Browsing by master's degree program "Mikrobiologian ja mikrobibiotekniikan maisteriohjelma"
Now showing items 41-60 of 126
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(2019)Fungal wood-decayers play an important role in the recycling of biomass and circulation of nutrients in nature. Fungi are capable to convert cellulose, hemicellulose, pectin and lignin, by the action of carbohydrate-acting enzymes (CAZymes) secreted and also by non-enzymatic reactions, depending on the ecology and decay strategy of the fungus. In the present study, four Basidiomycota fungi with different decay strategies were studied to compare their enzyme activity profiles. The white rot fungus Phlebia radiata, brown rot fungus Fomitopsis pinicola and “grey rot” fungus Schizophyllum commune were cultivated on birch (Betula pendula) wood pieces for twelve weeks, whereas the litter-decomposing fungus Coprinopsis cinerea was cultivated on cut barley (Hordeum vulgare) straw for six weeks. All fungi were also cultivated on liquid medium (malt extract 2%) for four weeks. Laccase, manganese peroxidase (MnP), β-glucosidase, xylanase and endoglucanase activities were followed weekly by measuring the absorbances on 96-well plates. The pH and the production of organic acids at each time point were also followed. The results showed that P. radiata produced high laccase and MnP activities. Additionally, high amounts of succinic acid in the aqueous phase of the solid-state cultivations were detected. F. pinicola had a notable production of xylanase activity on birch, in contrast to the moderate β-glucosidase and endoglucanase activities observed on the same substrate. S. commune was a strong producer of β-glucosidase, but especially xylanase activity on solid substrate. Lastly, the litter-decomposer C. cinerea seemed to have a poor performance in enzymatically decomposing the lignin portion from barley straw, whereas a preference on hemicellulose decomposition was observed. Overall, the results indicated the ability of the studied fungi in decomposing the components of the plant cell wall to different extents according to their decay strategy, which is key in the understanding of the ecophysiology of wood-decay and litter-decomposing fungi, and the potential of fungal enzymes for biotechnological applications.
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(2021)Cryptosporidium, a parasitic eukaryote, is the causative agent of cryptosporidiosis, a diarrheal disease with the potential to be life threatening in immunocompromised people. The number of cases has been rising as diagnostics have improved, suggesting the disease is more common than previously thought. Currently in Finland, samples are only typed to the genus level. The objective of the study was to establish a Cryptosporidium typing protocol for outbreak investigation nationally at the Finnish Institute of Health and Welfare with the long-term goal of preventing outbreaks. Reliable typing methods could also help find new reasons behind the increase in cases, monitor the emergence of new subtypes, and reveal new transmission routes. Typing was set up with samples from the Helsinki and Uusimaa Hospital District laboratory as well as the Finnish Food Authority. DNA was extracted from stool samples, species were identified with real-time PCR of the 18S rRNA gene, and gp60 subtypes were determined with nested PCR as well as Sanger sequencing. The Zymo QuickDNA Fecal/Soil Microbe Miniprep Kit was suitable for extracting troublesome parasite DNA. Out of the successfully subtyped samples, the majority was identified as Cryptosporidium parvum, out of which 60 % belonged to the IIaA15G2R1 subtype. The typing method described in this study was successful in typing 19 of 22 samples and will be utilised by the Finnish Institute of Health and Welfare for outbreak investigation on the national level.
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(2024)Antimicrobial resistance (AMR) is a crucial problem in the clinical field worldwide. The need for novel antimicrobials to tackle AMR is imminent. Image-derived data can be used to understand the mechanism of action (MoA) of newly discovered compounds and classify them within known classes of antibiotics. This study aimed to develop a simple image-based measurement method for screening new antimicrobials and simultaneously defining the MoA. In the process, Escherichia coli ATCC 25292 was selected as a model strain from the Gram-negative bacterial group. This strain was treated with antibiotics belonging to different classes. Treated bacteria were imaged with Cytation 5 Cell Imaging Reader and analyzed using ImageJ2 software. The developed workflow was validated by testing the pipeline to be applied for some ESKAPE strains categorized as antibiotic-resistant bacteria. The results revealed that this pipeline enables us to observe the bacterial single-cell phenotypic changes in response to antimicrobials, such as the elongation caused by ampicillin and ciprofloxacin treatments, which are cell wall synthesis and DNA replication inhibitors, respectively. The treated bacterial cells were significantly longer than untreated cells from the sample without antibiotic. Thus, the image-based-high throughput assay can support the drug discovery by identifying the preliminary MoA of new antimicrobials against AMR bacteria. Promising data obtained on E. coli and some pathogenic bacteria allow for pursuing similar approach with other AMR Gram-negative bacterial species.
