Genome Sequence of Klebsiella pneumoniae Ecl8, a Reference Strain for Targeted Genetic Manipulation

We report the genome sequence of Klebsiella pneumoniae subsp. pneumoniae Ecl8, a spontaneous streptomycin-resistant mutant of strain ECL4, derived from NCIB 418. K. pneumoniae Ecl8 has been shown to be genetically tractable for targeted gene deletion strategies and so provides a platform for in-depth analyses of this species.

Multidrug resistance protein MdtM adds to the repertoire of antiporters involved in alkaline pH homeostasis in Escherichia coli

Background: In neutralophilic bacteria, monovalent metal cation/H+ antiporters play a key role in pH homeostasis. In Escherichia coli, only four antiporters (NhaA, NhaB, MdfA and ChaA) are identified to function in maintenance of a stable cytoplasmic pH under conditions of alkaline stress. We hypothesised that the multidrug resistance protein MdtM, a recently characterised homologue of MdfA and a member of the major facilitator superfamily, also functions in alkaline pH homeostasis.
Results: Assays that compared the growth of an E. coli ΔmdtM deletion mutant transformed with a plasmid encoding wild-type MdtM or the dysfunctional MdtM D22A mutant at different external alkaline pH values (ranging from pH 8.5 to 10) revealed a potential contribution by MdtM to alkaline pH tolerance, but only when millimolar concentrations of sodium or potassium was present in the growth medium. Fluorescence-based activity assays using inverted vesicles generated from transformants of antiporter-deficient (ΔnhaA, ΔnhaB, ΔchaA) E. coli TO114 cells defined MdtM as a low-affinity antiporter that catalysed electrogenic exchange of Na+, K+, Rb+ or Li+ for H+. The K+/H+ antiport reaction had a pH optimum at 9.0, whereas the Na+/H+ exchange activity was optimum at pH 9.25. Measurement of internal cellular pH confirmed MdtM as contributing to maintenance of a stable cytoplasmic pH, acid relative to the external pH, under conditions of alkaline stress.
Conclusions: Taken together, the results support a role for MdtM in alkaline pH tolerance. MdtM can therefore be added to the currently limited list of antiporters known to function in pH homeostasis in the model organism E. coli.

Transfer of amiprophosmethyl resistance from a Nicotiana plumbaginifolia mutant by somatic hybridisation

Transfer of resistance to the phosphorothioamidate herbicide, amiprophosmethyl (APM), from the P-tubulin mutant of Nicotiana plumbaginifolia to the interspecific N, plumbaginifolia (+) N, sylvestris is and to the intertribal N, plumbaginifolia (+) Atropa belladonna somatic hybrids has been demonstrated. Transfer to the recipient species was accomplished by: (1) symmetric hybridisation and (2) asymmetric hybridisation using gamma-irradiation of donor protoplasts. Cytogenetic analysis confirmed the hybrid origin of the hybrids obtained. It was established that most of them typically inherited no more than three donor chromosomes, although it was possible to obtain symmetric hybrids in the case of symmetric fusion. Immunofluorescent microscopy analysis has shown that protoplasts of the mutant, and of the N. plumbagini-folia (+) N. sylvestris and N. plumbaginifolia (+) A. belladonna hybrids, retained the normal structure of interphase microtubule (MT) arrays and mitotic figures after treatment with 5 mu M APM, whereas MTs of protoplasts of the recipients were destroyed under these conditions. It was also shown that hybrid clones contained an altered beta-tubulin isoform originating from the N. plumbaginifolia mutant. The selected hybrid clones were characterised by cross-resistance to trifluralin, a dinitroaniline herbicide with the same mode of anti-MT action. Some of the somatic hybrids which could flower were fertile. It was established that seeds of some fertile hybrids were able to germinate in the presence of 5 mu M APM. The results obtained thus support the conclusion that the technique of somatic hybridisation, especially asymmetric fusion, can be used to transfer APM resistance from the N. plumbaginifolia mutant to different (related and remote) plant species of the Solanaceae, including important crops.

