Membrane Insertion of C-tail Anchored Proteins

The correct localization of proteins is essential for cell viability. In order to achieve correct protein localization to cellular membranes, conserved membrane targeting and translocation mechanisms have evolved. The focus of this work was membrane targeting and translocation of a group of proteins that circumvent the known targeting and translocation mechanisms, the C-tail anchored protein family. Members of this protein family carry out a wide range of functions, from protein translocation and recognition events preceding membrane fusion, to the regulation of programmed cell death. In this work, the mechanisms of membrane insertion and targeting of two C-tail anchored proteins were studied utilizing in vivo and in vitro methods, in yeast and mammalian cell systems. The proteins studied were cytochrome b(5), a well characterized C-tail anchored model protein, and N-Bak, a novel member of the Bcl-2 family of regulators of programmed cell death. Membrane insertion of cytochrome b(5) into the endoplasmic reticulum membrane was found to occur independently of the known protein conducting channels, through which signal peptide-containing polypeptides are translocated. In fact, the membrane insertion process was independent of any protein components and did not require energy. Instead membrane insertion was observed to be dependent on the lipid composition of the membrane. The targeting of N-Bak was found to depend on the cellular context. Either the mitochondrial or endoplasmic reticulum membranes were targeted, which resulted in morphological changes of the target membranes. These findings indicate the existence of a novel membrane insertion mechanism for C-tail anchored proteins, in which membrane integration of the transmembrane domain, and the translocation of C-terminal fragments, appears to be spontaneous. This mode of membrane insertion is regulated by the target membrane fluidity, which depends on the lipid composition of the bilayer, and the hydrophobicity of the transmembrane domain of the C-tail anchored protein, as well as by the availability of the C-tail for membrane integration. Together these mechanisms enable the cell to achieve spatial and temporal regulation of sub-cellular localization of C-tail anchored proteins.

Genotyping for genetic association studies: Methods and applications

In this thesis, two separate single nucleotide polymorphism (SNP) genotyping techniques were set up at the Finnish Genome Center, pooled genotyping was evaluated as a screening method for large-scale association studies, and finally, the former approaches were used to identify genetic factors predisposing to two distinct complex diseases by utilizing large epidemiological cohorts and also taking environmental factors into account. The first genotyping platform was based on traditional but improved restriction-fragment-length-polymorphism (RFLP) utilizing 384-microtiter well plates, multiplexing, small reaction volumes (5 µl), and automated genotype calling. We participated in the development of the second genotyping method, based on single nucleotide primer extension (SNuPeTM by Amersham Biosciences), by carrying out the alpha- and beta tests for the chemistry and the allele-calling software. Both techniques proved to be accurate, reliable, and suitable for projects with thousands of samples and tens of markers. Pooled genotyping (genotyping of pooled instead of individual DNA samples) was evaluated with Sequenom s MassArray MALDI-TOF, in addition to SNuPeTM and PCR-RFLP techniques. We used MassArray mainly as a point of comparison, because it is known to be well suited for pooled genotyping. All three methods were shown to be accurate, the standard deviations between measurements being 0.017 for the MassArray, 0.022 for the PCR-RFLP, and 0.026 for the SNuPeTM. The largest source of error in the process of pooled genotyping was shown to be the volumetric error, i.e., the preparation of pools. We also demonstrated that it would have been possible to narrow down the genetic locus underlying congenital chloride diarrhea (CLD), an autosomal recessive disorder, by using the pooling technique instead of genotyping individual samples. Although the approach seems to be well suited for traditional case-control studies, it is difficult to apply if any kind of stratification based on environmental factors is needed. Therefore we chose to continue with individual genotyping in the following association studies. Samples in the two separate large epidemiological cohorts were genotyped with the PCR-RFLP and SNuPeTM techniques. The first of these association studies concerned various pregnancy complications among 100,000 consecutive pregnancies in Finland, of which we genotyped 2292 patients and controls, in addition to a population sample of 644 blood donors, with 7 polymorphisms in the potentially thrombotic genes. In this thesis, the analysis of a sub-study of pregnancy-related venous thromboses was included. We showed that the impact of factor V Leiden polymorphism on pregnancy-related venous thrombosis, but not the other tested polymorphisms, was fairly large (odds ratio 11.6; 95% CI 3.6-33.6), and increased multiplicatively when combined with other risk factors such as obesity or advanced age. Owing to our study design, we were also able to estimate the risks at the population level. The second epidemiological cohort was the Helsinki Birth Cohort of men and women who were born during 1924-1933 in Helsinki. The aim was to identify genetic factors that might modify the well known link between small birth size and adult metabolic diseases, such as type 2 diabetes and impaired glucose tolerance. Among ~500 individuals with detailed birth measurements and current metabolic profile, we found that an insertion/deletion polymorphism of the angiotensin converting enzyme (ACE) gene was associated with the duration of gestation, and weight and length at birth. Interestingly, the ACE insertion allele was also associated with higher indices of insulin secretion (p=0.0004) in adult life, but only among individuals who were born small (those among the lowest third of birth weight). Likewise, low birth weight was associated with higher indices of insulin secretion (p=0.003), but only among carriers of the ACE insertion allele. The association with birth measurements was also found with a common haplotype of the glucocorticoid receptor (GR) gene. Furthermore, the association between short length at birth and adult impaired glucose tolerance was confined to carriers of this haplotype (p=0.007). These associations exemplify the interaction between environmental factors and genotype, which, possibly due to altered gene expression, predisposes to complex metabolic diseases. Indeed, we showed that the common GR gene haplotype associated with reduced mRNA expression in thymus of three individuals (p=0.0002).