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(2023)Carbapenemase-producing Enterobacteriaceae (CPE) resist carbapenem class of antibiotics in addition to the other type of antibiotic resistances they usually carry. The blaKPC-3 gene is one of the genes causing the carbapenemase production in bacteria. The aim of this study was to establish the Oxford Nanopore Technologies MinION sequencing method and integrate it with Illumina sequencing into a hybrid assembly for investigating the location of the blaKPC-3 gene in the bacterial genome. We used 14 isolates suspected of plasmid-mediated gene transfer and prepared three libraries using different DNA extraction methods. The contigs from the hybrid assembly sequences were used to annotate the target genes, which were then included in a phylogenetic tree and distance matrix analysis. DNA extraction method had an impact on the amount of data and the length of reads produced in MinION and the Smart DNA prep extraction kit with InnuPure C16 touch extraction device produced the best results. The blaKPC-3 gene was located on plasmids in each isolate, and the similarity of the plasmid sequences indicated that horizontal plasmid-mediated gene transfer has likely occurred between different species. However, the clonal spread cannot be excluded, and further research is needed to confirm these findings.
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Exploring the potential of lactic acid bacteria for sensory improvement of plant protein ingredients (2022)One of the greatest challenges of our time is securing the global protein supply for the growing population in a sustainable manner. Fermentation with lactic acid bacteria has a long history of successful employment for the production of fermented foods and beverages. During this study, the ability of diverse lactic acid bacteria for fermentation and sensory improvement of leguminous and cereal protein concentrates was investigated.The main aim of this study was to overcome the sensory limitations of these plant protein ingredients by finding suitable candidates for the design of new starter cultures for their fermentation. A collection of 82 lactic acid bacteria was screened for fermentation of leguminous and cereal protein concentrates with different nutrient supplementations. Most strains required additional nutrients to adequately acidify the leguminous protein concentrate during a 24-h fermentation, while the cereal-based substrate appeared to be a more complete growth substrate. Descriptive sensory analysis also revealed differences in the aroma perceived by a panel depending on the matrix, supplementation and fermenting strain employed. Three of the strains that produced the most desirable aromas and acidified sufficiently the test matrixes were further studied. All three strains preferentially fermented glucose to lactic acid rather than any other sugar. The concentration of hexanal, one of the volatile compounds involved in grassy and beany off-flavor formation, reduced during fermentations in favor of 1-hexanol, a compound with a significantly higher odor threshold. However, only two of the cultures were able to prevent the growth of contaminating bacteria during fermentation. The results of this study can provide guidance for selecting potential starter cultures and fermentation substrate composition to improve the aroma of plant protein ingredients. Two of the selected strains especially have shown potential to be used as starter cultures for the leguminous protein concentrate. Further studies are required to optimize the performance of the selected strains in the test matrixes and to quantitatively characterize their effect on the substrates’ volatile profile, taste and antinutritional factor content
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(2023)Membrane-bound pyrophosphatases (M-PPases) catalyse the reversible hydrolysis of pyrophosphate into two inorganic phosphate molecules. This hydrolysis is coupled to the transport of protons and/or sodium ions across the biological membrane, generating an electrochemical gradient, which can be utilized by the host organism as an energy source under different stress conditions. The essential physiological roles of M-PPases in agriculturally relevant plants and various human pathogens, such as Bacteroides fragilis, Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma brucei, make them an important research area. Despite the biochemical studies conducted with several M-PPases and structural characterization of Thermotoga maritima and Vigna radiata M-PPases, there remain several questions regarding the biochemistry and catalytic mechanism of M-PPases, one being the basis of K+ dependence. Mutational studies have suggested that a single residue at position 495 is the sole determinant of K+ dependence in M-PPases. In K+-dependent M-PPases this residue is an alanine and a K+ ion binding in close proximity of it, has been shown of having stimulatory effect on K+- dependent M-PPases by increasing the maximal rate of pyrophosphate hydrolysis. In K+-independent M-PPases, a lysine residue resides at 495 position, where the ε-NH3+ group of the lysine residue has been suggested, based on structural modelling and AlaàLys (A495K) mutational studies, to structurally and functionally mimic the activating function of K+ in K+-dependent M-PPases. In order to provide structural basis to this, in this study, a M-PPase belonging to a hyperthermophilic deep- sea bacterium T. maritima containing a single A495K point mutation was expressed in Saccharomyces cerevisiae, solubilized using the high-temperature (“Hot-solve”) method, purified with metal affinity chromatography and structurally characterized using X-ray crystallography. As suggested, the ε-NH3+ group of the introduced lysine was shown to occupy the same space and form the same interactions at the active site as the K+ ion in K+-dependent M-PPases. However, these structural findings were in contradiction with the conducted hydrolytic activity assay which showed that the A495K mutation, besides abolishing K+ dependence, significantly reduced the overall activity compared to the wild type by ~2-10-fold, depending on the K+ concentration. These findings suggest that additional factor(s) besides the 495 residue determine the K+ dependence in M-PPases.