Putrescine Reduces Antibiotic-Induced Oxidative Stress as a Mechanism of Modulation of Antibiotic Resistance in Burkholderia cenocepacia

Communication of antibiotic resistance among bacteria via small molecules is implicated in transient reduction of bacterial susceptibility to antibiotics, which could lead to therapeutic failures aggravating the problem of antibiotic resistance. Released putrescine from the extremely antibiotic resistant bacterium Burkholderia cenocepacia protects less resistant cells from different species against the antimicrobial peptide polymyxin B (PmB). Exposure of B. cenocepacia to sub-lethal concentrations of PmB and other bactericidal antibiotics induce reactive oxygen species (ROS) production and expression of the oxidative stress response regulator OxyR. We evaluated whether putrescine alleviates antibiotic-induced oxidative stress. The accumulation of intracellular ROS such as superoxide ion and hydrogen peroxide was assessed fluorometrically with dichlorofluorescein diacetate, while the expression of OxyR and putrescine synthesis enzymes was determined in luciferase assays using chromosomal promoter-lux reporter system fusions. We evaluated wild type and isogenic deletion mutant strains with defects in putrescine biosynthesis after exposure to sub-lethal concentrations of PmB and other bactericidal antibiotics. Exogenous putrescine protected against oxidative stress induced by PmB and other antibiotics, whereas reduced putrescine synthesis resulted in increased ROS generation, and a parallel increased sensitivity to PmB. Of the 3 B. cenocepacia putrescine synthesizing enzymes, PmB induced only BCAL2641, an ornithine decarboxylase. This study exposes BCAL2641 as a critical component of the putrescine-mediated communication of antibiotic resistance, and as a plausible target for designing inhibitors that would block the communication of such resistance among different bacteria, ultimately reducing the window of therapeutic failure in treating bacterial infections.

Electron Transfer Involving the Phylloquinone (A1) Cofactor of Photosystem I Examined with Time Resolved Absorbance and Electron Paramagnetic Resonance Spectroscopy

The dependence of the electron transfer (ET) rate on the Photosystem I (PSI) cofactor phylloquinone (A1) is studied by time-resolved absorbance and electron paramagnetic resonance (EPR) spectroscopy. Two active branches (A and B) of electron transfer converge to the FX cofactor from the A1A and A1B quinone. The work described in Chapter 5 investigates the single hydrogen bond from the amino acid residue PsaA-L722 backbone nitrogen to A1A for its effect on the electron transfer rate to FX. Room temperature transient EPR measurements show an increase in the rate for the A1A- to FX for the PsaA-L722T mutant and an increased hyperfine coupling to the 2-methyl group of A1A when compared to wild type. The Arrhenius plot of the A1A- to FX ET in the PsaA-L722T mutant suggests that the increased rate is probably the result of a slight change in the electronic coupling between A1A- and FX. The reasons for the non-Arrhenius behavior are discussed. The work discussed in Chapter 6 investigates the directionality of ET at low temperature by blocking ET to the iron-sulfur clusters FX, FA and FB in the menB deletion mutant strain of Synechocyctis sp. PCC 6803, which is unable to synthesize phylloquinone, by incorporating the high midpoint potential (49 mV vs SHE) 2,3-dichloro-1,4-naphthoquinone (Cl2NQ) into the A1A and A1B binding sites. Various EPR spectroscopic techniques were implemented to differentiate between the spectral features created from A and B- branch electron transfer. The implications of this result for the directionality of electron transfer in PS I are discussed. The work discussed in Chapter 7 was done to study the dependence of the heterogeneous ET at low temperature on A1 midpoint potential. The menB PSI mutant contains plastiquinone-9 in the A1 binding site. The solution midpoint potential of the quinone measures 100 mV more positive then wild-type phylloquinone. The irreversible ET to the terminal acceptors FA and FB at low temperature is not controlled by the forward step from A1 to FX as expected due to the thermodynamic differences of the A1 cofactor in the two active branches A and B. Alternatives for the ET heterogeneity are discussed.