Oncolytic adenoviruses for treatment of ovarian cancer

Virotherapy, the use of oncolytic properties of viruses for eradication of tumor cells, is an attractive strategy for treating cancers resistant to traditional modalities. Adenoviruses can be genetically modified to selectively replicate in and destroy tumor cells through exploitation of molecular differences between normal and cancer cells. The lytic life cycle of adenoviruses results in oncolysis of infected cells and spreading of virus progeny to surrounding cells. In this study, we evaluated different strategies for improving safety and efficacy of oncolytic virotherapy against human ovarian adenocarcinoma. We examined the antitumor efficacy of Ad5/3-Δ24, a serotype 3 receptor-targeted pRb-p16 pathway-selective oncolytic adenovirus, in combination with conventional chemotherapeutic agents. We observed synergistic activity in ovarian cancer cells when Ad5/3-Δ24 was given with either gemcitabine or epirubicin, common second-line treatment options for ovarian cancer. Our results also indicate that gemcitabine reduces the initial rate of Ad5/3-Δ24 replication without affecting the total amount of virus produced. In an orthotopic murine model of peritoneally disseminated ovarian cancer, combining Ad5/3-Δ24 with either gemcitabine or epirubicin resulted in greater therapeutic benefit than either agent alone. Another useful approach for increasing the efficacy of oncolytic agents is to arm viruses with therapeutic transgenes such as genes encoding prodrug-converting enzymes. We constructed Ad5/3-Δ24-TK-GFP, an oncolytic adenovirus encoding the thymidine kinase (TK) green fluorescent protein (GFP) fusion protein. This novel virus replicated efficiently on ovarian cancer cells, which correlated with increased GFP expression. Delivery of prodrug ganciclovir (GCV) immediately after infection abrogated viral replication, which might have utility as a safety switch mechanism. Oncolytic potency in vitro was enhanced by GCV in one cell line, and the interaction was not dependent on scheduling of the treatments. However, in murine models of metastatic ovarian cancer, administration of GCV did not add therapeutic benefit to this highly potent oncolytic agent. Detection of tumor progression and virus replication with bioluminescence and fluorescence imaging provided insight into the in vivo kinetics of oncolysis in living mice. For optimizing protocols for upcoming clinical trials, we utilized orthotopic murine models of ovarian cancer to analyze the effect of dose and scheduling of intraperitoneally delivered Ad5/3-Δ24. Weekly administration of Ad5/3-Δ24 did not significantly enhance antitumor efficacy over a single treatment. Our results also demonstrate that even a single intraperitoneal injection of only 100 viral particles significantly increased the survival of mice compared with untreated animals. Improved knowledge of adenovirus biology has resulted in creation of more effective oncolytic agents. However, with more potent therapy regimens an increase in unwanted side-effects is also possible. Therefore, inhibiting viral replication when necessary would be beneficial. We evaluated the antiviral activity of chlorpromazine and apigenin on adenovirus replication and associated toxicity in fresh human liver samples, normal cells, and ovarian cancer cells. Further, human xenografts in mice were utilized to evaluate antitumor efficacy, viral replication, and liver toxicity. Our data suggest that these agents can reduce replication of adenoviruses, which could provide a safety switch in case of replication-associated side-effects. In conclusion, we demonstrate that Ad5/3-Δ24 is a useful oncolytic agent for treatment of ovarian cancer either alone or in combination with conventional chemotherapeutic drugs. Insertion of genes encoding prodrug-converting enzymes into the genome of Ad5/3-Δ24 might not lead to enhanced antitumor efficacy with this highly potent oncolytic virus. As a safety feature, viral activity can be inhibited with pharmacological substances. Clinical trials are however needed to confirm if these preclinical results can be translated into efficacy in humans. Promising safety data seen here, and in previous publications suggest that clinical evaluation of the agent is feasible.