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(2019)Finding and developing new antimicrobial compounds against clinically important antimicrobial drug resistant bacterial pathogens is necessary to counter the threats to global health, food security and development caused by these organisms. One potential source for leads for novel antimicrobial agents are bacteriophages, whose genomes hold vast numbers of genes encoding for proteins that are able to inhibit bacteria in yet uncharacterized ways. Characterization of these proteins and their functions is likely to aid the discovery of new antimicrobial drugs. This study aimed to optimize the heterologous production of three bacteria-inhibiting proteins from bacteriophage φR1-RT for the characterization of the proteins and their interactions with the bacterial host cell. Expression plasmids were successfully constructed for the heterologous production of the proteins in both Lactococcus lactis and Escherichia coli -based expression systems. The L. lactis expression system utilized a tightly regulated nisin-controlled promoter and featured a lactococcal SSusp45 secretion leader to target the produced protein to extracellular secretion. The E. coli expression system used a tightly regulated arabinose-inducible promoter to control the expression of the bacteriotoxic proteins. Despite the successful construction of the expression plasmids, the bacteriophage φR1-RT proteins were not able to be produced in quantities suitable for protein purification in either of the expression systems used in this study. The lack of protein expression is likely due to either codon bias or the harmful effects of the bacteriotoxic proteins that build up inside the bacterial cells. Codon optimized genes or a eukaryotic expression system could be tried to produce enough protein for purification and further characterization.
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(2022)The popularity of fermented beverages is on the rise due to signature flavours, associated health and nutritional benefits and a 100% natural label. Research in this sector is currently focused on industrial-scale production of traditional homemade fermented beverages such as Kombucha, Kefir and Kvass. To expand consumer choice beyond these traditional beverages and to provide more nutritional and flavor diversity, it is essential to develop novel products by using new microbial communities and new substrates. The industrial scale-up of fermented beverages produced using microbial communities is challenging as the flavour complexity and functionality of the beverage depends on the complex fermentation processes and interactions between the microbiota species. Fermentation systems that can separate the metabolic stages into separate fermentation steps would be needed to simplify and make the complex fermentation more efficient, scalable, and reliable. The aim of the thesis was to develop and compare different fermentation strategies to control the complex fermentation of previously isolated microbes to produce a bio-flavoured, low-alcohol, malt beverage with a signature fruity flavour and aroma. During the study, green-malt microbial species: Lactiplantibacillus plantarum, Saccharomyces cerevisiae and Saprochaete suaveolens were identified as significant contributors to the development of aroma and flavour compounds in the malt fermentates. Using the optimal cell concentration of the selected species, three different fermentation strategies: simultaneous inoculation, sequential inoculation and sequential fermentation were adopted to design five different fermentation systems. Cocktail blends of individual fermentates were also created and tested for flavour and aroma. All potential production methods were compared in contrast for parameters such as ease of operation, time-efficiency, flavour and aroma, and future scalability. The results showed that complex fermentation of the novel and low alcohol malt beverage could be controlled by selecting organoleptically significant microorganisms from the complex community, controlling the time and order of inoculation and using a stagewise or modular fermentation system. Sequential fermentation produced the desired low alcohol level and flavorful, fruity malt beverages. However, this system required centrifugation at each step and thus resulted in limited ease of operation. Sequential inoculation was an optimal and efficient method of controlling the fermentation since it required a single vessel, and the metabolic stages were separated by inoculating microorganisms sequentially with a 24 h time interval between each inoculation. Creating cocktail blends from individual fermentates also produced bioflavoured, fruity, aromatic, low alcohol malt beverages. This method was time-efficient with maximum ease of operation. The resulting beverages from these different fermentation systems were novel and had fruity flavours and aroma from the metabolites synthesized by organisms S. suaveolens, L. plantarum and S. cerevisiae. Thus, bio-flavoured, low-alcohol, malt beverages with signature fruity flavour and aroma were created at VTT. For the first time S. suaveolens was used in combination with L. plantarum and S. cerevisiae for beverage production using novel three-microbe fermentation systems to control complex fermentation.
