Petrus Tang

The genome sequence of Salmonella enterica serovar Choleraesuis, a highly invasive and resistant zoonotic pathogen

Salmonella enterica serovar Choleraesuis (S.Choleraesuis), a highly invasive serovar among non-typhoidal Salmonella, usually causes sepsis or extra-intestinal focal infections in humans. S.Choleraesuis infections have now become particularly difficult to treat because of the emergence of resistance to multiple antimicrobial agents. The 4.7 Mb genome sequence of a multidrug-resistant S.Choleraesuis strain SC-B67 was determined. Genome wide comparison of three sequenced Salmonella genomes revealed that more deletion events occurred in S.Choleraesuis SC-B67 and S.Typhi CT18 relative to S.Typhimurium LT2. S.Choleraesuis has 151 pseudogenes, which, among the three Salmonella genomes, include the highest percentage of pseudogenes arising from the genes involved in bacterial chemotaxis signal-transduction pathways. Mutations in these genes may increase smooth swimming of the bacteria, potentially allowing more effective interactions with and invasion of host cells to occur. A key regulatory gene of TetR/AcrR family, acrR, was inactivated through the introduction of an internal stop codon resulting in overexpression of AcrAB that appears to be associated with ciprofloxacin resistance. While lateral gene transfer providing basic functions to allow niche expansion in the host and environment is maintained during the evolution of different serovars of Salmonella, genes providing little overall selective benefit may be lost rapidly. Our findings suggest that the formation of pseudogenes may provide a simple evolutionary pathway that complements gene acquisition to enhance virulence and antimicrobial resistance in S.Choleraesuis.

Genome-wide Association Study Reveals Multiple Nasopharyngeal Carcinoma-Associated Loci within the HLA Region at Chromosome 6p21.3

Nasopharyngeal carcinoma (NPC) is a multifactorial malignancy closely associated with genetic factors and Epstein-Barr virus infection. To identify the common genetic variants linked to NPC susceptibility, we conducted a genome-wide association study (GWAS) in 277 NPC patients and 285 healthy controls within the Taiwanese population, analyzing 480,365 single-nucleotide polymorphisms (SNPs). Twelve statistically significant SNPs were identified and mapped to chromosome 6p21.3. Associations were replicated in two independent sets of case-control samples. Two of the most significant SNPs (rs2517713 and rs2975042; p(combined) = 3.9 x 10(-20) and 1.6 x 10(-19), respectively) were located in the HLA-A gene. Moreover, we detected significant associations between NPC and two genes: specifically, gamma aminobutyric acid b receptor 1 (GABBR1) (rs29232; p(combined) = 8.97 x 10(-17)) and HLA-F (rs3129055 and rs9258122; p(combined) = 7.36 x 10(-11) and 3.33 x 10(-10), respectively). Notably, the association of rs29232 remained significant (residual p < 5 x 10(-4)) after adjustment for age, gender, and HLA-related SNPs. Furthermore, higher GABA(B) receptor 1 expression levels can be found in the tumor cells in comparison to the adjacent epithelial cells (p < 0.001) in NPC biopsies, implying a biological role of GABBR1 in NPC carcinogenesis. To our knowledge, it is the first GWAS report of NPC showing that multiple loci (HLA-A, HLA-F, and GABBR1) within chromosome 6p21.3 are associated with NPC. Although some of these relationships may be attributed to linkage disequilibrium between the loci, the findings clearly provide a fresh direction for the study of NPC development.

Comparative and Targeted Proteomic Analyses of Urinary Microparticles from Bladder Cancer and Hernia Patients

Bladder cancer is a common urologic cancer whose incidence continues to rise annually. Urinary microparticles are an attractive material for noninvasive bladder cancer biomarker discovery. In this study, we applied isotopic dimethylation labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to discover bladder cancer biomarkers in urinary microparticles isolated from hernia (control) and bladder cancer patients. This approach identified 2964 proteins based on more than two distinct peptides, of which 2058 had not previously been reported as constituents of human urine exosomes/microparticles. A total of 107 differentially expressed proteins were identified as candidate biomarkers. Differences in the concentrations of 29 proteins (41 signature peptides) were precisely quantified by LC-MRM/MS in 48 urine samples of bladder cancer, hernia, and urinary tract infection/hematuria. Concentrations of 24 proteins changed significantly (p<0.05) between bladder cancer (n=28) and hernia (n=12), with area-under-the-curve values ranging from 0.702 to 0.896. Finally, we quantified tumor-associated calcium-signal transducer 2 (TACSTD2) in raw urine specimens (n=221) using a commercial ELISA and confirmed its potential value for diagnosis of bladder cancer. Our study reveals a strong association of TACSTD2 with bladder cancer and highlights the potential of human urinary microparticles in the noninvasive diagnosis of bladder cancer.

