scholarly journals Dolosigranulum pigrum cooperation and competition in human nasal microbiota

2019 ◽  
Author(s):  
Silvio D. Brugger ◽  
Sara M. Eslami ◽  
Melinda M. Pettigrew ◽  
Isabel F. Escapa ◽  
Matthew T. Henke ◽  
...  
Keyword(s):  
Lactic Acid Production ◽  
Genomic Analysis ◽  
Positive Association ◽  
Gene Clusters ◽  
Upper Respiratory Tract ◽  
Microbe Interactions ◽  
Clinical Experiments ◽  
Nasal Microbiota

AbstractBackgroundMultiple epidemiological studies identify Dolosigranulum pigrum as a candidate beneficial bacterium based on its positive association with health, including negative associations with nasal/nasopharyngeal colonization by the pathogenic species Staphylococcus aureus and Streptococcus pneumoniae.ResultsUsing a multipronged approach to gain new insights into D. pigrum function, we observed phenotypic interactions and predictions of genomic capacity that support a role for microbe-microbe interactions involving D. pigrum in shaping the composition of human nasal microbiota. We identified in vivo community-level and in vitro phenotypic cooperation by specific nasal Corynebacterium species. Also, D. pigrum inhibited S. aureus growth in vitro. Whereas, robust inhibition of S. pneumoniae required both D. pigrum and a nasal Corynebacterium together, and not either alone. D. pigrum L-lactic-acid production was insufficient to account for these inhibitions. Genomic analysis of 11 strains revealed that D. pigrum has a small genome (average 1.86 Mb) and multiple predicted auxotrophies consistent with D. pigrum relying on its human host and cocolonizing bacteria for key nutrients. Further, the accessory genome of D. pigrum encoded a diverse repertoire of biosynthetic gene clusters, some of which may have a role in microbe-microbe interactions.ConclusionsThese new insights into D. pigrum’s functions advance the field from compositional analysis to genomic and phenotypic experimentation on a potentially beneficial bacterial resident of the human upper respiratory tract and lay the foundation for future animal and clinical experiments.

scholarly journals Dolosigranulum pigrum Cooperation and Competition in Human Nasal Microbiota

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Silvio D. Brugger ◽  
Sara M. Eslami ◽  
Melinda M. Pettigrew ◽  
Isabel F. Escapa ◽  
Matthew T. Henke ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Streptococcus Pneumoniae ◽  
Genomic Analysis ◽  
Upper Respiratory Tract ◽  
Content Type ◽  
Microbe Interactions ◽  
Clinical Experiments ◽  
Nasal Microbiota

ABSTRACT Multiple epidemiological studies identify Dolosigranulum pigrum as a candidate beneficial bacterium based on its positive association with health, including negative associations with nasal/nasopharyngeal colonization by the pathogenic species Staphylococcus aureus and Streptococcus pneumoniae. Using a multipronged approach to gain new insights into D. pigrum function, we observed phenotypic interactions and predictions of genomic capacity that support the idea of a role for microbe-microbe interactions involving D. pigrum in shaping the composition of human nasal microbiota. We identified in vivo community-level and in vitro phenotypic cooperation by specific nasal Corynebacterium species. Also, D. pigrum inhibited S. aureus growth in vitro, whereas robust inhibition of S. pneumoniae required both D. pigrum and a nasal Corynebacterium together. D. pigrum l-lactic acid production was insufficient to account for these inhibitions. Genomic analysis of 11 strains revealed that D. pigrum has a small genome (average 1.86 Mb) and multiple predicted auxotrophies consistent with D. pigrum relying on its human host and on cocolonizing bacteria for key nutrients. Further, the accessory genome of D. pigrum harbored a diverse repertoire of biosynthetic gene clusters, some of which may have a role in microbe-microbe interactions. These new insights into D. pigrum’s functions advance the field from compositional analysis to genomic and phenotypic experimentation on a potentially beneficial bacterial resident of the human upper respiratory tract and lay the foundation for future animal and clinical experiments. IMPORTANCE Staphylococcus aureus and Streptococcus pneumoniae infections cause significant morbidity and mortality in humans. For both, nasal colonization is a risk factor for infection. Studies of nasal microbiota identify Dolosigranulum pigrum as a benign bacterium present when adults are free of S. aureus or when children are free of S. pneumoniae. Here, we validated these in vivo associations with functional assays. We found that D. pigrum inhibited S. aureus in vitro and, together with a specific nasal Corynebacterium species, also inhibited S. pneumoniae. Furthermore, genomic analysis of D. pigrum indicated that it must obtain key nutrients from other nasal bacteria or from humans. These phenotypic interactions support the idea of a role for microbe-microbe interactions in shaping the composition of human nasal microbiota and implicate D. pigrum as a mutualist of humans. These findings support the feasibility of future development of microbe-targeted interventions to reshape nasal microbiota composition to exclude S. aureus and/or S. pneumoniae.

