Characterization of intracellular growth regulator icgR by utilizing transcriptomics to identify mediators of pathogenesis in Shigella flexneri.

Shigella species Gram-negative bacteria which cause a diarrheal disease, known as shigellosis, by invading and destroying the colonic mucosa and inducing a robust inflammatory response. With no vaccine available, shigellosis annually kills over 600,000 children in developing countries. This study demonstrates the utility of combining high-throughput bioinformatic methods with in vitro and in vivo assays to provide new insights into pathogenesis. Comparisons of in vivo and in vitro gene expression identified genes associated with intracellular growth. Additional bioinformatics analyses identified genes that are present in S. flexneri isolates but not in the three other Shigella species. Comparison of these two analyses revealed nine genes that are differentially expressed during invasion and that are specific to S. flexneri. One gene, a DeoR family transcriptional regulator with decreased expression during invasion, was further characterized and is now designated icgR, for intracellular growth regulator. Deletion of icgR caused no difference in growth in vitro but resulted in increased intracellular replication in HCT-8 cells. Further in vitro and in vivo studies using high-throughput sequencing of RNA transcripts (RNA-seq) of an isogenic ΔicgR mutant identified 34 genes that were upregulated under both growth conditions. This combined informatics and functional approach has allowed the characterization of a gene and pathway previously unknown in Shigella pathogenesis and provides a framework for further identification of novel virulence factors and regulatory pathways.

Draft genome sequences of the diarrheagenic Escherichia coli collection.

We report the draft genome sequences of the collection referred to as the Escherichia coli DECA collection, which was assembled to contain representative isolates of the 15 most common diarrheagenic clones in humans (http://shigatox.net/new/). These genomes represent a valuable resource to the community of researchers who examine these enteric pathogens.

Shigella flexneri effectors OspE1 and OspE2 mediate induced adherence to the colonic epithelium following bile salts exposure.

Shigella flexneri is a Gram-negative pathogen that invades the colonic epithelium. While invasion has been thoroughly investigated, it is unknown how Shigella first attaches to the epithelium. Previous literature suggests that Shigella utilizes adhesins that are induced by environmental signals, including bile salts, encountered in the small intestine prior to invasion. We hypothesized that bile would induce adherence factors to facilitate attachment to colonic epithelial cells. To test our hypothesis, S. flexneri strain 2457T was subcultured in media containing bile salts, and the ability of the bacteria to adhere to the apical surface of polarized T84 epithelial cells was measured. We observed a significant increase in adherence, which was absent in a virulence plasmid-cured strain and a type-III secretion system mutant. Microarray expression analysis indicated that the ospE1/ospE2 genes were induced in the presence of bile, and bile-induced adherence was lost in a ΔospE1/ΔospE2 mutant. Further studies demonstrated that the OspE1/OspE2 proteins were localized to the bacterial outer membrane following exposure to bile salts. The data presented are the first demonstration that the OspE1/OspE2 proteins promote initial adherence to the intestinal epithelium. The adhesins required for Shigella attachment to the colonic epithelium may serve as ideal targets for vaccine development.

Genomic characterization of enteroaggregative Escherichia coli from children in Mali.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is a cause of epidemic and sporadic diarrhea, yet its role as an enteric pathogen is not fully understood. METHODS: We characterized 121 EAEC strains isolated in 2008 as part of a case-control study of moderate to severe acute diarrhea among children 0-59 months of age in Bamako, Mali. We applied multiplex polymerase chain reaction and comparative genome hybridization to identify potential virulence factors among the EAEC strains, coupled with classification and regression tree modeling to reveal combinations of factors most strongly associated with illness. RESULTS: The gene encoding the autotransporter protease SepA, originally described in Shigella species, was most strongly associated with diarrhea among the EAEC strains tested (odds ratio, 5.6 [95% confidence interval, 1.92-16.17]; P = .0006). In addition, we identified 3 gene combinations correlated with diarrhea: (1) a clonal group positive for sepA and a putative hemolysin; (2) a group harboring the EAST-1 enterotoxin and the flagellar type H33 but no other previously identified EAEC virulence factor; and (3) a group carrying several of the typical EAEC virulence genes. CONCLUSION: Our data suggest that only a subset of EAEC strains are pathogenic in Mali and suggest that sepA may serve as a valuable marker for the most virulent isolates.

