Pharmacogenetics of Between-Individual Variability in Plasma Clearance of Bedaquiline and Clofazimine in South Africa.

BACKGROUND: Plasma bedaquiline clearance is reportedly more rapid with African ancestry. Our objective was to determine whether genetic polymorphisms explained between-individual variability in plasma clearance of bedaquiline, its M2 metabolite, and clofazimine in a cohort of patients treated for drug-resistant tuberculosis in South Africa. METHODS: Plasma clearance was estimated with nonlinear mixed-effects modeling. Associations between pharmacogenetic polymorphisms, genome-wide polymorphisms, and variability in clearance were examined using linear regression models. RESULTS: Of 195 cohort participants, 140 were evaluable for genetic associations. Among 21 polymorphisms selected based on prior genome-wide significant associations with any drug, rs776746 (CYP3A5∗3) was associated with slower clearance of bedaquiline (P = .0017) but not M2 (P = .25). CYP3A5∗3 heterozygosity and homozygosity were associated with 15% and 30% slower bedaquiline clearance, respectively. The lowest P value for clofazimine clearance was with VKORC1 rs9923231 (P = .13). In genome-wide analyses, the lowest P values for clearance of bedaquiline and clofazimine were with RFX4 rs76345012 (P = 6.4 × 10-7) and CNTN5 rs75285763 (P = 2.9 × 10-8), respectively. CONCLUSIONS: Among South Africans treated for drug-resistant tuberculosis, CYP3A5∗3 was associated with slower bedaquiline clearance. Different CYP3A5∗3 frequencies among populations may help explain the more rapid bedaquiline clearance reported in Africans. Associations with RFX4 and CNTN5 are likely by chance alone.

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom.

The ecological prominence of diatoms in the ocean environment largely results from their superior competitive ability for dissolved nitrate (NO3-). To investigate the cellular and genetic basis of diatom NO3- assimilation, we generated a knockout in the nitrate reductase gene (NR-KO) of the model pennate diatom Phaeodactylum tricornutum In NR-KO cells, N-assimilation was abolished although NO3- transport remained intact. Unassimilated NO3- accumulated in NR-KO cells, resulting in swelling and associated changes in biochemical composition and physiology. Elevated expression of genes encoding putative vacuolar NO3- chloride channel transporters plus electron micrographs indicating enlarged vacuoles suggested vacuolar storage of NO3- Triacylglycerol concentrations in the NR-KO cells increased immediately following the addition of NO3-, and these increases coincided with elevated gene expression of key triacylglycerol biosynthesis components. Simultaneously, induction of transcripts encoding proteins involved in thylakoid membrane lipid recycling suggested more abrupt repartitioning of carbon resources in NR-KO cells compared with the wild type. Conversely, ribosomal structure and photosystem genes were immediately deactivated in NR-KO cells following NO3- addition, followed within hours by deactivation of genes encoding enzymes for chlorophyll biosynthesis and carbon fixation and metabolism. N-assimilation pathway genes respond uniquely, apparently induced simultaneously by both NO3- replete and deplete conditions.

Mapping gastrointestinal gene expression patterns in wild primates and humans via fecal RNA-seq.

BACKGROUND: Limited accessibility to intestinal epithelial tissue in wild animals and humans makes it challenging to study patterns of intestinal gene regulation, and hence to monitor physiological status and health in field conditions. To explore solutions to this limitation, we have used a noninvasive approach via fecal RNA-seq, for the quantification of gene expression markers in gastrointestinal cells of free-range primates and a forager human population. Thus, a combination of poly(A) mRNA enrichment and rRNA depletion methods was used in tandem with RNA-seq to quantify and compare gastrointestinal gene expression patterns in fecal samples of wild Gorilla gorilla gorilla (n = 9) and BaAka hunter-gatherers (n = 10) from The Dzanga Sangha Protected Areas, Central African Republic. RESULTS: Although only a small fraction (< 4.9%) of intestinal mRNA signals was recovered, the data was sufficient to detect significant functional differences between gorillas and humans, at the gene and pathway levels. These intestinal gene expression differences were specifically associated with metabolic and immune functions. Additionally, non-host RNA-seq reads were used to gain preliminary insights on the subjects' dietary habits, intestinal microbiomes, and infection prevalence, via identification of fungi, nematode, arthropod and plant RNA. CONCLUSIONS: Overall, the results suggest that fecal RNA-seq, targeting gastrointestinal epithelial cells can be used to evaluate primate intestinal physiology and gut gene regulation, in samples obtained in challenging conditions in situ. The approach used herein may be useful to obtain information on primate intestinal health, while revealing preliminary insights into foraging ecology, microbiome, and diet.