Lost in Translation : Translation Mechanisms in Production of Cocksfoot Mottle Virus Proteins

The present study focuses on the translational strategies of Cocksfoot mottle virus (CfMV, genus Sobemovirus), which infects monocotyledonous plants. CfMV RNA lacks the 5'cap and the 3'poly(A) tail that ensure efficient translation of cellular messenger RNAs (mRNAs). Instead, CfMV RNA is covalently linked to a viral protein VPg (viral protein, genome-linked). This indicates that the viral untranslated regions (UTRs) must functionally compensate for the lack of the cap and poly(A) tail. We examined the efficacy of translation initiation in CfMV by comparing it to well-studied viral translational enhancers. Although insertion of the CfMV 5'UTR (CfMVe) into plant expression vectors improved gene expression in barley more than the other translational enhancers examined, studies at the RNA level showed that CfMVe alone or in combination with the CfMV 3'UTR did not provide the RNAs translational advantage. Mutation analysis revealed that translation initiation from CfMVe involved scanning. Interestingly, CfMVe also promoted translation initiation from an intercistronic position of dicistronic mRNAs in vitro. Furthermore, internal initiation occurred with similar efficacy in translation lysates that had reduced concentrations of eukaryotic initiation factor (eIF) 4E, suggesting that initiation was independent of the eIF4E. In contrast, reduced translation in the eIF4G-depleted lysates indicated that translation from internally positioned CfMVe was eIF4G-dependent. After successful translation initiation, leaky scanning brings the ribosomes to the second open reading frame (ORF). The CfMV polyprotein is produced from this and the following overlapping ORF via programmed -1 ribosomal frameshift (-1 PRF). Two signals in the mRNA at the beginning of the overlap program approximately every fifth ribosome to slip one nucleotide backwards and continue translation in the new -1 frame. This leads to the production of C-terminally extended polyprotein, which encodes the viral RNA-dependent RNA polymerase (RdRp). The -1 PRF event in CfMV was very efficient, even though it was programmed by a simple stem-loop structure instead of a pseudoknot, which is usually required for high -1 PRF frequencies. Interestingly, regions surrounding the -1 PRF signals improved the -1 PRF frequencies. Viral protein P27 inhibited the -1 PRF event in vivo, putatively by binding to the -1 PRF site. This suggested that P27 could regulate the occurrence of -1 PRF. Initiation of viral replication requires that viral proteins are released from the polyprotein. This is catalyzed by viral serine protease, which is also encoded from the polyprotein. N-terminal amino acid sequencing of CfMV VPg revealed that the junction of the protease and VPg was cleaved between glutamate (E) and asparagine (N) residues. This suggested that the processing sites used in CfMV differ from the glutamate and serine (S) or threonine (T) sites utilized in other sobemoviruses. However, further analysis revealed that the E/S and E/T sites may be used to cleave out some of the CfMV proteins.

Genetic diversity and microcystin production by Anabaena in the Gulf of Finland, Baltic Sea