scholarly journals Dual and Triple Epithelial Coculture Model Systems with Donor-Derived Microbiota and THP-1 Macrophages To Mimic Host-Microbe Interactions in the Human Sinonasal Cavities

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Charlotte De Rudder ◽  
Marta Calatayud Arroyo ◽  
Sarah Lebeer ◽  
Tom Van de Wiele
Keyword(s):  
Microbial Community ◽  
Epithelial Cells ◽  
Respiratory Tract ◽  
Model Systems ◽  
Upper Respiratory Tract ◽  
Microbe Interactions ◽  
Upper Respiratory ◽  
Host Microbe Interactions ◽  
Nasal Microbiota

ABSTRACT The epithelium of the human sinonasal cavities is colonized by a diverse microbial community, modulating epithelial development and immune priming and playing a role in respiratory disease. Here, we present a novel in vitro approach enabling a 3-day coculture of differentiated Calu-3 respiratory epithelial cells with a donor-derived bacterial community, a commensal species (Lactobacillus sakei), or a pathobiont (Staphylococcus aureus). We also assessed how the incorporation of macrophage-like cells could have a steering effect on both epithelial cells and the microbial community. Inoculation of donor-derived microbiota in our experimental setup did not pose cytotoxic stress on the epithelial cell layers, as demonstrated by unaltered cytokine and lactate dehydrogenase release compared to a sterile control. Epithelial integrity of the differentiated Calu-3 cells was maintained as well, with no differences in transepithelial electrical resistance observed between coculture with donor-derived microbiota and a sterile control. Transition of nasal microbiota from in vivo to in vitro conditions maintained phylogenetic richness, and yet a decrease in phylogenetic and phenotypic diversity was noted. Additional inclusion and coculture of THP-1-derived macrophages did not alter phylogenetic diversity, and yet donor-independent shifts toward higher Moraxella and Mycoplasma abundance were observed, while phenotypic diversity was also increased. Our results demonstrate that coculture of differentiated airway epithelial cells with a healthy donor-derived nasal community is a viable strategy to mimic host-microbe interactions in the human upper respiratory tract. Importantly, including an immune component allowed us to study host-microbe interactions in the upper respiratory tract more in depth. IMPORTANCE Despite the relevance of the resident microbiota in sinonasal health and disease and the need for cross talk between immune and epithelial cells in the upper respiratory tract, these parameters have not been combined in a single in vitro model system. We have developed a coculture system of differentiated respiratory epithelium and natural nasal microbiota and incorporated an immune component. As indicated by absence of cytotoxicity and stable cytokine profiles and epithelial integrity, nasal microbiota from human origin appeared to be well tolerated by host cells, while microbial community composition remained representative for that of the human (sino)nasal cavity. Importantly, the introduction of macrophage-like cells enabled us to obtain a differential readout from the epithelial cells dependent on the donor microbial background to which the cells were exposed. We conclude that both model systems offer the means to investigate host-microbe interactions in the upper respiratory tract in a more representative way.

scholarly journals T1000: A reduced toxicogenomics gene set for improved decision making

2019 ◽  
Author(s):  
Othman Soufan ◽  
Jessica Ewald ◽  
Charles Viau ◽  
Doug Crump ◽  
Markus Hecker ◽  
...  
Keyword(s):  
Dose Response ◽  
Gene Clusters ◽  
Gene Set ◽  
Chemical Hazards ◽  
Gene Sets ◽  
Toxicological Research ◽  
Multiple Species ◽  
Response Modeling

