Lantibiotic Nisin and Its Detection Methods

The type A lantibiotic nisin produced by several Lactococcus lactis strains, and one Streptococcus uberis strainis a small antimicrobial peptide that inhibits the growth of a wide range of gram-positive bacteria, such as Bacillus, Clostridium, Listeria and Staphylococcus species. It is nontoxic to humans and used as a food preservative (E234) in more than 50 countries including the EU, the USA, and China. National legislations concerning maximum addition levels of nisin in different foods vary greatly. Therefore, there is a demand for non-laborious and sensitive methods to identify and quantify nisin reliably from different food matrices. The horizontal inhibition assay, based on the inhibitory effect of nisin to Micrococcus luteus is the base for most quantification methods developed so far. However, the sensitivity and accuracy of the agar diffusion method is affected by several parameters. Immunological tests have also been described. Taken into account the sensitivity of immunological methods to interfering substances within sample matrices, and possible cross-reactivities with lantibiotics structurally close to nisin, their usefulness for nisin detection from food samples remains limited. The proteins responsible for nisin biosynthesis, and producer self-immunity are encoded by genes arranged into two inducible operons, nisA/Z/QBTCIPRK and nisFEG, which also contain internal, constitutive promoters PnisI and PnisR. The transmembrane histidine kinase NisK and the response regulator NisR form a two-component signal transduction system, in which NisK autophosphorylates after exposure to extra cellular nisin, and subsequently transfers the phosphate to NisR. The phosphorylated NisR then relays the signal downstream by binding to two regulated promoters in the nisin gene cluster, i.e the nisA/Z/Qand the nisF promoters, thus activating transcription of the structural gene nisA/Z/Q and the downstream genes nisBTCIPRK from the nisA/Z/Q promoter, and the genes nisFEG from the nisF promoter. In this work two novel and highly sensitive nisin bioassays were developed. Both of these quantification methods were based on NisRK mediated, nisin induced Green Fluorescent Protein (GFP) fluorescence. The suitabilities of these assays for quantifica¬tion of nisin from food samples were evaluated in several food matrices. These bioassays had nisin sensitivities in the nanogram or picogram levels. In addition, shelf life of nisin in cooked sausages and retainment of the induction activity of nisin in intestinal chyme (intestinal content) was assessed.

Lactic acid bacteria and their antimicrobial peptides : Induction,detection,partial characterization, and potential applications

Bacteriocin-producing lactic acid bacteria and their isolated peptide bacteriocins are of value to control pathogens and spoiling microorganisms in foods and feed. Nisin is the only bacteriocin that is commonly accepted as a food preservative and has a broad spectrum of activity against Gram-positive organisms including spore forming bacteria. In this study nisin induction was studied from two perspectives, induction from inside of the cell and selection of nisin inducible strains with increased nisin induction sensitivity. The results showed that a mutation in the nisin precursor transporter NisT rendered L. lactis incapable of nisin secretion and lead to nisin accumulation inside the cells. Intracellular proteolytic activity could cleave the N-terminal leader peptide of nisin precursor, resulting in active nisin in the cells. Using a nisin sensitive GFP bioassay it could be shown, that the active intracellular nisin could function as an inducer without any detectable release from the cells. The results suggested that nisin can be inserted into the cytoplasmic membrane from inside the cell and activate NisK. This model of two-component regulation may be a general mechanism of how amphiphilic signals activate the histidine kinase sensor and would represent a novel way for a signal transduction pathway to recognize its signal. In addition, nisin induction was studied through the isolation of natural mutants of the GFPuv nisin bioassay strain L. lactis LAC275 using fl uorescence-activated cell sorting (FACS). The isolated mutant strains represent second generation of GFPuv bioassay strains which can allow the detection of nisin at lower levels. The applied aspect of this thesis was focused on the potential of bacteriocins in chicken farming. One aim was to study nisin as a potential growth promoter in chicken feed. Therefore, the lactic acid bacteria of chicken crop and the nisin sensitivity of the isolated strains were tested. It was found that in the crop Lactobacillus reuteri, L. salivarius and L. crispatus were the dominating bacteria and variation in nisin resistance level of these strains was found. This suggested that nisin may be used as growth promoter without wiping out the dominating bacterial species in the crop. As the isolated lactobacilli may serve as bacteria promoting chicken health or reducing zoonoosis and bacteriocin production is one property associated with probiotics, the isolated strains were screened for bacteriocin activity against the pathogen Campylobacter jejuni. The results showed that many of the isolated L. salivarius strains could inhibit the growth of C. jejuni. The bacteriocin of the L. salivarius LAB47 strain, with the strongest activity, was further characterized. Salivaricin 47 is heat-stable and active in pH range 3 to 8, and the molecular mass was estimated to be approximately 3.2 kDa based on tricine SDS-PAGE analysis.