Assessment of Insertion Sequence Mobilization as an Adaptive Response to Oxidative Stress in Acinetobacter baumannii Using IS-seq.

Insertion sequence (IS) elements are found throughout bacterial genomes and contribute to genome variation by interrupting genes or altering gene expression. Few of the more than 30 IS elements described in Acinetobacter baumannii have been characterized for transposition activity or expression effects. A targeted sequencing method, IS-seq, was developed to efficiently map the locations of new insertion events in A. baumannii genomes and was used to identify novel IS sites following growth in the presence of hydrogen peroxide, which causes oxidative stress. Serial subculture in the presence of subinhibitory concentrations of hydrogen peroxide led to rapid selection of cells carrying an ISAba1 element upstream of the catalase-peroxidase gene katG Several additional sites for the elements ISAba1, ISAba13, ISAba25, ISAba26, and ISAba125 were found at low abundance after serial subculture, indicating that each element is active and contributes to genetic variation that may be subject to selection. Following hydrogen peroxide exposure, rapid changes in gene expression were observed in genes related to iron homeostasis. The IS insertions adjacent to katG resulted in more than 20-fold overexpression of the gene and increased hydrogen peroxide tolerance.IMPORTANCE Insertion sequences (IS) contribute to genomic and phenotypic variation in many bacterial species, but little is known about how transposition rates vary among elements or how selective pressure influences this process. A new method for identifying new insertion locations that arise under experimental growth conditions in the genome, termed IS-seq, was developed and tested with cells grown in the presence of hydrogen peroxide, which causes oxidative stress. Gene expression changes in response to hydrogen peroxide exposure are similar to those observed in other species and include genes that control free iron concentrations. New IS insertions adjacent to a gene encoding a catalase enzyme confirm that IS elements can rapidly contribute to adaptive variation in the presence of selection.

Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis.

Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle.

Prophage induction is enhanced and required for renal disease and lethality in an EHEC mouse model.

Enterohemorrhagic Escherichia coli (EHEC), particularly serotype O157:H7, causes hemorrhagic colitis, hemolytic uremic syndrome, and even death. In vitro studies showed that Shiga toxin 2 (Stx2), the primary virulence factor expressed by EDL933 (an O157:H7 strain), is encoded by the 933W prophage. And the bacterial subpopulation in which the 933W prophage is induced is the producer of Stx2. Using the germ-free mouse, we show the essential role 933W induction plays in the virulence of EDL933 infection. An EDL933 derivative with a single mutation in its 933W prophage, resulting specifically in that phage being uninducible, colonizes the intestines, but fails to cause any of the pathological changes seen with the parent strain. Hence, induction of the 933W prophage is the primary event leading to disease from EDL933 infection. We constructed a derivative of EDL933, SIVET, with a biosensor that specifically measures induction of the 933W prophage. Using this biosensor to measure 933W induction in germ-free mice, we found an increase three logs greater than was expected from in vitro results. Since the induced population produces and releases Stx2, this result indicates that an activity in the intestine increases Stx2 production.

Validating a Promoter Library for Application in Plasmid-Based Diatom Genetic Engineering.

While diatoms are promising synthetic biology platforms, there currently exists a limited number of validated genetic regulatory parts available for genetic engineering. The standard method for diatom transformation, nonspecific introduction of DNA into chromosomes via biolistic particle bombardment, is low throughput and suffers from clonal variability and epigenetic effects. Recent developments in diatom engineering have demonstrated that autonomously replicating episomal plasmids serve as stable expression platforms for diverse gene expression technologies. These plasmids are delivered via bacterial conjugation and, when combined with modular DNA assembly technologies, provide a flexibility and speed not possible with biolistic-mediated strain generation. In order to expand the current toolbox for plasmid-based engineering in the diatom Phaeodactylum tricornutum, a conjugation-based forward genetics screen for promoter discovery was developed, and application to a diatom genomic DNA library defined 252 P. tricornutum promoter elements. From this library, 40 promoter/terminator pairs were delivered via conjugation on episomal plasmids, characterized in vivo, and ranked across 4 orders of magnitude difference in reporter gene expression levels.

Synthesis of lipoteichoic acids in Bacillus anthracis.