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(2019)Mycosporines and mycosporine-like amino acids (MAAs) are small-molecules that provide UV protection in a broad range of organisms. Cyanobacteria produce a diverse set of MAA chemical variants, many of which are glycosylated. Even though the biosynthetic pathway for the production of a common cyanobacterial MAA, shinorine, is known, the biosynthetic origins of the glycosylated variants remains unclear. In this work, bioinformatics analyses were performed to catalogue the genetic diversity encoded in the MAA gene clusters in cyanobacterial genomes and identify a set of enzymes that might be involved in MAA biosynthesis. A total of 211 cyanobacterial genomes were found to contain the MAA gene cluster, with six containing glycosyltransferase genes within the gene cluster. Afterwards, 38 strains from the University of Helsinki Culture Collection were tested for the production of MAAs using QTOF-LC/MS analyses. This resulted in the identification of several novel glycosylated MAA chemical variants from Nostoc sp. UHCC 0302, which contained a 7.4 kb MAA biosynthetic gene cluster consisting of 7 genes, including two for glycosyltransferases and one for dioxygenase. Heterologous expression of this gene cluster in Escherichia coli TOP10 resulted in the production of a glycosylated porphyra-334 variant of 509 m/z by the transformant cells, showing that colanic acid biosynthesis glycosyltransferases can catalyse the addition of hexose to MAAs. These results suggested a biosynthetic route for the production of glycosylated MAAs in cyanobacteria and allowed to propose a putative role for dioxygenases in MAA biosynthesis. Further characterization of additional glycosyltransferases is necessary to improve our understanding of glycosylated MAA biosynthesis and functionality, which could be applied to large scale processes and be used in industrial applications.
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(2020)Parkinson’s disease is a common neurodegenerative disorder that affects the movement. Although the disease has been known for a long time and intensive studies on this subject have been carried out, the cause of the disease is still unidentified. Interestingly, certain metallic molecules have been found in the neurodegenerative tissues. The metabolism of certain bacterial species has been found to be responsible for the accumulation of these molecules. In this study, we investigated the association between five specific species of gut bacteria, as well as their putative role in the accumulation of the metallic molecules and Parkinson’s disease. The methods comprised of molecular assays for detection and quantification of the bacteria, respectively, from feces samples derived from healthy individuals (n=20) and patients with the disease (n=20). The outcome of molecular assays was verified by traditional microbiological methods. The results suggested that more studies should be done to verify any role of these bacteria in Parkinson ́s disease.
