Project description:Background: Yersinia outer protein (Yop) H is a secreted virulence factor of Yersinia enterocolitica which inhibits phagocytosis of Y. enterocolitica and promotes virulence of Y. enterocolitica (Ye) in mice. The aim of this study was to address whether and how YopH affects the innate immune response against Ye in mice. Results: For this purpose mice were infected with wild type Ye (pYV+) or a YopH-deficient Ye mutant strain (DyopH). CD11b+ cells were isolated from infected spleen and subjected to gene expression analysis using microarrays. Despite attenuation of DyopH in vivo, by variation of infection doses we were able to achieve conditions that allow comparison of gene expression in pYV+ and DyopH infections at either comparable infection courses or splenic bacterial burden. Gene expression analysis provided evidence that expression levels of several immune response genes including IFN-g and IL-6 are high after pYV+ infection but low after sublethal DyopH infection. In line with these findings, infection of IFN-gR-/- and IL-6-/- mice with pYV+ or DyopH revealed that these cytokines are dispensable for control of DyopH, but not pYV+ infection. Consistently, in bacteria killing assays with BMM in vitro, stimulation of BMM with IFN-g is required for killing of pYV+ but not DyopH. Conclusion: In conclusion, this data suggest that IFN-g counteracts YopH-mediated virulence mechanisms of Ye which in Ye wild type infection contribute to evasion of the innate immune response including killing by macrophages. Keywords: Comparison of gene expression due to bacterial virulence factors
Project description:Aflatoxins are toxic and carcinogenic polyketide metabolites produced by fungal species, including Aspergillus flavus and A. parasiticus. The biosynthesis of aflatoxins is modulated by many environmental factors, including the availability of a carbon source. The gene expression profile of A. parasiticus was evaluated during a shift from a medium with low concentration of simple sugars, Yeast Extract (YE), to a similar medium with sucrose, Yeast Extract Sucrose (YES). Total RNA and aflatoxins (B1, B2, G1, and G2) were quantified from fungal mycelia harvested pre- and post-shifting. When compared to YE media, YES caused temporary reduction of the aflatoxin levels detected at 3 hours post-shifting and they remained low well past 12 hours post-shift. Aflatoxin levels did not exceed the levels in YE until 24 hrs post shift, at which time point a 10-fold increase was observed over YE. Microarray analysis comparing the RNA samples from the 48 hr YE culture to the YES samples identified a total of 2120 genes that were expressed across all experiments, including most of the aflatoxin biosynthesis genes. One-way Analysis of Variance (ANOVA) identified 56 genes that were expressed with significant variation across all time points. Three genes responsible for converting norsolorinic acid to averantin were identified among these significantly expressed genes. The potential role of these genes involvement in the regulation of aflatoxin biosynthesis is discussed. Keywords: time course, media shift, Aspergillus, aflatoxin
Project description:The pod is the main edible part of Phaseolus vulgaris L. (common bean). The commercial use of the pods is mainly affected by their color. Consumers seem to prefer golden pods. However, planters suffer economic losses because of pod color instability. The aim of the present study was to identify the gene responsible for the golden pod trait in the common bean. ‘A18-1’ (a golden bean line) and ‘Renaya’ (a green bean line) were chosen as the experimental materials. Genetic analysis indicated that a single recessive gene, pv-ye, controls the golden pod trait. A candidate region of 4.24-Mb was mapped to chromosome A02 using bulked-segregant analysis coupled to whole genome sequencing. In this region, linkage analysis in an F2 population localized the pv-ye gene to an interval of 182.9-kb between the simple sequence repeat markers SSR77 and SSR93. This region comprised 16 genes in this region, comprising 12 annotated genes from the P. vulgaris database, and 4 functionally unknown genes. Combined with transcriptome sequencing, we identified Phvul.002G006200 as the potential candidate gene for pv-ye. Sequencing of Phvul.002G006200 identified a single nucleotide polymorphism (SNP) in pv-ye. This SNP is located in the coding region and is responsible for substituting a glutamic acid with an glutamine at position 416 of the pv-ye protein (E416Q). A pair of primers covering the SNP was designed and the fragment was sequenced to screen 316 F2 plants with the ‘A18-1’ phenotype, based on the different site. Our findings showed that the among the 316 mapped individuals, the SNP cosegregated with the ‘A18-1’ phenotype. The findings presented here could form the basis to reveal the mechanism of the golden pod trait in the common bean at the molecular level.
Project description:Background:; Yersinia outer protein (Yop) H is a secreted virulence factor of Yersinia enterocolitica which inhibits phagocytosis of Y. enterocolitica and promotes virulence of Y. enterocolitica (Ye) in mice. The aim of this study was to address whether and how YopH affects the innate immune response against Ye in mice. Results:; For this purpose mice were infected with wild type Ye (pYV+) or a YopH-deficient Ye mutant strain (DyopH). CD11b+ cells were isolated from infected spleen and subjected to gene expression analysis using microarrays. Despite attenuation of DyopH in vivo, by variation of infection doses we were able to achieve conditions that allow comparison of gene expression in pYV+ and DyopH infections at either comparable infection courses or splenic bacterial burden. Gene expression analysis provided evidence that expression levels of several immune response genes including IFN-g and IL-6 are high after pYV+ infection but low after sublethal DyopH infection. In line with these findings, infection of IFN-gR-/- and IL-6-/- mice with pYV+ or DyopH revealed that these cytokines are dispensable for control of DyopH, but not pYV+ infection. Consistently, in bacteria killing assays with BMM in vitro, stimulation of BMM with IFN-g is required for killing of pYV+ but not DyopH. Conclusion:; In conclusion, this data suggest that IFN-g counteracts YopH-mediated virulence mechanisms of Ye which in Ye wild type infection contribute to evasion of the innate immune response including killing by macrophages. Experiment Overall Design: In this study microarray analyses were performed to define differences in gene expression of cells associated with innate immune response (CD11b+ cells) after infection of mice with a sublethal and lethal infection with wildtype Yersinia enterocolitica compared to uninfected mice. In addition, we wanted to investigate whether differences in gene expression can be defined which are due to the virulence factor YopH. Moroeover, we were interested whether gene expression pattern of sublethal and lethal infected mice are different. Allover all five samples were compared. Number of replicates 1.