Origins of the E. coli strain causing an outbreak of hemolytic-uremic syndrome in Germany.

BACKGROUND: A large outbreak of diarrhea and the hemolytic-uremic syndrome caused by an unusual serotype of Shiga-toxin-producing Escherichia coli (O104:H4) began in Germany in May 2011. As of July 22, a large number of cases of diarrhea caused by Shiga-toxin-producing E. coli have been reported--3167 without the hemolytic-uremic syndrome (16 deaths) and 908 with the hemolytic-uremic syndrome (34 deaths)--indicating that this strain is notably more virulent than most of the Shiga-toxin-producing E. coli strains. Preliminary genetic characterization of the outbreak strain suggested that, unlike most of these strains, it should be classified within the enteroaggregative pathotype of E. coli. METHODS: We used third-generation, single-molecule, real-time DNA sequencing to determine the complete genome sequence of the German outbreak strain, as well as the genome sequences of seven diarrhea-associated enteroaggregative E. coli serotype O104:H4 strains from Africa and four enteroaggregative E. coli reference strains belonging to other serotypes. Genomewide comparisons were performed with the use of these enteroaggregative E. coli genomes, as well as those of 40 previously sequenced E. coli isolates. RESULTS: The enteroaggregative E. coli O104:H4 strains are closely related and form a distinct clade among E. coli and enteroaggregative E. coli strains. However, the genome of the German outbreak strain can be distinguished from those of other O104:H4 strains because it contains a prophage encoding Shiga toxin 2 and a distinct set of additional virulence and antibiotic-resistance factors. CONCLUSIONS: Our findings suggest that horizontal genetic exchange allowed for the emergence of the highly virulent Shiga-toxin-producing enteroaggregative E. coli O104:H4 strain that caused the German outbreak. More broadly, these findings highlight the way in which the plasticity of bacterial genomes facilitates the emergence of new pathogens.

Genomic characterization of asymptomatic Escherichia coli isolated from the neobladder.

The replacement of the bladder with a neobladder made from ileal tissue is the prescribed treatment in some cases of bladder cancer or trauma. Studies have demonstrated that individuals with an ileal neobladder have recurrent colonization by Escherichia coli and other species that are commonly associated with urinary tract infections; however, pyelonephritis and complicated symptomatic infections with ileal neobladders are relatively rare. This study examines the genomic content of two E. coli isolates from individuals with neobladders using comparative genomic hybridization (CGH) with a pan-E. coli/Shigella microarray. Comparisons of the neobladder genome hybridization patterns with reference genomes demonstrate that the neobladder isolates are more similar to the commensal, laboratory-adapted E. coli and a subset of enteroaggregative E. coli than they are to uropathogenic E. coli isolates. Genes identified by CGH as exclusively present in the neobladder isolates among the 30 examined isolates were primarily from large enteric isolate plasmids. Isolations identified a large plasmid in each isolate, and sequencing confirmed similarity to previously identified plasmids of enteric species. Screening, via PCR, of more than 100 isolates of E. coli from environmental, diarrhoeagenic and urinary tract sources did not identify neobladder-specific genes that were widely distributed in these populations. These results taken together demonstrate that the neobladder isolates, while distinct, are genomically more similar to gastrointestinal or commensal E. coli, suggesting why they can colonize the transplanted intestinal tissue but rarely progress to acute pyelonephritis or more severe disease.