Traditional medicinal uses and biological activities of some plant extracts of African Combretum Loefl., Terminalia L. and Pteleopsis Engl. species

In Africa various species of Combretum, Terminalia and Pteleopsis are used in traditional medicine. Despite of this, some species of these genera have still not been studied for their biological effects to validate their traditional uses. The aim of this work has been to document the ethnomedicinal uses of several species of Combretum and Terminalia in Mbeya region, south-western Tanzania, and to use this information for finding species with good antimicrobial and cytotoxic potential. During a five weeks expedition to Tanzania in spring 1999 sixteen different species of Combretum and Terminalia, as well as Pteleopsis myrtifolia were collected from various locations in the districts of Mbeya, Iringa and Dar-es-Salaam. Traditional healers in seven different villages in the Mbeya region were interviewed in Swahili and Nyakyusa on the medicinal uses of Combretum and Terminalia species shown to them. A questionnaire was used during the interviews. The results of the interviews correlated well between different villages, the same species being used in similar ways in different villages. Of the ten species shown to the healers six were frequently used for treatment of skin diseases, bacterial infections, diarrhea, oedema and wounds. The dried plants were most commonly prepared into hot water decoctions or mixed into maize porridge, Ugali. Infusions made from dried or fresh plant material were also common. Wounds and topical infections were treated with ointments made from the dried plant material mixed with sheep fat. Twenty-one extracts of six species of Combretum and four of Terminalia, collected from Tanzania, were screened for their antibacterial effects against two gram-negative and five gram-positive bacteria, as well as the yeast, Candida albicans, using an agar diffusion method. Most of the screened plants showed substantial antimicrobial activity. A methanolic root extract of T. sambesiaca showed the most potent antibacterial effects of all the plant species screened, and gave a MIC value of 0.9 mg/ml against Enterobacter aerogenes. Also root extracts of T. sericea and T. kaiserana gave excellent antimicrobial effects, and notably a hot water extract of T. sericea was as potent as extracts of this species made from EtOH and MeOH. Thus, the traditional way of preparing T. sericea into hot water decoctions seems to extract antimicrobial compounds. Thirty-five extracts of five species of Terminalia, ten of Combretum and Pteleopsis myrtifolia were screened for their antifungal effects against five species of yeast (Candida spp.) and Cryptococcus neoformans. The species differed from each other to their antifungal effects, some being very effective whereas others showed no antifungal effects. The most effective extracts showed antifungal effects comparable to the standard antibiotics itraconazol and amphotericin B. Species of Terminalia gave in general stronger antifungal effects than those of Combretum. The best effects were obtained with methanolic root extracts of T. sambesiaca, T. sericea and T. kaiserana, and this investigation indicates that decoctions of these species might be used for treatment of HIV-related fungal infections. Twenty-seven crude extracts of eight species of Combretum, five of Terminalia and Pteleopsis myrtifolia were evaluated for their cytotoxic effects against human cancer cell lines (HeLa, cervical carcinoma; MCF 7, breast carcinoma, T 24 bladder carcinoma) and one endothelial cell line (BBCE, bovine brain capillary endothelial cells). The most outstanding effects were obtained with a leaf extract of Combretum fragrans, which nearly totally inhibited the proliferation of T 24 and HeLa cells at a concentration of 25 ug/ml and inhibited 60 % of the growth of the HeLa cells at a concentration of 4.3 ug/ml. The species of Terminalia were less cytotoxically potent than the Combretum species, although T. sericea and T. sambesiaca gave good cytotoxic effects (< 30 % proliferation). In summary this study indicates that some of the species of Terminalia, Combretum and Pteleopsis, used in Tanzanian traditional medicine, are powerful inhibitors of both microbial and cancer cell growth. In depth studies would be needed to find the active compounds behind these biological activities.