Project description:(from abstract): Iron oxidation is a desirable trait of biomining microorganisms, although the mechanism is not well-understood in extreme thermoacidophiles. The complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, ~2300 ORFs) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins (in)directly involved with bioleaching. As expected, the M. sedula genome encodes genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation. The fox gene cluster, involved in iron oxidation in the thermoacidophilic archaeon Sulfolobus metallicus, could also be identified. These iron-oxidizing components are missing from genomes of non-leaching Sulfolobales like Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius DSM 639. Whole genome transcriptional response analysis showed that 88 ORFs were up-regulated 2-fold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized. Dye flip of Mse cells includes two samples, yeast exact (YE) and yeast extract + ferrous sulfate (YEF). The first slide, Y3F5, has YE RNA labeled with cy3 and YEF RNA labeled with cy5, while the second slide, F3Y5, has YEF RNA labeled with cy3 and YE RNA labeled with cy5. YE serves as the reference condition, with the expectation that ORFs involved with Fe2+ oxidation (and SO4 metabolism) will be upregulated on YEF (log2 fold change of YE-YEF < -1).
Project description:We show that ZS1 in the medium supplemented with YE (YE-medium) produces more cell biomass but less rhamnolipid than it does in Glc-medium. To elucidate the transcriptional regulation of genes that are involved in biosynthesis of rhamnolipids and its precursors, RNA-seq-based transcriptional profiling of ZS1 cells in response to reciprocal change of YEand Glc-media is performed. Based on the assembly of ZS1 transcriptome using the reference PAO1 genome, we show that genes involved in energy metabolic pathways in ZS1 strain are highly transcribed in YE medium but not in Glc-medium, in agreement with their cell mass production. Similarly, transcription of quorum sensing systems genes lasI-lasR, rhlI-rhlR, and pqsH-mvfR are downregulated in Glc-medium. On the other hand, we show that two of the three enzymes RhlB and RhlC essential for rhamnolipid biosynthesis are transcriptionally upregulated, independent of quorum sensing signals. Notably, three of the four enzymes involved in dTDP-L-rhamnose, a precursor for the rhamnolipid biosynthesis, are downregulated in Glc except for RmlD that catalyzes the last reaction in the pathway. Together, our results indicate that increased rhamnolipid production in ZS1 cells is independent of quorum sensing signals. We propose that quorum sensing-independent rhamnolipid production in ZS1 Glc-culture is achieved by transcriptional re-programming of the minimum number of genes involved in rhamnolipid biosynthesis.
Project description:DRUG-Seq is a miniatruized and multiplexed RNA-Seq protocol described in Ye et al., 2018 (https://www.nature.com/articles/s41467-018-06500-x) To determine transcriptional changes upon drug perturbation in LN-229 glioblastoma cells, we profiled 20 drug responses across two time-points (6 and 22 hours) by DRUG-Seq
Project description:The outer membrane (OM) of Gram-negative bacteria efficiently protects from harmful environmental stresses such as antibiotics, disinfectants or dryness. Main constituents of the OM are integral OM β-barrel proteins (OMPs). In Gram-negative bacteria such as Escherichia coli (Ec), Yersinia enterocolitica (Ye) and Pseudomonas aeruginosa (Pa), the insertion of OMPs depends on a sophisticated biogenesis pathway. This comprises the SecYEG translocon, which enables inner membrane (IM) passage, the chaperones SurA, Skp and DegP, which facilitate the passage of β-barrel OMPs through the periplasm, and the β-barrel assembly machinery (BAM) which facilitates insertion into the OM. In Ec, Ye and Pa the deletion of SurA is particularly detrimental and leads to a loss of OM integrity, sensitization to antibiotics, and hypovirulence. In search of targets that could be exploited to develop compounds that interfere with OM integrity in Acinetobacter baumannii (Ab), we employed the multidrug-resistant strain AB5075 to generate single gene knockout strains lacking individual periplasmic chaperones. In contrast to Ec, Ye and Pa, AB5075 tolerates the lack of SurA, Skp or DegP with only weak mutant phenotypes. While the double knockout strains surAskp and surAdegP are conditionally lethal in Ec, all double deletions were well tolerated by AB5075. Strikingly, even a triple knockout strain of AB5075, lacking surA, skp and degP, was viable. Our findings underline the extreme adaptibility of Ab and suggest the existence of mechanisms bypassing or acting redundantly to the known OMP biogenesis pathway comprising SurA, Skp and DegP.