There is growing interest within regulatory agencies and toxicological research communities to develop, test, and apply new approaches, such as toxicogenomics, to more efficiently evaluate chemical hazards. Given the complexity of analyzing thousands of genes simultaneously, there is a need to identify reduced gene sets.Though several gene sets have been defined for toxicological applications, few of these were purposefully derived using toxicogenomics data. Here, we developed and applied a systematic approach to identify 1000 genes (called Toxicogenomics-1000 or T1000) highly responsive to chemical exposures. First, a co-expression network of 11,210genes was built by leveraging microarray data from the Open TG-GATEs program. This network was then re-weighted based on prior knowledge of their biological (KEGG, MSigDB) and toxicological (CTD) relevance. Finally, weighted correlation network analysis was applied to identify 258 gene clusters. T1000 was defined by selecting genes from each cluster that were most associated with outcome measures. For model evaluation, we compared the performance of T1000 to that of other gene sets (L1000, S1500, Genes selected by Limma, and random set) using two external datasets. Additionally, a smaller (T384) and a larger version (T1500) of T1000 were used for dose-response modeling to test the effect of gene set size. Our findings demonstrated that the T1000 gene set is predictive of apical outcomes across a range of conditions (e.g.,in vitroand in vivo, dose-response, multiple species, tissues, and chemicals), and generally performs as well, or better than other gene sets available.

Abstract 14532: Computational Drug Repurposing Identifies a Piperlongumine Analog That Controls a Gstp1-iscu Pathogenic Axis in Pulmonary Hypertension

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Jimin Yang
Keyword(s):  
Drug Repurposing ◽  
Gene Clusters ◽  
Loss Of Function ◽  
Sequencing Data ◽  
Glutathione S Transferase ◽  
Computational Screening ◽  
Therapeutic Development ◽  
Pulmonary Arterial

Background and Hypothesis: Pulmonary arterial hypertension (PAH) is an incurable vascular disease for which chemotherapies are being considered for therapeutic development. There is no method reported to date for effective computational screening of these drugs for this disease. Big data analyses that leverage the molecular parallels between cancer and PH may define novel pathogenic mechanisms and facilitate repurposing of chemotherapies for PAH. More specifically, while functional deficiency of the iron-sulfur (Fe-S) biogenesis gene ISCU and oxidative metabolism in human pulmonary arterial endothelial cells (PAECs) is known to drive PAH, the pathogenic regulation of ISCU is not fully defined, and no tailored drugs have been identified to bolster ISCU activity. Methods and Results: We applied a computational algorithm EDDY (Evaluating Differential DependencY), which analyzes RNA sequencing data from 810 cancer cell lines exposed to 368 small molecules, in order to identify chemotherapeutics that depended upon rewired PH-related gene clusters. The top ranked drug was a piperlongumine (PL) analog (BRD2889) that was predicted to extensively rewire dependencies across PH gene clusters, mediated by ISCU. In vitro, coupling gain- and loss-of-function analyses of GSTP1 with BRD2889 exposure in PAECs, we found that BRD2889 inhibits glutathione S-transferase P1 (GSTP1), an enzyme which in turn catalyzes ISCU glutathionylation and increases its stability in hypoxia. Consequently, BRD2889 and GSTP1 knockdown phenocopy one another by increasing Fe-S-dependent Complex I activity and mitochondrial oxygen consumption while ameliorating pathogenic apoptosis. Consistent with these computational and in vitro results, in a mouse model of PAH (IL-6 transgenic mice in hypoxia), BRD2889 improved hemodynamic and molecular disease manifestations in vivo. Conclusions: Using a novel computational platform, we identified a coordinated connection between BRD342289 and GSTP1-ISCU axis, crucial to PAEC metabolism. This study offers insight to fundamental PH pathobiology and sets the stage for accelerated repurposing of chemotherapies such as BRD342289 in PH.

scholarly journals Influence of apolipoprotein E genotype and dietary α-tocopherol on redox status and C-reactive protein levels in apolipoprotein E3 and E4 targeted replacement mice

2008 ◽  
Vol 100 (1) ◽  
pp. 44-53 ◽  
Author(s):  
Laia Jofre-Monseny ◽  
Patricia Huebbe ◽  
Inken Stange ◽  
Christine Boesch-Saadatmandi ◽  
Jan Frank ◽  
...  
Keyword(s):  
Apoe Genotype ◽  
Positive Association ◽  
Thiobarbituric Acid ◽  
Redox Status ◽  
C Reactive Protein ◽  
Cvd Risk ◽  
Reactive Protein ◽  
Antioxidant Defence System