Pentitol phosphate dehydrogenases: Discovery, characterization and use in D-arabitol and xylitol production by metabolically engineered Bacillus subtilis

The ultimate goal of this study has been to construct metabolically engineered microbial strains capable of fermenting glucose into pentitols D-arabitol and, especially, xylitol. The path that was chosen to achieve this goal required discovery, isolation and sequencing of at least two pentitol phosphate dehydrogenases of different specificity, followed by cloning and expression of their genes and characterization of recombinant arabitol and xylitol phosphate dehydrogenases. An enzyme of a previously unknown specificity, D-arabitol phosphate dehydrogenase (APDH), was discovered in Enterococcus avium. The enzyme was purified to homogenity from E. avium strain ATCC 33665. SDS/PAGE revealed that the enzyme has a molecular mass of 41 ± 2 kDa, whereas a molecular mass of 160 ± 5 kDa was observed under non-denaturing conditions implying that the APDH may exist as a tetramer with identical subunits. Purified APDH was found to have narrow substrate specificity, converting only D-arabitol 1-phosphate and D-arabitol 5-phosphate into D-xylulose 5-phosphate and D-ribulose 5-phosphate, respectively, in the oxidative reaction. Both NAD+ and NADP+ were accepted as co-factors. Based on the partial protein sequences, the gene encoding APDH was cloned. Homology comparisons place APDH within the medium chain dehydrogenase family. Unlike most members of this family, APDH requires Mn2+ but no Zn2+ for enzymatic activity. The DNA sequence surrounding the gene suggests that it belongs to an operon that also contains several components of phosphotransferase system (PTS). The apparent role of the enzyme is to participate in arabitol catabolism via the arabitol phosphate route similar to the ribitol and xylitol catabolic routes described previously. Xylitol phosphate dehydrogenase (XPDH) was isolated from Lactobacillus rhamnosus strain ATCC 15820. The enzyme was partially sequenced. Amino acid sequences were used to isolate the gene encoding the enzyme. The homology comparisons of the deduced amino acid sequence of L. rhamnosus XPDH revealed several similar enzymes in genomes of various species of Gram-positive bacteria. Two enzymes of Clostridium difficile and an enzyme of Bacillus halodurans were cloned and their substrate specificities together with the substrate specificity of L. rhamnosus XPDH were compared. It was found that one of the XPDH enzymes of C. difficile and the XPDH of L. rhamnosus had the highest selectivity towards D-xylulose 5-phosphate. A known transketolase-deficient and D-ribose-producing mutant of Bacillus subtilis (ATCC 31094) was further modified by disrupting its rpi (D-ribose phosphate isomerase) gene to create D-ribulose- and D-xylulose-producing strain. Expression of APDH of E. avium and XPDH of L. rhamnosus and C. difficile in D-ribulose- and D-xylulose-producing strain of B. subtilis resulted in strains capable of converting D-glucose into D-arabitol and xylitol, respectively. The D-arabitol yield on D-glucose was 38 % (w/w). Xylitol production was accompanied by co-production of ribitol limiting xylitol yield to 23 %.

Oxazolidine-2-thiones: A molecular modeling study

Two oxazolidine-2-thiones, thio-analogs of linezolid, were synthesized and their antibacterial properties evaluated. Unlike oxazolidinones, the thio-analogs did not inhibit the growth of Gram positive bacteria. A molecular modeling study has been carried out to aid understanding of this unexpected finding.

The BglG group of antiterminators: a growing family of bacterial regulators

The product of the bglG gene of Escherichia coli was among the first bacterial antiterminators to be identified and characterized. Since the elucidation ten years ago of its role in the regulation of the bgl operon of E. coli,a large number of homologies have been discovered in both Gram-positive and Gram-negative bacteria. Often the homologues of BglG in other organisms are also involved in regulating β-glucoside utilization. Surprisingly, in many cases, they mediate antitermination to regulate a variety of other catabolic functions. Because of the high degree of conservation of the cis-acting regulatory elements, antiterminators from one organism can function in another. Generally the antiterminator protein itself is negatively regulated by phosphorylation by a component of the phosphotransferase system. This family of proteins thus represents a highly evolved regulatory system that is conserved across evolutionarily distant genuses.

