There is a constant need of new synthetic emulsifiers in the food industry. Sugar esters are widely used as food grade synthetic emulsifiers, amongst which sucrose esters are the most common. Although sucrose esters are used very frequently, little is known about the use of lactose esters in food. There is a need for characterization of lactose esters before they can be used in foods. The objective of this study was to characterize a lactose ester, lactose monolaurate (LML) as an antimicrobial agent on food pathogens, evaluate its effect on 20 % oil-in-water emulsions as an emulsifier, and to explore its effect on crystallization behavior of anhydrous milk fat. In the first study (Chapter 3), the effect of LML was evaluated on survival of some Gram-positive and Gram-negative bacteria. For Listeria monocytogenes, a concentration of 1 mg/ml showed some inhibition in growth media whereas the cells were completely killed at 5 mg/ml. For Mycobacteria, an LML concentration between 0.1-1mg/ml was lethal. Scanning electron microscopy was also conducted to examine any changes in the morphology of cells. Listeria exhibited a change in morphology and a wrinkling effect was shown in Mycobacteria. In the second study (Chapter 4), the effect of LML as an emulsifier was evaluated in 20 % oil-in-water emulsions. The use level of LML was comparable to commercially available emulsifier polysorbate 20, and produced comparable stabilization in the emulsions upon use. In this study, an attempt was also made to optimize the synthesis of LML with respect to the immobilized enzyme and solvent combination. It was concluded that for 20 % oil-in-water emulsions, LML is a promising emulsifier at 0.5%. In the third study (Chapter 5), the effect of LML was evaluated at two concentrations on the crystallization behavior of anhydrous milk fat at two temperatures with high and low supercooling. On application of high intensity ultrasound (HIU) to anhydrous milk fat (AMF) at 31°C and 0.05 % LML the effect on viscosity of sample and crystallization behavior was evaluated. It was concluded that the viscosity of AMF decreased with the addition of 0.05% LML. The lower viscosity of anhydrous milk fat on addition of LML could be restored with the application of HIU.

No description available.

L. monocytogenes is ubiquitous in environment and can grow and survive in a wide range of environmental conditions. It contaminates foods via raw materials or food processing environments. However, the current knowledge of its ecology and in particular, the mode of environmental survival and transmission of L. monocytogenes remains limited. One important aspect of environmental survival of L. monocytogenes may be contact with other microorganisms, including amoebae, which naturally feed on bacteria as a source of nutrients. In this context, research has shown that several intra-cellular pathogens are able to survive or replicate within free-living amoebae. In view of the potential for amoebae to act as environmental reservoirs for bacteria, the interaction of L. monocytogenes with freeliving Acanthamoeba spp. and the impact of plasmid-associated genes on their interaction were investigated. Several strains of environmental and clinical isolates of L. monocytogenes were used for co-culture with amoebae. Axenic amoebae were isolated from environmental sources (water, soil) by cultivation in PYG supplemented with antibiotics. L. monocytogenes strains were co-cultured with amoebae on plates, in trays (as monolayers of amoeba cells) and in flasks to provide qualitative and quantitative assessments of the survival of bacteria and amoebae. Bacteriological methods and microscopy (fluorescence, TEM and phase contrast) were used to track the fate of internalized bacteria. A vector that allowed GFP expression under control of the prfA promoter was used to assess the expression of Listeria virulence genes within amoeba cells. The role plasmid encoded genes in interactions of L. monocytogenes with amoebae was assessed using plasmid-cured versus wild type in the co-cultures. Finally the mechanisms of bacterial uptake and killing by A. polyphaga were assessed using chemical inhibitors that affected actin polymerization (Cytochalasin D, Wortmannin), phagosome-lysosome fusion (Suramin) and phagosomal acidification (ammonium chloride, Bafilomycin A and Monensin). Sequence analysis of a section of the large plasmid from strain DRDC8 revealed a high level of similarity of gene organization and DNA sequences with plasmid-borne genes found in other Listeria spp. and Gram-positive bacteria. While the majority of environmental isolates of L. monocytogenes contained a large plasmid, it was absent in clinical isolates. Further, plasmid of DRDC8 was lost during serial passage in HeLa cells. This data indicated that the plasmid may be readily lost during isolation procedures or during growth within host animals/cells and thus plasmid instability may explain the absence of plasmid in clinical isolates. Co-culture of L. monocytogenes with Acanthamoeba spp. showed these amoebae are able to actively phagocytose and kill bacteria within 2-5 h irrespective of temperature used. Amoebae killed both plasmid cured and