Browsing by master's degree program "Mikrobiologian ja mikrobibiotekniikan maisteriohjelma"
Now showing items 1-20 of 126
-
(2024)Coxsackievirus A9 (CVA9) is an enterovirus in the family Picornaviridae. While most infections are asymptomatic, it can cause diseases ranging from mild to serious, especially in neonates where it is known to cause aseptic meningitis and sepsis. There are currently no available vaccines or specific antiviral therapies against coxsackievirus infections. CVA9 has a 30 nm icosahedrally-symmetric capsid comprised of four viral proteins, VP1-4. These four proteins associate together to form a single protomer which is repeated 60 times to make the complete capsid. VP1 forms the five-fold vertex, at the base of which is located a hydrophobic pocket. This pocket is typically occupied by the pocket factor, palmitate. Upon internalization in an endosome, the pocket factor disassociates from the pocket, leading to pocket collapse and subsequently capsid expansion and loss of VP4 and the viral genome. Antiviral therapeutics against CVA9 have focused on stabilizing the hydrophobic pocket to ultimately prevent genome release, but there are currently no approved treatments. Ongoing research has expanded the search to the N-phenyl benzamides class of compounds, which have demonstrated antiviral activity against CVA9. An in-situ docking analysis proposed the hydrophobic pocket as one of several potential binding sites. Here, I used cryogenic electron microscopy and single particle processing to determine the binding site of one such compound, CL301, validating one proposed binding site and the antiviral mechanism of action. A dataset of 24,208 particles was reconstructed resulting in a 2.6 Å resolution electron density map. Atomic modelling showed that an averaged density contributed by both CL301 and palmitate occupied the VP1 hydrophobic pocket. There is not sufficient space for both molecules to occupy the pocket at once, so there is a mixed population, which could be individual virions having all 60 pockets occupied with one or the other molecule, or only a fraction of pockets per virion being occupied. As there is little conformational difference between the two, the statistical analysis used to remove heterogeneity in the dataset was not sensitive enough. In conclusion, CL301 is effective through capsid stabilization preventing expansion and release of the viral genome. Future work should concentrate on improving the affinity of this class of compounds.
-
(2023)Norovirukset ovat merkittäviä akuutin gastroenteriitin aiheuttajia maailmassa. Ne aiheuttavat noin 700 miljoonaa infektiota ja 200,000 kuolemaa vuosittain. Tämän vuoksi on noroviruksen mahdollisesta ilmateitse leviämisestä tullut merkittävä kiinnostuksen kohde. Tässä tutkimuksessa kehitettiin aerosolisysteemi, jota käytettiin tutkimaan, miten hiiren noroviruksen (MNV) infektiokyky säilyy aerosoloinnin seurauksena. Koe toteutettiin tuottamalla aerosoleja MNV-virusliuoksesta sumuttimen avulla (nebulizer), jonka jälkeen aerosolit kerättiin ohjaamalla ne suppilon kautta petrimaljalla olevalle DMEM-kasvatusliuokselle sekä RAW 264.7 soluille. Mikroskopoimalla soluja todettiin viruksen onnistuneen infektoimaan solut suoraan aerosoleista. DMEM-kasvatusliuoksesta määritettiin infektiivisten virusten pitoisuus sekä tehtiin RT-qPCR-analyysi. Tulosten perusteella pystyttiin havaitsemaan, että hiiren noroviruksen infektiokyky heikkenee aerosoloinnin seurauksena. Lisäksi havaittiin, että isoa osaa viruksista ei pystytty keräämään, kun aerosolit mahdollisesti tarttuivat letkuihin ja aerosolisysteemin seinämiin. Tulevaisuudessa tarvitaan aerosolilaitteita, jotka käsittelevät viruksia hellemmin, keräävät aerosoleja tehokkaammin, sekä matkivat ihmisen elimistöä paremmin.
-
(2022)Phage lysins are enzymes that degrade bacterial cell wall. A wild-type Lactococcus lactis strain LAC460 secretes three phage lysins, LysL, LysP, and LysT, encoded by three different prophages. Unlike common phage lysins, these enzymes do not break down the host's cell wall. Therefore, these lysins can attack other L. lactis strains and behave like bacteriocins, antimicrobial proteins. The binding of a phage lysin to bacterial cell wall requires a specific cell wall binding domain (CBD) in the lysin. However, nothing about the CBDs of LysL, LysP and LysT is known. This study aimed to determine the CBDs of these three lysins and the target specificity of the lysins. Putative CBD regions of the lysins were fused with green fluorescent protein (GFPuv). GFPuv-CBD-LysL and GFPuv-CBD-LysT were ligated into the pASG-IBA4 vector and cloned in Escherichia coli DH5α. After all, only the construction of the GFPuv-CBD- LysL was successful resulting in fluorescent transformants. To analyse the binding of GFPuv- CBD-LysL to cells of different L. lactis strains, the fusion proteins were mixed with the LysL sensitive L. lactis MG1614, LysL resistant L. lactis LM0230, and the LysL producing LAC460 cells. With fluorescence microscope it could be seen that the GFPuv-CBD-LysL decorated the cell surface of L. lactis MG1614 with green fluorescence, but LM0230 and LAC460 cells remained non-fluorescent. The fluorescence of the cells was also measured with a fluorometer, showing strong fluorescence from MG1614, but nothing from the other two strains. This showed that the fusion protein specifically bound to the MG1614 cell surface, but it did not bind to the LysL resistant strain LM0230 or the LysL producer LAC460 cell. In conclusion, the results demonstrate that the C-terminus of LysL contains a specific cell wall binding domain. In addition, the results provide an explanation for how LAC460 can secrete LysL without autolysis, as phage lysins not able to bind onto peptidoglycan are unable to lyse cells.