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(2019)Inflammatory bowel disease (IBD) is a globally increasing chronic disease, for which the pathogenesis still is unclear. The most common subtypes of IBD are Crohn’s disease (CD) and ulcerative colitis (UC). It is widely known that, in addition to the genetics, an altered immune response against the gut microbiome plays an important role in the development of the disease. For the IBD patients, to whom conventional medication is not sufficient, the TNF-α blocker infliximab, is given. However, about one third of the patients receiving infliximab treatment, do not respond to the drug, or lose response over time. Since there to this day are no reliable diagnostic markers available, the finding of such is of great importance. The goal of this study was to investigate possible markers for drug response in the gut mycobiota composition of IBD patients. The gut mycobiota composition of 72 IBD patients receiving infliximab was studied by MiSeq sequencing of fungal DNA from fecal samples, collected during one year. The sequencing data was analyzed using the mare package in R. In addition, anti-Saccharomyces cerevisiae antibody (ASCA) concentrations were measured from baseline serum samples by ELISA. Finally, calprotectin concentrations were measured from baseline and twelve weeks post infliximab serum samples by ELISA to study whether serum samples could be used instead of fecal samples for measuring calprotectin values. Results show an increase of the Candida and Spiromyces genera in the gut mycobiota of non-responding patients at baseline. At all timepoints, the Spiromyces genus was observed at a higher abundance, compared to the group of patients responding well or partially to the medication. Interestingly, the increase of Candida was seen only in Crohn’s disease patients, when looking at the composition at all timepoints. ASCA values did not differ between the response groups. The serum calprotectin values did not correlate with fecal calprotectin, and serum calprotectin can thus not be used as a marker of gut inflammation. In conclusion, the gut mycobiota can offer predictive markers for drug response prediction to infliximab in IBD patients, which can with further studies offer a clinical diagnostic tool for prediction of drug response.
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(2019)Propionibacterium freudenreichii is a common bacterium used in food industry. Despite of its wide use it has not been extensively studied yet. P. freudenreichii is usually considered as an anaerobe although it carries all the genes required for aerobic respiration in its genome. In my study, I examined the effect of oxygen on cell physiology and metabolism of P. freudenreichii. Cells were grown in bioreactors under anaerobic and microaerobic conditions as well as using a two-step cultivation with first an anaerobic and then a microaerobic phase. The partial pressure of oxygen was 0 kPa in anaerobic conditions and 4 kPa in microaerobic conditions. The growth rates and amount of ATP were studied, the metabolic activity of the cells was analysed, production of acetic, pyruvic, lactic and propionic acid was examined and finally I studied the expression of the atpA and recA genes. According to the results, P. freudenreichii grows with about two times shorter generation time in microaerobic conditions than in anaerobic conditions. The cells were metabolically most active in exponential phase when conditions were microaerobic. The amount of ATP was found to be rather static under the conditions used. Propionic acid was produced in anaerobic conditions but when conditions switched to microaerobic, its concentration diminished. Pyruvic acid was found to accumulate in microaerobic conditions. The atpA gene was expressed more efficiently in microaerobic conditions than in anaerobic conditions. P. freudenreichii probably utilizes reversible Wood-Werkman -fermentation route to produce ATP by oxidative phosphorylation when oxygen serves as an electron acceptor. Substrate-level phosphorylation did not increase in microaerobic conditions. The growth rate of P. freudenreichii and the final cell densities it reaches can be increased by cultivation in microaerobic conditions instead of anaerobic conditions.
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(2023)Phage lysins are enzymes, which hydrolyze peptidoglycan on bacterial cell wall. Most phage lysins have a modular structure, where N-terminal enzymatically active domain is linked to a C-terminal cell binding domain (CBD). Wild-type Lactococcus lactis LAC460 secretes three prophage lysins, LysL, LysP, and LysT, of which LysL and LysP act as antimicrobial agents killing other closely related bacteria. Of these lysins, only LysL have been shown to contain a CBD in its C-terminus. The aim of this study was to characterize the LysP and LysT enzyme activities by producing them in a heterologous Lactococcus cremoris strain, and to investigate the existence of CBDs in their C-termini. Antimicrobial activities were studied against indicator cells on agar plates, in cell suspensions, and in zymographic gel. Putative CBDs were fused with green fluorescent protein (GFP) to measure fluorescence after binding the fusion proteins onto indicator cells. It was revealed that LysP and LysT antimicrobial activities were detected from heterologous producers’ cell-free supernatants and cell lysates. The lysins were shown to function independently without the need of other phage proteins, and their unusual slow migration in zymogram gel was confirmed. L. cremoris production of the GFP-CBD fusion proteins resulted in low fluorescence, which was insufficient for detecting the proteins’ binding onto indicator cells. In conclusion, heterologous expression and characterization of LysP and LysT were successful, while the existence of CBDs remained to be elucidated.