Process Improvement of Coir Pith Degradation Involving White Rot Fungi By Substitution of Urea With Nitrogen Fixing Bacteria and Application of Biodegraded Coir Pith as Plant Growth Medium

In this study, a novel improved technology could be developed to convert the recalcitrant coir pith into environmental friendly organic manure. The standard method of composting involves the substitution of urea with nitrogen fixing bacteria viz. Azotobacter vinelandii and Azospirillum brasilense leading to the development of an improved method of coir pith. The combined action of the microorganisms could enhance the biodegradation of coir pith. In the present study, Pleurotus sajor caju, an edible mushroom which has the ability to degrade coir pith, and the addition of nitrogen fixing bacteria like Azotobacter vinelandii and Azospirillum brasilense could accelerate the action of the fungi on coir pith. The use of these microorganisms brings about definite changes in the NPK, Ammonia, Organic Carbon and Lignin contents in coir pith. This study will encourage the use of biodegraded coir pith as organic manure for agri/horti purpose to get better yields and can serve as a better technology to solve the problem of accumulated coir pith in coir based industries

The CtsR regulator of Listeria monocytogenes contains a variant glycine repeat region that affects piezotolerance, stress resistance, motility and virulence

A spontaneous high hydrostatic pressure (HHP)-tolerant mutant of Listeria monocytogenes ScottA, named AK01, was isolated previously. This mutant was immotile and showed increased resistance to heat, acid and H2O2 compared with the wild type (wt) (Karatzas, K.A.G. and Bennik, M.H.J. 2002 Appl Environ Microbiol 68: 3183–3189). In this study, we conclusively linked the increased HHP and stress tolerance of strain AK01 to a single codon deletion in ctsR (class three stress gene repressor) in a region encoding a highly conserved glycine repeat. CtsR negatively regulates the expression of the clp genes, including clpP, clpE and the clpC operon (encompassing ctsR itself), which belong to the class III heat shock genes. Allelic replacement of the ctsR gene in the wt background with the mutant ctsR gene, designated ctsRΔGly, rendered mutants with phenotypes and protein expression profiles identical to those of strain AK01. The expression levels of CtsR, ClpC and ClpP proteins were significantly higher in ctsRΔGly mutants than in the wt strain, indicative of the CtsRΔGly protein being inactive. Further evidence that the CtsRΔGly protein lacks its repressor function came from the finding that the Clp proteins in the mutant were not further induced upon heat shock, and that HHP tolerance of a ctsR deletion strain was as high as that of a ctsRΔGly mutant. The high HHP tolerance possibly results from the increased expression of the clp genes in the absence of (active) CtsR repressor. Importantly, the strains expressing CtsRΔGly show significantly attenuated virulence compared with the wt strain; however, no indication of disregulation of PrfA in the mutant strains was found. Our data highlight an important regulatory role of the glycine-rich region of CtsR in stress resistance and virulence.

Deletion 5q12: Delineation of a Phenotype Including Mental Retardation and Ocular Defects

Array-CGH enables the detection of submicroscopic chromosomal deletions and duplications and leads to an accurate delineation of the imbalances, raising the possibility of correlating genotype to phenotype and mapping minimal critical regions associated with particular patterns of clinical features. We report here on four patients sharing common clinical features (psychomotor retardation, coarse facies and ocular anomalies), with proximal 5q deletions identified by oligo array-CGH. The deletions range from 5.75 to 17.26-Mb in size and occurred de novo. A common 2.63-Mb region between the deletions described here can be defined in 5q12.1 (59,390,122-62,021,754 bp bp from 5pter, hg18) and includes 12 genes. Among them, KIF2A, which encodes a kinesin superfamily protein, is a particularly interesting candidate for the phenotype, as it suppresses the growth of axonal collateral branches and is involved in normal brain development. Ocular defects, albeit unspecific, seem to be common in the 5q12.1 deletion. Identification of additional cases of deletions involving the 5q12.1 region will allow more accurate genotype-phenotype correlations. (C) 2011 Wiley-Liss, Inc.