-
(2022)Saccharomyces boulardii is a probiotic yeast related to Saccharomyces cerevisiae but with distinct genetic, taxonomic, and metabolic properties. S. cerevisiae has been used extensively in biotechnological applications. Currently, many strains are available, and multiple genetic tools have been developed, which allow the expression of several exogenous proteins of interest with applications in the fields of medicine, biofuels, the food industry, and scientific research, among others. Although S. boulardii has been widely studied due to its probiotic properties against several gastrointestinal tract disorders, very few studies addressed the use of this yeast as a vector for expression of foreign genes of interest with biotechnological applications. I studied the previously constructed S. boulardii SAC12, which secretes the anti-listerial bacteriocin leucocin C originating from Leuconostoc carnosum 4010. The objective was to study if the bacteriocin leucocin C producing S. boulardii could produce leucocin C in beer fermentation and if leucocin C containing beer can be used as marinade to control Listeria monocytogenes in raw chicken breast strips. The results showed that SAC12 has good ability to secrete LecC, and thus it was used to brew anti-listerial beer. According to results, beer could maintain its anti-listerial activity for 38 days. The anti-listerial effect of the beer stored for different times was analyzed through marinating chicken breast strips (spiked with L. monocytogenes) with the beer for overnight. Results indicated a positive impact of anti-listerial beer in reduction of the viable cells of L. monocytogenes by about 1.6 log from (2.2 ± 0.6) × 10⁷ CFU/g (beer from day 24), and 2.2 log from (1.8 ± 0.3) × 10⁵ CFU/g (beer from day 38). To sum up, the S. boulardii SAC12 efficiently secreted the bacteriocin leucocin C. Brewing beer with S. boulardii SAC12 resulted in beer containing leucocin C. Such beer showed anti-listerial effect when used as marinade for chicken breast strips.
-
(2021)The spread of antibiotic resistance is a global health threat. Hospitals are a potential source of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs), which may disseminate into the environment via wastewater. Hospital water environments, such as sink traps and shower drains, are known to harbor antibiotic-resistant bacteria, which might spread from the drains to the patients causing nosocomial infections that are hard to treat because of the limited number of treatments available. However, the current understanding of antibiotic resistance in the drains of residences, and how it relates to the situation in hospitals is limited. The aim of this study was to compare the microbial communities and ARGs in the water environments of homes and hospitals. The sink traps and shower drains of three hospital rooms and eighteen homes were sampled for metagenomic sequencing, and bioinformatic tools were used to detect the microbial taxa and ARGs in the metagenomes. The resistomes of hospital environments were distinct from those of homes and exhibited a higher diversity of ARGs. On the other hand, the microbial communities of homes and hospital rooms could not be clearly distinguished, although there were some differences in the abundances of certain taxa. The abundance of ARGs was higher in the hospital shower drains than in the corresponding samples in homes, but there was no statistical difference in the abundance of ARGs between the sink traps of homes and the hospital. Although the study had limitations, such as the low number of hospital samples, it indicates that the water environments of hospitals have a resistome that is distinct from that of homes and highlights the role of hospital sink traps and shower drains as potential hotspots of antibiotic resistance.
-
(2019)Lactic acid bacteria have a long history of use in food industry due to their favorable metabolic properties and health benefits for human health. Therefore, they are generally recognized as safe (GRAS) by FDA (U.S Food and Drug Administration) and have QPS (Qualified Presumption of Safety) status granted by EFSA (European Food Safety Authority). Nowadays, antimicrobial resistance (AMR) is a serious global risk and due to the increasing AMRs, more and more microbial infections have become more difficult to treat with antibiotics. AMR has mainly been of concern in relation to pathogenic microbes. However, since fermented foods are favorable environments for AMR gene transfer it should also be considered in the context of beneficial bacteria and their potential to spread AMR genes into pathogenic microbes. The aim of this study was to determine antibiotic susceptibilities of Lactobacillus plantarum, Lactobacillus rhamnosus, Leuconostoc sp. and Weissella sp. strains by E-test method and to detect selected specific antibiotic resistance genes by PCR. In addition, the goal was to define new cut-off values for Weissella strains since, so far, these have not been defined by EFSA. Antibiotic susceptibilities were determined against eight antibiotics: ampicillin, chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, streptomycin and tetracycline. The detected AMR genes were blaZ, mecA, cat, lnuA, tetK and tetM. Most of the determined strains were observed to exhibit a notable resistance to kanamycin. Several Leuconostoc sp. and L. rhamnosus strains showed also resistance to chloramphenicol. Interestingly, one L. rhamnosus strain was observed to exhibit multiresistance to chloramphenicol and clindamycin. Moreover, 48% Leuconostoc strains had higher MIC value for streptomycin than the cut-off value defined by EFSA. Any of the selected AMR genes were not detected even though a notable resistance during the phenotypic testing was observed. However, this might be explained by the small amount of detected AMR genes. The results obtained in the present study provided more information about the antibiotic susceptibility and the safety of L. plantarum, L. rhamnosus, Leuconostoc sp. and Weissella sp. strains. Moreover, new cut-off values were proposed for Weissella sp. strains.