Microbe-induced activation of inflammatory cytokine response in human cells

Human body is in continuous contact with microbes. Although many microbes are harmless or beneficial for humans, pathogenic microbes possess a threat to wellbeing. Antimicrobial protection is provided by the immune system, which can be functionally divided into two parts, namely innate and adaptive immunity. The key players of the innate immunity are phagocytic white blood cells such as neutrophils, monocytes, macrophages and dendritic cells (DCs), which constantly monitor the blood and peripheral tissues. These cells are armed for rapid activation upon microbial contact since they express a variety of microbe-recognizing receptors. Macrophages and DCs also act as antigen presenting cells (APCs) and play an important role in the development of adaptive immunity. The development of adaptive immunity requires intimate cooperation between APCs and T lymphocytes and results in microbe-specific immune responses. Moreover, adaptive immunity generates immunological memory, which rapidly and efficiently protects the host from reinfection. Properly functioning immune system requires efficient communication between cells. Cytokines are proteins, which mediate intercellular communication together with direct cell-cell contacts. Immune cells produce inflammatory cytokines rapidly following microbial contact. Inflammatory cytokines modulate the development of local immune response by binding to cell surface receptors, which results in the activation of intracellular signalling and modulates target cell gene expression. One class of inflammatory cytokines chemokines has a major role in regulating cellular traffic. Locally produced inflammatory chemokines guide the recruitment of effector cells to the site of inflammation during microbial infection. In this study two key questions were addressed. First, the ability of pathogenic and non-pathogenic Gram-positive bacteria to activate inflammatory cytokine and chemokine production in different human APCs was compared. In these studies macrophages and DCs were stimulated with pathogenic Steptococcus pyogenes or non-pathogenic Lactobacillus rhamnosus. The second aim of this thesis work was to analyze the role of pro-inflammatory cytokines in the regulation of microbe-induced chemokine production. In these studies bacteria-stimulated macrophages and influenza A virus-infected lung epithelial cells were used as model systems. The results of this study show that although macrophages and DCs share several common antimicrobial functions, these cells have significantly distinct responses against pathogenic and non-pathogenic Gram-positive bacteria. Macrophages were activated in a nearly similar fashion by pathogenic S. pyogenes and non-pathogenic L. rhamnosus. Both bacteria induced the production of similar core set of inflammatory chemokines consisting of several CC-class chemokines and CXCL8. These chemokines attract monocytes, neutrophils, dendritic cells and T cells. Thus, the results suggest that bacteria-activated macrophages efficiently recruit other effector cells to the site of inflammation. Moreover, macrophages seem to be activated by all bacteria irrespective of their pathogenicity. DCs, in contrast, were efficiently activated only by pathogenic S. pyogenes, which induced DC maturation and production of several inflammatory cytokines and chemokines. In contrast, L. rhamnosus-stimulated DCs matured only partially and, most importantly, these cells did not produce inflammatory cytokines or chemokines. L. rhamnosus-stimulated DCs had a phenotype of "semi-mature" DCs and this type of DCs have been suggested to enhance tolerogenic adaptive immune responses. Since DCs have an essential role in the development of adaptive immune response the results suggest that, in contrast to macrophages, DCs may be able to discriminate between pathogenic and non-pathogenic bacteria and thus mount appropriate inflammatory or tolerogenic adaptive immune response depending on the microbe in question. The results of this study also show that pro-inflammatory cytokines can contribute to microbe-induced chemokine production at multiple levels. S. pyogenes-induced type I interferon (IFN) was found to enhance the production of certain inflammatory chemokines in macrophages during bacterial stimulation. Thus, bacteria-induced chemokine production is regulated by direct (microbe-induced) and indirect (pro-inflammatory cytokine-induced) mechanisms during inflammation. In epithelial cells IFN- and tumor necrosis factor- (TNF-) were found to enhance the expression of PRRs and components of cellular signal transduction machinery. Pre-treatment of epithelial cells with these cytokines prior to virus infection resulted in markedly enhanced chemokine response compared to untreated cells. In conclusion, the results obtained from this study show that pro-inflammatory cytokines can enhance microbe-induced chemokine production during microbial infection by providing a positive feedback loop. In addition, pro-inflammatory cytokines can render normally low-responding cells to high chemokine producers via enhancement of microbial detection and signal transduction.