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(2023)Continuous exposure to ultraviolet radiation can cause detrimental health effects in humans, including erythema, hyperpigmentation, photo-aging, and skin cancer. Sunscreens provide important protection of skin against the harmful effects of ultraviolet radiation. However, chemical and physical sunblock filters can be harmful for both human beings and the environment. There is a need for natural alternatives to commercial filters currently used in sunscreens. Mycosporine-like amino acids (MAAs) are natural microbial sunscreens produced mainly by marine organisms to protect themselves from ultraviolet radiation. There has been over three decades of interest in these natural compounds for applications in the cosmetic industry. However, low production levels in nature have hindered large-scale processes for industrial applications. Here, we demonstrates the successful expression of a MAA biosynthetic pathway from cyanobacteria in a metabolically engineered strain of Escherichia coli. Heterologous expression of the artificial codon-optimized MAA biosynthetic pathway resulted in high-level production of porphyra-334 and shinorine. These two MAAs have exceptional high molar extinction coefficients and are found in a number of commercial sunscreen formulations that rely on algal extracts. The expression of a gene encoding an ABC-transporter, which is associated with cryptic MAA biosynthetic gene clusters in cyanobacteria, in Escherichia coli resulted in the effective transport of both porphyra-334 and shinorine outside the cell. Together, these two advances improve the possibility of biotechnological production of these microbial sunscreens in industrial microbial hosts.
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(2021)Anoplocephala perfoliata, an equine tapeworm, is an intestinal parasite of horses. Tapeworms colonize and live attached to the mucosal surfaces of ileum, caecum, and colon. The lifecycle of an equine tapeworm is indirect which means it requires an intermediate host in addition to its definitive host (a horse). The intermediate host is a mite (Oribatidae) which lives in pastures. The infection occurs when the horse digests the mites containing the infective larval forms while grazing. Infection is usually asymptomatic but can cause lesions in the intestines, and colic. The current coprological techniques often fail to recognise the infection, as they are based on observing the helminth eggs in faeces. Unlike many other intestinal parasites equine tapeworms do not release their eggs into the faecal matter. Instead, they are carried into the environment within the segments of the worm. This causes issues with diagnostics. In general, anthelmintics are recommended to be used only on horses that are diagnosed as having the need for a medication in order to prevent anthelmintic resistance from spreading. Problems in diagnosing the infections lead to decisions about medication having to be made without a proper diagnosis of a tapeworm infection. A reliable coprological test would identify the horses in need of medical care. Because of difficulties in detecting tapeworm eggs, antigens secreted by the worm could be used as a basis for test development. The aim of this study was to characterise secreted and somatic antigens and to localise them in the structures of the tapeworm, in order to obtain more information concerning the mechanisms of infection and to recognise potential antigen candidates for diagnostic purposes. Antigenic proteins were characterised using immunoblotting. Proteins were first separated based on their molecular weights and then treated with horse serum antibodies to indicate antigenic proteins. Immunohistochemical staining was used to localise the antigenic proteins in the structures of the tapeworm. Serum and worms collected from previously euthanised or slaughtered horses were used as material for this study. None of the horses were euthanised or slaughtered because of this study. Several of the secreted or somatic proteins were characterised as antigenic. The molecular weights of the observed characterised antigens ranged from 10 to 150 kDa (kilodaltons). Although these antigens were not specifically localised in the structures of the worm, the eggs and surface structures of the worm seemed to be antigenic. Exact locations could not be confirmed due to unspecific binding. Antigenic proteins characterised in this study by using immunoblotting will be identified using mass spectrometry in further studies. The end goal is to find a suitable antigen that can be used as a base for developing a diagnostic ELISA-test.
