Genomic islands from five strains of Burkholderia pseudomallei

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia pseudomallei </it>is the etiologic agent of melioidosis, a significant cause of morbidity and mortality where this infection is endemic. Genomic differences among strains of <it>B. pseudomallei </it>are predicted to be one of the major causes of the diverse clinical manifestations observed among patients with melioidosis. The purpose of this study was to examine the role of genomic islands (GIs) as sources of genomic diversity in this species.</p> <p>Results</p> <p>We found that genomic islands (GIs) vary greatly among <it>B. pseudomallei </it>strains. We identified 71 distinct GIs from the genome sequences of five reference strains of <it>B. pseudomallei</it>: K96243, 1710b, 1106a, MSHR668, and MSHR305. The genomic positions of these GIs are not random, as many of them are associated with tRNA gene loci. In particular, the 3' end sequences of tRNA genes are predicted to be involved in the integration of GIs. We propose the term "tRNA-mediated site-specific recombination" (tRNA-SSR) for this mechanism. In addition, we provide a GI nomenclature that is based upon integration hotspots identified here or previously described.</p> <p>Conclusion</p> <p>Our data suggest that acquisition of GIs is one of the major sources of genomic diversity within <it>B. pseudomallei </it>and the molecular mechanisms that facilitate horizontally-acquired GIs are common across multiple strains of <it>B. pseudomallei</it>. The differential presence of the 71 GIs across multiple strains demonstrates the importance of these mobile elements for shaping the genetic composition of individual strains and populations within this bacterial species.</p

Identification of Melioidosis Outbreak by Multilocus Variable Number Tandem Repeat Analysis

One-sentence summary for table of contents: This analysis can identify a clonal outbreak of this disease within 8 hours of receipt of bacterial isolates

Phylogeographic reconstruction of a bacterial species with high levels of lateral gene transfer

Background Phylogeographic reconstruction of some bacterial populations is hindered by low diversity coupled with high levels of lateral gene transfer. A comparison of recombination levels and diversity at seven housekeeping genes for eleven bacterial species, most of which are commonly cited as having high levels of lateral gene transfer shows that the relative contributions of homologous recombination versus mutation for Burkholderia pseudomallei is over two times higher than for Streptococcus pneumoniae and is thus the highest value yet reported in bacteria. Despite the potential for homologous recombination to increase diversity, B. pseudomallei exhibits a relative lack of diversity at these loci. In these situations, whole genome genotyping of orthologous shared single nucleotide polymorphism loci, discovered using next generation sequencing technologies, can provide very large data sets capable of estimating core phylogenetic relationships. We compared and searched 43 whole genome sequences of B. pseudomallei and its closest relatives for single nucleotide polymorphisms in orthologous shared regions to use in phylogenetic reconstruction. Results Bayesian phylogenetic analyses of >14,000 single nucleotide polymorphisms yielded completely resolved trees for these 43 strains with high levels of statistical support. These results enable a better understanding of a separate analysis of population differentiation among >1,700 B. pseudomallei isolates as defined by sequence data from seven housekeeping genes. We analyzed this larger data set for population structure and allele sharing that can be attributed to lateral gene transfer. Our results suggest that despite an almost panmictic population, we can detect two distinct populations of B. pseudomallei that conform to biogeographic patterns found in many plant and animal species. That is, separation along Wallace's Line, a biogeographic boundary between Southeast Asia and Australia. Conclusion We describe an Australian origin for B. pseudomallei, characterized by a single introduction event into Southeast Asia during a recent glacial period, and variable levels of lateral gene transfer within populations. These patterns provide insights into mechanisms of genetic diversification in B. pseudomallei and its closest relatives, and provide a framework for integrating the traditionally separate fields of population genetics and phylogenetics for other bacterial species with high levels of lateral gene transfer

Within-Host Evolution of Burkholderia pseudomallei in Four Cases of Acute Melioidosis