Reticulon 3 binds the 2C protein of enterovirus 71 and is required for viral replication

Enterovirus 71 is an enterovirus of the family Picornaviridae. The 2C protein of poliovirus, a relative of enterovirus 71, is essential for viral replication. The poliovirus 2C protein is associated with host membrane vesicles, which form viral replication complexes where viral RNA synthesis takes place. We have now identified a host-encoded 2C binding protein called reticulon 3, which we found to be associated with the replication complex through direct interaction with the enterovirus 71-encoded 2C protein. We observed that the N terminus of the 2C protein, which has both RNA- and membrane-binding activity, interacted with reticulon 3. This region of interaction was mapped to its reticulon homology domain, whereas that of 2C was encoded by the 25th amino acid, isoleucine. Reticulon 3 could also interact with the 2C proteins encoded by other enteroviruses, such as poliovirus and coxsackievirus A16, implying that it is a common factor for such viral replication. Reduced production of reticulon 3 by RNA interference markedly reduced the synthesis of enterovirus 71-encoded viral proteins and replicative double-stranded RNA, reducing plaque formation and apoptosis. Furthermore, reintroduction of nondegradable reticulon 3 into these knockdown cells rescued enterovirus 71 infectivity, and viral protein and double-stranded RNA synthesis. Thus, reticulon 3 is an important component of enterovirus 71 replication, through its potential role in modulation of the sequential interactions between enterovirus 71 viral RNA and the replication complex.

DSAP: deep-sequencing small RNA analysis pipeline.

DSAP is an automated multiple-task web service designed to provide a total solution to analyzing deep-sequencing small RNA datasets generated by next-generation sequencing technology. DSAP uses a tab-delimited file as an input format, which holds the unique sequence reads (tags) and their corresponding number of copies generated by the Solexa sequencing platform. The input data will go through four analysis steps in DSAP: (i) cleanup: removal of adaptors and poly-A/T/C/G/N nucleotides; (ii) clustering: grouping of cleaned sequence tags into unique sequence clusters; (iii) non-coding RNA (ncRNA) matching: sequence homology mapping against a transcribed sequence library from the ncRNA database Rfam (http://rfam.sanger.ac.uk/); and (iv) known miRNA matching: detection of known miRNAs in miRBase (http://www.mirbase.org/) based on sequence homology. The expression levels corresponding to matched ncRNAs and miRNAs are summarized in multi-color clickable bar charts linked to external databases. DSAP is also capable of displaying miRNA expression levels from different jobs using a log2-scaled color matrix. Furthermore, a cross-species comparative function is also provided to show the distribution of identified miRNAs in different species as deposited in miRBase. DSAP is available at http://dsap.cgu.edu.tw.

Genome evolution driven by host adaptations results in a more virulent and antimicrobial-resistant Streptococcus pneumoniae serotype 14

BackgroundStreptococcus pneumoniae serotype 14 is one of the most common pneumococcal serotypes that cause invasive pneumococcal diseases worldwide. Serotype 14 often expresses resistance to a variety of antimicrobial agents, resulting in difficulties in treatment. To gain insight into the evolution of virulence and antimicrobial resistance traits in S. pneumoniae from the genome level, we sequenced the entire genome of a serotype 14 isolate (CGSP14), and carried out comprehensive comparison with other pneumococcal genomes. Multiple serotype 14 clinical isolates were also genotyped by multilocus sequence typing (MLST).ResultsComparative genomic analysis revealed that the CGSP14 acquired a number of new genes by horizontal gene transfer (HGT), most of which were associated with virulence and antimicrobial resistance and clustered in mobile genetic elements. The most remarkable feature is the acquisition of two conjugative transposons and one resistance island encoding eight resistance genes. Results of MLST suggested that the major driving force for the genome evolution is the environmental drug pressure.ConclusionThe genome sequence of S. pneumoniae serotype 14 shows a bacterium with rapid adaptations to its lifecycle in human community. These include a versatile genome content, with a wide range of mobile elements, and chromosomal rearrangement; the latter re-balanced the genome after events of HGT.

Identification of microRNA in the protist Trichomonas vaginalis

MicroRNAs (miRNAs) are a class of small noncoding RNAs that have important regulatory roles in multicellular organisms. However, miRNA has never been identified experimentally in protist. Direct cloning of 438 expressed miRNA tags by microRNA serial analysis of gene expression from the parasitic protist Trichomonas vaginalis identified nine candidate miRNAs. Bioinformatics analysis of the corresponding genomic region revealed that these miRNA candidates contain a classical stem-loop-stem structure of pre-microRNAs. Analysis of the 20 nt long mature tva-miR-001 showed that it is an intergenic miRNA located at the scaffold DS113596. Tva-miR-001 was differentially expressed in the trophozoite, pseudocyst and amoeboid stages. Based on the experimental results of the present study, we provided solid evidence that protist possesses a miRNA regulating network comparable with multicellular organisms for the first time.