-
(2022)Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new respiratory virus capable of transmitting between interspecies and has caused outbreaks in mink farms since April 2020. The infection in mink farms has been a concern due to the overcrowded mink population which allows viral transmission and mutation to rapidly develop. The development of SARS-CoV-2 vaccines for mink is needed to prevent an outbreak in mink farms. In this study, we tested the safety and efficacy of our SARS-CoV-2 vaccine in a mink model. Our vaccine tests reveal no side effects of the vaccine on vaccinated mink, of which 40 mg/dose was found to be the optimal dosage, and was used as a standard for the later infection experiments. Microneutralization tests indicated that the neutralizing antibodies from vaccinated mink can protect cells line against early 2020 dominant variants (Wuhan, and Alpha), but poorly against later dominant strains (Beta, and Delta). The vaccinated mink were further observed the changes in antibody levels by enzyme-linked immunosorbent assay (ELISA), and the results suggested that the antibodies could be detected in sera samples of vaccinated subjects for at least 23 weeks after receiving two dosages of the vaccine. The presence of the virus was monitored in collected saliva samples throughout the infection days to study the effect of the vaccine on preventing the mink from SARS-CoV-2 infection by reverse transcription (RT)PCR. The quantification cycles (Cq) values were similar between vaccinated and non-vaccinated mink of both genders and between different gender, indicating that the vaccine could not help with preventing SARS-CoV-2 infection. Although the vaccine does not protect the vaccinated mink from the infection, the monitoring of clinical signs suggested that it helps the mink by reducing the severity of the disease. In conclusion, the vaccine showed promising results in inducing the mink body to produce neutralizing antibodies against the SARS-CoV-2 and relieved the symptoms of the disease.
-
(2023)Alzheimer’s disease (AD) is the seventh leading cause of death worldwide. One hallmark of AD includes the amyloid beta (Aβ1-42) peptide that accumulates into oligomers, fibrils, and plaques. Aβ1-42 has been shown to be structurally and functionally similar to antimicrobial peptides (AMPs). Publications have reported that Borrelia burgdorferi can be found in the brain of AD patients. B. burgdorferi and B. garinii cause Lyme disease (LD). B. duttonii is responsible for relapsing fever (RF), a disease characterized by recurrent episodes of high fever. The aim of this research was to study whether synthetic Aβ1-42 binds to LD and RF Borrelia sp. and several bacterial molecules important for their virulence, and whether Borrelia sp. have evolved strategies to evade Aβ1-42-mediated killing. Binding of Aβ1-42 to B. burgdorferi, B. garinii and B. duttonii and several microbial molecules was studied by ELISA and immunoblotting. Bacterial culturing and microscopy were used to study survival, agglutination, and phagocytosis of Borrelia sp. in the presence of Aβ1-42 and microglia. In this research, Aβ1-42 was able to bind and agglutinate all of the three studied Borrelia sp. However, Aβ1-42 reduced the survival and increased the phagocytosis of B. duttonii. while B. burgdorferi and B. garinii were unaffected. In addition, potential Aβ1-42 binding molecules were detected from several bacterial species, including FhbA expressed by B. duttonii. In conclusion, this study suggests that some restricted species of bacteria may evade Aβ1-42 entrapment and thus may be involved in the ability of the species to invade the CNS that may trigger neuroinflammation related to AD.