Syanobakteerit (sinilevät) ovat olleet Itämeressä koko nykymuotoisen Itämeren ajan, sillä paleolimnologiset todisteet niiden olemassaolosta Itämeren alueella ovat noin 7000 vuoden takaa. Syanobakteerien massaesiintymät eli kukinnat ovat kuitenkin sekä levinneet laajemmille alueille että tulleet voimakkaimmiksi viimeisten vuosikymmenien aikana. Tähän on osasyynä ihmisten aiheuttama kuormitus, joka rehevöittää Itämerta. Suomenlahti, jota tämä tutkimus käsittelee, on kärsinyt tästä rehevöitymiskehityksestä muita Itämeren altaita enemmän. Syanobakteerit muodostavat jokakesäisiä kukintoja Suomenlahdella - niin sen avomerialueilla kuin rannoillakin. Yleisimmät kukintoja muodostavat syanobakteerisuvut ovat Nodularia, Anabaena ja Aphanizomenon. Kukinnat aiheuttavat paitsi esteettistä haittaa myös terveydellisen riskitekijän. Niiden myrkyllisyys liitetään usein Nodularia-suvun tuottamaan nodulariini-maksamyrkkyyn. Itämeren Aphanizomenon-suvun on todettu olevan myrkytön. Vaikka Itämeren kukintoja aiheuttavista Nodularia- ja Aphanizomenon-syanobakteereista tiedetään varsin paljon, on molekyylimenetelmiin pohjautuva syanobakteeritutkimus ohittanut Itämeren Anabaena-suvun monelta osin. Tämän työn tarkoituksena oli syventää käsitystämme Itämeren Anabaena-syanobakteerista, sen mahdollisesta myrkyllisyydestä, geneettisestä monimuotoisuudesta ja fylogeneettisista sukulaisuussuhteista. Tässä työssä eristettiin 49 planktista Anabaena-kantaa, joista viisi tuottivat mikrokystiinejä. Tämä oli ensimmäinen yksiselitteinen todiste, että Itämeren Anabaena tuottaa maksamyrkyllisiä mikrokystiini-yhdisteitä. Jokainen eristetty myrkyllinen Anabaena-kanta tuotti useita mikrokystiini-variantteja. Lisäksi mikrokystiinejä löydettiin kukintanäytteistä, joissa oli myrkkyä syntetisoivia geenejä sisältäneitä Anabaena-syanobakteereita. Myrkkyjä löydettiin molempina tutkimusvuosina 2003 ja 2004. Myrkkyjen esiintyminen ei siten ollut vain yksittäinen ilmiö. Tässä työssä saimme viitteitä siitä, että maksamyrkyllinen Anabaena-syanobakteeri esiintyisi vähäsuolaisissa vesissä. Tämä riippuvuussuhde jää kuitenkin tulevien tutkimuksien selvitettäväksi. Tässä työssä havaittiin mikrokystiinisyntetaasi-geenien inaktivoituminen Itämeren Anabaena-kannassa ja kukintanäytteissä. Kuvasimme Anabaena-kannan mikrokystiinisyntetaasigeenien sisältä insertioita, jotka hyvin todennäköisesti inaktivoivat myrkyntuoton. Insertion sisältäneeltä kannalta löysimme kuitenkin kaikki mikrokystiinisyntetaasigeenit osoittaen, että geenien olemassaolo ei välttämättä varmista kannan mikrokystiinintuottoa. Mielenkiintoista oli se, että inaktivaation aiheuttavia insertioita löytyi kukintanäytteistä molemmilta tutkimusvuosilta. Vastaavia insertioita ei kuitenkaan löydetty makean veden Anabaena-kannoista tai järvinäytteistä. On yleistä, että syanobakteerikukinnoista löytyy usean syanobakteerisuvun edustajia. Myrkyllisiä sukuja tai lajeja ei voida kuitenkaan erottaa mikroskooppisesti myrkyttömistä. Käsillä olevassa tutkimuksessa kehitettiin molekyylimenetelmä, jolla on mahdollista määrittää kukinnan mahdollisesti maksamyrkylliset syanobakteerisuvut. Tätä menetelmää sovellettiin Itämeren kukintojen tutkimiseen. Itämeren pintavesistä ja ranta-alueiden pohjasta eristetyt Anabaena-kannat osoittautuivat geneettisesti monimuotoisiksi. Tämä Anabaena-syanobakteerien geneettinen monimuotoisuus vahvistettiin monistamalla geenejä suoraan kukintanäytteistä ilman kantojen eristystä. Makeiden vesien ja Itämeren Anabaena-kannat ovat geneettisesti hyvin samankaltaisia. Geneettisissä vertailuissa kävi kuitenkin ilmi, että pohjassa elävien Anabaena-kantojen geneettinen monimuotoisuus oli suurempaa kuin pintavesistä eristettyjen kantojen. Itämeren Anabaena-kantojen sekvenssit muodostivat omia ryhmiä sukupuun sisällä, jolloin on mahdollista, että nämä edustavat Itämeren omia Anabaena-ekotyyppejä. Tämä tutkimus oli ensimmäinen, jossa uusin molekyylimenetelmin systemaattisesti selvitettiin Itämeren Anabaena-syanobakteerin geneettistä populaatiorakennetta, fylogeniaa ja myrkyntuottoa. Tulevaisuudessa monitorointitutkimuksissa on otettava huomioon myös Itämeren Anabaena-syanobakteerin mahdollinen maksamyrkyntuotto – erityisesti vähäsuolaisemmilla rannikkovesillä.

A New Rare-Gas Compound: HXeOXeH

Rare-gas chemistry is of growing interest, and the recent advances include the "insertion" of a Xe atom into OH and water in the rare-gas hydrides HXeO and HXeOH. The insertion of Xe atoms into the H-C bonds of hydrocarbons was also demonstrated for HXeCC, HXeCCH and HXeCCXeH, the last of which was the first rare-gas hydride containing two rare-gas atoms. We describe the preparation and characterization of a new rare-gas compound, HXeOXeH. HXeOXeH was prepared in solid xenon by photolysis of a suitable precursor, for example water, and subsequent mobilization of the photoproducts. The experimental identification was carried out by FTIR spectroscopy, isotopic substitution and by use of various precursors. The photolytical and thermal stability of the new rare-gas hydride was also studied. The experimental work was supported by extensive quantum chemical calculations provided by our co-workers. HXeOXeH forms in a cryogenic xenon matrix from neutral O and H atoms in a two-step diffusion-controlled process involving HXeO as an intermediate [reactions (1) and (2)]. This formation mechanism is unique in that a rare-gas hydride is formed from another rare-gas hydride. H + Xe + O → HXeO (1) HXeO + Xe + H → HXeOXeH (2) Similarly to other rare-gas hydrides, HXeOXeH has a strongly IR-active H-Xe stretching vibration, allowing its spectral detection at 1379.3 cm-1. HXeOXeH is a very high-energy metastable species, yet thermally more stable than many other rare-gas hydrides. The calculated bending barrier of 0.57 eV, is not enough to explain the observed stability, and HXeOXeH might be affected by additional stabilization from the solid xenon environment. Chemical bonding between xenon and environmentally abundant species like water is of particular importance due to the “missing-xenon” problem. The relatively high thermal stability of HXeOXeH compared to other oxygen containing rare-gas compounds is relevant in this respect. Our work also raises the possibility of polymeric (–Xe–O)n networks, similarly to the computationally studied (XeCC)n polymers.