Pkc1 acts through Zds1 and Gic1 to suppress growth and cell polarity defects of a yeast eIF5A mutant

eIF5A is a highly conserved putative eukaryotic translation initiation factor that has been implicated in translation initiation, nucleocytoplasmic transport, mRNA decay, and cell proliferation, but with no precise function assigned so far. We have previously shown that high-copy PKCI suppresses the phenotype of tif51A-1, a temperature-sensitive mutant of eIF5A in S. cerevisiae. Here, in an attempt to further understand how Pkc1 functionally interacts with eIF-5A, it was determined that PKCI suppression of tif51A-1 is independent of the cell integrity MAP kinase cascade. Furthermore, two new suppressor genes, ZDS1 and GIC1, were identified. We demonstrated that ZDS1 and ZDS2 are necessary for PKC1, but not for GIC1 suppression. Moreover, high-copy GIC1 also suppresses the growth defect of a PKCI mutant (stt1), suggesting the existence of a Pkc1-Zds1-Gic1 pathway. Consistent with the function of Gic1 in actin organization, the tif51A-1 strain shows an actin polarity defect that is partially recovered by overexpression of Pkc1 and Zds1 as well as Gic1. Additionally, PCL1 and BNI1, important regulators of yeast cell polarity, also suppress tif51A-1 temperature sensitiviiy Taken together, these data strongly Support the correlated involvement of Pkc1 and eIF5A in establishing actin polarity, which is essential for bud formation and G1/S transition in S. cerevisiae.

Deletion of Epstein-Barr virus latent membrane protein 1 gene in United States and Brazilian Hodgkin's disease and reactive lymphoid tissue: High frequency of a 30-bp deletion

A 30-basepair (bp) deletion in the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene has been reported in nasopharyngeal carcinoma and EBV-associated malignant lymphomas. Prior studies have found the deletion in about 10% to 28% of cases of Hodgkin's disease (HD), particularly in cases with aggressive histology. We studied the prevalence of 30-bp LMP1 gene deletion in EBV-positive HD in the United States (US) (12 cases) and Brazil (26 cases) with comparison to reactive lymphoid tissues (21 cases) and HD without EBV-positive Reed-Sternberg cells (15 cases). We studied the status of the LMP1 gene by Southern blot hybridization of polymerase chain reaction (PCR) products obtained after amplification with primers spanning the site of the deletion. We also performed EBV typing, EBER1 in situ hybridization, and LMP1 protein immunohistochemistry. EBV was detected in 12/26 (46%) cases of HD from the US and 26/27 (96%) cases of Brazilian HD. The 30-bp LMP1 gene deletion was observed in 4/12 (33%) cases of EBV-positive HD from US, and 12/26 (46%) cases of Brazilian EBV-positive HD, including 3 cases of type B EBV, as compared with 12/21 (57%) reactive lymphoid tissues and 9/15 (60%) cases of EBV-negative HD. US and Brazilian HD showed a higher prevalence of the 30-bp LMP1 gene deletion, compared with studies of others. The unexpected finding of high incidence of 30-bp deletion in LMP1 gene in reactive lymphoid tissue and HD without EBV-positive Reed-Sternberg cells suggests that this deletion may not be relevant to HD pathogenesis in most cases. Copyright (C) 1997 by W.B. Saunders Company.

The Deoxyhypusine Synthase Mutant dys1-1 Reveals the Association of eIF5A and Asc1 with Cell Wall Integrity

The putative eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein among archaea and eukaryotes that has recently been implicated in the elongation step of translation. eIF5A undergoes an essential and conserved posttranslational modification at a specific lysine to generate the residue hypusine. The enzymes deoxyhypusine synthase (Dys1) and deoxyhypusine hydroxylase (Lia1) catalyze this two-step modification process. Although several Saccharomyces cerevisiae eIF5A mutants have importantly contributed to the study of eIF5A function, no conditional mutant of Dys1 has been described so far. In this study, we generated and characterized the dys1-1 mutant, which showed a strong depletion of mutated Dys1 protein, resulting in more than 2-fold decrease in hypusine levels relative to the wild type. The dys1-1 mutant demonstrated a defect in total protein synthesis, a defect in polysome profile indicative of a translation elongation defect and a reduced association of eIF5A with polysomes. The growth phenotype of dys1-1 mutant is severe, growing only in the presence of 1 M sorbitol, an osmotic stabilizer. Although this phenotype is characteristic of Pkc1 cell wall integrity mutants, the sorbitol requirement from dys1-1 is not associated with cell lysis. We observed that the dys1-1 genetically interacts with the sole yeast protein kinase C (Pkc1) and Asc1, a component of the 40S ribosomal subunit. The dys1-1 mutant was synthetically lethal in combination with asc1Δ and overexpression of TIF51A (eIF5A) or DYS1 is toxic for an asc1Δ strain. Moreover, eIF5A is more associated with translating ribosomes in the absence of Asc1 in the cell. Finally, analysis of the sensitivity to cell wall-perturbing compounds revealed a more similar behavior of the dys1-1 and asc1Δ mutants in comparison with the pkc1Δ mutant. These data suggest a correlated role for eIF5A and Asc1 in coordinating the translational control of a subset of mRNAs associated with cell integrity. © 2013 Galvão et al.

Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

Functional characterization of Streptococcus pyogenes pili

Group A Streptococcus is a Gram-positive human pathogen able to colonize both upper respiratory tract and skin. GAS is responsible for several acute diseases and autoimmune sequelae that account for half a million deaths worldwide every year (Cunningham et al., 2000). As other bacteria, GAS infections requires the capacity of the pathogen to adhere to host tissues and to form cell aggregates. The ability to persist in distinct host niches like the throat and the skin and to trigger infections is associated with the expression of different GAS virulence factors. GAS pili has been described as important virulence factors encoded by different FCT-operon regions. Based on this information, we decided to study the possible effect of environmental conditions that could regulate the pili expression. In this study we reported the influence of pH environment variations in biofilm formation for strains pertaining to a panel of different GAS FCT-types. The biofilm formation was promoted, excepted in the FCT-1 strains, by a changing in pH from physiological to acidic condition of growth in in vitro biofilm assay. By analyzing the possible association between biofilm formation and pH dependence, we have found that in FCT-2 and FCT-3 strains, the biofilm is promoted by pH reduction leading to an increase of pili expression. These data confirmed a direct link between pH dependent pilus expression and biofilm formation in GAS. As pili are a multi component structure we decided to investigate the functional role of one of its subunits, the AP-1 protein. AP-1 is highly conserved through the different FCT-types and suggests a possible essential role for the pili function. We focused our attention on the AP-1 protein encoded by the FCT-1 strains (M6). In particular this AP-1 protein contains the von Willebrand Factor A (VWFA) domain, which share an homology with the human VWFA domain that has been reported to be involved in adhesion process. We have demonstrated that the AP-1 protein binds to human epithelial cells by its VWFA domain, whereas the biofilm formation is mediated by the N-terminal region of AP-1 protein. Moreover, analyzing the importance of AP-1 in in vivo experiments we found a major capacity of tissue dissemination for the wild-type strain compared to the isogenic AP-1 deletion mutant. Pili have been also reported as potential vaccine candidates against Gram positive bacteria. For these reason we decided to investigate the relationship between cross reaction of sera raised against different GAS and GBS pilin subunits and the presence of a conserved Cna_B domain, in different pilin components. Our idea was to investigate if, using pilus conserved domains, a broad coverage vaccine against streptococcal infection could be possible.

Gamma-secretase modulators and inhibitors in Alzheimer's disease: influence of genetic background on efficacy and mechanism of action