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(2020)The Genome Encyclopedia of Spacecraft Associated Microbes (GESAM) project seeks to increase our knowledge of spacecraft associated microbes surviving in the extreme environments of spacecraft associated clean rooms. Planetary Protection researchers at NASA’s Jet Propulsion Laboratory have been collecting and archiving bacterial isolates from NASA Mars missions since the 1970s, however, only a small fraction of the entire bacterial collection has been taxonomically identified and even fewer have been functionally characterized. Previous efforts to taxonomically identify microbes employed 16S rRNA gene sequencing techniques, however, this method alone failed to provide the resolution required to consistently identify these isolates at a species and strain level, many of which are members of taxonomic groups further challenging identification. Samples collected from spacecraft surfaces and within cleanrooms throughout assembly testing and launch operations were subjected to NASA’s heat shock assay and stored for future analysis. In this thesis, for the first time, Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) has been used to identify bacterial strains from the Mars Odyssey and Mars Pathfinder missions. Due to its clinically biased design framework, the development of an in-house database of mass spectrum profiles complemented with 16S rRNA gene sequencing-based taxonomic IDs was necessary to identify this project’s isolates which reside in extreme environments of spacecraft assembly cleanrooms and are not well represented in existing shared spectral databases. MALDI-TOF MS was found to be a fast, reliable, and powerful method of identifying bacterial isolates at species and strain level. Despite MALDI-TOF’s high resolving power, its ability to identify space associated microbes residing in spacecraft assembly cleanrooms is hindered by the reliance of mass spectrum profile creation on 16S rRNA gene taxonomic identification, and the lack of shared spectrum data and publicly available databases. The resolution of taxonomic groups via composite correlation index analysis is paramount to the development of spectra libraries and the utilization of MALDI to its full potential. While MALDI is well suited to identifying the proxy representation of cultivatable spore-forming bacteria produced by NASA’s Standard Assay, a surprising number of non-spore-forming have been identified and many more are expected to be uncultivatable suggesting the need for less selective omics-based approaches in order to establish a more comprehensive idea of the communities present in spacecraft assembly cleanroom.
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(2021)Tuberculosis (TB) still ranks as one of the most dangerous infectious diseases around the world and it is accountable for over 1.5 million deaths every year. World Health Organization has estimated that one fourth of the world’s population is infected with Mycobacterium tuberculosis. The current treatment against TB has drawbacks and the only available vaccine against TB does not provide sufficient protection against the disease and therefore new treatments are much needed. There has also been a lack of good animal models, but the zebrafish (Danio rerio) have been recently found to be a good model to study especially granuloma formation, latency, and reactivation of TB. Their natural pathogen, Mycobacterium marinum causes similar infection in the fish than M. tuberculosis in humans. One characteristic of TB is the formation of granulomas, which are aggregates of immune cells that contain the bacteria. However, M. tuberculosis can escape the granuloma and in such a way spread in the host. The inflammasome is an innate immune system mechanism that activates the immunological response in an infection and has a role in the formation of granulomas. PYCARD is an adaptor protein that has a role in inflammasome activation, which makes it an interesting target when studying the immunological response against M. tuberculosis infection. In this study, granuloma formation in pycard-/- and pycard+/+ zebrafish were compared. The granulomas were studied for their size, location, structure and hypoxicity, and the number of granulomas in each fish was counted. Also, the number of free bacteria was assessed. No significant differences were found in any of these aspects between pycard-/- and pycard+/+ fish. Variation between individual fish was great in both groups.
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(2021)In this study the host range of cystoviruses was explored. Cystoviruses are bacteriophages belonging to the Cystoviridae family. They are enveloped, tri-segmented double-stranded RNA viruses which mainly infect plant pathogenic Pseudomonas syringae strains. Thus, they could potentially be used in phage therapy applications to treat bacterial infections. Before phage therapy can be implemented, the host range dynamics of the cystoviruses need to be understood to ensure that they do not affect the growth of harmless or beneficial bacteria. Cystoviruses have also been used as safe RNA virus models to study factors contributing to virus host range shifts and viral fitness. The first aim of this study was to analyse the host range of cystoviruses phi6, phiNN, phi8, phi12, phi13 and phi2954 by spot test assay. A reverse genetics method was used to detect whether the host range of cystoviruses expands, when the host entry step is bypassed. It was necessary to use derivatives of the potential host strains that contain a plasmid for T7 polymerase expression. As a control, such cell lines were also directly infected with the studied phages. Also, the ability of phages phi6 and phiNN to evolve to infect each other's isolation hosts was studied as well as the frequency and the fitness of the host range mutants was analysed by traditional plating assay. This study provides new information about the host ranges of the cystoviruses. All cystoviruses were shown to have a unique host range pattern with phi6 and phiNN having the widest host ranges. The reverse genetics method allowed cystoviruses phi6, phi8, phi13 and phi2954 to expand their host ranges but subsequent control experiments raised questions as to whether the plasmid containing T7 polymerase gene had a role in this. Both phi6 and phiNN evolved to infect each other´s isolation hosts but the frequency of these host range mutants was lower than has been reported for phi6 in previous studies. It was also demonstrated that the expansion of the host range often imposed fitness costs on the original host.