Mu in vitro transposition applications in protein engineering

Transposons, mobile genetic elements that are ubiquitous in all living organisms have been used as tools in molecular biology for decades. They have the ability to move into discrete DNA locations with no apparent homology to the target site. The utility of transposons as molecular tools is based on their ability to integrate into various DNA sequences efficiently, producing extensive mutant clone libraries that can be used in various molecular biology applications. Bacteriophage Mu is one of the most useful transposons due to its well-characterized and simple in vitro transposition reaction. This study establishes the properties of the Mu in vitro transposition system as a versatile multipurpose tool in molecular biology. In addition, this study describes Mu-based applications for engineering proteins by random insertional transposon mutagenesis in order to study structure-function relationships in proteins. We initially characterized the properties of the minimal Mu in vitro transposition system. We showed that the Mu transposition system works efficiently and accurately and produces insertions into a wide spectrum of target sites in different DNA molecules. Then, we developed a pentapeptide insertion mutagenesis strategy for inserting random five amino acid cassettes into proteins. These protein variants can be used especially for screening important sites for protein-protein interactions. Also, the system may produce temperature-sensitive variants of the protein of interest. Furthermore, we developed an efficient screening system for high-resolution mapping of protein-protein interfaces with the pentapeptide insertion mutagenesis. This was accomplished by combining the mutagenesis with subsequent yeast two-hybrid screening and PCR-based genetic footprinting. This combination allows the analysis of the whole mutant library en masse, without the need for producing or isolating separate mutant clones, and the protein-protein interfaces can be determined at amino acid accuracy. The system was validated by analysing the interacting region of JFC1 with Rab8A, and we show that the interaction is mediated via the JFC1 Slp homology domain. In addition, we developed a procedure for the production of nested sets of N- and C-terminal deletion variants of proteins with the Mu system. These variants are useful in many functional studies of proteins, especially in mapping regions involved in protein-protein interactions. This methodology was validated by analysing the region in yeast Mso1 involved in an interaction with Sec1. The results of this study show that the Mu in vitro transposition system is versatile for various applicational purposes and can efficiently be adapted to random protein engineering applications for functional studies of proteins.

Thyroid Hormone Control of Cardiac Substrate Metabolism

Thyroid hormone (TH) plays an important role in maintaining a homeostasis in all the cells of our body. It also has significant cardiovascular effects, and abnormalities of its concentration can cause cardiovascular disease and even morbidity. Especially development of heart failure has been connected to low levels of thyroid hormone. A decrease in TH levels or TH-receptor binding adversely effects cardiac function. Although, this occurs in part through alterations in excitation-contraction and transport proteins, recent data from our laboratory indicate that TH also mediates changes in myocardial energy metabolism. Thyroid dysfunction may limit the heart s ability to shift substrate pathways and provide adequate energy supply during stress responses. Our goals of these studies were to determine substrate oxidation pattern in systemic and cardiac specific hypothyroidism at rest and at higher rates of oxygen demand. Additionally we investigated the TH mediated mechanisms in myocardial substrate selection and established the metabolic phenotype caused by a thyroid receptor dysfunction. We measured cardiac metabolism in an isolated heart model using 13Carbon isotopomer analyses with MR spectroscopy to determine function, oxygen consumption, fluxes and fractional contribution of acetyl-CoA to the citric acid cycle (CAC). Molecular pathways for changes in cardiac function and substrate shifts occurring during stress through thyroid receptor abnormalities were determined by protein analyses. Our results show that TH modifies substrate selection through nuclear-mediated and rapid posttranscriptional mechanisms. It modifies substrate selection differentially at rest and at higher rates of oxygen demand. Chronic TH deficiency depresses total CAC flux and selectively fatty acid flux, whereas acute TH supplementation decreases lactate oxidation. Insertion of a dominant negative thyroid receptor (Δ337T) alters metabolic phenotype and contractive efficiency in heart. The capability of the Δ337T heart to increase carbohydrate oxidation in response to stress seems to be limited. These studies provided a clearer understanding of the TH role in heart disease and shed light to identification of the molecular mechanisms that will facilitate in finding targets for heart failure prevention and treatment.

Type III Secretion System of Phytopathogenic Bacterium Pseudomonas syringae:From Gene to Function