-
(2023)Filamentous basidiomycete fungi are an abundant source of laccase (benzedio:oxygen oxidoreductases, E.C.1.10.3.2) enzymes that oxidize phenolic compounds by a one electron removal resulting in free radicals with concomitant reduction of oxygen to water. Because of this, laccases are regarded as “green catalysts”, making them of great interest for industrial applications. Phenolic compounds of plant origin are of special interest to the food industry because of their low toxicity and high antioxidant, antimicrobial activities. Because of large number of laccase candidates present in fungal genomes and time demanding methods for their biochemical characterization, in silico methods for the prediction of their activity towards phenolic compounds are needed to be developed to better use the potential of fungal laccases in applications. The goal of this study was to utilize in silico predictions on 19 preselected basidiomycete laccases to determine their activities towards six selected phenolic compounds that are used in food related applications. Based on the structural and functional predictions, two laccases with the best docking characteristics, Pycnoporus cinnabarinus laccase 4273 and Trametes cingulata laccase 1498596, were then selected for recombinant production in the methylotrophic yeast Pichia pastoris, and their biochemical characteristics and activity towards the phenolic compounds were determined experimentally. The recombinant laccases presented optimal working pH in the acidic range from pH 2.0 to 6.0. Both laccases also presented good solvent stability in ethanol and DMSO. The T. cingulata laccase showed thermal tolerance up to 60°C while the P. cinnabarinus laccase was stable up to 40°C. Both recombinant laccases had activity towards 2,6-DMP and the phenolic compound sinapic acid, low activity (<1.9 µkat/L) was observed towards caffeic acid, and no activity (<1.1 µkat/L) was observed towards cinnamic acid, ferulic acid and p-couamaric acid. Experimental and in silico results were compared to evaluate if it is possible to accurately predict laccase activity towards the selected phenolic compounds using in silico approaches. From the in-silico predictions, it was suggested that hydrogen bonding between a substrate molecule and amino acid residue Ser-113 of P. cinnabarinus laccase 4273 and His-111 of T. cingulata laccase 1498596 may be important for the activity towards phenolic compounds. Formation of hydrogen bonds between other amino acid residues and the substrates might be detrimental for laccase activity. The predicted substrate binding site of the laccases differ from yet very close to that of crystal structures of Melanocarpus albomyces laccase. However, the activity towards selected phenolic compounds were somewhat consistent with predictions indicating a possible applicability of structural and functional predictions in the study of fungal laccase activities.
-
(2023)Aspergillus niger is a filamentous fungus that is known for its ability to degrade plant biomass polysaccharides. A total of 86 sugar transporters have been identified in A. niger, but only 10 of them have been thoroughly characterized. Sugar transporter proteins are crucial for fungi as they enable efficient utilization of sugars in their metabolism and therefore breakdown of plant biomass. Additionally, sugar transporters can be used in various biotechnological applications. L-arabinose is a pentose sugar present in plant biomass and A. niger can utilize it through the pentose catabolic pathway (PCP). Recently, a sugar transporter LatA was identified from A. niger, capable of transporting the PCP intermediate product L-arabitol into fungal cells. L-arabitol is a polyol similar to xylitol and can be used as a low-calorie sweetener in food and beverage industries. Although A. niger LatA has previously been shown to be specific to L-arabitol in vivo, its in vitro functional activity has not yet been described. This study aimed to in vitro characterize two potential L-arabitol transporters from A. niger, LatA and unpublished 9364, using the yeast Saccharomyces cerevisiae. As a platform strain, we used S. cerevisiae IMK1010 that is devoid of all hexose and disaccharide transporters, as well as disaccharide hydrolases. In addition, we used a disaccharide-polyol and a pentose metabolic strain which were generated from the IMK1010 strain. The metabolic strains carried pathways for maltose, saccharose, sorbitol and mannitol, and xylose and arabinose, respectively. This provided a controlled research environment for studying A. niger LatA and 9364 transporters. The sugar specificity of the transporters was tested through two different growth experiments on solid media with all the strains and in liquid media with IMK1010 strains. The tested sugars included D-glucose, D-fructose and D-mannose hexoses, D-xylose and L-arabinose pentoses, maltose and sucrose disaccharides, and D-mannitol and D-sorbitol polyols. In addition, LatA was examined through a disappearance assay, measuring the loss of sugar from the liquid growth medium. Altogether four different combination gene constructs, green fluorescent protein (GFP) gene fusions and plain sugar transporter gene constructs were successfully engineered and 21 different transformant yeast strains produced for this study. GFP gene fusions, were in addition to growth experiments, used to study the localization of the sugar transporters to the cell membrane. In strains containing combination gene constructs encoding sugar transporters and GFP, the sugar transporters were successfully localized to the cell membrane, showing already that the transporters potentially have transport activity in the heterologous expression system. Based on the results, A. niger 9364 transported the tested hexoses and maltose in the growth experiments but did not transport tested pentoses, disaccharides or D-mannitol and D-sorbitol polyols. As expected, A. niger LatA did not transport any of the tested sugars, confirming its specificity to L-arabitol polyol. However, in the disappearance assay LatA unexpectedly did not transport L-arabitol. This might be due to the possible toxicity of the polyols in high concentrations to yeast cells and many of them also serve as regulators of osmotic pressure in cells, which may lower the transport capacity of the sugar transporters. In the future the function of the transporters can be tested in different sugar concentrations and pH in disappearance assay. Alternatively, a L-arabitol metabolic strain could be constructed to investigate sugar specificity using the growth experiment instead of the disappearance assay. The study provided new information of A. niger 9364 and further insights into the sugar specificity of A. niger LatA. These sugar transporters could be used in various biotechnological applications in the future.
