Removal of Cadmium, Lead and Arsenic from Water by Lactic Acid Bacteria

A number of contaminants such as arsenic, cadmium and lead are released into the environment from natural and anthropogenic sources contaminating food and water. Chronic oral ingestion of arsenic, cadmium and lead is associated with adverse effects in the skin, internal organs and nervous system. In addition to conventional methods, biosorption using inactivated biomasses of algae, fungi and bacteria has been introduced as a novel method for decontamination of toxic metals from water. The aim of this work was to evaluate the applicability of lactic acid bacteria as tools for heavy metal removal from water and characterize their properties for further development of a biofilter. The results established that in addition to removal of mycotoxins, cyanotoxins and heterocyclic amines, lactic acid bacteria have a capacity to bind cationic heavy metals, cadmium and lead. The binding was found to be dependent on the bacterial strain and pH, and occurred rapidly on the bacterial surface, but was reduced in the presence of other cationic metals. The data demonstrates that the metals were bound by electrostatic interactions to cell wall components. Transmission electron micrographs showed the presence of lead deposits on the surface of biomass used in the lead binding studies, indicating involvement of another uptake/binding mechanism. The most efficient strains bound up to 55 mg Cd and 176 mg Pb / g dry biomass. A low removal of anionic As(V) was also observed after chemical modification of the cell wall. Full desorption of bound cadmium and lead using either dilute HNO₃ or EDTA established the reversibility of binding. Removal of both metals was significantly reduced when biomass regenerated with EDTA was used. Biomass regenerated with dilute HNO3 retained its cadmium binding capacity well, but lead binding was reduced. The results established that the cadmium and lead binding capacity of lactic acid bacteria, and factors affecting it, are similar to what has been previously observed for other biomasses used for the same purpose. However, lactic acid bacteria have a capacity to remove other aqueous contaminants such as cyanotoxins, which may give them an additional advantage over the other alternatives. Further studies focusing on immobilization of biomass and the removal of several contaminants simultaneously using immobilized bacteria are required.

Design and production of protein nanostructures for biomolecular detection

Particulate nanostructures are increasingly used for analytical purposes. Such particles are often generated by chemical synthesis from non-renewable raw materials. Generation of uniform nanoscale particles is challenging and particle surfaces must be modified to make the particles biocompatible and water-soluble. Usually nanoparticles are functionalized with binding molecules (e.g., antibodies or their fragments) and a label substance (if needed). Overall, producing nanoparticles for use in bioaffinity assays is a multistep process requiring several manufacturing and purification steps. This study describes a biological method of generating functionalized protein-based nanoparticles with specific binding activity on the particle surface and label activity inside the particles. Traditional chemical bioconjugation of the particle and specific binding molecules is replaced with genetic fusion of the binding molecule gene and particle backbone gene. The entity of the particle shell and binding moieties are synthesized from generic raw materials by bacteria, and fermentation is combined with a simple purification method based on inclusion bodies. The label activity is introduced during the purification. The process results in particles that are ready-to-use as reagents in bioaffinity. Apoferritin was used as particle body and the system was demonstrated using three different binding moieties: a small protein, a peptide and a single chain Fv antibody fragment that represents a complex protein including disulfide bridge.If needed, Eu3+ was used as label substance. The results showed that production system resulted in pure protein preparations, and the particles were of homogeneous size when visualized with transmission electron microscopy. Passively introduced label was stably associated with the particles, and binding molecules genetically fused to the particle specifically bound target molecules. Functionality of the particles in bioaffinity assays were successfully demonstrated with two types of assays; as labels and in particle-enhanced agglutination assay. This biological production procedure features many advantages that make the process especially suited for applications that have frequent and recurring requirements for homogeneous functional particles. The production process of ready, functional and watersoluble particles follows principles of “green chemistry”, is upscalable, fast and cost-effective.