The type III secretion system (T3SS) is an essential requirement for the virulence of many Gram-negative bacteria which infect plants, animals and men. Pathogens use the T3SS to deliver effector proteins from the bacterial cytoplasm to the eukaryotic host cells, where the effectors subvert host defenses. The best candidates for directing effector protein traffic are the bacterial type III-associated appendages, called needles or pili. In plant pathogenic bacteria, the best characterized example of a T3SS-associated appendage is the HrpA pilus of the plant pathogen Pseudomonas syringae pv. tomato DC3000. The components of the T3SS in plant pathogens are encoded by a cluster of hrp (hypersensitive reaction and pathogenicity) genes. Two major classes of T3SS-secreted proteins are: harpin proteins such as HrpZ which are exported into extracellular space, and avirulence (Avr) proteins such as AvrPto which are translocated directly to the plant cytoplasm. This study deals with the structural and functional characterization of the T3SS-associated HrpA pilus and the T3SS-secreted harpins. By insertional mutagenesis analysis of HrpA, we located the optimal epitope insertion site in the amino-terminus of HrpA, and revealed the potential application of the HrpA pilus as a carrier of antigenic determinants for vaccination. By pulse-expression of proteins combined with immuno-electron microscopy, we discovered the Hrp pilus assembly strategy as addition of HrpA subunits to the distal end of the growing pilus, and we showed for the first time that secretion of HrpZ occurs at the tip of the pilus. The pilus thus functions as a conduit delivering proteins to the extracellular milieu. By using phage-display and scanning-insertion mutagenesis methods we identified a conserved HrpZ-binding peptide and localized the peptide-binding site to the central domain of HrpZ. We also found that the HrpZ specifically interacts with a host bean protein. Taken together, the current results provide deeper insight into the molecular mechanism of T3SS-associated pilus assembly and effector protein translocation, which will be helpful for further studies on the pathogenic mechanisms of Gram-negative bacteria and for developing new strategies to prevent bacterial infection.

Characterization of genomic diversity in extraintestinal pathogenic Escherichia coli (ExPEC) and development of a diagnostic DNA microarray for the differentiation of ExPEC isolates causing urinary tract infections

Extraintestinal pathogenic Escherichia coli (ExPEC) represent a diverse group of strains of E. coli, which infect extraintestinal sites, such as the urinary tract, the bloodstream, the meninges, the peritoneal cavity, and the lungs. Urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC), the major subgroup of ExPEC, are among the most prevalent microbial diseases world wide and a substantial burden for public health care systems. UTIs are responsible for serious morbidity and mortality in the elderly, in young children, and in immune-compromised and hospitalized patients. ExPEC strains are different, both from genetic and clinical perspectives, from commensal E. coli strains belonging to the normal intestinal flora and from intestinal pathogenic E. coli strains causing diarrhea. ExPEC strains are characterized by a broad range of alternate virulence factors, such as adhesins, toxins, and iron accumulation systems. Unlike diarrheagenic E. coli, whose distinctive virulence determinants evoke characteristic diarrheagenic symptoms and signs, ExPEC strains are exceedingly heterogeneous and are known to possess no specific virulence factors or a set of factors, which are obligatory for the infection of a certain extraintestinal site (e. g. the urinary tract). The ExPEC genomes are highly diverse mosaic structures in permanent flux. These strains have obtained a significant amount of DNA (predictably up to 25% of the genomes) through acquisition of foreign DNA from diverse related or non-related donor species by lateral transfer of mobile genetic elements, including pathogenicity islands (PAIs), plasmids, phages, transposons, and insertion elements. The ability of ExPEC strains to cause disease is mainly derived from this horizontally acquired gene pool; the extragenous DNA facilitates rapid adaptation of the pathogen to changing conditions and hence the extent of the spectrum of sites that can be infected. However, neither the amount of unique DNA in different ExPEC strains (or UPEC strains) nor the mechanisms lying behind the observed genomic mobility are known. Due to this extreme heterogeneity of the UPEC and ExPEC populations in general, the routine surveillance of ExPEC is exceedingly difficult. In this project, we presented a novel virulence gene algorithm (VGA) for the estimation of the extraintestinal virulence potential (VP, pathogenicity risk) of clinically relevant ExPECs and fecal E. coli isolates. The VGA was based on a DNA microarray specific for the ExPEC phenotype (ExPEC pathoarray). This array contained 77 DNA probes homologous with known (e.g. adhesion factors, iron accumulation systems, and toxins) and putative (e.g. genes predictably involved in adhesion, iron uptake, or in metabolic functions) ExPEC virulence determinants. In total, 25 of DNA probes homologous with known virulence factors and 36 of DNA probes representing putative extraintestinal virulence determinants were found at significantly higher frequency in virulent ExPEC isolates than in commensal E. coli strains. We showed that the ExPEC pathoarray and the VGA could be readily used for the differentiation of highly virulent ExPECs both from less virulent ExPEC clones and from commensal E. coli strains as well. Implementing the VGA in a group of unknown ExPECs (n=53) and fecal E. coli isolates (n=37), 83% of strains were correctly identified as extraintestinal virulent or commensal E. coli. Conversely, 15% of clinical ExPECs and 19% of fecal E. coli strains failed to raster into their respective pathogenic and non-pathogenic groups. Clinical data and virulence gene profiles of these strains warranted the estimated VPs; UPEC strains with atypically low risk-ratios were largely isolated from patients with certain medical history, including diabetes mellitus or catheterization, or from elderly patients. In addition, fecal E. coli strains with VPs characteristic for ExPEC were shown to represent the diagnostically important fraction of resident strains of the gut flora with a high potential of causing extraintestinal infections. Interestingly, a large fraction of DNA probes associated with the ExPEC phenotype corresponded to novel DNA sequences without any known function in UTIs and thus represented new genetic markers for the extraintestinal virulence. These DNA probes included unknown DNA sequences originating from the genomic subtractions of four clinical ExPEC isolates as well as from five novel cosmid sequences identified in the UPEC strains HE300 and JS299. The characterized cosmid sequences (pJS332, pJS448, pJS666, pJS700, and pJS706) revealed complex modular DNA structures with known and unknown DNA fragments arranged in a puzzle-like manner and integrated into the common E. coli genomic backbone. Furthermore, cosmid pJS332 of the UPEC strain HE300, which carried a chromosomal virulence gene cluster (iroBCDEN) encoding the salmochelin siderophore system, was shown to be part of a transmissible plasmid of Salmonella enterica. Taken together, the results of this project pointed towards the assumptions that first, (i) homologous recombination, even within coding genes, contributes to the observed mosaicism of ExPEC genomes and secondly, (ii) besides en block transfer of large DNA regions (e.g. chromosomal PAIs) also rearrangements of small DNA modules provide a means of genomic plasticity. The data presented in this project supplemented previous whole genome sequencing projects of E. coli and indicated that each E. coli genome displays a unique assemblage of individual mosaic structures, which enable these strains to successfully colonize and infect different anatomical sites.

