70 research outputs found
Comparative genomics and transcriptomics of Escherichia coli isolates carrying virulence factors of both enteropathogenic and enterotoxigenic E. coli
AbstractEscherichia coli that are capable of causing human disease are often classified into pathogenic variants (pathovars) based on their virulence gene content. However, disease-associated hybrid E. coli, containing unique combinations of multiple canonical virulence factors have also been described. Such was the case of the E. coli O104:H4 outbreak in 2011, which caused significant morbidity and mortality. Among the pathovars of diarrheagenic E. coli that cause significant human disease are the enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC). In the current study we use comparative genomics, transcriptomics, and functional studies to characterize isolates that contain virulence factors of both EPEC and ETEC. Based on phylogenomic analysis, these hybrid isolates are more genomically-related to EPEC, but appear to have acquired ETEC virulence genes. Global transcriptional analysis using RNA sequencing, demonstrated that the EPEC and ETEC virulence genes of these hybrid isolates were differentially-expressed under virulence-inducing laboratory conditions, similar to reference isolates. Immunoblot assays further verified that the virulence gene products were produced and that the T3SS effector EspB of EPEC, and heat-labile toxin of ETEC were secreted. These findings document the existence and virulence potential of an E. coli pathovar hybrid that blurs the distinction between E. coli pathovars.</jats:p
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Cost effective, experimentally robust differential-expression analysis for human/mammalian, pathogen and dual-species transcriptomics.
As sequencing read length has increased, researchers have quickly adopted longer reads for their experiments. Here, we examine 14 pathogen or host-pathogen differential gene expression data sets to assess whether using longer reads is warranted. A variety of data sets was used to assess what genomic attributes might affect the outcome of differential gene expression analysis including: gene density, operons, gene length, number of introns/exons and intron length. No genome attribute was found to influence the data in principal components analysis, hierarchical clustering with bootstrap support, or regression analyses of pairwise comparisons that were undertaken on the same reads, looking at all combinations of paired and unpaired reads trimmed to 36, 54, 72 and 101 bp. Read pairing had the greatest effect when there was little variation in the samples from different conditions or in their replicates (e.g. little differential gene expression). But overall, 54 and 72 bp reads were typically most similar. Given differences in costs and mapping percentages, we recommend 54 bp reads for organisms with no or few introns and 72 bp reads for all others. In a third of the data sets, read pairing had absolutely no effect, despite paired reads having twice as much data. Therefore, single-end reads seem robust for differential-expression analyses, but in eukaryotes paired-end reads are likely desired to analyse splice variants and should be preferred for data sets that are acquired with the intent to be community resources that might be used in secondary data analyses
Candida albicans quorum-sensing molecule farnesol modulates staphyloxanthin production and activates the thiol-based oxidative-stress response in Staphylococcus aureus
Microbial species utilize secreted-signaling molecules to coordinate their behavior. Our previous investigations demonstrated a key role for the Candida albicans-secreted quorum-sensing molecule farnesol in modulating Staphylococcus aureus response to antimicrobials in mixed biofilms. In this study, we aimed to provide mechanistic insights into the impact of farnesol on S. aureus within the context of inter-species interactions. To mimic biofilm dynamics, farnesol-sensitized S. aureus cells were generated via sequential farnesol exposure. The sensitized phenotype exhibited dramatic loss of the typical pigment, which we identified as staphyloxanthin, an important virulence factor synthesized by the Crt operon in S. aureus. Additionally, farnesol exposure exerted oxidative-stress as indicated by transcriptional analysis demonstrating alterations in redox-sensors and major virulence regulators. Paradoxically, the activated stress-response conferred S. aureus with enhanced tolerance to H2O2 and phagocytic killing. Since expression of enzymes in the staphyloxanthin biosynthesis pathway was not impacted by farnesol, we generated a theoretical-binding model which indicated that farnesol may block staphyloxanthin biosynthesis via competitive-binding to the CrtM enzyme crucial for staphyloxanthin synthesis, due to high structural similarity to the CrtM substrate. Finally, mixed growth with C. albicans was found to similarly induce S. aureus depigmentation, but not during growth with a farnesol-deficient C. albicans strain. Collectively, the findings demonstrate that a fungal molecule acts as a redox-cycler eliciting a bacterial stress response via activation of the thiol-based redox system under the control of global regulators. Therefore, farnesol-induced transcriptional modulations of key regulatory networks in S. aureus may modulate the pathogenesis of C. albicans- S. aureus co-infections