Lipid-Containing Icosahedral dsDNA Bacteriophages: Entry, Exit and Structure

In this thesis three icosahedral lipid-containing double-stranded (ds) deoxyribonucleic acid (DNA) bacteriophages have been studied: PRD1, Bam35 and P23-77. The work focuses on the entry, exit and structure of the viruses. PRD1 is the type member of the Tectiviridae family, infecting a variety of Gram-negative bacteria. The PRD1 receptor binding complex, consisting of the penton protein P31, the spike protein P5 and the receptor binding protein P2 recognizes a specific receptor on the host surface. In this study we found that the transmembrane protein P16 has an important stabilization function as the fourth member of the receptor binding complex and protein P16 may have a role in the formation of a tubular membrane structure, which is needed in the ejection of the genome into the cell. Phage Bam35 (Tectiviridae), which infects Gram-positive hosts, has been earlier found to resemble PRD1 in morphology and genome organization The uncharacterized early and late events in the Bam35 life cycle were studied by electrochemical methods. Physiological changes in the beginning of the infection were found to be similar in both lysogenic and nonlysogenic cell lines, Bam35 inducing a temporal decrease of membrane voltage and K+ efflux. At the end of the infection cycle physiological changes were observed only in the nonlysogenic cell line. The strong K+ efflux 40 min after infection and the induced premature cell lysis propose that Bam35 has a similar holin-endolysin lysis system to that of PRD1. Thermophilic icosahedral dsDNA Thermus phages P23-65H, P23-72 and P23-77 have been proposed to belong to the Tectiviridae family. In this study these phages were compared to each other. Analysis of structural protein patterns and stability revealed these phages to be very similar but not identical. The most stable of the studied viruses, P23-77, was further analyzed in more detail. Cryo-electron microscopy and three-dimensional image reconstruction was used to determine the structure of virus to 14 Å resolution. Results of thin layer chromatography for neutral lipids together with analysis of the three dimensional reconstruction of P23-77 virus particle revealed the presence of an internal lipid membrane. The overall capsid architecture of P23-77 is similar to PRD1 and Bam35, but most closely it resembles the structure of the capsid of archaeal virus SH1. This complicates the classification of dsDNA, internal lipid-containing icosahedral viruses.

Lactobacillus bacteremia, with special focus on the safety of probiotic Lactobacillus rhamnosus GG

Lactobacilli are gram positive rods, which belong to normal oropharyngeal, gastrointestinal and urogenital flora. They are widely used in food industry and as food additives. Although their virulence is presumed to be very low, opportunistic bacteremic infections, especially in immunocompromised hosts, have been detected. In the present study, the possible effects of increasing probiotic use of Lactobacillus rhamnosus GG (LGG) on the occurrence of bacteremia due to lactobacilli was evaluated on population level. In Finland, 90 Lactobacillus bacteremia cases were reported to the National Infectious Disease Register maintained by National Public Health Institute, during 1995-2000. Their proportion of all bacteremia cases was on average 0.24%, corresponding to 0.3 cases/100 000 inhabitants annually. In the Helsinki University Central Hospital district the corresponding proportion of all bacteremia cases was observed during 1990-2000. Despite LGG intake increased six folded no increasing trend in the occurrence of lactobacilli bacteremia was seen. A total of 85 Lactobacillus sp. blood isolates collected from different human bacteremic cases were characterised and compared with the commercial probiotic LGG strain. In species characterisation 46 L. rhamnosus strains, 12 L. fermentum and L. casei strains each, three each of L. gasseri, L. salivarius and L. jensenii species, two L. curvatus, and one each of L. plantarum, L. sakei, L. zeae and L. reuteri species were detected. Nearly half of the L. rhamnosus findings turned out to be indistinguishable from the probiotic LGG strain. Common predisposing factors to Lactobacillus bacteremia were immunosuppression, prior prolonged hospitalisation and prior surgical interventions. Severe or fatal comorbidities were found in 82% of the patients. Mortality at one month was 26% and severe underlying diseases were a significant predictor of death (OR 15.8). Antimicrobial susceptibility of Lactobacillus strains was species dependent. The Lactobacillus isolates were generally susceptible to imipenem, piperacillin-tazobactam, clindamycin and erythromycin, whereas all other than L. gasseri and L. jensenii species were not at all susceptible to vancomycin. The susceptibility to cephalosporin varied greatly even within species why they might not be recommended for treatment of Lactobacillus infections. The effect and safety of probiotic LGG preparation in amelioration of gastric symptoms and diarrhea in HIV-infected patients was evaluated. No significant differences in gastrointestinal symptoms or diarrhoea in LGG treated patients compared to placebo could be found. LGG was well tolerated with no adverse effects including bacteremic outbreaks could be observed. The use of probiotic LGG can be regarded safe in this immunocompromised patient group.

