- Volume 4, Issue 7, 2022

Methanogens, the archaea uniquely detoxifying fermentative hydrogen into methane in the digestive tract, are increasingly being detected in pathology situations, rendering their rapid identification mandatory. We improved the experimental protocol to identify broth-cultured methanogens by matrix-assisted laser desorption time-of-flight MS (MALDI-TOF-MS). A database incorporating 34 reference spectra derived from 16 methanogen reference strains representative of eight species supported further identification of 21 Methanobrevibacter smithii and 14 Methanobrevibacter oralis isolates broth-cultured from human stool and oral fluid, respectively, with scores >2. In addition, MALDI-TOF-MS differentiated five Methanobrevibacter smithii genotypes incorporated in the study. The data reported here found MALDI-TOF-MS as a first-line identification method for methanogens recovered from microbiota and clinical samples.

The microbiome is intricately linked to human health and, when dysregulated, can cause disease. It has been shown that specific members of the intestinal microbiota regulate immunity, a finding that offers an approach for treating autoimmune disease through microbiome engineering. In previously published work, we identified Clostridium immunis, a new human-derived commensal bacterial species that protects mice against colitis. To understand the host immunological response to C. immunis, we performed comprehensive flow cytometric analysis of the colonic immune system of C. immunis-treated mice. We observed a significant decrease in the number of group 3 innate lymphoid cells (ILC3s), a rare but critical tissue-resident immune cell population that has been implicated in autoimmune diseases. We hypothesize that C. immunisproduces a bioactive molecule that downregulates ILC3s. To identify the genetic determinants of ILC3 downregulation by C. immunis, we compared the ability of closely related clostridia to modulate ILC3-produced cytokines in a colonic explant model. By excluding genes encoded by two inactive clostridia strains, we managed to identify 40 genes that are associated with ILC3 modulation. We similarly observed that C. immunis culture supernatant contained activity. By excluding genes that have predicted intracellular products, we further narrowed this list to 8 candidate genes. Critically, we have recently developed a method to perform targeted genetic knockouts in C. immunis, and are currently generating mutants for these genes. This work will therefore identify the gene product required for ILC3 modulation and potentially identify novel classes of bacterially derived molecules to treat ILC3-driven disease.

Propioni bacterium propionicum is an anaerobic gram positive bacteria. It’s rarely described as to cause brain abscess. We report the isolation of this organism from a brain abscess and emphasise the problems associated with it’s diagnosis. A 62 year old lady with a history of acromegaly underwent emergency surgery for recurrent pituitary lesion felt to be apoplexy. Four weeks later she presented with headache and confusion. Co morbidities included autoimmune diseases on long term steroids and previous use of immunosuppressants. Clinical examination showed neck stiffness. Brain MRI demonstrated an enhancing lesion in the fourth ventricle causing secondary obstructive hydrocephalus requiring endoscopic third ventriculostomy. Blood tests, CSF analysis and whole body PET scan were unable to differentiate between infection and malignancy. Excision of the lesion was felt very high risk as deep in the tectal area. A sporadic trial of antibiotics, high dose steroids and immunotherapy was tried. Patient developed worsening headache with progression of the lesion. Underwent craniotomy and pus was found which isolated P. propionicum sensitive to penicillin and betalactams. Treated with intravenous meropenem for a year. There was significant reduction in the size of the abscess and hydrocephalus on brain MRI at 8 months post-diagnosis. P. propionicum has been isolated and associated with lesions of the lacrimal glands, lung and abdomen. We could find only two case reports of brain abscess due to P. propionicum. Identification of P.propionicum in the microbiology laboratory can be difficult requiring anaerobic growth conditions and extended cultures. Treatment is prolonged antimicrobial therapy.