FastAnnotator- an efficient transcript annotation web tool

BackgroundRecent developments in high-throughput sequencing (HTS) technologies have made it feasible to sequence the complete transcriptomes of non-model organisms or metatranscriptomes from environmental samples. The challenge after generating hundreds of millions of sequences is to annotate these transcripts and classify the transcripts based on their putative functions. Because many biological scientists lack the knowledge to install Linux-based software packages or maintain databases used for transcript annotation, we developed an automatic annotation tool with an easy-to-use interface.MethodsTo elucidate the potential functions of gene transcripts, we integrated well-established annotation tools: Blast2GO, PRIAM and RPS BLAST in a web-based service, FastAnnotator, which can assign Gene Ontology (GO) terms, Enzyme Commission numbers (EC numbers) and functional domains to query sequences.ResultsUsing six transcriptome sequence datasets as examples, we demonstrated the ability of FastAnnotator to assign functional annotations. FastAnnotator annotated 88.1% and 81.3% of the transcripts from the well-studied organisms Caenorhabditis elegans and Streptococcus parasanguinis, respectively. Furthermore, FastAnnotator annotated 62.9%, 20.4%, 53.1% and 42.0% of the sequences from the transcriptomes of sweet potato, clam, amoeba, and Trichomonas vaginalis, respectively, which lack reference genomes. We demonstrated that FastAnnotator can complete the annotation process in a reasonable amount of time and is suitable for the annotation of transcriptomes from model organisms or organisms for which annotated reference genomes are not avaiable.ConclusionsThe sequencing process no longer represents the bottleneck in the study of genomics, and automatic annotation tools have become invaluable as the annotation procedure has become the limiting step. We present FastAnnotator, which was an automated annotation web tool designed to efficiently annotate sequences with their gene functions, enzyme functions or domains. FastAnnotator is useful in transcriptome studies and especially for those focusing on non-model organisms or metatranscriptomes. FastAnnotator does not require local installation and is freely available at http://fastannotator.cgu.edu.tw.

Antioxidant defense is one of the mechanisms by which mosquito cells survive dengue 2 viral infection

Dengue viruses (DENVs) generally induce apoptosis in mammalian cells but cause only minor damage in mosquito cells. To find genes involved in determining the cell fate, datasets derived from expressed sequence tags (ESTs) of C6/36 cells with and without infection were established. Of overexpressed genes found in infected dataset, chaperone proteins were validated significantly upregulated in C6/36 cells at 24 hpi. It suggests that DENV-2 in mosquito cells activates the unfolded protein response to cope with endoplasmic reticular stress. Changes in the mitochondrial membrane potential and generation of superoxide provided further evidence that DENV-2 induces oxidative stress in both C6/36 and BHK-21 cells. Significant elevation of glutathione S-transferase (GST) activity was shown in infected C6/36, but not BHK-21, cells, while suppression of GST produced superoxide at 36 hpi and increased the cell death rate at 48 hpi. This indicates that mosquito cells protect themselves against viral infection through antioxidant defenses.

Trichomonas vaginalis vast BspA-like gene family: evidence for functional diversity from structural organisation and transcriptomics.

BackgroundTrichomonas vaginalis is the most common non-viral human sexually transmitted pathogen and importantly, contributes to facilitating the spread of HIV. Yet very little is known about its surface and secreted proteins mediating interactions with, and permitting the invasion and colonisation of, the host mucosa. Initial annotations of T. vaginalis genome identified a plethora of candidate extracellular proteins.ResultsData mining of the T. vaginalis genome identified 911 BspA-like entries (TvBspA) sharing TpLRR-like leucine-rich repeats, which represent the largest gene family encoding potential extracellular proteins for the pathogen. A broad range of microorganisms encoding BspA-like proteins was identified and these are mainly known to live on mucosal surfaces, among these T. vaginalis is endowed with the largest gene family. Over 190 TvBspA proteins with inferred transmembrane domains were characterised by a considerable structural diversity between their TpLRR and other types of repetitive sequences and two subfamilies possessed distinct classic sorting signal motifs for endocytosis. One TvBspA subfamily also shared a glycine-rich protein domain with proteins from Clostridium difficile pathogenic strains and C. difficile phages. Consistent with the hypothesis that TvBspA protein structural diversity implies diverse roles, we demonstrated for several TvBspA genes differential expression at the transcript level in different growth conditions. Identified variants of repetitive segments between several TvBspA paralogues and orthologues from two clinical isolates were also consistent with TpLRR and other repetitive sequences to be functionally important. For one TvBspA protein cell surface expression and antibody responses by both female and male T. vaginalis infected patients were also demonstrated.ConclusionsThe biased mucosal habitat for microbial species encoding BspA-like proteins, the characterisation of a vast structural diversity for the TvBspA proteins, differential expression of a subset of TvBspA genes and the cellular localisation and immunological data for one TvBspA; all point to the importance of the TvBspA proteins to various aspects of T. vaginalis pathobiology at the host-pathogen interface.