Browsing by master's degree program "Master 's Programme in Microbiology and Microbial Biotechnology"

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.

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.

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.

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.

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.

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.

Preserving viral nucleic acids is of outmost importance to capture the viral diversity in metagenomic studies. In my master’s thesis, I compare viromes of genus Culex mosquitos stored in two different virus storage media and empty tubes. The mosquitos were collected from Kalajärvi in Espoo, Finland in the summer of 2020 as larvae and were grown to adults in laboratory conditions. Eight pools of five female mosquitos each were stored in each of the two media as well as empty tubes and the samples were homogenized The homogenates were filtered, and the RNA was extracted from them with TRIzol reagent. RNA was then reverse-transcribed to cDNA and amplified with a whole transcriptome amplification kit. The PCR product was prepared with a library preparation kit for sequencing with Illumina Next Generation Sequencer. The resulting reads were processed with a bioinformatic pipeline for identifying viruses from metagenomic sequence data. The results show a clear difference in virus species distribution by storage media. We identified 34 virus species from at least 13 families. Samples stored in ∑-Virocult had the highest yield of viral reads (70.40% of all reads from the pools) as well as the widest variety of mosquito species (n=26). Samples stored in empty tubes had the second most mosquito species (n=10) but the lowest viral read yield (1.25%). RNAlater stored samples had the least virus species (n=7) but a higher viral read percentage than those stored in empty tubes (3.26%). The results indicate the importance of choice of storage media. Since ∑-Virocult had the highest amount of reads and the widest variety, it might be the most useful storage media for our purposes. However, some viruses were found in other samples but not in ∑-Virocult stored samples, indicating a need of different storages conditions for different viruses. It is also important to be consistent in the use of media as it may affect virome results. More work needs to be done to assess if these results are true for other mosquito species as well.

Emerging research suggests that bacteriophages (phages) may exhibit alternative infection strategies that deviate from the preconceived lytic or lysogenic life cycles. Carrier cell infection is an alternative phage life cycle where complete virus particles are formed and remain within host cells, without cell lysis or integration into the host genome. Phage Φ6 (Φ6), the type member of the double-stranded RNA (dsRNA) virus family Cystoviridae, is a lytic phage that can also establish a carrier cell within its plant pathogenic host, Pseudomonas syringae pathovar (pv.) phaseolicola strain HB10Y (HB10Y). This thesis contributes to current limited knowledge and provides an insight on the underlying mechanisms of the Φ6 carrier cell infection. This study has agricultural and ecological relevance and may contribute to future plant therapeutic options. Synthetic carrier cell lines harboring Φ6 tri-segmented genome or Φ6 genomic constructs in which the coding regions in the S- and/or M- segments were replaced by heterologous sequences from tobacco mosaic virus (TMV) were created using a reverse genetics method. Spontaneous Φ6 carrier cell lines were also isolated from HB10Y after exposure of the host to excess phage. Spontaneous carrier cells were not stable, but rather occasionally released phage into liquid culture. Synthetic carrier cell lines were subjected to secondary phage infection and were found to be less susceptible than wild type (WT) to Φ6 but not Φ8, a more distant member of Cystoviridae. Studies suggest that carrier cell resistance to secondary infection (superinfection exclusion) is exhibited through the Φ6 S-segment gene 8. To test how temperature affects the stability of Φ6 carrier cells, spontaneous carrier cell line culture was incubated at RT and 30°C, and phage productivity was compared. Elevated temperature induced carrier cell stability. Comparison of the growth curves between Φ6 synthetic and spontaneous carrier cell lines and their respective WT strains showed that Φ6 carrier cell infection does not greatly affect host growth.

In the past 20 years, three known disease emergence events of highly pathogenic coronaviruses have highlighted the importance of monitoring wildlife for the presence of these viruses. Their peculiar characteristics, like high mutation and recombination rate, have increased their potential for species adaptation and interspecies transmission. Understanding the diversity of these viruses in wildlife and increased surveillance might be key to predicting and preventing future spillovers and pandemics. Studies on wildlife coronaviruses commonly focus on the order Chiroptera, mainly in temperate and tropical regions of the Asian continent. Even though animals belonging to this order are considered the main reservoir, the importance of other small terrestrial mammals should not be overlooked. Rodents, for instance, are animals of great interest for many zoonoses, as they often host parasites, bacteria and other groups of viruses that cause diseases in humans. A recent description of several lineages of coronaviruses recovered from rodents from China highlighted and suggested the presence of these viruses in small terrestrial rodents. In this project, we aimed to investigate the presence of coronaviruses in small mammals from France. Samples were collected during spring 2021 in twelve different locations, within two regions of eastern France, Auvergne Rhône-Alpes and Franche Comté. A total of 448 rodents, 13 shrews and 416 bat samples were collected. The samples were screened and coronaviruses sequences were recovered in 20 different samples. Nine Betacoronavirus genus sequences were recovered from rodent colon samples, and one Alpha- and ten Betacoronavirus sequences from bat guano. These results confirmed previous evidence of these viruses’ presence in small mammals from France and provide the first evidence of betacoronaviruses circulating in wild French bats. The study covers two eastern regions that have not been surveilled in previously released studies therefore this highlights the need to increase the efforts in monitoring these viruses and their wildlife host

Lactobacilli especially Limosilactobacillus reuteri’s strains inhabit the GI tract of humans with glycerol/diol dehydratase activity metabolizing glycerol and producing a broad-spectrum antimicrobial system called reuterin. It consists of 3-hydroxypropanal (3-HPA), acrolein, and its derivatives. Due to the toxic activity of ubiquitous acrolein, an analytical toolbox to determine acrolein formation by food cultures is needed. We developed assays to estimate microbial formation of acrolein using a colorimetric method based on tryptophan and a fluorescence-based approach with 2-amino-1-methyl benzimidazole (AMBI) as a probe. We compared tryptophan and AMBI-based quantification of reuterin produced by resting cells or during the growth of Lb. reuteri DSM 20016. With 600 mM glycerol, resting cells produced 329 ± 35 mM 3-HPA as quantified by HPLC-RI, and 390 ± 13 mM of 3-HPA/acrolein based on the colorimetric method with 3-HPA as standard. Acrolein (40 ± 11 mM) was detected using an AMBI probe. We also detected 3-HPA and acrolein formation during the exponential growth phase in the presence of 50 mM glycerol in different media. Also, as acrolein induces redox stress, redox potential and sensitivity to reuterin/acrolein of the engineered green fluorescence protein (roGFP2) were determined. Ultimately, the roGFP2 gene was tried to clone in E. coli (JM109) using the pTH1mp constitutive expression vector to establish as a biosensor for acrolein detection. Our results suggest that quantification of acrolein by fluorescence-based approaches and biosensors constitute novel methods to estimate any risk of acrolein formation in presence of glycerol/diol dehydratase-positive microbes and glycerol during food fermentation.