Lipoteichoic acid (LTA), a glycerol phosphate polymer, is a component of the envelope of Gram-positive bacteria that has hitherto not been identified in Bacillus anthracis, the causative agent of anthrax. LTA synthesis in Staphylococcus aureus and other microbes is catalyzed by the product of the ltaS gene, a membrane protein that polymerizes polyglycerol phosphate from phosphatidyl glycerol. Here we identified four ltaS homologues, designated ltaS1 to -4, in the genome of Bacillus anthracis. Polyglycerol phosphate-specific monoclonal antibodies were used to detect LTA in the envelope of B. anthracis strain Sterne (pXO1(+) pXO2(-)) vegetative forms. B. anthracis mutants lacking ltaS1, ltaS2, ltaS3, or ltaS4 did not display defects in growth or LTA synthesis. In contrast, B. anthracis strains lacking both ltaS1 and ltaS2 were unable to synthesize LTA and exhibited reduced viability, altered envelope morphology, aberrant separation of vegetative forms, and decreased sporulation efficiency. Expression of ltaS1 or ltaS2 alone in B. anthracis as well as in other microbes was sufficient for polyglycerol phosphate synthesis. Thus, similar to S. aureus, B. anthracis employs LtaS enzymes to synthesize LTA, an envelope component that promotes bacterial growth and cell division.

Activation of a prophage-encoded tyrosine kinase by a heterologous infecting phage results in a self-inflicted abortive infection.

Bacteria in their struggle for survival have evolved or acquired defences against attacking phage. However, phage often contribute to this defence through mechanisms in which a prophage protects the bacterial population from attack by another, often unrelated, phage. The 933W prophage, which carries Shiga toxin genes that enhance pathogenicity of enterohaemorrhagic Escherichia coli strain O157:H7, also carries the stk gene encoding a eukaryotic-like tyrosine kinase that excludes (aborts) infection by phage HK97. This exclusion requires the kinase activity of Stk. Little, if any, protein tyrosine phosphorylation can be detected in a 933W lysogen prior to infection with HK97, while extensive Stk-mediated tyrosine phosphorylation is evident following infection. This includes autophosphorylation that stabilizes Stk protein from degradation. Although increased levels of Stk are found following HK97 infection, these higher levels are not necessary or sufficient for exclusion or protein phosphorylation. An HK97 open reading frame, orf41, is necessary for exclusion and Stk kinase activity. We hypothesize that interaction with gp41 stimulates Stk kinase activity. Exclusion of HK97 appears to be specific since other phages tested, λ, φ80, H-19B, λ-P22dis and T4rII, were not excluded. Infection of the 933W lysogen with a non-excluded phage fails to induce Stk-determined phosphorylation.

Dinoflagellate-cyanobacterium communication may determine the composition of phytoplankton assemblage in a mesotrophic lake.

The reasons for annual variability in the composition of phytoplankton assemblages are poorly understood but may include competition for resources and allelopathic interactions. We show that domination by the patch-forming dinoflagellate, Peridinium gatunense, or, alternatively, a bloom of a toxic cyanobacterium, Microcystis sp., in the Sea of Galilee may be accounted for by mutual density-dependent allelopathic interactions. Over the last 11 years, the abundance of these species in the lake displayed strong negative correlation. Laboratory experiments showed reciprocal, density-dependent, but nutrient-independent, inhibition of growth. Application of spent P. gatunense medium induced sedimentation and, subsequently, massive lysis of Microcystis cells within 24 hr, and sedimentation and lysis were concomitant with a large rise in the level of McyB, which is involved in toxin biosynthesis by Microcystis. P. gatunense responded to the presence of Microcystis by a species-specific pathway that involved a biphasic oxidative burst and activation of certain protein kinases. Blocking this recognition by MAP-kinase inhibitors abolished the biphasic oxidative burst and affected the fate (death or cell division) of the P. gatunense cells. We propose that patchy growth habits may confer enhanced defense capabilities, providing ecological advantages that compensate for the aggravated limitation of resources in the patch. Cross-talk via allelochemicals may explain the phytoplankton assemblage in the Sea of Galilee.

Initial genome sequencing of the sugarcane CP 96-1252 complex hybrid.

The CP 96-1252 cultivar of sugarcane is a complex hybrid of commercial importance. DNA was extracted from lab-grown leaf tissue and sequenced. The raw Illumina DNA sequencing results provide 101 Gbp of genome sequence reads. The dataset is available from https://www.ncbi.nlm.nih.gov/bioproject/PRJNA345486/.

Intestinal fungi contribute to development of alcoholic liver disease.