-
(2023)Puumala orthohantavirus (PUUV) is a single stranded negative sense RNA virus, carried by the bank vole (Myodes glareolus). Like other orthohantaviruses, it does not cause visible symptoms in the host species, but when transmitted to humans, it can cause a mild version of hemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica (NE). PUUV is the only pathogenic orthohantavirus that is endemic to Finland, where it has a relatively high incidence of approximately 35 in 100 000 inhabitants or 1000 to 3000 diagnosed cases annually. Here we describe a miniaturized immunofluorescence assay (mini-IFA) for measuring antibody response against PUUV from bank vole whole blood and heart samples as well as from patient serum samples. The method outline was based on the work done by Pietiäinen et al., (2022), but it was adapted for the detection of PUUV antibodies. Transfected cells expressing the PUUV structural proteins (N, GPC, Gn and Gc) were used instead of PUUV infected cells, which allowed for performing all steps outside of bio-safety level 3 (BSL3) conditions. This method also enables the simultaneous measurement of IgM, IgA and IgG antibody response from each sample in a more efficient and higher output manner, when compared to traditional immunofluorescence methods. Our results show that the method is effective for testing large amounts of samples for PUUV antibodies and it allows for quick and convenient access to high-quality images that can be used for both detecting interesting targets for future studies, as well as producing a visual archive of the test results.
-
(2024)In recent years, icosahedral internal membrane-containing single-stranded DNA (ssDNA) phages have been identified. Phages phi18:4 and phi48:2, both infecting Cellulophaga baltica bacteria and isolated from the Baltic Sea, are part of this phage group. Circular ssDNA genomes of these phages are different in length, but they are packed into capsids of uniform size which is unusual among viruses. Initially, the phages were cultivated using their original C. baltica host strains #18 and #48 but during the study, spontaneously induced #18ind virus was observed to originate from the #18 strain and possessed a double-stranded DNA genome. These observations led to an examination of the phage’s host range, ultimately resulting in the use of the #48 strain as a host for both phages. The life cycle strategy of both phages was lytic and efficiently produced viral progeny in the #48 strain. Zymogram analysis was utilized to investigate the presence of peptidoglycan-degrading enzymes in the phage structure, assisting in host cell penetration. Two possible peptidoglycan hydrolyzing enzymes were observed for each phage, although sequence data suggested only one enzyme. The formation of virions was analyzed by exposing the viruses to biochemical conditions. Freezing and thawing, along with guanidine hydrochloride treatment, turned out to separate the lipid membranes and capsid proteins of the virions but require further optimization for a detailed examination of the protein composition of the membrane. This study together with other research findings, clarifies the structure, life cycle, and evolutionary relationship of lipid-containing ssDNA phages in the context of other phages. In the future, phi18:4 and phi48:2 could serve as model systems for further investigations into other discovered Cellulophaga ssDNA phages.
-
(2022)The genetic and morphological diversity of viruses and more specifically membrane-containing bacteriophages (phages) with single-stranded DNA (ssDNA) genomes is largely unexplored. It can be difficult to detect evolutionary relationships of viruses using solely sequence-based methods due to their rapid sequence evolution. However, more distant evolutionary connections of viruses have been observed based on structure data. Here we introduce an icosahedral tailless ssDNA phage, Cellulophaga phage phi48:2, isolated from the Baltic Sea that has not been assigned to any virus family or taxa. Phage phi48:2 has been previously linked to the family Finnlakeviridae whose members are icosahedral, internal membrane-containing phages with circular ssDNA genomes. However, the presence of lipids in phi48:2 virion has not been studied. In this study, different buffer conditions were tested for infectivity and stability of phi48:2 allowing us to optimize the purification of the phage particles by rate zonal and equilibrium ultracentrifugation in sucrose. Solvent tests in chloroform and ether, as well as low buoyant density of the virion suggested the presence of lipids in the phi48:2 virion. Analysis of the phi48:2 lipids extracted from highly purified virions by thin-layer chromatography revealed that phi48:2 is a membrane-containing phage and acquires its lipids unselectively from its host bacterium Cellulophaga baltica. Lastly, cryogenic electron microscopy of the purified virions also proposed that lipids form a membrane vesicle under the capsid. Altogether our results show that phi48:2 is an icosahedral membrane-containing phage, thus connecting it further with FLiP, which is the sole member of family Finnlakeviridae. Moreover, FLiP and phi48:2 virions are both ~60 nm in diameter and showed some similarity in their major capsid protein sequences (~21% amino acid identity). To conclude, even though phi48:2 and FLiP share various similarities they cannot be placed within the same family due to the low similarity in their genome sequences. However, for now we can assume they are possible distant relatives. The diversity and abundancy of membrane-containing ssDNA phages is gradually starting to uncover and through their characterization and classification we might consequently understand better their significance in microbial ecology.