The molecular basis of the positive association between apoE4 genotype and CVD remains unclear. There is direct in vitro evidence indicating that apoE4 is a poorer antioxidant relative to the apoE3 isoform, with some indirect in vivo evidence also available. Therefore it was hypothesised that apoE4 carriers may benefit from α-tocopherol (α-Toc) supplementation. Targeted replacement mice expressing the human apoE3 and apoE4 were fed with a diet poor (0 mg/kg diet) or rich (200 mg/kg diet) in α-Toc for 12 weeks. Neither apoE genotype nor dietary α-Toc exerted any effects on the antioxidant defence system, including glutathione, catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase activities. In addition, no differences were observed in mitogen-induced lymphocyte proliferation. α-Toc concentrations were modestly higher in plasma and lower in tissues of apoE4 compared with apoE3 mice, with the greatest differences evident in the lung, suggesting that an apoE4 genotype may reduce α-Toc delivery to tissues. A tendency towards increased plasma F2-isoprostanes in apoE4 mice was observed, while liver thiobarbituric acid-reactive substances did not differ between apoE3 and apoE4 mice. In addition, C-reactive protein (CRP) concentrations were reduced in apoE4 mice indicating that this positive effect on CRP may in part negate the increased CVD risk associated with an apoE4 genotype.

scholarly journals A Novel Automethylation Reaction in the Aspergillus nidulans LaeA Protein Generates S-Methylmethionine

2013 ◽  
Vol 288 (20) ◽  
pp. 14032-14045 ◽  
Author(s):  
Alexander N. Patananan ◽  
Jonathan M. Palmer ◽  
Graeme S. Garvey ◽  
Nancy P. Keller ◽  
Steven G. Clarke
Keyword(s):  
Amino Acid ◽  
Aspergillus Nidulans ◽  
Point Mutations ◽  
Pathogenic Fungi ◽  
Gene Clusters ◽  
Methionine Residue ◽  
Cell Extracts ◽  
Animal Pathogens

The filamentous fungi in the genus Aspergillus are opportunistic plant and animal pathogens that can adapt to their environment by producing various secondary metabolites, including lovastatin, penicillin, and aflatoxin. The synthesis of these small molecules is dependent on gene clusters that are globally regulated by the LaeA protein. Null mutants of LaeA in all pathogenic fungi examined to date show decreased virulence coupled with reduced secondary metabolism. Although the amino acid sequence of LaeA contains the motifs characteristic of seven-β-strand methyltransferases, a methyl-accepting substrate of LaeA has not been identified. In this work we did not find a methyl-accepting substrate in Aspergillus nidulans with various assays, including in vivo S-adenosyl-[methyl-3H]methionine labeling, targeted in vitro methylation experiments using putative protein substrates, or in vitro methylation assays using whole cell extracts grown under different conditions. However, in each experiment LaeA was shown to self-methylate. Amino acid hydrolysis of radioactively labeled LaeA followed by cation exchange and reverse phase chromatography identified methionine as the modified residue. Point mutations show that the major site of modification of LaeA is on methionine 207. However, in vivo complementation showed that methionine 207 is not required for the biological function of LaeA. LaeA is the first protein to exhibit automethylation at a methionine residue. These findings not only indicate LaeA may perform novel chemistry with S-adenosylmethionine but also provide new insights into the physiological function of LaeA.

scholarly journals Genomic Analysis Identifies NovelPseudomonas aeruginosaResistance Genes under Selection during Inhaled Aztreonam TherapyIn Vivo

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Kathryn McLean ◽  
Duankun Lee ◽  
Elizabeth A. Holmes ◽  
Kelsi Penewit ◽  
Adam Waalkes ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Positive Selection ◽  
Single Gene ◽  
Genomic Analysis ◽  
Maximal Activity ◽  
Content Type ◽  
Cystic Fibrosis Airway ◽  
Bacterial Fitness

ABSTRACTInhaled aztreonam is increasingly used for chronicPseudomonas aeruginosasuppression in patients with cystic fibrosis (CF), but the potential for that organism to evolve aztreonam resistance remains incompletely explored. Here, we performed genomic analysis of clonally related pre- and posttreatment CF clinical isolate pairs to identify genes that are under positive selection during aztreonam therapyin vivo. We identified 16 frequently mutated genes associated with aztreonam resistance, the most prevalent beingftsIandampC, and 13 of which increased aztreonam resistance when introduced as single gene transposon mutants. Several previously implicated aztreonam resistance genes were found to be under positive selection in clinical isolates even in the absence of inhaled aztreonam exposure, indicating that other selective pressures in the cystic fibrosis airway can promote aztreonam resistance. Given its potential to confer plasmid-mediated resistance, we further characterized mutantampCalleles and performed artificial evolution ofampCfor maximal activity against aztreonam. We found that naturally occurringampCmutants conferred variably increased resistance to aztreonam (2- to 64-fold) and other β-lactam agents but that its maximal evolutionary capacity for hydrolyzing aztreonam was considerably higher (512- to 1,024-fold increases) and was achieved while maintaining or increasing resistance to other drugs. These studies implicate novel chromosomal aztreonam resistance determinants while highlighting that different mutations are favored during selectionin vivoandin vitro, show thatampChas a high maximal potential to hydrolyze aztreonam, and provide an approach to disambiguate mutations promoting specific resistance phenotypes from those more generally increasing bacterial fitnessin vivo.

