Project description:Introduction: Combined infections from Candida albicans and Staphylococcus aureus are a leading cause of death in the developed world. Evidence suggests that Candida enhances the virulence of Staphylococcus - hyphae penetrate through tissue barriers, while S. aureus tightly associates with the hyphae to obtain entry to the host organism. Indeed, in a biofilm state, C. albicans enhances the antimicrobial resistance characteristics of S. aureus. The association of these microorganisms is also associated with significantly increased morbidity and mortality. Due to this tight association we hypothesised that metabolic effects were also in evidence.</br> Objectives: To explore the interaction, we used a novel GC-Orbitrap – based mass spectrometer, the QExactive GC, which combines the high peak capacity and chromatographic resolution of gas chromatography with the sub-ppm mass accuracy of an Orbitrap system. This allows the capability to leverage the widely available electron ionisation libraries for untargeted applications, along with expanding accurate mass libraries and targeted matches based around authentic standards.</br> Methods: Optimised C. albicans and S. aureus mono- and co-cultured biofilms were analysed in addition to the fresh and spent bacterial growth media.</br> Results: The targeted analysis experiment was based around 36 sugars and sugar phosphates, 22 amino acids and five organic acids. We detected an additional 22 highly scoring compounds from untargeted analysis.</br> Conclusion: Many of the results were as expected – rapid consumption of glucose and fructose from the medium regardless of the cell type. We also detected trehalose from the untargeted data, only in medium that contained C. albicans, commensurate with it being a predominantly fungal sugar. Notable from the results is that the pentose phosphate pathway appears to be enhanced in the cells from co-cultured biofilms.
Project description:Biofilms are sessile microbial communities that are often resistant to conventional antimicrobial therapeutics and the host immune system. Candida albicans is an opportunistic pathogenic yeast and responsible for candidiasis. It readily colonizes host tissues and implant devices, and forms biofilms, which play an important role in pathogenesis and drug resistance. Its morphological transition from budding yeast to hyphal form and subsequent biofilm formation is regarded as the crucial factor for drug tolerance and virulence of Candida infections. In this study, nepodin (also called musizin) from Rumex japonicus root was investigated for antibiofilm, antihyphae, and antivirulence activities against fluconazole-resistant C. albicans strain. Nepodin at 2 µg/ml from Rumex plant effectively inhibited C. albicans biofilm formation by more than 90% but had no effect on planktonic cell growth. Also, Rumex root extract and nepodin inhibited hyphal growth and cell aggregation of C. albicans. Interestingly, nepodin also showed antibiofilm activity against Staphylococcus aureus or A. baumannii strains and two systems of dual biofilms of C. albicans and S. aureus or A. baumannii, respectively. Transcriptomic analysis using RNA-seq and qRT-PCR showed nepodin repressed the expressions of several hypha/biofilm related genes (ECE1, HWP1, and UME6) and overexpressed several transport genes (CDR4, CDR11, IFD6, and TPO2), which supported observed phenotypic changes.
Project description:Effect of the presence of Lactococcus lactis on Staphylococcus aureus transcriptome in cheese matrix. S. aureus was co-cultured with L. lactis LD61 in cheese matrix during 7 days. RNA samples were extracted at different time points (6 h, 8 h, 10 h, 24 h and 7 days) in order to monitor the dynamic response of S. aureus MW2 in cheese matrix in presence of L. lactis
Project description:Purpose: The purpose of this study was to simulataneously examine the host and fungal pathogen transcriptional profiles of four distinct infection fates during macrophage and Candida albicans interactions Methods: Membrane stained (Deep Red),primary, bone marrow derived, murine macrophages and Candida albicans expressing GFP and mCherry were exposed to each other over a four hour time course. Samples were collected at 0, 1, 2 and 4 hours and sorted for four infection subpopulations: 1. Macrophages which phagocytosed live C. albicans (GFP+ /mCherry+ /Deep Red +), 2. Macrophages which phagocytosed dead C. albicans (GFP- /mCherry+ /Deep Red +), 3. Uninfected macrophages(GFP- /mCherry- /Deep Red +) and 4. Unengulfed C. albicans (GFP+ /mCherry + /Deep Red -). Unexposed controls were also collected for some time points (i.e. macrophages never exposed to C. albicans and C. albicans never exposed to macrophages). Single macrophages infected with live or dead C. albicans were also sorted. Smart-seq2 was used to create libraries for both infection subpopulation and single, infected cell samples that were sequenced on Illumina’s Miseqand Nextseq. Basic quality assessment of Illumina reads and sample demultiplexing was done with Picard version 1.107 and Trimmomatic. Samples profiling exclusively the mouse transcriptional response were aligned to the mouse transcriptome generated from the v. Dec. 2011 GRCm38/mm10 and a collection of mouse rRNA sequences from the UCSC genome website. Samples profiling exclusively the yeast transcriptional response were aligned to the C. albicans transcriptome strain SC5314 version A21-s02-m09-r10 downloaded from Candida Genome Database. Samples containing both macrophages and C. albicans were aligned to a “composite transcriptome” made by combining the mouse transcriptome and C. albicans transcriptomes described above and alignment was done via BWA (version 0.7.10-r789.) Multi-reads (reads that aligned to both host and pathogen transcripts) were discarded. Then, each host or pathogen sample file were aligned to its corresponding reference using Bowtie2 and RSEM (v.1.2.21). Transcript abundance was estimated using transcripts per million (TPM). For subpopulation samples, TPM was calculated using edgeR, all as implemented in the Trinity package version 2.1.. Genes were considered differentially expressed only if they had a 4-fold change difference (> 4 FC) in TPM values and a false discovery rate below or equal to 0.001 (FDR < 0.001), unless specified otherwise. For single macrophages infected with C. albicans, samples were aligned to the combined transcriptome as described above and RSEM was used to calculate TPM. Results: We were able to simultaneously measure the host and fungal pathogen transcriptional profiles of four distinct infection fates during macrophage and Candida albicans interactions Conclusions: Our study represents an analysis of both distinct infection populations of macrophages and fungus.