Antimicrobial effects of ethanolic extracts from Cuscuta reflexa Roxb. (Convolvulaceae).

Ethanolic whole plant extracts obtained from Cuscuta reflexa Roxb were screened against Gram positive (Bacillus subtilis and Staphylococcus aureus) and Gram negative (Escherichia coli and Salmonella typhi) bacteria to evaluate their antimicrobial activity. Of the four concentrations of plant extract tested (200 µg/mL, 300 µg/mL, 400 µg/mL or 500 µg/mL), 500 µg/mL elicited the greatest zones of bacterial inhibition across three of the bacteria. In contrast, the growth of Salmonella typhi was not halted regardless of extract concentration. At 200 µg/mL, only the growth of E. coli was inhibited. Overall, although the greatest antimicrobial activity was demonstrated to be against E. coli at a concentration of 500 µg/mL (24.6±0.24), upon comparison to the other bacteria, both B. cereus and S. aureus educed similar zones of inhibition upon comparison to their positive antibiotic control.

Optical, Field-Emission, and Antimicrobial Properties of ZnO Nanostructured Films Deposited at Room Temperature by Activated Reactive Evaporation

ZnO nanostructures were deposited on flexible polymer sheet and cotton fabrics at room temperature by activated reactive evaporation. Room-temperature photoluminescence spectrum of ZnO nanostructured film exhibited a week intrinsic UV emission and a strong broad yellow-orange visible emission. TEM and HRTEM studies show that the grown nanostructures are crystalline in nature and their growth direction was indentified to be along [002]. ZnO nanostructures grown on the copper-coated flexible polymer sheets exhibited stable field-emissio characteristics with a threshold voltage of 2.74 V/mu m (250 mu A) and a very large field enhancement factor (beta) of 23,213. Cotton fabric coated with ZnO nanostructures show an excellent antimicrobial activity against Staphylococcus aureus bacteria (Gram positive), and similar to 73% reduction in the bacterial population is achieved compared to uncoated fabrics after 4 h in viability. Using a shadow mask technique, we also selectively deposited the nanostructures at room temperature on polymer substrates.

Antibacterial & antiplasmid activities of Helicteres isora L.

Background & objectives: The multiple drug resistance (MDR) is a serious health problem and major challenge to the global drug discovery programmes. Most of the genetic determinants that confer resistance to antibiotics are located on R-plasmids in bacteria. The present investigation was undertaken to investigate the ability of organic extract of the fruits of Helicteres isora to cure R-plasmids from certain clinical isolates. mMethods: Active fractions demonstrating antibacterial and antiplasmid activities were isolated from the acetone extracts of shade dried fruits of H. isora by bioassay guided fractionation. Minimal inhibitory concentration (MIC) of antibiotics and organic extracts was determined by agar dilution method. Plasmid curing activity of organic fractions was determined by evaluating the ability of bacterial colonies (pre treated with organic fraction for 18 h) to grow in the presence of antibiotics. The physical loss of plasmid DNA in the cured derivatives was further confirmed by agarose gel electrophoresis. Results: The active fraction did not inhibit the growth of either the clinical isolates or the strains harbouring reference plasmids even at a concentration of 400 mu g/ml. However, the same fraction could cure plasmids from Enterococcus faecalis, Escherichia coli, Bacillus cereus and E. coli (RP4) at curing efficiencies of 14, 26, 22 and 2 per cent respectively. The active fraction mediated plasmid curing resulted in the subsequent loss of antibiotic resistance encoded in the plasmids as revealed by antibiotic resistance profile of cured strains. The physical loss of plasmid was also confirmed by agarose gel electrophoresis. Interpretation & conclusions: The active fraction of acetone extract of H. isora fruits cured R-plasmids from Gram-positive and Gram-negative clinical isolates as well as reference strains. Such plasmid loss reversed the multiple antibiotic resistance in cured derivatives making them sensitive to low concentrations of antibiotics. Acetone fractions of H. isora may be a source to develop antiplasmid agents of natural origin to contain the development and spread of plasmid borne multiple antibiotic resistance.