-
(2023)Enterotoksigeeninen Escherichia coli (ETEC) -infektio on yksi merkittävimmistä kolibasilloosin aiheuttajista nuorilla eläimillä ja on siksi merkittävä syy E. colin aiheuttamaan ripuliin maailmanlaajuisesti. Välittömiä ratkaisuja tarvittaisiin tämän kriisin ratkaisemiseksi ja erityisesti faagiterapia olisi yksi mahdollinen vaihtoehto ETEC-infektioiden hoitoon. Jumbofageilla on hyvät ominaisuudet mahdolliseen terapiakäyttöön niiden laajan genomin ansiosta, mutta ne ovat vielä varsin huonosti tunnettuja. Tämän tutkielman tavoitteena oli karakterisoida äskettäin eristetty Escherichia coli faagi fPf-Eco01 keskittyen erityisesti faagiterapian kannalta keskeisiin ominaisuuksiin. Tavoitteena oli myös selvittää, soveltuuko faagi terapiakäyttöön. Faagin genomin koko oli 379 kb, mikä luokittelee sen jumbofageihin. fPf-Eco01 faagi on mahdollisesti Asteriusvirus-suvun edustaja kuuluen samalla Caudoviricetes-luokkaan. Genomisekvenssin analysoinnissa ei ilmennyt haitallisia geenejä, jotka voisivat koodata antibioottiresistenssiä tai toksiineja. Sekvenssin perusteella faagin elinkierto ei olisi lysogeeninen, mikä voisi estää faagin käytön faagiterapiassa. Läpäisyelektroni-mikroskopia paljasti, että faagi fPf-Eco01 on kooltaan jumbofagin kokoinen ja sillä on supistuva häntä sekä selkeä häntälevy ja häntäsäikeet. Faagi-partikkelin keskipituus on 227 nm. Faagilla on laaja isäntäkirjo, sillä se infektoi jopa 38 % testatuista kliinisistä E. coli -kannoista. Osa faagin isäntäkantana toimivista kannoista oli laajakirjoisia beetalaktamaasi (ESBL) kantoja, jotka olivat eristetty suomalaisilta sairaalapotilailta. Faagi ei kuitenkaan infektoinut muita ETEC-kantoja, kuin vain sen alkuperäisen eristysisännän. Faagi sieti matalia pH-olosuhteita ja selvisi infektiokykyisenä useissa säilytysliuoksissa, joita voidaan käyttää faagiterapiassa. Näin ollen faagi pysyisi mahdollisesti infektiokykyisenä, mikäli faagihoitoa annettaisiin suun kautta tai faageja annosteltaisiin suonensisäisen nesteen mukana. Faagi fPf-Eco01 vaikuttaa olevan hyvä kandidaatti faagiterapiakäyttöön. Faagin laajan isäntäkirjon vuoksi sitä voitaisiin mahdollisesti käyttää ihmisten E. coli -infektioiden hoidossa. Lisätutkimuksia tarvitaan, jotta voidaan selvittää faagin mahdollista käyttöä ETEC-infektioita vastaan.
-
(2023)In recent years, exceptionally large bacteriophages with genome sizes over 500 kilobase pairs (kbp), called megaphages, have been discovered from sequence data, but no previous publications discussing megaphage isolates have been published. In 2011, a phage infecting a Flavobacterium strain was isolated from the Kymijoki river. The phage, named FKy-1, was determined to have a genome size of 643 kbp, based on yet unpublished results, making it the first described megaphage isolate. In this study, we focused on characterizing megaphage FKy-1, by observing the virus morphology, determining the type and length of its life cycle, and measuring its stability in different temperatures and conditions. Purification of the phage by precipitation and ultracentrifugation in a sucrose density gradient resulted in separation of both virion and phage subcomplexes. Based on transmission electron microscopy and cryogenic electron microscopy, FKy-1 was observed to have typical myovirus morphology, with a large icosahedral head of around 160 nm in diameter, and a tail of around 180 nm in length. Molecular masses of the major proteins present in the virion and phage subcomplexes were estimated using sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 50-70 kDa for the major capsid protein, 60-70 kDa and 150-200 kDa for the major tail proteins. Digestion attempts with restriction endonucleases proved unsuccessful, indicating possible phage genome modifications or other defensive mechanisms. The phage was determined to have a lytic life cycle which takes over 3 h to cause cell lysis, resulting in the release of around 10 progenies per infected host cell. The phage proved to be quite stable, with minimal impact on infectivity measured at a temperature range of -20 °C to +40 °C, and in minimal buffer conditions. In summary, we proved that the purification method used here is well suited for megaphages, and that FKy-1 is of myovirus morphology, produces a low number of progenies per host, and is relatively stable. As no other publications regarding megaphages exist, this study acts as a good basis for future research regarding megaphage morphology, infection cycle and stability.
-
(2020)This study aimed to investigate the microbiological quality of the whole and gutted Baltic herring at different seasons by traditional culture-dependent methods combined with the identification of bacterial isolates by MALDI-TOF MS. Additionally, the microbiome of the herring was characterized by culture-independent 16S rRNA gene amplicon sequencing. Bacterial counts were within acceptable limits at all seasons although the H2S-producing bacteria levels were above the recommended level of 5 log10 CFU g-1 at two sampling points. With the culture-dependent methods and the sequencing of the 16S rRNA gene, the microbiome of the whole and gutted herring was dominated by the bacterial class Gammaproteobacteria. Shewanella, Pseudomonas, and Aeromonas were the most frequently isolated genera among the viable population identified with MALDI-TOF MS. With the culture-independent approach, Shewanella followed by Psychrobacter were the most abundant genera. Additionally, a high relative abundance of the phyla Firmicutes and Actinobacteria and, in some samples, Epsilonbacteriaeota represented by the genus Arcobacter, was detected. Variances in the microbiological quality of different herring batches observed in this study could not be attributed to the season. Therefore future research through a longer period was proposed, including data on the environmental factors, such as the fishing location and the water quality, possibly affecting the quality of the herring.