Chronic liver disease with cirrhosis is the 12th leading cause of death in the United States, and alcoholic liver disease accounts for approximately half of all cirrhosis deaths. Chronic alcohol consumption is associated with intestinal bacterial dysbiosis, yet we understand little about the contribution of intestinal fungi, or mycobiota, to alcoholic liver disease. Here we have demonstrated that chronic alcohol administration increases mycobiota populations and translocation of fungal ß-glucan into systemic circulation in mice. Treating mice with antifungal agents reduced intestinal fungal overgrowth, decreased ß-glucan translocation, and ameliorated ethanol-induced liver disease. Using bone marrow chimeric mice, we found that ß-glucan induces liver inflammation via the C-type lectin-like receptor CLEC7A on Kupffer cells and possibly other bone marrow-derived cells. Subsequent increases in IL-1ß expression and secretion contributed to hepatocyte damage and promoted development of ethanol-induced liver disease. We observed that alcohol-dependent patients displayed reduced intestinal fungal diversity and Candida overgrowth. Compared with healthy individuals and patients with non-alcohol-related cirrhosis, alcoholic cirrhosis patients had increased systemic exposure and immune response to mycobiota. Moreover, the levels of extraintestinal exposure and immune response correlated with mortality. Thus, chronic alcohol consumption is associated with an altered mycobiota and translocation of fungal products. Manipulating the intestinal mycobiome might be an effective strategy for attenuating alcohol-related liver disease.

Genome-Scale Model Reveals Metabolic Basis of Biomass Partitioning in a Model Diatom.

Diatoms are eukaryotic microalgae that contain genes from various sources, including bacteria and the secondary endosymbiotic host. Due to this unique combination of genes, diatoms are taxonomically and functionally distinct from other algae and vascular plants and confer novel metabolic capabilities. Based on the genome annotation, we performed a genome-scale metabolic network reconstruction for the marine diatom Phaeodactylum tricornutum. Due to their endosymbiotic origin, diatoms possess a complex chloroplast structure which complicates the prediction of subcellular protein localization. Based on previous work we implemented a pipeline that exploits a series of bioinformatics tools to predict protein localization. The manually curated reconstructed metabolic network iLB1027_lipid accounts for 1,027 genes associated with 4,456 reactions and 2,172 metabolites distributed across six compartments. To constrain the genome-scale model, we determined the organism specific biomass composition in terms of lipids, carbohydrates, and proteins using Fourier transform infrared spectrometry. Our simulations indicate the presence of a yet unknown glutamine-ornithine shunt that could be used to transfer reducing equivalents generated by photosynthesis to the mitochondria. The model reflects the known biochemical composition of P. tricornutum in defined culture conditions and enables metabolic engineering strategies to improve the use of P. tricornutum for biotechnological applications.

A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses.

Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories.

Designer diatom episomes delivered by bacterial conjugation.

Eukaryotic microalgae hold great promise for the bioproduction of fuels and higher value chemicals. However, compared with model genetic organisms such as Escherichia coli and Saccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement has been hampered by the inefficient genetic tools. To date, many algal species are transformable only via particle bombardment, and the introduced DNA is integrated randomly into the nuclear genome. Here we describe the first nuclear episomal vector for diatoms and a plasmid delivery method via conjugation from Escherichia coli to the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. We identify a yeast-derived sequence that enables stable episome replication in these diatoms even in the absence of antibiotic selection and show that episomes are maintained as closed circles at copy number equivalent to native chromosomes. This highly efficient genetic system facilitates high-throughput functional characterization of algal genes and accelerates molecular phytoplankton research.

A putative sensor kinase, Hik31, is involved in the response of Synechocystis sp. strain PCC 6803 to the presence of glucose.

The reason(s) for glucose sensitivity in certain cyanobacterial strains is poorly understood. Inactivation of genes encoding the putative sensor kinase Hik31 in Synechocystis sp. strain PCC 6803 resulted in a mutant unable to grow in the presence of D-glucose. Sensitivities to D-glucose, its analogue 2-deoxy-D-glucose, and fructose, were alleviated in mutants in which glcP, encoding the glucose transporter, was inactivated. These data indicate that permeation of these substrates is required to inflict cell death. The mutant Deltahik31, and the glucose-sensitive strain of Synechocystis, do not possess glucokinase activity, although a transcript originating from glk, encoding glucokinase, is present. Inactivation of glk led to severe sensitivity to glucose, indicating that the presence of glucose itself, within the cells, inflicted this sensitivity. On the other hand, sensitivity to 2-deoxy-D-glucose was lower in Deltaglk, thus distinguishing between the effect of glucose itself and that of its analogue, which, in the absence of glucokinase activity, may not be phosphorylated. Addition of glucose led to a small rise in glucose-6-phosphate dehydrogenase activity in the wild type, but constitutive activity was observed in the Deltahik31 mutant regardless of the presence of glucose. Microarray analyses showed only small changes in the abundance of global transcripts in Synechocystis following glucose addition, but the transcription levels of several genes, including icfG, but not glk, were strongly affected by inactivation of hik31. The mechanism(s) whereby Hik31 is involved in glucose sensing and response is discussed.