Genome-wide diversity and gene expression profiling of Babesia microti isolates identify polymorphic genes that mediate host-pathogen interactions
Babesia microti, a tick-transmitted, intraerythrocytic protozoan parasite circulating mainly among small mammals, is the primary cause of human babesiosis. While most cases are transmitted by Ixodes ticks, the disease may also be transmitted through blood transfusion and perinatally. A comprehensive analysis of genome composition, genetic diversity, and gene expression profiling of seven B. microti isolates revealed that genetic variation in isolates from the Northeast United States is almost exclusively associated with genes encoding the surface proteome and secretome of the parasite. Furthermore, we found that polymorphism is restricted to a small number of genes, which are highly expressed during infection. In order to identify pathogen-encoded factors involved in host-parasite interactions, we screened a proteome array comprised of 174 B. microti proteins, including several predicted members of the parasite secretome. Using this immuno-proteomic approach we identified several novel antigens that trigger strong host immune responses during the onset of infection. The genomic and immunological data presented herein provide the first insights into the determinants of B. microti interaction with its mammalian hosts and their relevance for understanding the selective pressures acting on parasite evolution
Rapid transcriptome sequencing of an invasive pest, the brown marmorated stink bug Halyomorpha halys
Halyomorpha halys (Stål) (Insecta:Hemiptera;Pentatomidae), commonly known as the Brown Marmorated Stink Bug (BMSB), is an invasive pest of the mid-Atlantic region of the United States, causing economically important damage to a wide range of crops. Native to Asia, BMSB was first observed in Allentown, PA, USA, in 1996, and this pest is now well-established throughout the US mid-Atlantic region and beyond. In addition to the serious threat BMSB poses to agriculture, BMSB has become a nuisance to homeowners, invading home gardens and congregating in large numbers in human-made structures, including homes, to overwinter. Despite its significance as an agricultural pest with limited control options, only 100 bp of BMSB sequence data was available in public databases when this project began. Transcriptome sequencing was undertaken to provide a molecular resource to the research community to inform the development of pest control strategies and to provide molecular data for population genetics studies of BMSB. Using normalized, strand-specific libraries, we sequenced pools of all BMSB life stages on the Illumina HiSeq. Trinity was used to assemble 200,000 putative transcripts in >100,000 components. A novel bioinformatic method that analyzed the strand-specificity of the data reduced this to 53,071 putative transcripts from 18,573 components. By integrating multiple other data types, we narrowed this further to 13,211 representative transcripts. Bacterial endosymbiont genes were identified in this dataset, some of which have a copy number consistent with being lateral gene transfers between endosymbiont genomes and Hemiptera, including ankyrin-repeat related proteins, lysozyme, and mannanase. Such genes and endosymbionts may provide novel targets for BMSB-specific biocontrol. This study demonstrates the utility of strand-specific sequencing in generating shotgun transcriptomes and that rapid sequencing shotgun transcriptomes is possible without the need for extensive inbreeding to generate homozygous lines. Such sequencing can provide a rapid response to pest invasions similar to that already described for disease epidemiology.https://doi.org/10.1186/1471-2164-15-73
A secondary mechanism of action for triazole antifungals in Aspergillus fumigatus mediated by hmg1
Triazole antifungals function as ergosterol biosynthesis inhibitors and are frontline therapy for invasive fungal infections, such as invasive aspergillosis. The primary mechanism of action of triazoles is through the specific inhibition of a cytochrome P450 14-α-sterol demethylase enzyme, Cyp51A/B, resulting in depletion of cellular ergosterol. Here, we uncover a clinically relevant secondary mechanism of action for triazoles within the ergosterol biosynthesis pathway. We provide evidence that triazole-mediated inhibition of Cyp51A/B activity generates sterol intermediate perturbations that are likely decoded by the sterol sensing functions of HMG-CoA reductase and Insulin-Induced Gene orthologs as increased pathway activity. This, in turn, results in negative feedback regulation of HMG-CoA reductase, the rate-limiting step of sterol biosynthesis. We also provide evidence that HMG-CoA reductase sterol sensing domain mutations previously identified as generating resistance in clinical isolates of Aspergillus fumigatus partially disrupt this triazole-induced feedback. Therefore, our data point to a secondary mechanism of action for the triazoles: induction of HMG-CoA reductase negative feedback for downregulation of ergosterol biosynthesis pathway activity. Abrogation of this feedback through acquired mutations in the HMG-CoA reductase sterol sensing domain diminishes triazole antifungal activity against fungal pathogens and underpins HMG-CoA reductase-mediated resistance
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