-
(2024)Drainage and global warming increase the threat of carbon-rich northern peatlands drying up, which may cause a positive feedback loop, where the global warming is further accelerated. To better predict the feedback effects, the diversity and functions of soil microbial communities should be investigated. In particular, the role of intracellular parasites of soil is only little studied despite their high abundance. Bdellovibrio and like organisms (BALOs) is a phylogenetically heterogeneous group of obligate bacterial predators of Gram-negative bacteria. Due to their wide distribution and host range, BALOs may play an important role in regulating the structure and dynamics of microbial communities in soil and affect cycling processes. The ability to attack biofilms and prevent biofilm formation makes BALOs also potential candidates for various industrial, medical, agricultural, and biotechnological applications. The aim of the thesis was to characterize two BALOs previously isolated from the subarctic peat soil, Pallas, Finland: 2/ANJLi2 infecting Mucilaginibacter lappiensis and 2/K2C7 infecting Janthinobacterium sp., by using both culture-dependent and bioinformatics-based methods. Transmission electron microscopy (TEM) showed that the isolates display rod-shaped free-swimming cells with a single polar flagellum, morphology typical for BALOs. From a set of 40 strains representing 10 genera, two additional hosts were confirmed for 2/ANJLi2 and four additional hosts for 2/K2C7. In both cases, additional host strains belonged to the same genus, as the respective original isolation host, suggesting that the host range of the isolates is narrow. No host-independence growth was observed. Under TEM, different phases of the isolates’ life cycles could be observed, including free swimming BALO cells and the ones developing inside their host cells, which indicates a periplasmic life cycle. When 2/ANJLi2 and 2/K2C7 were incubated in liquid cultures with their original hosts, both BALOs obtained high titers of 108—109 plaque forming units per ml by 72 and 48 h post-infection, respectively, suggesting that the two isolates have effective but relatively slow life cycles. 2/K2C7 infection resulted in a clear drop of viable host cells, but 2/ANJLi2 infection seems to be less deleterious to its host cells. Genome analyses showed that the isolates are distantly related to each other and are new species of BALOs: 2/ANJLi2 is suggested to belong to the Bacteriovoracaceae family in the class Bacteriovoracia, while 2/K2C7 belongs to the Bdellovibrio genus in the class Bdellovibrionia. Identified gene functions were mostly related to protein synthesis, energy metabolism, and cell cycle. According to metabolic profiling, BALOs had fewer metabolic functions compared to their hosts. Many gene functions remained unknown, highlighting undersampling of soil BALOs and a need for more detailed future studies.
-
(2023)Abstract Faculty: Faculty of Agriculture and Forestry* and Faculty of Medicine *coordination Degree programme: Master′s program in Microbiology and Microbial Biotechnology Author: Johanna Potila Title: Characterization of potentially therapeutic bacteria from a healthy fecal donor. Level: Master′s thesis Month and year: August 2023 Number of pages: 40 Keywords: Clostridioides difficile, dysbiosis, FMT, next-generation probiotics, adhesion, anti-inflammatory Supervisors: PhD Kaisa Hiippala, PhD, Docent Reetta Satokari and PhD Pauliina Lankinen Where deposited: E-thesis University of Helsinki Abstract: Recurrent Clostridioides difficile infection (rCDI) is a healthcare-associated infection related to antibiotic use, that causes significant morbidity and mortality. Fecal microbiota transplantation (FMT) is the most effective treatment for rCDI and it is successful in nearly 90% of patients. However, there are some risks related to FMT use such as the potential risk of transferring pathogens or other phenotypes despite donor screening. Defined bacterial mixtures consisting of endogenous commensal gut microbes with beneficial properties could be used instead of FMT to mitigate the risks and improve the availability of the treatment. 12 bacterial strains previously isolated from a healthy fecal donor were characterized in this study. At first, oxygen tolerance and culturability of the isolates in several different media were examined. The second aim was to investigate if these isolates are safe for bacteriotherapeutic use by testing hemolytic properties, antibiotic susceptibilities and proinflammatory properties. The third objective was to investigate potential beneficial properties such as adherence of the isolates to mucus and epithelial cell lines and anti-inflammatory effects on epithelial cells. Caco-2 and HT-29 cell lines were used as a model of intestinal epithelial cells. Growth was abundant on standard brain heart infusion (BHI) medium supplemented with 0,5% yeast extract and more than half of the isolates tolerated the 4-hour oxygen exposure. These results suggest that many of the strains have good production characteristics. All 12 isolates were non-hemolytic and most of them were susceptible to many commonly used anti-microbials such as amoxycillin/clavulanic acid and piperacillin/tazobactam. Low induction of interleukin-8 (IL-8) release from HT-29 cells was observed for all the isolates which indicates no pro-inflammatory effect. These safety tests suggest that the isolates are safe for therapeutical use. Adhesion to mucus and intestinal epithelial cells (HT-29, Caco-2) was low to moderate (2-7%), which can potentially promote their colonization in the gut. No attenuation of Escherichia coli lipopolysaccharide (LPS)-induced IL-8 release from HT-29 cells was observed, which indicates that characterized strains do not have anti-inflammatory effects on epithelial cells. However, it is likely that they have some other important roles in the gut e.g., in cross-feeding networks and can thus help with restoration of a healthy, diverse gut ecosystem. In conclusion, the characterized isolates could be suitable for bacteriotherapeutic use in the treatment of rCDI.