Polymicrobial oral biofilms, which consist of fermentative-bacteria, are associated with periodontitis, gingivitis and cause systemic diseases1. Unlike aerobic-respiration, fermentation does not require electron acceptors like O2; and redox-cycling of biological electron-carriers, like NADH, drives the intracellular oxidation and reduction of organic-substrates2. Thus, the energy gain is potentially lower than that of respiratory metabolism; however, the high pathogenic activity in anaerobic conditions remained ambiguous3. Afew studies have shown that fermentative gut microbes are capable ofreducing external electron acceptors viaextracellular electron transfer (EET)4-7. EET, a phenomenon initially found in environmental-bacteria, where metabolically generated electrons are transferred to external electron-acceptors through an outer-membraneredoxprotein complex8-9. Thus, the pathogens colonization in the human microbiome may be supported by their EET capability and is important to explore such potentiality. Here, we electrochemically characterized oral-biofilm pathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, to examine their EET ability with lactate/glucose. Both strains showed current production on an electrode surface, associated with consumption of substrate10. The addition of antibiotics that suppress the biosynthesis of membrane or protein showed a significant current decrease, demonstrating that current production reflects the cellular-activity. Further, transmission-electron-microscopy of 3,3‡-diaminobenzidine (DAB) stained cells revealed the presence of redox-enzymes on the cell-membrane suggesting a potential EET mechanism via membrane proteins9. These results could be a basis to reevaluate human oral pathogens from an electroactive point of view. The identified EET activity of the two strains can be utilized for an effective test for assessing the impact of antibacterial compounds on the pathogen cellular-activity on an electrode11.

Anaerobic incubation methods are widely used to cultivate pathogenic anaerobes, but recent years have seen increased interest in potentially therapeutic species originating from the normal intestinal microbiota. We compared the abilities of selected anaerobic pathogens, “normal microbiota” and more recently characterized potentially therapeutic strains to grow on agar at 37°C in the presence of increasing oxygen concentrations, using a variable atmosphere workstation to control oxygen concentration in increments of 0.1%. In initial screening with a high inoculum of 105 to 106 cfu on streak plates, Bacteroides fragilis and Clostridioides difficile strains grew in the presence of up to 2.4% v/v oxygen. Bifidobacterium, Fusobacterium and Finegoldia strains tolerated 0.5 – 1.0% and Eggerthella lenta tolerated 0.1%. Strains of Roseburia, Alistipes, Blautia and Faecalibacterium grew only in strictly anaerobic conditions (<0.01% oxygen) and not in 0.1% oxygen. For strains tolerating >0.1% oxygen in initial experiments, percentage recoveries of smaller inocula (100 – 300 cfu on surface spread plates) were determined in atmospheric oxygen concentrations increasing by 0.1% increments, in comparison with strictly anaerobic colony counts. In 2.0% v/v oxygen, inoculum recovery for 2 × B. fragilis and 1 × C. difficile, was >90%, while recovery of a second C. difficile strain was 25%. For F. magna, inoculum recovery ranged from approximately 100% in 0.1% oxygen to <1% in 0.5% oxygen. These findings demonstrate the variable oxygen tolerance of obligately anaerobic bacteria and emphasize the need for stringent anaerobiosis when culturing the more recently characterized strains currently being developed as live biotherapeutic products.

In eukaryotic cells, secretory and transmembrane proteins fold in the endoplasmic reticulum (ER) before they exit the ER. Accumulation of unfolded proteins activatesIre1 to promote the unfolded protein response (UPR). In the yeast Saccharomyces cerevisiae, activation of Ire1 leads tosplicing of the mRNA for the transcription factor Hac1. Wehave usedb-galactosidase reporter assaysto demonstrateexpression of a UPRE-lacZreporter genethat is positively regulated by the Ire1–Hac1 signalling pathway. In this study, the protein kinase domain was subjected to mutations to alter the catalytic aspartate D797 and lysine K799, which interacts with the terminal phosphate group of ATP, to alanine. Also, point mutations in the Mg+2coordinating loop converted asparagine N802 and aspartic acid D828 to alanine. The expression of theUPRE-lacZreporter gene in all single mutants K799, N802 and D797 was decreased compared to WT-Ire1. Under conditions of various ER stress inducing drugs the ability of the D797A mutant strain to survive was reduced in growth assays compared to other mutants in theMg+2 coordinating loop and catalytic domain. D797-Ire1 mutant has less inb-galactosidase activity, survival of ER stress and HAC1 splicing. Expression of Ire1 carrying double mutations D797A-N802A increasedb-galactosidase activity significantly and restored growth compared to the single mutant D797A-Ire1. WT and protein kinase mutants express to the same level. This study suggeststhat introducing a second mutation such as K799A-Ire1 or N802A-Ire1 to the single mutant D797A-Ire1, restores signaling activity of Ire1.