scholarly journals Cell membrane shape control--effects of chloromethyl ketone peptides

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 1203-1208 ◽  
Author(s):  
E Alhanaty ◽  
MP Sheetz
Keyword(s):  
Shape Control ◽  
Shape Recovery ◽  
Cultured Cells ◽  
Shape Change ◽  
Lactic Acid Production ◽  
Recovery Process ◽  
Chloromethyl Ketone ◽  
Membrane Shape

Abstract The shape of the human erythrocyte is normally maintained in vivo as a biconcave disc for 120 days. In vitro, the cell shape can be altered readily by amphipathic compounds; however, given time and an energy source, the cells can recover the discoid morphology. An active shape control mechanism is postulated to regulate erythrocyte shape. The shape recovery process is a necessary element in reversing perturbations of shape and is basic to our understanding of how membrane shape is altered. We report here that the process of shape recovery from crenation is dramatically accelerated upon pretreatment of the cells with micromolar (20–100 microM) concentrations of chloromethyl ketone peptides [such as N-alpha-tosyl-L-phenylalanine- chloromethyl ketone (tos-pheCH2Cl)]. Such pretreatments do not appear to affect cellular viability, as judged by their normal biconcave disc shape, their sensitivity to crenators, their lactic acid production, or the ATP-dependent shape change of the purified membranes. Treatment with high concentrations of tos-pheCH2Cl does cause normal cells to become stomatocytic by an energy-requiring process, i.e., it requires glucose, incubation at 37 degrees C, and will not occur in ATP-depleted cells. We suggest that the chloromethyl ketone peptides affect a metabolic process that is associated with the hexose monophosphate (HMP) shunt. Through the alteration of the HMP shunt metabolism, they modify an active stomatocytic process in the erythrocyte that can correct for the perturbation caused by crenators. Implications of these findings for analogous phenomena in cultured cells are discussed.

scholarly journals Bacillus velezensis 83 a bacterial strain from mango phyllosphere, useful for biological control and plant growth promotion

AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Karina A. Balderas-Ruíz ◽  
Patricia Bustos ◽  
Rosa I. Santamaria ◽  
Víctor González ◽  
Sergio Andrés Cristiano-Fajardo ◽  
...  
Keyword(s):  
Biological Control ◽  
Plant Growth ◽  
Biological Control Agent ◽  
Gene Clusters ◽  
In Vitro Assays ◽  
Shoot Biomass ◽  
Plant Growth Promoting Bacteria ◽  
Bacillus Velezensis

Abstract Bacillus velezensis 83 was isolated from mango tree phyllosphere of orchards located in El Rosario, Sinaloa, México. The assessment of this strain as BCA (biological control agent), as well as PGPB (plant growth-promoting bacteria), were demonstrated through in vivo and in vitro assays. In vivo assays showed that B. velezensis 83 was able to control anthracnose (Kent mangoes) as efficiently as chemical treatment with Captan 50 PH™ or Cupravit hidro™. The inoculation of B. velezensis 83 to the roots of maize seedlings yielded an increase of 12% in height and 45% of root biomass, as compared with uninoculated seedlings. In vitro co-culture assays showed that B. velezensis 83 promoted Arabidopsis thaliana growth (root and shoot biomass) while, under the same experimental conditions, B. velezensis FZB42 (reference strain) had a suppressive effect on plant growth. In order to characterize the isolated strain, the complete genome sequence of B. velezensis 83 is reported. Its circular genome consists of 3,997,902 bp coding to 3949 predicted genes. The assembly and annotation of this genome revealed gene clusters related with plant-bacteria interaction and sporulation, as well as ten secondary metabolites biosynthetic gene clusters implicated in the biological control of phytopathogens. Despite the high genomic identity (> 98%) between B. velezensis 83 and B. velezensis FZB42, they are phenotypically different. Indeed, in vitro production of compounds such as surfactin and bacillomycin D (biocontrol activity) and γ-PGA (biofilm component) is significantly different between both strains.

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