A comparative genomic analysis of diverse clonal types of enterotoxigenic Escherichia coli reveals pathovar-specific conservation.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal illness in children less than 5 years of age in low- and middle-income nations, whereas it is an emerging enteric pathogen in industrialized nations. Despite being an important cause of diarrhea, little is known about the genomic composition of ETEC. To address this, we sequenced the genomes of five ETEC isolates obtained from children in Guinea-Bissau with diarrhea. These five isolates represent distinct and globally dominant ETEC clonal groups. Comparative genomic analyses utilizing a gene-independent whole-genome alignment method demonstrated that sequenced ETEC strains share approximately 2.7 million bases of genomic sequence. Phylogenetic analysis of this "core genome" confirmed the diverse history of the ETEC pathovar and provides a finer resolution of the E. coli relationships than multilocus sequence typing. No identified genomic regions were conserved exclusively in all ETEC genomes; however, we identified more genomic content conserved among ETEC genomes than among non-ETEC E. coli genomes, suggesting that ETEC isolates share a genomic core. Comparisons of known virulence and of surface-exposed and colonization factor genes across all sequenced ETEC genomes not only identified variability but also indicated that some antigens are restricted to the ETEC pathovar. Overall, the generation of these five genome sequences, in addition to the two previously generated ETEC genomes, highlights the genomic diversity of ETEC. These studies increase our understanding of ETEC evolution, as well as provide insight into virulence factors and conserved proteins, which may be targets for vaccine development.

High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns.

BACKGROUND: Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression. RESULTS: A high throughput pipeline was developed to generate promoter-reporter (GFP) transgenic lines for Arabidopsis genes expressed at very low levels and to examine their expression patterns in vivo. The promoter region from a total of 627 non- or low-expressed genes in Arabidopsis based on Arabidopsis annotation release 5 were amplified and cloned into a Gateway vector. A total of 353 promoter-reporter (GFP) constructs were successfully transferred into Agrobacterium (GV3101) by triparental mating and subsequently used for Arabidopsis transformation. Kanamycin-resistant transgenic lines were obtained from 266 constructs and among them positive GFP expression was detected from 150 constructs. Of these 150 constructs, multiple transgenic lines exhibiting consistent expression patterns were obtained for 112 constructs. A total 81 different regions of expression were discovered during our screening of positive transgenic plants and assigned Plant Ontology (PO) codes. CONCLUSIONS: Many of the genes tested for which expression data were lacking previously are indeed expressed in Arabidopsis during the developmental stages screened. More importantly, our study provides plant researchers with another resource of gene expression information in Arabidopsis. The results of this study are captured in a MySQL database and can be searched at http://www.jcvi.org/arabidopsis/qpcr/index.shtml. Transgenic seeds and constructs are also available for the research community.

A community resource for high-throughput quantitative RT-PCR analysis of transcription factor gene expression in Medicago truncatula.

BACKGROUND: Medicago truncatula is a model legume species that is currently the focus of an international genome sequencing effort. Although several different oligonucleotide and cDNA arrays have been produced for genome-wide transcript analysis of this species, intrinsic limitations in the sensitivity of hybridization-based technologies mean that transcripts of genes expressed at low-levels cannot be measured accurately with these tools. Amongst such genes are many encoding transcription factors (TFs), which are arguably the most important class of regulatory proteins. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is the most sensitive method currently available for transcript quantification, and one that can be scaled up to analyze transcripts of thousands of genes in parallel. Thus, qRT-PCR is an ideal method to tackle the problem of TF transcript quantification in Medicago and other plants. RESULTS: We established a bioinformatics pipeline to identify putative TF genes in Medicago truncatula and to design gene-specific oligonucleotide primers for qRT-PCR analysis of TF transcripts. We validated the efficacy and gene-specificity of over 1000 TF primer pairs and utilized these to identify sets of organ-enhanced TF genes that may play important roles in organ development or differentiation in this species. This community resource will be developed further as more genome sequence becomes available, with the ultimate goal of producing validated, gene-specific primers for all Medicago TF genes. CONCLUSION: High-throughput qRT-PCR using a 384-well plate format enables rapid, flexible, and sensitive quantification of all predicted Medicago transcription factor mRNAs. This resource has been utilized recently by several groups in Europe, Australia, and the USA, and we expect that it will become the 'gold-standard' for TF transcript profiling in Medicago truncatula.

Development of Arabidopsis whole-genome microarrays and their application to the discovery of binding sites for the TGA2 transcription factor in salicylic acid-treated plants.