LLO mutants with the same rate for the parental bacteria. Fluorescence microscopy and TEM of bacteria within trophozoites showed the bacteria become confined within tight vacuolar structures surrounded by lysosomes and mitochondria and degraded after 4 to 5 h post phagocytosis. This data indicated that although L. monocytogenes is an effective pathogen of mammalian cells, it could not escape from phagosomes and evade the killing mechanisms of amoeba trophozoites. Consequently, bacteria are killed within phagolysosome in trophozoites before they express their virulence genes to escape from phagosomes and get access to cytoplasm. This was confirmed by observing no GFP expression by bacteria carried prfA::gfp construct in co-culture with amoebae whereas it was observed during co-culture with HeLa cells. Using inhibitors, the mechanisms involved in phagocytosis, and killing of L. monocytogenes cells by A. polyphaga were assessed. The results showed that the uptake of bacterial cells is mediated by the trophozoites but not the bacteria and mannose binding protein is not involved in this process. Furthermore, pre-treatment of trophozoites with inhibitors indicated both phagosomal acidification and phagosome lysosome fusion are involved in killing of bacteria. These results suggest that compared to mammalian cells e.g. HeLa cells, amoeba trophozoites are better able to effectively inactivate and destroy internalized L. monocytogenes cells. In conclusion, Acanthamoeba spp. are able to uptake and kill L. monocytogenes cells in phagolysosome compartments. However, bacteria can saprophyticaly grow on materials released from amoeba trophozoites. Thus this group of amoebae is not able to harbour L. monocytogenes cells, or act as environmental reservoirs for this opportunistic pathogen under the laboratory conditions tested. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1292868 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2007

Listeriosis is a potentially lethal disease caused by the Gram-positive facultative intracellular pathogen Listeria monocytogenes (L.m.). L.m. is found ubiquitously in the environment and infects humans via ingestion of contaminated food. Contaminated products are usually derived from ruminants and involve dairy products and different kinds of processed meat. Listeriosis is a potential lifethreatening disease with a total mortality rate of 20-30 %. The development of listeriosis may lead to meningitis and septicemia or other invasive diseases. Pregnant women are of increased risk of developing listeriosis and a materno-fetal infection commonly lead to spontaneous abortion or still-birth. Regulation of gene expression, and specifically virulence gene expression, is essential for pathogenic bacteria to be equipped for handling counteractions from the host as well as thriving in the often hostile environment. In pathogenic Listeria, virulence gene expression is under the control of the global virulence gene regulator PrfA. The expression of prfA is highly regulated at the transcriptional, post-transcriptional and post- translational level. We have identified a novel type of post-transcriptional regulation of prfA-mRNA by a trans-acting riboswitch element (SreA). By binding to the leader region of prfA-mRNA, SreA negatively regulates the expression of prfA. To our knowledge, this is the first description of a cis-acting riboswitch capable of functioning as a small RNA in trans, regulating targets on distant sites. To date, there have been around 100 sRNAs identified in Listeria monocytogenes, but experimental data is still limited. We have characterized a blood induced sRNA, Rli38, which is important for full virulence during oral infection of mice. Our data suggest that Rli38 regulates the expression of at least two proteins; OppD (Oligopeptide transport protein) and IsdG (heme degrading monooxygenase). Both of these proteins have been implicated in the infectious cycle of L.m. We speculate that the virulence phenotype of an ∆rli38 mutant is possibly mediated through the effect of these proteins. L.m. is a complex pathogen, able to infect and replicate in a variety of organs and cause several distinctive forms of disease. These qualities of L.m. generate difficulties in simulating human listeriosis in animal models, as entailed by the multitude of models used in the field. In this work, we have evaluated the use of an alternative animal model in studying listeriosis. Our results describe the differentiated virulence potential of wildtype bacteria and a ∆prfA mutant strain in the chicken embryo by live/death screening and organ colonization. Large differences in mean time to death were found between wild-type and the ∆prfA strain and ∆prfA cells displayed a considerable defect in colonization of the embryonal liver. The results presented in this thesis show that the chicken embryo infection model is a valuable and convenient tool in studying end-outcome and organ colonization of Listeria monocytogenes. Taken together, this thesis describes the characterization of two previously unknown sRNAs in the human pathogen Listeria monocytogenes and the use of an alternative infection model for simulating listeriosis.