According to the amyloid hypothesis, Alzheimer’s disease (AD) is caused by aberrant production or clearance of the amyloid-β (Aβ) peptides, and in particular of the longer more aggregation-prone Aβ42. The Aβ peptides are generated through successive proteolytic cleavage of the amyloid precursor protein (APP) by the β-site APP cleaving enzyme (BACE) and γ-secretase. γ-secretase produces Aβ peptides with variable C-termini ranging from Aβ34 to Aβ48, presumably by sequential trimming of longer into shorter peptides. γ-secretase is a multiprotein complex consisting of at least four different proteins and the presenilin proteins (PS1 or PS2) contain the catalytic center of the complex. In 2001 several non-steroidal anti-inflammatory drugs were identified as the founding members of a new class of γ-secretase modulators (GSMs) that can selectively reduce production of Aβ42. Concomitantly, these GSMs increase Aβ38 production indicating closely coordinated generation of Aβ42 and Aβ38 and a potential precursor-product relationship between these peptides. GSMs seem to exert their activity by direct modulation of γ-secretase. Support for this hypothesis is drawn from the finding that some PS mutations associated with early-onset familial AD (FAD) can modulate the cellular response to GSMs and to γ-secretase inhibitors (GSIs), which inhibit production of all Aβ peptides and are known to directly interact with PS. A particularly interesting FAD PS mutation is PS1-ΔExon9, a complex deletion mutant that blocks endoproteolysis of PS1 and renders cells completely non-responsive to GSMs. Studies presented in this thesis show that the diminished response of PS1-ΔExon9 to GSMs is mainly caused by its lack of endoproteolytic cleavage. Furthermore, we were able to demonstrate that a reduced response to GSMs and GSIs is not limited to PS1-ΔExon9 but is a common effect of aggressive FAD-associated PS1 mutations. Surprisingly, we also found that while the Aβ42 response to GSMs is almost completely abolished by these PS1 mutations, the accompanying Aβ38 increase was indistinguishable to wild-type PS1. Finally, the reduced response to GSIs was confirmed in a mouse model with transgenic expression of an aggressive FAD-associated PS1 mutation as a highly potent GSI failed to reduce Aβ42 levels in brain of these mice. Taken together, our findings provide clear evidence for independent generation of Aβ42 and Aβ38 peptides, and argue that the sequential cleavage model might be an oversimplification of the molecular mechanism of γ-secretase. Most importantly, our results highlight the significance of genetic background in drug discovery efforts aimed at γ-secretase, and indicate that the use of cellular models with transgenic expression of FAD-associated PS mutations might confound studies of the potency and efficacy of GSMs and GSIs. Therefore, such models should be strictly avoided in the ongoing preclinical development of these promising and potentially disease-modifying therapeutics for AD.

Analysing the possible influence of transposon TCl4.7 insertion on the function of the genome of Cydia pomonella granulovirus

CpGV-MCp5 is a natural mutant of the Cydia pomonella Granulovirus (Mexican isolate) (CpGV-M) that harbors an insect host transposon termed TCl4.7 in its genome. TCl4.7 is located between the open reading frames Cp15 and Cp16 and separates two homologous regions hr3 and hr4, which have been recently shown to be origins of replication of CpGV-M. The MCp5 has a significant replication disadvantage in the presence of the wild-type CpGV-M. In this study, the possible effects of TCl4.7 transposon insertion on the genome function of its insertion site has been analysed. The role of Cp15 and Cp16 in the context of the virus infection cycle was examined by generating a CpGV-Bacmid (CpBAC) and Cp15 knock-out (CpBACCp15KO) and Cp16 knock-out (CpBACCp16KO) mutants. The mutant CpBACCp15KO was not able to replicate in CM larvae suggesting that Cp15 was essential for virus replication. In contrast, the mutant CpBACCp16KO infected CM larvae and produced viable occlusion bodies (OBs) demonstrating that Cp16 is a non-essential gene for virus in vivo infection of C. pomonella. The temporal transcription of Cp15 and Cp16, as well as of Cp31 (F protein) as a control, was analysed using RT-PCR and quantitative real-time PCR. It suggested a general delay or reduction of gene transcription of MCp5 compared to the parental CpGV-M. Western blot analyses using anti-Cp15 and anti-Cp16 polyclonal antibodies, however, did not show any immuno-reactive response. Thus, a direct influence of TCl4.7 on the expression of Cp15 and Cp16 could not be substantiated. To investigate whether the interruption of hr3 and hr4 palindromes affects the virus replication, two mutant bacmids with a deletion of hr3 and hr4 (CpBAChr3/hr4-KO) and another with an insertion of a Kanamycin resistance cassette between hr3 and hr4 (CpBAChr3-kan-hr4) were generated. Both mutant bacmids replicated and produced infectious virus OBs, which did not significantly differ in their median lethal concentration (LC50) and median survival time (ST50) compared to the parental CpBAC. Interestingly, the mutant CpBAChr3-kan-hr4 was very effectively out-competed by parental CpBAC, when CM larvae were co-infected with known ratios of OBs of CpBAC and the mutant CpBAChr3-kan-hr4. These observations suggested a functional co-operation between hr3 and hr4 which was interrupted by the KanR insertion in CpBAChr3-kan-hr4 and possibly by TCl4.7 transposon insertion in the mutant MCp5. This hypothesis may explain the observed replication disadvantage of the mutants MCp5 and CpBAChr3-kan-hr4 in the presence of the parental viruses CpGV-M and CpBAC, respectively.