The interaction between two model organisms, the fruit fly Drosophila melanogaster, and the budding yeast Saccharomyces cerevisiae, is widely perceived as mutually beneficial. The bridge of this biological connection is the volatile compounds from yeast fermentation, which can be attractive to the flies. The generation of volatile molecules derives from complex metabolism and different yeast will generate different combinations of volatiles. The attractiveness to fruit flies can therefore be considered a complex trait. In this study, six strains from SGRP were selected for fermenting commercial grape juice. The attractiveness of these ferments to a wild fruit fly line was then tested. Flies prefer the ferments of the Wine European (WE) strain over the North America (NA) strain. The preference assay was then applied to ferments of genotyped F1 progeny of WE crossed to NA for QTL mapping of genetic variation in attractiveness. GC-MS profiling of all ferments demonstrated that NA could be easily distinguished from other strains. 15 compounds were further quantified and the concentrations in F1 progeny ferments were correlated with genotypes using our r/QTL pipeline. Candidate genes from QTL mapping were tested by reciprocal hemizygosity analysis, validating the impact of two genes, PTC6 and YFL040W. The alleles of these validated genes from NA has better effect on the preference assay than the allele from WE, which is can be considered as antagonistic QTLs. The identification variation in these two genes underpinning metabolic differences, demonstrates how WE and NA have different attraction to one wild fruit fly line.

YML003w. a gene of unknown function is defined on a truncated gene in S288C. The next annotated ORF, YML002w, is therefore not expressed in S288C. Previous studies suggest that these two genes is a large ORF in wild type strains named VRL1. Based on our previous study, we speculate YML003w might be involved in a DNA repair process and its null mutant is sensitive to DNA damage agents. No spontaneous or UV induced mutations are seen in the deletion of YML003w, while overexpression of YML003w results in an increase in spontaneous and UV-induced mutations compared to the wild type frequencies. We then created a series of double deletion strains with yml003w- and other DNA repair related genes. The deletion of YML003w results in loss of spontaneous mutations in most deletions, with the exception of rad5- and mms2-. UV sensitivity of many of these mutants is reversed in the absence of YML003w. Our findings suggest that YML003w plays an important role in mutagenesis and UV sensitivity and potentially interacts with Rad5 mediated pathways.

The Tup1-Cyc8 (Ssn6) co-repressor complex is a powerful epigenetic regulator of genes in the yeast Saccharomyces cerevisiae. The highly conserved complex brings about a repressive chromatin structure at regulatory regions of its target genes or prevents the recruitment of the factors needed for activation of transcription. The FLO family of genes are repressed by the Tup1-Cyc8 complex, these genes encode the proteins required for flocculation, a stress response in yeast where the cells aggregate, or form flocs, to protect cells within the floc. Interestingly each mutant strain (tup1, cyc8 and tup1 cyc8) has a distinct flocculant phenotype. The tup1 strain displays large, dense flocs compared to smaller, more dispersed flocs associated with the cyc8 strain, whereas the tup1 cyc8 strain displays an intermediate flocculant phenotype. RT-qPCR showed that FLO1, considered to be the dominant member of this family of genes, is highly de-repressed in the tup1 and tup1 cyc8 deletion strains. This suggests that Tup1 makes the dominant contribution to repression of this gene. However, this pattern is not seen at all target genes. The results of RNA-Sequencing show a core set of 429 genes significantly upregulated in all three mutant strains. These genes, on average, show the highest de-repression in the cyc8 and tup1 cyc8 mutant strains. Indicating that Cyc8 makes the dominant contribution to repression at the majority of target genes. Together these results indicate that each of the subunits of the Tup1-Cyc8 complex may be dominant in bringing about repression at different sets of genes.

Candida albicansis an opportunistic pathogenic yeast commonly found as part of the gut microbiome, which is thought to be the major reservoir of C. albicans in humans. The ability of C. albicans to adhere to, invade and damage epithelium, along with subsequent escape from the gut lumen into the bloodstream, can lead to life-threatening disseminated fungal infections. Examining the interactions of C. albicans with the intestinal epithelial barrier is vital to understanding its ability to disseminate and cause systemic infections.

A set of seven C. albicans strains (six of gut origin: one healthy donor, one cancer patient, one Crohn’s disease patient and three unknown health status; alongside ‘non-gut’ reference strain SC5314) was profiled for virulence traits. C. albicans strains were profiled for biofilm formation (crystal violet staining), hyphal formation (microscopy) and cytotoxicity (lactate dehydrogenase release; during coculture with confluent Caco-2 gut epithelial cells). Additionally, the ability of C. albicans strains to translocate across epithelial cell layers was assessed during coculture with differentiated Caco-2 monolayers on Transwell™ permeable supports.