Probiotic lactobacilli and bifidobacteria in the mouth – in vitro studies on saliva-mediated functions and acid production

Probiotic lactobacilli and bifidobacteria in the mouth – in vitro studies on saliva-mediated functions and acid production Probiotics are viable bacteria which, when used in adequate amounts, are beneficial to the health of the host. Although most often related to intestinal health, probiotic bacteria can be found also in the mouth after consumption of products that contain them. This study aimed at evaluating the oral effects of probiotic bacteria already in commercial use. In a series of in vitro studies, the oral colonisation potential of different probiotic bacteria, their acid production and potential saliva-mediated effects on oral microbial ecology were investigated. The latter included effects on the salivary pellicle, the adhesion of other bacteria, and the activation of the peroxidase system. Streptococcus mutans, Streptococcus gordonii, Aggregatibacter actinomycetemcomitans and Helicobacter pylori were used as bacterial indicators of the studied phenomena. There were significant differences between the probiotic strains in their colonisation potential. They all were acidogenic, although using different sugars and sugar alcohols. However, their acid production could be inhibited by the peroxidase system. Based on the results, it can be suggested that probiotic bacteria might influence the oral microbiota by different, partly species or strain-specific means. These include the inhibition of bacterial adhesion, modification of the enamel pellicle, antimicrobial activity, and activation of the peroxidase system. To conclude, probiotic strains differed from each other in their colonisation potential and other oral effects as evaluated in vitro. Both positive and potentially harmful effects were observed, but the significance of the perceived results needs to be further evaluated in vivo.

Inactive endosialidase-based detection of bacterial and oncofetal polysialic acid

Polysialic acid is a carbohydrate polymer which consist of N-acetylneuraminic acid units joined by alpha2,8-linkages. It is developmentally regulated and has an important role during normal neuronal development. In adults, it participates in complex neurological processes, such as memory, neural plasticity, tumor cell growth and metastasis. Polysialic acid also constitutes the capsule of some meningitis and sepsis-causing bacteria, such as Escherichia coli K1, group B meningococci, Mannheimia haemolytica A2 and Moraxella nonliquefaciens. Polysialic acid is poorly immunogenic; therefore high affinity antibodies against it are difficult to prepare, thus specific and fast detection methods are needed. Endosialidase is an enzyme derived from the E. coli K1 bacteriophage, which specifically recognizes and degrades polysialic acid. In this study, a novel detection method for polysialic acid was developed based on a fusion protein of inactive endosialidase and the green fluorescent protein. It utilizes the ability of the mutant, inactive endosialidase to bind but not cleave polysialic acid. Sequencing of the endosialidase gene revealed that amino acid substitutions near the active site of the enzyme differentiate the active and inactive forms of the enzyme. The fusion protein was applied for the detection of polysialic acid in bacteria and neuroblastoma. The results indicate that the fusion protein is a fast, sensitive and specific reagent for the detection of polysialic acid. The use of an inactive enzyme as a specific molecular tool for the detection of its substrate represents an approach which could potentially find wide applicability in the specific detection of diverse macromolecules.

A platform for anti-biofilm assays combining viability, biomass and matrix quantifications in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms

Bacteria can exist as planktonic, the lifestyle in which single cells exist in suspension, and as biofilms, which are surface-attached bacterial communities embedded in a selfproduced matrix. Most of the antibiotics and the methods for antimicrobial work have been developed for planktonic bacteria. However, the majority of the bacteria in natural habitats live as biofilms. Biofilms develop dauntingly fast high resistance towards conventional antibacterial treatments and thus, there is a great need to meet the demands of effective anti-biofilm therapy. In this thesis project it was attempted to fill the void of anti-biofilm screening methods by developing a platform of assays that evaluate the effect that screened compounds have on the total biomass, viability and the extracellular polysaccharide (EPS) layer of the biofilms. Additionally, a new method for studying biofilms and their interactions with compounds in a continuous flow system was developed using capillary electrochromatography (CEC). The screening platform was utilized with a screening campaign using a small library of cinchona alkaloids. The assays were optimized to be statistically robust enough for screening. The first assay, based on crystal violet staining, measures total biofilm biomass, and it was automated using a liquid handling workstation to decrease the manual workload and signal variation. The second assay, based on resazurin staining, measures viability of the biofilm, and it was thoroughly optimized for the strain used, but was then a very simple and fast method to be used for primary screening. The fluorescent resazurin probe is not toxic to the biofilms. In fact, it was also shown in this project that staining the biofilms with resazurin prior to staining with crystal violet had no effect on the latter and they can be used in sequence on the same screening plate. This sequential addition step was indeed a major improvement on the use of reagents and consumables and also shortened the work time. As a third assay in the platform a wheat germ agglutinin based assay was added to evaluate the effect a compound has on the EPS layer. Using this assay it was found that even if compounds might have clear effect on both biomass and viability, the EPS layer can be left untouched or even be increased. This is a clear implication of the importance of using several assays to be able to find “true hits” in a screening setting. In the pilot study of screening for antimicrobial and anti-biofilm effects using a cinchona alkaloid library, one compound was found to have antimicrobial effect against planktonic bacteria and prevent biofilm formation at low micromolar concentration. To eradicate biofilms, a higher concentration was needed. It was also shown that the chemical space occupied by the active compound was slightly different than the rest of the cinchona alkaloids as well as the rest of the compounds used for validatory screening during the optimization processes of the separate assays.

Virulence properties of Aggregatibacter actinomycetemcomitans biofilm and characterisation of its putative cytokine exploitation

Biofilms are surface-attached multispecies microbial communities that are embedded by their self-produced extracellular polymeric substances. This lifestyle enhances the survival of the bacteria and plays a major role in many chronic bacterial infections. For instance, periodontitis is initiated by multispecies biofilms. The phases of active periodontal tissue destruction and notably increased levels of proinflammatory mediators, such as the key inflammatory mediator interleukin (IL)-1beta, are typical of the disease. The opportunistic periodontal pathogen Aggregatibacter actinomycetemcomitans is usually abundant at sites of aggressive periodontitis. Despite potent host immune system responses to subgingival invaders, A. actinomycetemcomitans is able to resist clearance attempts. Moreover, some strains of A. actinomycetemcomitans can generate genetic diversity through natural transformation, which may improve the species’ adjustment tothe subgingival environment in the long term. Some biofilm forming species are known to bind and sense human cytokines. As a response to cytokines, bacteria may increase biofilm formation and alter their expression of virulence genes. Specific outer membrane receptors for interferon-γ or IL-1β have been characterised in two Gram-negative pathogens. Because little is known about periodontal pathogens’ ability to sense cytokines, we used A. actinomycetemcomitans as a model organism to investigate how the species responds to IL-1beta. The main aims of this thesis were to explore cytokine binding on single-species A. actinomycetemcomitans biofilms and to determine the effects of cytokines on the biofilm formation and metabolic activity of the species. Additionally, the cytokine’s putative internalisation and interaction with A. actinomycetemcomitans proteins were studied. The possible impact of biofilm IL-1beta sequestering on the proliferation and apoptosis of gingival keratinocyte cells was evaluated in an organotypic mucosa co-culture model. Finally, the role of the extramembranous domain of the outer membrane protein HofQ (emHofQ) in DNA binding linked to DNA uptake in A. actinomycetemcomitans was examined. Our main finding revealed that viable A. actinomycetemcomitans biofilms can bind and take up the IL-1β produced by gingival cells. At the sites of pathogen-host interaction, the proliferation and apoptosis of gingival keratinocytes decreased slightly. Notably, the exposure of biofilms to IL-1beta caused their metabolic activity to drop, which may be linked to the observed interaction of IL-1beta with the conserved intracellular proteins DNA binding protein HU and the trimeric form of ATP synthase subunit beta. A Pasteurellaceaespecific lipoprotein, which had no previously determined function, was characterized as an IL-1beta interacting membrane protein that was expressed in the biofilm cultures of all tested A. actinomycetemcomitans strains. The use of a subcellular localisation tool combined with experimental analyses suggested that the identified lipoprotein, bacterial interleukin receptor I (BilRI), may be associated with the outer membrane with a portion of the protein oriented towards the external milieu. The results of the emHofQ study indicated that emHofQ has both the structural and functional capability to bind DNA. This result implies that emHofQ plays a role in DNA assimilation. The results from the current study also demonstrate that the Gram-negative oral species appears to sense the central proinflammatory mediator IL-1beta.