The bacterium Listeria monocytogenes can cause the disease listeriosis in both humans and animals. For the epidemiological investigation of listeriosis detection and characterisation of the organism are important steps. Paper I. There are few reports on the incidence of L. monocytogenes in clinical samples from humans in India. Therefore, we investigated 144 samples from immunocompromised patients. L. monocytogenes was isolated from two placental bits from women with poor obstetric history, one patient with renal failure and three other patients. Five isolates were positive for the virulence genes hlyA, actA and iap. The sixth isolate was positive for hlyA and actA genes. Paper II. Characterisation of 601 human L. monocytogenes isolates causing invasive listeriosis during the period 1986 to 2007 in Sweden reveals a decrease in serovar 4b strains. Since 1996, serovar 1/2a has become the predominant serovar causing human listeriosis: PFGE analysis revealed two clusters including different serotypes suggesting that we need more studies on genetic relatedness among clinical isolates. Paper III. The incidence of Listeria species in seafood from markets in Goa was studied. One hundred and fifteen raw/fresh seafoods bought at the fish markets were sampled and tested for presence of Listeria spp. L. monocytogenes was detected in 10 samples. L. monocytogenes in raw seafood may pose a health risk in kitchen if contaminating ready-to-eat food. Paper IV. Gravad and cold-smoked salmon are associated with human listeriosis in Sweden. L. monocytogenes was isolated from 11 of 56 products. Serovar 1/2a was predominant, followed by 4b. REA/PFGE typing of the isolates identified five types of L. monocytogenes. One type was identical to a human type, two other were closely related.These findings suggest that gravad and cold-smoked salmon are still possible sources of listeriosis in Sweden. Paper V. Many outbreaks of listeriosis have been related to consumption of dairy-associated products. Therefore, 123 farm bulk milk samples in India and 20 cervico-vaginal samples from dairy cows with reproductive disorders were investigated for L. monocytogenes. L. monocytogenes was isolated from 17.9% of bulk milk samples and from 10% of cervico-vaginal swabs. The virulence gene hlyA was detected in all isolates. These findings represent a public health risk where unpasteurised milk and milk products are largely consumed. Paper VI. Isolates of L. monocytogenes (n=177) from 22 animal species were characterized and compared with human strains isolated between 1986-2006 in Sweden. Although many animals and humans shared pulsovars, they did not appear at the same time or with the same proportion of strains. The pulsovars shared by both animals and humans may indicate that there is an exchange of L. monocytogenes strains between these two groups due to either direct or indirect transmission. / <p>The work is done in cooperation with the School of Hospitality, Culinary Arts &amp; Meal Science, Örebro UniversityVishal Singh Parihar, Örebro University, Department of Restaurant and Culinary Arts, Sörälgsvägen 2, SE-712 60 Grythyttan, Sweden or ICAR Research Complex for Goa, Ela, Old Goa – 403402, Goa, India. Phone 0832-2284678/79; Fa:0832-2285649. E-mail: drvishu@yahoo.co.in</p>

The Gram-positive human pathogen Listeria monocytogenes uses a wide range of virulence factors for its pathogenesis. The majority of its virulence genes are encoded on a 9-kb pathogenicity island and are controlled by the transcriptional activator PrfA. Expression of these genes is maximal at 37°C and minimal at 30°C in a mechanism involving an RNA thermosensor. This thesis brings up different aspects of RNA-mediated regulation, including regulatory RNA structures within coding mRNA controlling expression to 5-untranslated RNA (5´-UTR) that controls downstream genes (cis-acting) as well as small non-coding RNAs (ncRNAs) that bind other target RNA (trans-acting). We investigated the importance of the coding region of the prfA-mRNA for its expression. Various