Lower Gastrointestinal Microbiota in Health and Irritable Bowel Syndrome : Characterisation and Effect of Probiotic Intervention

The human gastrointestinal (GI) microbiota is a complex ecosystem that lives in symbiosis with its host. The growing awareness of the importance of the microbiota to the host as well as the development of culture-free laboratory techniques and computational methods has enormously expanded our knowledge of this microbial community. Irritable bowel syndrome (IBS) is a common functional bowel disorder affecting up to a fifth of the Western population. To date, IBS diagnosis has been based on GI symptoms and the exclusion of organic diseases. The GI microbiota has been found to be altered in this syndrome and probiotics can alleviate the symptoms, although clear links between the symptoms and the microbiota have not been demonstrated. The aim of the present work was to characterise IBS related alterations in the intestinal microbiota, their relation to IBS symptoms and their responsiveness to probiotic theraphy. In this thesis research, the healthy human microbiota was characterised by cloning and sequencing 16S rRNA genes from a faecal microbial community DNA pool that was first profiled and fractionated according to its guanine and cytosine content (%G+C). The most noticeable finding was that the high G+C Gram-positive bacteria (the phylum Actinobacteria) were more abundant compared to a corresponding library constructed from the unfractionated DNA pool sample. Previous molecular analyses of the gut microbiota have also shown comparatively low amounts of high G+C bacteria. Furthermore, the %G+C profiling approach was applied to a sample constructed of faecal DNA from diarrhea-predominant IBS (IBS-D) subjects. The phylogenetic microbial community comparison performed for healthy and IBS-D sequence libraries revealed that the IBS-D sample was rich in representatives of the phyla Firmicutes and Proteobacteria whereas Actinobacteria and Bacteroidetes were abundant in the healthy subjects. The family Lachnospiraceae within the Firmicutes was especially prevalent in the IBS-D sample. Moreover, associations of the GI microbiota with intestinal symptoms and the quality of life (QOL) were investigated, as well as the effect of probiotics on these factors. The microbial targets that were analysed with the quantitative real-time polymerase chain reaction (qPCR) in this study were phylotypes (species definition according to 16S rRNA gene sequence similarity) previously associated with either health or IBS. With a set of samples, the presence or abundance of a phylotype that had 94% 16S rRNA gene sequence similarity to Ruminococcus torques (R. torques 94%) was shown to be associated with the severity of IBS symptoms. The qPCR analyses for selected phylotypes were also applied to samples from a six-month probiotic intervention with a mixture of Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99. The intervention had been previously reported to alleviate IBS symptoms, but no associations with the analysed microbiota representatives were shown. However, with the phylotype-specific assays applied here, the abundance of the R. torques 94% -phylotype was shown to be lowered in the probiotic-receiving group during the probiotic supplementation, whereas a Clostridium thermosuccinogenes 85% phylotype, previously associated with a healthy microbiota, was found to be increased compared to the placebo group. To conclude, with the combination of methods applied, higher abundance of Actinobacteria was detected in the healthy gut than found in previous studies, and significant phylum-level microbiota alterations could be shown in IBS-D. Thus, the results of this study provide a detailed overview of the human GI microbiota in healthy subjects and in subjects with IBS. Furthermore, the IBS symptoms were linked to a particular clostridial phylotype, and probiotic supplementation was demonstrated to alter the GI microbiota towards a healthier state with regard to this and an additional bacterial phylotype. For the first time, distinct phylotype-level alterations in the microbiota were linked to IBS symptoms and shown to respond to probiotic therapy.