Pp2a: At The Crossroads Between Growth and Defence In Plants

Living organisms manage their resources in well evolutionary-preserved manner to grow and reproduce. Plants are no exceptions, beginning from their seed stage they have to perceive environmental conditions to avoid germination at wrong time or rough soil. Under favourable conditions, plants invest photosynthetic end products in cell and organ growth to provide best possible conditions for generation of offspring. Under natural conditions, however, plants are exposed to a multitude of environmental stress factors, including high light and insufficient light, drought and flooding, various bacteria and viruses, herbivores, and other plants that compete for nutrients and light. To survive under environmental challenges, plants have evolved signaling mechanisms that recognise environmental changes and perform fine-tuned actions that maintain cellular homeostasis. Controlled phosphorylation and dephosphorylation of proteins plays an important role in maintaining balanced flow of information within cells. In this study, I examined the role of protein phosphatase 2A (PP2A) on plant growth and acclimation under optimal and stressful conditions. To this aim, I studied gene expression profiles, proteomes and protein interactions, and their impacts on plant health and survival, taking advantage of the model plant Arabidopsis thaliana and the mutant approach. Special emphasis was made on two highly similar PP2A-B regulatory subunits, B’γ and B’ζ. Promoters of B’γ and B’ζ were found to be similarly active in the developing tissues of the plant. In mature leaves, however, the promoter of B’γ was active in patches in leaf periphery, while the activity of B’ζ promoter was evident in leaf edges. The partially overlapping expression patterns, together with computational models of B’γ and B’ζ within trimeric PP2A holoenzymes suggested that B’γ and B’ζ may competitively bind into similar PP2A trimmers and thus influence each other’s actions. Arabidopsis thaliana pp2a-b’γ and pp2a-b’γζ double mutants showed dwarfish phenotypes, indicating that B’γ and B’ζ are needed for appropriate growth regulation under favorable conditions. However, while pp2a-b’γ displayed constitutive immune responses and appearance of premature yellowings on leaves, the pp2a-b’γζ double mutant supressed these yellowings. More detailed analysis of defense responses revealed that B’γ and B’ζ mediate counteracting effects on salicylic acid dependent defense signalling. Associated with this, B’γ and B’ζ were both found to interact in vivo with CALCIUM DEPENDENT PROTEIN KINASE 1 (CPK1), a crucial element of salicylic acid signalling pathway against pathogens in plants. In addition, B’γ was shown to modulate cellular reactive oxygen species (ROS) metabolism by controlling the abundance of ALTERNATIVE OXIDASE 1A and 1D in mitochondria. PP2A B’γ and B’ζ subunits turned out to play crucial roles in the optimization of plant choices during their development. Taken together, PP2A allows fluent responses to environmental changes, maintenance of plant homeostasis, and grant survivability with minimised cost of redirection of resources from growth to defence.

Serpulina-suvun bakteereiden diagnostiikka sekä sioista vuosina 1993-1995 eristettyjen Serpulina-suvun bakteereiden tarkastelua

Summary: The diagnostics of Serpulina bacteria and a review on Finnish swine colonic spirochaetes isolated during 1993-1995

Targeted ablation of gonadotropin dependent endocrine tumors in transgenic mice through their luteinizing hormone receptor (LHR)