We have developed two long-oligonucleotide microarrays for the analysis of genome features in Arabidopsis thaliana, in particular for the high-throughput identification of transcription factor-binding sites. The first platform contains 190,000 probes representing the 2-kb regions upstream of all annotated genes at a density of seven probes per promoter. The second platform is divided into three chips, each of over 390,000 features, and represents the entire Arabidopsis genome at a density of one probe per 90 bases. Protein-DNA complexes resulting from the formaldehyde fixation of leaves of plants 2 h after exposure to 1 mm salicylic acid (SA) were immunoprecipitated using antibodies against the TGA2 transcription factor. After reversal of the cross-links and amplification, the resulting ChIP sample was hybridized to both platforms. High signal ratios of the ChIP sample versus raw chromatin for clusters of neighboring probes provided evidence for 51 putative binding sites for TGA2, including the only previously confirmed site in the promoter of PR-1 (At2g14610). Enrichment of several regions was confirmed by quantitative real-time PCR. Motif search revealed that the palindromic octamer TGACGTCA was found in 55% of the enriched regions. Interestingly, 15 of the putative binding sites for TGA2 lie outside the presumptive promoter regions. The effect of the 2-h SA treatment on gene expression was measured using Affymetrix ATH1 arrays, and SA-induced genes were found to be significantly over-represented among genes neighboring putative TGA2-binding sites.

Analysis of the cDNAs of hypothetical genes on Arabidopsis chromosome 2 reveals numerous transcript variants.

In the fully sequenced Arabidopsis (Arabidopsis thaliana) genome, many gene models are annotated as "hypothetical protein," whose gene structures are predicted solely by computer algorithms with no support from either expressed sequence matches from Arabidopsis, or nucleic acid or protein homologs from other species. In order to confirm their existence and predicted gene structures, a high-throughput method of rapid amplification of cDNA ends (RACE) was used to obtain their cDNA sequences from 11 cDNA populations. Primers from all of the 797 hypothetical genes on chromosome 2 were designed, and, through 5' and 3' RACE, clones from 506 genes were sequenced and cDNA sequences from 399 target genes were recovered. The cDNA sequences were obtained by assembling their 5' and 3' RACE polymerase chain reaction products. These sequences revealed that (1) the structures of 151 hypothetical genes were different from their predictions; (2) 116 hypothetical genes had alternatively spliced transcripts and 187 genes displayed polyadenylation sites; and (3) there were transcripts arising from both strands, from the strand opposite to that of the prediction and possible dicistronic transcripts. Promoters from five randomly chosen hypothetical genes (At2g02540, At2g31270, At2g33640, At2g35550, and At2g36340) were cloned into report constructs, and their expressions are tissue or development stage specific. Our results indicate at least 50% of hypothetical genes on chromosome 2 are expressed in the cDNA populations with about 38% of the gene structures differing from their predictions. Thus, by using this targeted approach, high-throughput RACE, we revealed numerous transcripts including many uncharacterized variants from these hypothetical genes.

Development and evaluation of an Arabidopsis whole genome Affymetrix probe array.

We describe the development of a high-density Arabidopsis'whole genome' oligonucleotide probe array for expression analysis (the Affymetrix ATH1 GeneChip probe array) that contains approximately 22 750 probe sets. Precedence on the array was given to genes for which either expression evidence or a credible database match existed. The remaining space was filled with 'hypothetical' genes. The new ATH1 array represents approximately 23 750 genes of which 60% were detected in RNA from cultured seedlings. Sensitivity of the array, determined using spiking controls, was approximately one transcript per cell. The array demonstrated high technical reproducibility and concordance with real-time PCR results. Indole-3 acetic acid (IAA)-induced changes in gene expression were used for biological validation of the array. A total of 222 genes were significantly upregulated and 103 significantly downregulated by exposure to IAA. Of the genes whose products could be functionally classified, the largest specific classes of upregulated genes were transcriptional regulators and protein kinases, many fewer of which were represented among the downregulated genes. Over one-third of the auxin-regulated genes have no known function, although many belong to gene families with members that have previously been shown to be auxin regulated. For the 6714 genes represented both on this and the earlier Arabidopsis Genome (AG) array, both signal intensities and gene expression ratios were very similar. Mapping of the oligonucleotides on the ATH1 array to the latest (version 4.0) annotation showed that over 95% of the probe sets (based on version 2.0 annotation) still fully represented their original target genes.