Project description:Mytilus coruscus were injected with microbes into the adductor muscle. The microbes are Staphylococcus aureus, Vibrio alginolyticus, and Candida albicans. After microbial challenge, hemolymph was collected from the adductor muscle of M. coruscus at 2h post bacterial challenge. The hemolymph was isolated by 12% SDS-PAGE and the differential bands were collected for LC-MS/MS proteomic analysis.
Project description:C.albicans induces the upregulation of inflammation related genes at the same time it also induces TGF-ß signalling pathway related genes from human blood derived monocytes. RNA sequencing was prerformed from Candida albicans co-incubated monoyctes from 3 different donors. Candida albicans significantly upregulates 6363 genes in human blood derived monocytes in 1h of co-incubation.
Project description:Sepsis is a clinical syndrome that can be caused by bacteria or fungi. Early knowledge on the nature of the causative agent is a prerequisite for targeted anti-microbial therapy. Besides currently used detection methods like blood culture and PCR-based assays, the analysis of the transcriptional response of the host to infecting organisms holds great promise. In this study, we aim to examine the transcriptional footprint of infections caused by the bacterial pathogens Staphylococcus aureus and Escherichia coli and the fungal pathogens Candida albicans and Aspergillus fumigatus in a human whole-blood model. Moreover, we use the expression information to build a random forest classifier to determine if the pathogen is bacterial, fungal or neither of the two. After normalizing the transcription intensities using stably expressed reference genes, we filtered the gene set for biomarkers of bacterial or fungal blood infections. This selection is based on differential expression and an additional gene relevance measure. In this way, we identified 38 biomarker genes, including IL6, SOCS3, and IRG1 which were already associated to sepsis by other studies. Using these genes, we trained the classifier and assessed its performance. It yielded a 96% accuracy (sensitivities >93%, specificities >97%) for a 10-fold stratified cross-validation and a 92% accuracy (sensitivities and specificities >83%) for an additional dataset comprising Cryptococcus neoformans infections. Furthermore, the noise-robustness of the classifier suggests high rates of correct class predictions on datasets of new species. In conclusion, this genome-wide approach demonstrates an effective feature selection process in combination with the construction of a well-performing classification model. Further analyses of genes with pathogen-dependent expression patterns can provide insights into the systemic host responses, which may lead to new anti-microbial therapeutic advances. Analysis of innate immune activation on the basis of gene expression of whole blood cells during ex vivo whole blood infection with bacterial (Staphylococcus aureus, Escherichia coli) and fungal pathogens (Candida albicans, Aspergillus fumigatus) in comparison to mock-treated blood.
Project description:Young adult fer-15;fem-1 Caenorhabditis elegans were infected with Staphylococcus aureus for 8 h to determine the transcriptional host response to Staphylococcus aureus. Analysis of differential gene expression in C. elegans young adults exposed to two different bacteria: E. coli strain OP50 (control), wild-type Staphylococcus aureus RN6390. Samples were analyzed at 8 hours after exposure to the different bacteria. These studies identified C. elegans genes induced by pathogen infection. Keywords: response to pathogen infection, innate immunity, host-pathogen interactions
Project description:Candida albicans were cultured under normal and hypoxic conditions and collected secretome from the culture supernatant with centrifugation. The secretome contains secretory proteins, extracellular vehicles, and others.
Project description:Staphylococcus aureus and Pseudomonas aeruginosa are bacterial pathogens that have been shown to co-exist in biofilms related to numerous infections. Although the interaction between these two species is competitive, both partially benefit from the coexistence. In this study, we exhaustively characterized the interaction between Staphylococcus aureus and Pseudomonas aeruginosa by utilizing a proteomics approach, individually targeting the surface-associated proteins (surfaceome), and proteins secreted or otherwise liberated to the extracellular space (exoproteome). To that end, the conditions to co-culture S. aureus and P. aeruginosa in vitro were optimized and a high-resolution proteomics approach was applied to compare surface-associated and extracellular protein profiles between mono- and co-cultured biofilms.