Gonadal somatic cell and adrenocortical endocrine tumors are rare. The incidence of adrenocortical carcinomas is only 1-2/1000000 a year. However, they are aggressive, especially in adulthood and currently surgery is the only curative treatment. Cytotoxic agents are in use in advanced cancers, but side effects and multidrug resistance are often problems. Thus there is a need for novel curative treatment methods. In contrast, ovarian granulosa cell tumors and testicular Leydig cell tumors are usually benign, especially at a younger age. The aim of the present thesis was to study a novel targeted treatment method through luteinizing hormone/chorionic gonadotropin receptor (LHCGR) in a transgenic mouse tumor model. The cytotoxic agent was lytic peptide Hecate-CGbeta conjugate where 23 amino acid Hecate, a synthetic form of honeybee venom melittin, was conjugated to 15 amino acid fragment of human chorionic gonadotropin β subunit. Lytic peptides are known to act only on negatively charged cells, such as bacteria and cancer cells and hereby, due to hCGbeta fragment, the conjugate is able to bind directly to LHCGR bearing cancer cells, saving the healthy ones. The experiments were carried out in inhibin-alpha-Simian Virus 40-T-antigen transgenic mice that are known to express LHCGR-bearing gonadal tumors, namely Leydig and granulosa cell tumors by 100% penetrance. If the mice are gonadectomized prepubertally they form adrenocortical tumors instead. Transgenic and wild type mice were treated for three consecutive weeks with control vehicle, Hecate or Hecate-CGbeta conjugate. GnRH antagonist or estradiol was given to a group of mice with or without Hecate-CGbeta conjugate to analyze the additive role of gonadotropin blockage in adrenocortical tumor treatment efficacy. Hecate-CGbeta conjugate was able to diminish the gonadal and adrenal tumor size effectively in males. No treatment related side effects were found. Gonadotropin blockage through GnRH antagonist was the best treatment in female adrenal tumors. The mode of cell death by Hecate-CGbeta conjugate was proven to be through necrosis. LHCGR and GATA-4 were co-expressed in tumors, where the treatment down-regulated their expression simultaneously, suggesting their possible use as tumor markers. In conclusion, the present thesis showed that Hecate-CGbeta conjugate targets its action selectively through LHCGR and selectively kills the LHCGR bearing tumor cells. It works both in gonadal somatic and in ectopic LHCGR bearing adrenal tumors. These results establish a more general principle that receptors expressed ectopically in malignant cells can be exploited in targeted cytotoxic therapies without affecting the normal healthy cells.

Functional studies on bacterial nucleotide-regulated inorganic pyrophosphatases

CBS domains are ~60 amino acid tandemly repeated regulatory modules forming a widely distributed domain superfamily. Found in thousands of proteins from all kingdoms of life, CBS domains have adopted a variety of functions during evolution, one of which is regulation of enzyme activity through binding of adenylate-containing compounds in a hydrophobic cavity. Mutations in human CBS domain-containing proteins cause hereditary diseases. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes, which pull pyrophosphate (PPi) producing reactions forward by hydrolyzing PPi into phosphate. Of the two nonhomologous soluble PPases, dimeric family II PPases, belonging to the DHH family of phosphoesterases, require a transition metal and magnesium for maximal activity. A quarter of the almost 500 family II PPases, found in bacteria and archaea, contain a 120-250 amino acid N-terminal insertion, comprised of two CBS domains separated in sequence by a DRTGG domain. These enzymes are thus named CBS-PPases. The function of the DRTGG domain in proteins is unknown. The aim of this PhD thesis was to elucidate the structural and functional differences of CBS-PPases in comparison to family II PPases lacking the regulatory insert. To this end, we expressed, purified and characterized the CBS-PPases from Clostridium perfringens (cpCBS-PPase) and Moorella thermoacetica (mtCBS-PPase), the latter lacking a DRTGG domain. Both enzymes are homodimers in solution and display maximal activity against PPi in the presence of Co2+ and Mg2+. Uniquely, the DRTGG domain was found to enable tripolyphosphate hydrolysis at rates similar to that of PPi. Additionally, we found that AMP and ADP inhibit, while ATP and AP4A activate CBSPPases, thus enabling regulation in response to changes in cellular energy status. We then observed substrate- and nucleotide-induced conformational transitions in mtCBS-PPase and found that the enzyme exists in two differentially active conformations, interconverted through substrate binding and resulting in a 2.5-fold enzyme activation. AMP binding was shown to produce an alternate conformation, which is reached through a different pathway than the substrate-induced conformation. We solved the structure of the regulatory insert from cpCBS-PPase in complex with AMP and AP4A and proposed that conformational changes in the loops connecting the catalytic and regulatory domains enable activity regulation. We examined the effects of mutations in the CBS domains of mtCBS-PPase on catalytic activity, as well as, nucleotide binding and inhibition.