Obstetric and gynaecological aspects of HIV infection

The purpose of the present study was to examine the outcome of pregnancies among HIV-infected women in Helsinki, use of the levonorgestrel-releasing intrauterine system (LNG-IUS) among HIV-infected women and the prevalence and risk factors of cytological and histologically proven cervical lesions in this population. Between 1993 and 2003 a total of 45 HIV-infected women delivered 52 singleton infants. HIV infection was diagnosed during pregnancy in 40% of the mothers. Seventeen of the mothers received antiretroviral (ARV) medication prior to pregnancy and in 34 cases, the medication was started during pregnancy. A good virological response (i.e. HIV RNA load <1000/mL during the last trimester) to ARV medication was achieved in 36/40 (90%) of the patients in whom HI viral load measurements were performed. Of the infants, 92% were born at term, and their mean (±SD) birth weight was 3350±395 g. The Caesarean section rate was low, 25%. All newborns received ARV medication and none of the infants born to mothers with pre-delivery diagnosis of maternal HIV infection were infected. The safety and advantages of the LNG-IUS were studied prospectively (n=12) and retrospectively (n=6). The LNG-IUS was well tolerated and no cases of PID or pregnancy were noted. Menstrual bleeding was reduced significantly during use of the LNG-IUS; this was associated with a slight increase in haemoglobin levels. Serum oestradiol concentrations remained in the follicular range in all subjects. The key finding was that genital shedding of HIV RNA did not change after the insertion of the LNG-IUS. The mean annual prevalence of low-grade squamous intraepithelial lesions (SIL) was 15% and that of high-grade SIL was 5% among 108 systematically followed HIV-infected women during 1989 2003. A reduced CD4 lymphocyte count was associated with an increased prevalence of SIL, whereas duration of HIV infection, use of ARV medication and HI viral load were not. The cumulative risk of any type of SIL was 17% after one year and 48% after five years among patients with initially normal Pap smears. The risk of developing SIL was associated with young age and a high initial HI viral load. During the follow-up 51 subjects (n=153) displayed cervical intraepithelial neoplasia (CIN), (16% CIN1 and 18% CIN 2-3). Only one case of cancer of the uterine cervix was detected. Pap smears were reliable in screening for CIN. Both nulliparity (p<0.01) and bacterial vaginosis (p<0.04) emerged as significant risk factors of CIN. In conclusion, a combination of universal antenatal screening and multidisciplinary management allows individualized treatment and prevents vertical transmission of HIV. Use of the LNG-IUS is safe among HIV-infected women and cervicovaginal shedding of HIV RNA is not affected by use of the LNG-IUS. The risk of cervical pre-malignant lesions is high among HIV-infected women despite systematic follow-up.