Common virulence traits of hyphal formation and cytotoxicity were reduced in gut C. albicanscompared to non-gut reference strain SC5314, while biofilm formation compared to SC5314 was variable. Translocation assays revealed that gut strains of C. albicans are capable of translocating across Caco-2 epithelial cell layers in greater numbers than non-gut reference strain SC5314, with the highest levels of translocation observed from a Crohn’s disease isolate. These insights suggest that gut-specific adaptations may influence luminal escape and pathogenicity of C. albicans.

The conserved protein kinase complex TORC1 is a central regulator of protein homeostasis. TORC1 is active under nutrient replete, unstressed conditions and promotes bulk protein synthesis, cell growth, and proliferation. Many stresses inactivate TORC1, including nutrient starvation. When TORC1 is inactivated, bulk protein synthesis is inhibited, and stress-responsive protein synthesis activated. Degradation of damaged or unwanted proteins is increased through autophagy activation and enhanced proteasome assembly.

The 26S proteasome is composed of the 20S core particle (CP), capped with one or two 19S regulatory particles (RP). Following TORC1 inhibition, the Mpk1 kinase is activated. Mpk1 facilitates increased translation of proteasome regulatory particle assembly chaperones (RPACs), leading to increased proteasome assembly. The mechanisms underlying the RPAC translation increase remain unclear, however.

Here, we show that actin depolarisation upon TORC1 inhibition causes RPAC mRNAs to associate more with actin patch structures. Increasing this interaction enhances translation further. We identified Ede1 as a protein which binds translating RPAC mRNA and show that Ede1 is required to increase RPAC mRNA association with actin patches, RPAC translation and proteasome assembly upon stress.

The combination of microfluidics and time-lapse microscopy is a powerful one, with the potential for the generation of time-series data for single-cell measurements across a whole population of cells. This project aims to employ these tools for the development of technologies and strategies that facilitate the study of stochastic events at the single-cell level. A key focus of the work is the development of microfluidic and image analysis techniques.

Such developments within this project are being applied to study the specific example of noise within the expression of the yeast GCN4 transcriptional regulator. This is activated during amino acid starvation, through a novel post-transcriptional regulatory system. The majority of research into gene expression noise thus far has focused on transcriptional noise, so the impact of post-transcriptional regulation on noise is relatively poorly understood.

Biological systems show many apparently deterministic behaviours at the macroscopic level, but their underlying mechanisms are often stochastic in nature. Noise in gene expression generates heterogeneity across clonal cell populations, and is an important factor in many cellular processes (e.g. circadian rhythms). Even within the well-known “repressilator” synthetic gene circuit, noise had a significant impact on the circuit output. An appreciation of stochasticity in gene expression is therefore crucial for a complete understanding of biology, and will also be essential for the successful design of future synthetic biological systems.

TheS. cerevisiaegenome contains five highly homologous PRS genes, each capable of encoding PRPP (phosphoribosyl-pyrophosphate) synthetase, a key enzyme essential for the synthesis of purine and pyrimidine nucleotides. PRS1andPRS5may have arisen from the prototype Prs-encoding genes, PRS2, PRS3andPRS4by duplication followed by acquisition of non-homologous regions (NHRs), shown to link primary metabolism with intracellular signalling. ThePRSgenes are highly conserved from yeast to human. We have created genocopies in PRS1 associated with the human neuropathies, Arts syndrome and CMTX5 which caused increased caffeine sensitivity and elevated Rlm1 expression in the mutated strains. The insertions in Prs1 and Prs5 serve as a physical link to the cell wall integrity (CWI) pathway and explain why cell viability requires specific heterodimers for the provision of PRPP and the maintenance of CWI integrity. The interaction between Prs1 and Mpk1/Slt2, a component of the CWI pathway was demonstrated by immunoprecipitation. Prs3 contains a NLS (nuclear localisation site), deletion of which causes caffeine sensitivity, reduced Rlm1 expression and loss of the Prs1/Prs3 heterodimer. The contribution of Prs5 to CWI is shown by increased phosphorylation of Mpk1/Slt2 following mutation of the three phosphosites located within NHR5-2. We now intend to investigate the signalling pathway connecting the conserved master regulator TOR (target of rapamycin) with PRPP synthetase, thus adding a new dimension to the application of yeast research in the discovery of novel therapeutic targets for the treatment of human neuropathies, CMTX5 and Arts syndrome.