Switchable lanthanide fluorescence probes in homogenous molecular diagnostics

The number of molecular diagnostic assays has increased tremendously in recent years.Nucleic acid diagnostic assays have been developed, especially for the detection of human pathogenic microbes and genetic markers predisposing to certain diseases. Closed-tube methods are preferred because they are usually faster and easier to perform than heterogenous methods and in addition, target nucleic acids are commonly amplified leading to risk of contamination of the following reactions by the amplification product if the reactions are opened. The present study introduces a new closed-tube switchable complementation probes based PCR assay concept where two non-fluorescent probes form a fluorescent lanthanide chelate complex in the presence of the target DNA. In this dual-probe PCR assay method one oligonucleotide probe carries a non-fluorescent lanthanide chelate and another probe a light absorbing antenna ligand. The fluorescent lanthanide chelate complex is formed only when the non-fluorescent probes are hybridized to adjacent positions into the target DNA bringing the reporter moieties in close proximity. The complex is formed by self-assembled lanthanide chelate complementation where the antenna ligand is coordinated to the lanthanide ion captured in the chelate. The complementation probes based assays with time-resolved fluorescence measurement showed low background signal level and hence, relatively high nucleic acid detection sensitivity (low picomolar target concentration). Different lanthanide chelate structures were explored and a new cyclic seven dentate lanthanide chelate was found suitable for complementation probe method. It was also found to resist relatively high PCR reaction temperatures, which was essential for the PCR assay applications. A seven-dentate chelate with two unoccupied coordination sites must be used instead of a more stable eight- or nine-dentate chelate because the antenna ligand needs to be coordinated to the free coordination sites of the lanthanide ion. The previously used linear seven-dentate lanthanide chelate was found to be unstable in PCR conditions and hence, the new cyclic chelate was needed. The complementation probe PCR assay method showed high signal-to-background ratio up to 300 due to a low background fluorescence level and the results (threshold cycles) in real-time PCR were reached approximately 6 amplification cycles earlier compared to the commonly used FRET-based closed-tube PCR method. The suitability of the complementation probe method for different nucleic acid assay applications was studied. 1) A duplex complementation probe C. trachomatis PCR assay with a simple 10-minute urine sample preparation was developed to study suitability of the method for clinical diagnostics. The performance of the C. trachomatis assay was equal to the commercial C. trachomatis nucleic acid amplification assay containing more complex sample preparation based on DNA extraction. 2) A PCR assay for the detection of HLA-DQA1*05 allele, that is used to predict the risk of type 1 diabetes, was developed to study the performance of the method in genotyping. A simple blood sample preparation was used where the nucleic acids were released from dried blood sample punches using high temperature and alkaline reaction conditions. The complementation probe HLA-DQA1*05 PCR assay showed good genotyping performance correlating 100% with the routinely used heterogenous reference assay. 3) To study the suitability of the complementation probe method for direct measurement of the target organism, e.g., in the culture media, the complementation probes were applied to amplificationfree closed-tube bacteriophage quantification by measuring M13 bacteriophage ssDNA. A low picomolar bacteriophage concentration was detected in a rapid 20- minute assay. The assay provides a quick and reliable alternative to the commonly used and relatively unreliable UV-photometry and time-consuming culture based bacteriophage detection methods and indicates that the method could also be used for direct measurement of other micro-organisms. The complementation probe PCR method has a low background signal level leading to a high signal-to-background ratio and relatively sensitive nucleic acid detection. The method is compatible with simple sample preparation and it was shown to tolerate residues of urine, blood, bacteria and bacterial culture media. The common trend in nucleic acid diagnostics is to create easy-to-use assays suitable for rapid near patient analysis. The complementation probe PCR assays with a brief sample preparation should be relatively easy to automate and hence, would allow the development of highperformance nucleic acid amplification assays with a short overall assay time.