Little is currently known about bacterial pathogen evolution and adaptation within the host during acute infection. Previous studies of Burkholderia pseudomallei, the etiologic agent of melioidosis, have shown that this opportunistic pathogen mutates rapidly both in vitro and in vivo at tandemly repeated loci, making this organism a relevant model for studying short-term evolution. In the current study, B. pseudomallei isolates cultured from multiple body sites from four Thai patients with disseminated melioidosis were subjected to fine-scale genotyping using multilocus variable-number tandem repeat analysis (MLVA). In order to understand and model the in vivo variable-number tandem repeat (VNTR) mutational process, we characterized the patterns and rates of mutations in vitro through parallel serial passage experiments of B. pseudomallei. Despite the short period of infection, substantial divergence from the putative founder genotype was observed in all four melioidosis cases. This study presents a paradigm for examining bacterial evolution over the short timescale of an acute infection. Further studies are required to determine whether the mutational process leads to phenotypic alterations that impact upon bacterial fitness in vivo. Our findings have important implications for future sampling strategies, since colonies in a single clinical sample may be genetically heterogeneous, and organisms in a culture taken late in the infective process may have undergone considerable genetic change compared with the founder inoculum

Novel Histone Demethylase Inhibitors Synergistically Enhance the Effects of a DNA Hypomethylating Agent in Breast Cancer Cells

DNA methylation and histone methylation function together in the epigenetic regulation of gene expression, but these processes can be altered in cancer. Recently, the H3K4 demethylase, KDM5B, was shown to be amplified and overexpressed in luminal breast cancer, making it an ideal target for chemotherapeutic intervention. In this study, we characterized the phenotypic and molecular effects of a novel group of KDM5 inhibitors, either alone or in combination with the DNA demethylating agent 5-Aza-2’-deoxycytidine (DAC), in luminal breast cancer cells. We found that KDM5 inhibitors and DAC synergistically inhibit cell proliferation and induce apoptosis relative to each drug alone. Additionally, microarray analysis indicated that combination treatment with KDM5 inhibitors and DAC resulted in the significant upregulation of hundreds of genes relative to DAC alone. Among these targets was an enrichment for genes in immunomodulatory pathways which are upregulated after exposure to the DNA demethylating agent 5-azacytidine. We then analyzed whole genome DNA methylation levels using the Infinium 450k microarray and, when compared to DAC treatment alone, found no additional loss of DNA methylation in the combination treatment. Instead, upregulaton of target genes appears to be mediated, at least in part, by a specific increase in H3K4 trimethylation levels at the at target promoters following exposure to the KDM5 inhibitors. Our results indicate that target genes are regulated by both DNA methylation and histone methylation in breast cancer cells. Upregulation of these genes via combined KDM5 inhibitor and DAC treatment leads to the synergistic inhibition of proliferation and may represent an exciting new application for epigenetic therapy in the treatment of breast cancer

Novel Histone Demethylase Inhibitors Synergistically Enhance the Effects of a DNA Hypomethylating Agent in Breast Cancer Cells

DNA methylation and histone methylation function together in the epigenetic regulation of gene expression, but these processes can be altered in cancer. Recently, the H3K4 demethylase, KDM5B, was shown to be amplified and overexpressed in luminal breast cancer, making it an ideal target for chemotherapeutic intervention. In this study, we characterized the phenotypic and molecular effects of a novel group of KDM5 inhibitors, either alone or in combination with the DNA demethylating agent 5-Aza-2’-deoxycytidine (DAC), in luminal breast cancer cells. We found that KDM5 inhibitors and DAC synergistically inhibit cell proliferation and induce apoptosis relative to each drug alone. Additionally, microarray analysis indicated that combination treatment with KDM5 inhibitors and DAC resulted in the significant upregulation of hundreds of genes relative to DAC alone. Among these targets was an enrichment for genes in immunomodulatory pathways which are upregulated after exposure to the DNA demethylating agent 5-azacytidine. We then analyzed whole genome DNA methylation levels using the Infinium 450k microarray and, when compared to DAC treatment alone, found no additional loss of DNA methylation in the combination treatment. Instead, upregulaton of target genes appears to be mediated, at least in part, by a specific increase in H3K4 trimethylation levels at the at target promoters following exposure to the KDM5 inhibitors. Our results indicate that target genes are regulated by both DNA methylation and histone methylation in breast cancer cells. Upregulation of these genes via combined KDM5 inhibitor and DAC treatment leads to the synergistic inhibition of proliferation and may represent an exciting new application for epigenetic therapy in the treatment of breast cancer

Fine-Scale Genetic Diversity among Burkholderia pseudomallei Soil Isolates in Northeast Thailand▿

Burkholderia pseudomallei soil isolates from northeast Thailand were genotyped using multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) and multilocus sequence typing (MLST). MLVA identified 19 genotypes within three clades, while MLST revealed two genotypes. These close genetic relationships imply a recent colonization followed by localized expansion, similar to what occurs in an outbreak situation