Moonlighting Proteins of Lactobacillus crispatus : Extracellular Localization, Cell Wall Anchoring and Interactions with the Host

Moonlighting functions have been described for several proteins previously thought to localize exclusively in the cytoplasm of bacterial or eukaryotic cells. Moonlighting proteins usually perform conserved functions, e. g. in glycolysis or as chaperonins, and their traditional and moonlighting function(s) usually localize to different cell compartments. The most characterized moonlighting proteins in Grampositive bacteria are the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which function in bacteria-host interactions, e. g. as adhesins or plasminogen receptors. Research on bacterial moonlighting proteins has focused on Gram-positive bacterial pathogens, where many of their functions have been associated with bacterial virulence. In this thesis work I show that also species of the genus Lactobacillus have moonlighting proteins that carry out functions earlier associated with bacterial virulence only. I identified enolase, GAPDH, glutamine synthetase (GS), and glucose-6-phosphate isomerase (GPI) as moonlighting proteins of Lactobacillus crispatus strain ST1 and demonstrated that they are associated with cell surface and easily released from the cell surface into incubation buffer. I also showed that these lactobacillar proteins moonlight either as adhesins with affinity for basement membrane and extracellular matrix proteins or as plasminogen receptors. The mechanisms of surface translocation and anchoring of bacterial moonlighting proteins have remained enigmatic. In this work, the surface localization of enolase, GAPDH, GS and GPI was shown to depend on environmental factors. The members of the genus Lactobacillus are fermentative organisms that lower the ambient pH by producing lactic acid. At acidic pH enolase, GAPDH, GS and GPI were associated with the cell surface, whereas at neutral pH they were released into the buffer. The release did not involve de novo protein synthesis. I showed that purified recombinant His6-enolase, His6-GAPDH, His6-GS and His6-GPI reassociate with cell wall and bind in vitro to lipoteichoic acids at acidic pH. The in-vitro binding of these proteins localizes to cell division septa and cell poles. I also show that the release of moonlighting proteins is enhanced in the presence of cathelicidin LL- 37, which is an antimicrobial peptide and a central part of the innate immunity defence. I found that the LL-37-induced detachment of moonlighting proteins from cell surface is associated with cell wall permeabilization by LL-37. The results in this thesis work are compatible with the hypothesis that the moonlighting proteins of L. crispatus associate to the cell wall via electrostatic or ionic interactions and that they are released into surroundings in stress conditions. Their surface translocation is, at least in part, a result from their release from dead or permeabilized cells and subsequent reassociation onto the cell wall. The results of this thesis show that lactobacillar cells rapidly change their surface architecture in response to environmental factors and that these changes influence bacterial interactions with the host.

Crystallization and preliminary X-ray diffraction studies of sortase A from Streptococcus pneumoniae

Sortases are cell-membrane-anchored cysteine transpeptidases that are essential for the assembly and anchoring of cell-surface adhesins in Gram-positive bacteria. Thus, they play critical roles in virulence, infection and colonization by pathogens. Sortases have been classified into four types based on their primary sequence and the target-protein motifs that they recognize. All Gram-positive bacteria express a class A housekeeping sortase (SrtA). Sortase A from Streptococcus pneumoniae (NP_358691) has been crystallized in two crystal forms. Diamond-shaped crystals of Delta N(59)SrtA diffracted to 4.0 angstrom resolution and belonged to a tetragonal system with unit-cell parameters a = b = 122.8, c = 86.5 angstrom, alpha = beta = gamma = 90 degrees, while rod-shaped crystals of Delta N(81)SrtA diffracted to 2.91 angstrom resolution and belonged to the monoclinic space group P2(1) with unit-cell parameters a = 66.8, b = 103.47, c = 74.79 angstrom, alpha = gamma = 90, beta = 115.65 degrees. The Matthews coefficient (V(M) = 2.77 angstrom(3) Da(-1)) with similar to 56% solvent content suggested the presence of four molecules in the asymmetric unit for Delta N(81)SrtA. Also, a multi-copy search using a monomer as a probe in the molecular-replacement method resulted in the successful location of four sortase molecules in the asymmetric unit, with statistics R = 41.61, R(free) = 46.44, correlation coefficient (CC) = 64.31, CC(free) = 57.67.