Childhood otitis media - risk factors and surgical management

Background: Otitis media (OM) is one of the most common childhood diseases. Approximately every third child suffers from recurrent acute otitis media (RAOM), and 5% of all children have persistent middle ear effusion for months during their childhood. Despite numerous studies on the prevention and treatment of OM during the past decades, its management remains challenging and controversial. In this study, the effect of adenoidectomy on the risk for OM, the potential risk factors influencing the development of OM and the frequency of asthma among otitis-prone children were investigated. Subjects and methods: One prospective randomized trial and two retrospective studies were conducted. In the prospective trial, 217 children with RAOM or chronic otitis media with effusion (COME) were randomized to have tympanostomy with or without adenoidectomy. The age of the children at recruitment was between 1 and 4 years. RAOM was defined as having at least 3 episodes of AOM during the last 6 months or at least 5 episodes of AOM during the last 12 months. COME was defined as having persistent middle ear effusion for 2-3 months. The children were followed up for one year. In the first retrospective study, the frequency of childhood infections and allergy was evaluated by a questionnaire among 819 individuals. In the second retrospective study, data of asthma diagnosis were analysed from hospital discharge records of 1616 children who underwent adenoidectomy or had probing of the nasolacrimal duct. Results: In the prospective randomized study, adenoidectomy had no beneficial effect on the prevention of subsequent episodes of AOM. Parental smoking was found to be a significant risk factor for OM even after the insertion of tympanostomy tubes. The frequencies of exposure to tobacco smoke and day-care attendance at the time of randomization were similar among children with RAOM and COME. However, the frequencies of allergy to animal dust and pollen and parental asthma were lower among children with COME than those with RAOM. The questionnaire survey and the hospital discharge data revealed that children who had frequent episodes of OM had an increased risk for asthma. Conclusions: The first surgical intervention to treat an otitis-prone child younger than 4 years should not include adenoidectomy. Interventions to stop parental smoking could significantly reduce the risk for childhood RAOM. Whether an otitis-prone child develops COME or RAOM, seems to be influenced by genetic predisposition more strongly than by environmental risk factors. Children who suffer from repeated upper respiratory tract infections, like OM, may be at increased risk for developing asthma.

Functional role of the Mso1p-Sec1p complex in membrane fusion regulation

Sec1/Munc18 (SM) protein family members are evolutionary conserved proteins. They perform an essential, albeit poorly understood function in SNARE complex formation in membrane fusion. In addition to the SNARE complex components, only a few SM protein binding proteins are known. Typically, their binding modes to SM proteins and their contribution to the membrane fusion regulation is poorly characterised. We identified Mso1p as a novel Sec1p interacting partner. It was shown that Mso1p and Sec1p interact at sites of polarised secretion and that this localisation is dependent on the Rab GTPase Sec4p and its GEF Sec2p. Using targeted mutagenesis and N- and C-terminal deletants, it was discovered that the interaction between an N-terminal peptide of Mso1p and the putative Syntaxin N-peptide binding area in Sec1p domain 1 is important for membrane fusion regulation. The yeast Syntaxin homologues Sso1p and Sso2p lack the N-terminal peptide. Our results show that in addition to binding to the putative N-peptide binding area in Sec1p, Mso1p can interact with Sso1p and Sso2p. This result suggests that Mso1p can mimic the N-peptide binding to facilitate membrane fusion. In addition to Mso1p, a novel role in membrane fusion regulation was revealed for the Sec1p C-terminal tail, which is missing in its mammalian homologues. Deletion of the Sec1p-tail results in temperature sensitive growth and reduced sporulation. Using in vivo and in vitro experiments, it was shown that the Sec1p-tail mediates SNARE complex binding and assembly. These results propose a regulatory role for the Sec1p-tail in SNARE complex formation. Furthermore, two novel interaction partners for Mso1p, the Rab GTPase Sec4p and plasma membrane phospholipids, were identified. The Sec4p link was identified using Bimolecular Fluorescence Complementation assays with Mso1p and the non-SNARE binding Sec1p(1-657). The assay revealed that Mso1p can target Sec1p(1-657) to sites of secretion. This effect is mediated via the Mso1p C-terminus, which previously has been genetically linked to Sec4p. These results and in vitro binding experiments suggest that Mso1p acts in cooperation with the GTP-bound form of Sec4p on vesicle-like structures prior to membrane fusion. Mso1p shares homology with the PIP2 binding domain of the mammalian Munc18 binding Mint proteins. It was shown both in vivo and in vitro that Mso1p is a phospholipid inserting protein and that this insertion is mediated by the conserved Mso1p amino terminus. In vivo, the Mso1p phospholipid binding is needed for sporulation and Mso1p-Sec1p localisation at the sites of secretion at the plasma membrane. The results reveal a novel layer of membrane fusion regulation in exocytosis and propose a coordinating role for Mso1p in connection with membrane lipids, Sec1p, Sec4p and SNARE complexes in this process.

Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS

Reactive oxygen species (ROS) have important functions in plant stress responses and development. In plants, ozone and pathogen infection induce an extracellular oxidative burst that is involved in the regulation of cell death. However, very little is known about how plants can perceive ROS and regulate the initiation and the containment of cell death. We have identified an Arabidopsis thaliana protein, GRIM REAPER (GRI), that is involved in the regulation of cell death induced by extracellular ROS. Plants with an insertion in GRI display an ozone-sensitive phenotype. GRI is an Arabidopsis ortholog of the tobacco flower-specific Stig1 gene. The GRI protein appears to be processed in leaves with a release of an N-terminal fragment of the protein. Infiltration of the N-terminal fragment of the GRI protein into leaves caused cell death in a superoxide-and salicylic acid-dependent manner. Analysis of the extracellular GRI protein yields information on how plants can initiate ROS-induced cell death during stress response and development.