lengths of prfA-mRNA were fused with reporter genes. Our finding suggested that the first 20 codons of prfA-mRNA were essential for efficient translation in Listeria monocytogenes. Translation of the shorter constructs was shown to be reduced. The expression level showed an inverse correlation with the RNA secondary structure stability in the beginning of the coding region. Riboswitches have previously been known to control expression of their downstream mRNA in a cis-acting manner. A trans-acting S-adenosylmethionine-binding riboswitch termed SreA was identified in Listeria monocytogenes. It was found to control the expression of the virulence regulator PrfA, by binding to the prfA-UTR and thereby affecting its translation. We examined the RNA locus encoding different virulence factors in Listeria monocytogenes. Several of them were preceded by 5´-UTRs of various lengths. We speculate that these 5´-UTRs could control expression of the downstream mRNA, provided they are of sufficient length. These findings prompted us to examine where and when Listeria monocytogenes switches on gene expression. Tiling array was used to compare RNAs isolated from wild-type and mutant bacteria grown at different growth conditions. Antisense RNAs covering parts of or whole open-reading frames as well as 29 new ncRNAs were identified. Several novel riboswitches possibly functioning as upstream terminators were also found. My thesis work compiles together a variety of novel RNA-mediated gene regulatory entities. A first coordinated transcriptional map of Listeria monocytogenes has been set up. My work has also revealed that the expression of the virulence regulator PrfA is controlled at several levels, indicating the importance of both the 5´-UTR and the coding RNA for regulated expression.

There is a constant need of new synthetic emulsifiers in the food industry. Sugar esters are widely used as food grade synthetic emulsifiers, amongst which sucrose esters are the most common. Although sucrose esters are used very frequently, little is known about the use of lactose esters in food. There is a need for characterization of lactose esters before they can be used in foods. The objective of this study was to characterize a lactose ester, lactose monolaurate (LML) as an antimicrobial agent on food pathogens, evaluate its effect on 20 % oil-in-water emulsions as an emulsifier, and to explore its effect on crystallization behavior of anhydrous milk fat. In the first study (Chapter 3), the effect of LML was evaluated on survival of some Gram-positive and Gram-negative bacteria. For Listeria monocytogenes, a concentration of 1 mg/ml showed some inhibition in growth media whereas the cells were completely killed at 5 mg/ml. For Mycobacteria, an LML concentration between 0.1-1mg/ml was lethal. Scanning electron microscopy was also conducted to examine any changes in the morphology of cells. Listeria exhibited a change in morphology and a wrinkling effect was shown in Mycobacteria. In the second study (Chapter 4), the effect of LML as an emulsifier was evaluated in 20 % oil-in-water emulsions. The use level of LML was comparable to commercially available emulsifier polysorbate 20, and produced comparable stabilization in the emulsions upon use. In this study, an attempt was also made to optimize the synthesis of LML with respect to the immobilized enzyme and solvent combination. It was concluded that for 20 % oil-in-water emulsions, LML is a promising emulsifier at 0.5%. In the third study (Chapter 5), the effect of LML was evaluated at two concentrations on the crystallization behavior of anhydrous milk fat at two temperatures with high and low supercooling. On application of high intensity ultrasound (HIU) to anhydrous milk fat (AMF) at 31°C and 0.05 % LML the effect on viscosity of sample and crystallization behavior was evaluated. It was concluded that the viscosity of AMF decreased with the addition of 0.05% LML. The lower viscosity of anhydrous milk fat on addition of LML could be restored with the application of HIU.