-
(2019)Antibiotic-resistant bacteria are an increasing threat to global health, caused by the excessive use of antibiotics and the lack of new antimicrobial agents being introduced to the market. New approaches to prevent and cure bacterial infections are needed to halt the growing crisis. One of the most promising alternatives is phage therapy which utilizes bacteriophages to target and kill pathogens with specificity. Pseudomonas aeruginosa is a common opportunistic pathogen that is intrinsically resistant to antibiotics, making it one of the most heavily studied targets of phage therapy. In this study, I characterized four P. aeruginosa phages, fHo-Pae01, PA1P1, PA8P1 and PA11P1, and evaluate their potency in therapeutic applications. Bioinformatic analysis of the genomes revealed the phages to be genetically highly similar and belonging to the Pbunavirus genus of the Myoviridae family. No genes encoding harmful toxins, antibiotic-resistance, or lysogeny were predicted. On the other hand, many of the predicted genes had unknown functions. The host ranges of the phages were assessed using 47 clinical P. aeruginosa strains and predicted host receptor binding tail proteins were compared. Some correlation between the host ranges and mutations in the tail proteins were observed but this alone was not sufficient to explain the differences in the host ranges. The recently isolated vB_PaeM_fHoPae01 (fHo-Pae01) phage was further characterized by a one-step growth curve and imaged with a promising atomic force microscopy method that had not been used before in the Skurnik group. Though the imaging results failed to provide any further knowledge of the phage, the 70-minute-long latent period of infection could be determined from the growth curve. Anion- exchange chromatography was found inefficient in purifying the fHo-Pae01 phage, so alternative methods such as endotoxin columns should be used when purifying these phages for patient use. In conclusion, all four phages appeared to be safe for therapeutic use based on current knowledge, and PA1P1 and PA11P1 were the most promising candidates due to their broad host ranges.
-
(2024)Aspergillus niger is a well-studied filamentous ascomycete fungus and one of the most used fungal species in various biotechnological sectors. Recently the research focus on A. niger has shifted towards production of biochemicals and biomaterials from plant biomass residues. However, there are still many aspects of the plant biomass conversion process by A. niger that are not known in detail, including its sugar uptake systems. Sugar uptake in fungi relies on sugar transporter (ST) proteins that control the uptake of different sugar molecules. Fungal STs are abundant and diverse in their function, which is why many of them remain uncharacterized. Identification of fungal STs for substrates like xylose can improve the industrial production of lignocellulose-based bioproducts by e.g. improving the substrate uptake rate in fungal cell factories. In this thesis, four putative xylose STs from A. niger, XltD, XltE, XltG, and XltH, were characterized both physiologically and functionally in A. niger and Saccharomyces cerevisiae, respectively, together with two previously functionally characterized xylose transporters, XltA and XltB. In addition, a fifth putative xylose transporter XltF was characterized functionally in S. cerevisiae. For the physiological characterization, A. niger 6Δxlt strain was generated by deleting xltA, xltB, xltD, xltE, xltG, and xltH genes using CRISPR/Cas9 methodology. The physiological analysis of the 6Δxlt strain revealed the presence of additional xylose transporters in the A. niger genome, which still remain to be discovered. The functional characterization of the putative A. niger xylose STs was carried out by creating six recombinant S. cerevisiae IMK1010 strains producing the STs fused with a green fluorescent protein. Functional characterization confirmed the correct localization of the STs within plasma membrane, except for XltG which accumulated inside the yeast cells. This indicated potential localization of XltG within endoplasmic reticulum and a putative role in intracellular sugar transport. Growth assays of the recombinant yeast strains demonstrated variable affinities of the A. niger STs for hexoses. The results showed XltF not to be a xylose ST, but instead a hexose ST with low-affinity for fructose and dual-affinity for glucose. The STs were further tested for their substrate specificity and affinity for xylose in a competitive assay between xylose and glucose. XltE displayed a preference for xylose over glucose identifying it as a new low-affinity xylose transporter. Although further research is needed to elucidate the roles of the studied A. niger STs, XltE is a promising candidate for enhancing xylose uptake in fungal cell factories.
Now showing items 1-20 of 126