asian cohorts. We first performed an inverse-variance genomewide association meta-analysis of 4,043 cases and 21,729 controls of European ancestry. In a secondary trans-ancestry meta-analysis, we included 734 cases and 9,846 controls of East Asian ancestry. Candidate causal genes were prioritized by 5 complementary methods: expression quantitative trait loci, transcriptome-wide association, fine-mapping of causal gene sets, genetically driven DNA methylation, and effects on protein structure. Gene set and tissue enrichment analyses, genetic correlation, genetic risk score analysis, and Mendelian randomization were part of the downstream analyses. Results: The estimated single nucleotide polymorphism (SNP)-based heritability of cluster headache was 14.5%. We identified 9 independent signals in 7 genomewide significant loci in the primary meta-analysis, and one additional locus in the trans-ethnic meta-analysis. Five of the loci were previously known. The 20 genes prioritized as potentially causal for cluster headache showed enrichment to artery and brain tissue. Cluster headache was genetically correlated with cigarette smoking, risk-taking behavior, attention deficit hyperactivity disorder (ADHD), depression, and musculoskeletal pain. Mendelian randomization analysis indicated a causal effect of cigarette smoking intensity on cluster headache. Three of the identified loci were shared with migraine. Interpretation: This first genomewide association study meta-analysis gives clues to the biological basis of cluster headache and indicates that smoking is a causal risk factor

Geospatial Semantics

Geospatial semantics is a broad field that involves a variety of research areas. The term semantics refers to the meaning of things, and is in contrast with the term syntactics. Accordingly, studies on geospatial semantics usually focus on understanding the meaning of geographic entities as well as their counterparts in the cognitive and digital world, such as cognitive geographic concepts and digital gazetteers. Geospatial semantics can also facilitate the design of geographic information systems (GIS) by enhancing the interoperability of distributed systems and developing more intelligent interfaces for user interactions. During the past years, a lot of research has been conducted, approaching geospatial semantics from different perspectives, using a variety of methods, and targeting different problems. Meanwhile, the arrival of big geo data, especially the large amount of unstructured text data on the Web, and the fast development of natural language processing methods enable new research directions in geospatial semantics. This chapter, therefore, provides a systematic review on the existing geospatial semantic research. Six major research areas are identified and discussed, including semantic interoperability, digital gazetteers, geographic information retrieval, geospatial Semantic Web, place semantics, and cognitive geographic concepts.Comment: Yingjie Hu (2017). Geospatial Semantics. In Bo Huang, Thomas J. Cova, and Ming-Hsiang Tsou et al. (Eds): Comprehensive Geographic Information Systems, Elsevier. Oxford, U

Black-Box Adversarial Attack on Time Series Classification

With the increasing use of deep neural network (DNN) in time series classification (TSC), recent work reveals the threat of adversarial attack, where the adversary can construct adversarial examples to cause model mistakes. However, existing researches on the adversarial attack of TSC typically adopt an unrealistic white-box setting with model details transparent to the adversary. In this work, we study a more rigorous black-box setting with attack detection applied, which restricts gradient access and requires the adversarial example to be also stealthy. Theoretical analyses reveal that the key lies in: estimating black-box gradient with diversity and non-convexity of TSC models resolved, and restricting the l0 norm of the perturbation to construct adversarial samples. Towards this end, we propose a new framework named BlackTreeS, which solves the hard optimization issue for adversarial example construction with two simple yet effective modules. In particular, we propose a tree search strategy to find influential positions in a sequence, and independently estimate the black-box gradients for these positions. Extensive experiments on three real-world TSC datasets and five DNN based models validate the effectiveness of BlackTreeS, e.g., it improves the attack success rate from 19.3% to 27.3%, and decreases the detection success rate from 90.9% to 6.8% for LSTM on the UWave dataset

Aerobic heterogeneous palladium-catalyzed oxidative allenic C−H arylation : Benzoquinone as a direct redox mediator between O2 and Pd

Transition metal-catalyzed aerobic oxidative reactions using molecular oxygen as the terminal oxidant play a significant role in organic synthesis. Benzoquinone (BQ) has been widely used as an electron-transfer mediator (ETM) in biomimetic palladium-catalyzed aerobic oxidative reactions, but always together with an ETM between O2 and BQ, such as a macrocyclic metal complex. Herein, we report on a heterogeneous palladium-catalyzed allenic C(sp3)-H arylation with only catalytic amounts of BQ under air without the need of an additional ETM. A range of multisubstituted 1,3-dienes were synthesized under mild reaction conditions. Mechanistic studies reveal the bifunctional role of BQ as a ligand for reductive elimination and as an ETM between Pd(0) and O2. This new regime of oxidation has important implications for further development of other transition metal-catalyzed aerobic oxidative reactions

Application of an O-Linked Glycosylation System in Yersinia enterocolitica Serotype O:9 to Generate a New Candidate Vaccine against Brucella abortus

Brucellosis is a major zoonotic public health threat worldwide, causing veterinary morbidity and major economic losses in endemic regions. However, no efficacious brucellosis vaccine is yet available, and live attenuated vaccines commonly used in animals can cause human infection. N- and O-linked glycosylation systems have been successfully developed and exploited for the production of successful bioconjugate vaccines. Here, we applied an O-linked glycosylation system to a low-pathogenicity bacterium, Yersinia enterocolitica serotype O:9 (Y. enterocolitica O:9), which has repeating units of O-antigen polysaccharide (OPS) identical to that of Brucella abortus (B. abortus), to develop a bioconjugate vaccine against Brucella. The glycoprotein we produced was recognized by both anti-B. abortus and anti-Y. enterocolitica O:9 monoclonal antibodies. Three doses of bioconjugate vaccine-elicited B. abortus OPS-specific serum IgG in mice, significantly reducing bacterial loads in the spleen following infection with the B. abortus hypovirulent smooth strain A19. This candidate vaccine mitigated B. abortus infection and prevented severe tissue damage, thereby protecting against lethal challenge with A19. Overall, the results indicated that the bioconjugate vaccine elicited a strong immune response and provided significant protection against brucellosis. The described vaccine preparation strategy is safe and avoids large-scale culture of the highly pathogenic B. abortus

Comparative proteomics of Shigella flexneri 2a strain 301 using a rabbit ileal loop model reveals key proteins for bacterial adaptation in host niches

Objectives: Many studies focusing on changes in the host following Shigella spp invasion have been reported in recent years. However, the key factors required for the adaptation of these pathogens to host niches have usually been neglected. Methods: In this study, a comparative proteomic analysis was performed to examine changes in the protein expression profile of Shigella flexneri within the host using a rabbit ileal loop model to reveal proteins that are associated with pathogenic adaptation. Results: The protein expression profiles of bacteria isolated from the ileum and colon were very similar, although they differed slightly from that of bacteria isolated from the cecum. When compared with the sample in vitro, the expressions of seven proteins were found to be upshifted in vivo (OmpA, YgiW, MglB, YfiD, MetK, TktA, and AhpF), while two proteins were down-regulated (ElaB and GlnH). Conclusions: The abundance of nine proteins changed in vivo, suggesting that these proteins may contribute to adaptation to the intestinal lumen