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(2022)S. aureus infections are prominent worldwide, and with the rapid increase in antimicrobial resistant variants such as methicillin-resistant MRSA, the need for new treatment alternatives is imminent (Monaco et al., 2017). Lytic bacteriophages are continually evolving new methods for the destruction of bacterial cells while avoiding their defence mechanisms. Screening hypothetical proteins of unknown function (HPUFs) from bacteriophages for toxic activity against bacteria may provide new and potentially life-saving approaches to combat bacterial infections (Liu et al., 2004, Singh et al., 2019). The Stab21 phage of Staphylococcus is a recently described lytic phage with over 85 % of its open reading frames annotated as HPUFs (Oduor et al., 2019). The successful identification of potentially toxic gene products could facilitate the discovery of novel bacterial targets for the development of new antimicrobials. It could also provide treatment options to multi-drug resistant S. aureus caused infections where no effective drugs are currently available. To reduce unnecessary screening of phage particle associated yet poorly annotated proteins, total proteins of phage particle were previously identified by LC-MS. Similar studies have previously been performed with Yersinia phage fR1-RT and Klebsiella phage fHe-Kpn01, where a handful of toxic proteins were discovered (Mohanraj et al., 2019, Spruit et al., 2020). To accelerate the screening process, a next-generation sequencing (NGS) high-throughput screening method was further developed by Kasurinen et al. (2021). In this study, 96 true HPUFs were selected and screened for their bactericidal activity in E. coli using the NGS-based approach. Fourteen potentially bacteriotoxic Stab-21 gene products were identified through toxicity screening in E. coli. Of these, three had a particularly low ratio of isolated plasmid after transformation while having a significant number of reads over each joint sequence, indicating their potentially high toxicity. The three most promising candidates were the gene products of g008, g081c and g175 of the Stab21 bacteriophage.
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(2020)Human norovirus (HuNoV) is the leading cause of foodborne illness globally. Especially minimally processed goods such as berries and shellfish are common sources of HuNoV outbreaks. High pressure processing (HPP) is a relatively novel food processing technology that can both inactivate foodborne pathogens and extend the shelf-life of food items in cold temperatures. HPP is especially suitable for fresh purees, juices and sauces. In this study, we used murine norovirus strain MNV-1 to model the inactivation of HuNoV by HPP in three berry puree matrices and phosphate-buffered saline (PBS). We assessed the effect of HPP by cell-based TCID50 infectivity assay and real-time reverse transcription quantitative PCR (RT-qPCR) with RNase and porcine gastric mucin (PGM) binding assay. We strived to find if there were differences between distinct pressures (4500 and 6000 bars) and hold times (3, 6, and 9 minutes) to efficiently inactivate MNV-1 in berry purees. We observed that the matrix type affected the survival of MNV-1 significantly both during HPP and transportation. During transportation, MNV-1 survived better in PBS than in berry purees. MNV-1 was efficiently inactivated in PBS leading to >3-log10 reductions in the number of infectious particles (TCID50/ml) at both 4500 and 6000 bars. In berry puree matrices, MNV-1 was most efficiently inactivated in blackcurrant puree resulting in ≈3-log10 reductions in genome equivalents. The efficacy of pressures and hold-times could not be differentiated in any of the used matrices. MNV-1 in raspberry puree showed no infectivity in RAW 264.7 cells but displayed ≈2-log10 reductions in genome equivalents. MNV-1 in strawberry puree displayed <1-log10 reductions in RAW 264.7 cells. Our results imply that PGM binding assay and RNase as pre-RT-qPCR treatments have problems in selecting infectious MNV-1 particles for amplification. Hence when using these pre-treatments, concluding on MNV-1 infectivity should be done cautiously.
Now showing items 41-60 of 126