Cancer vaccines can fail despite the induction of large numbers of CD8+ T cells. Two categories of memory CD8+ T cells have been defined; central memory (TCM, IL-7RαhighCD44highCD62Lhigh) and effector memory (TEM, IL-7RαhighCD44highCD62Llow). It is clear that the memory phenotype of CD8+ T cells can affect vaccine potential; however methods to augment a beneficial phenotype are not clear. I have compared three vaccine delivery systems: Listeria monocytogenes, Salmonella enterica serovar Typhimurium and the particulate liposomal adjuvant, archaeosomes, for their efficacy to protect against murine melanoma. My study revealed that the anti-tumour response is strongly influenced by the kinetics, phenotype, and lymph node homing potential of CD8+ T cells. Listeria monocytogenes-ovalbumin (LM-OVA) induced TCM cells were adept at long lasting protection against B16-OVA melanoma due to their increased homeostatic and antigen-induced proliferation, interleukin-2 production, and ability to extravasate into tumour draining lymph nodes. Conversely, although Salmonella Typhimurium-ovalbumin (ST-OVA) induced TEM, produced IFN-γ, and killed target cells, this was insufficient for long-term tumour protection. Selectin-ligand engagements of TCM cells influenced their homing potential and efficacy against murine melanoma. Fucosyltransferase deficient (FtDKO) mice, lacking functional selectin ligands, were vaccinated with LM-OVA; despite the activation of cytotoxic CD8+ T cells, there was a reduced protection against murine melanoma compared to wild-type. FtDKO CD8+ T cells exhibited reduced extravasation into FtDKO lymph nodes compared to wild-type. Additionally, fewer FtDKO CD8+ T cells compared to wild-type migrated into tumour sites. Archaeosome vaccination was used to compare the influence of CD8+ T cell quantity versus phenotype. Single or multiple therapeutic vaccinations with archaeosome-OVA yielded transient melanoma tumour protection, despite an increased frequency of circulating and tumour infiltrating CD8+ T cells. This correlated with increased expression of Program death receptor-1 (PD-1) on CD8+ T cells and induction of regulatory T cells. Prophylactic archaeosome-OVA vaccination resulted in a maximal frequency of antigen-specific CD8+ T cells of ~50-60 % with just three injections, and ~50 % of the mice were of mice were afforded long-term tumour protection (> 90 days). Overall, my study shows that the choice of vaccine adjuvant and/or vector can profoundly influence CD8+ T cell quality and cancer vaccine efficacy.

Cancer vaccines can fail despite the induction of large numbers of CD8+ T cells. Two categories of memory CD8+ T cells have been defined; central memory (TCM, IL-7RαhighCD44highCD62Lhigh) and effector memory (TEM, IL-7RαhighCD44highCD62Llow). It is clear that the memory phenotype of CD8+ T cells can affect vaccine potential; however methods to augment a beneficial phenotype are not clear. I have compared three vaccine delivery systems: Listeria monocytogenes, Salmonella enterica serovar Typhimurium and the particulate liposomal adjuvant, archaeosomes, for their efficacy to protect against murine melanoma. My study revealed that the anti-tumour response is strongly influenced by the kinetics, phenotype, and lymph node homing potential of CD8+ T cells. Listeria monocytogenes-ovalbumin (LM-OVA) induced TCM cells were adept at long lasting protection against B16-OVA melanoma due to their increased homeostatic and antigen-induced proliferation, interleukin-2 production, and ability to extravasate into tumour draining lymph nodes. Conversely, although Salmonella Typhimurium-ovalbumin (ST-OVA) induced TEM, produced IFN-γ, and killed target cells, this was insufficient for long-term tumour protection. Selectin-ligand engagements of TCM cells influenced their homing potential and efficacy against murine melanoma. Fucosyltransferase deficient (FtDKO) mice, lacking functional selectin ligands, were vaccinated with LM-OVA; despite the activation of cytotoxic CD8+ T cells, there was a reduced protection against murine melanoma compared to wild-type. FtDKO CD8+ T cells exhibited reduced extravasation into FtDKO lymph nodes compared to wild-type. Additionally, fewer FtDKO CD8+ T cells compared to wild-type migrated into tumour sites. Archaeosome vaccination was used to compare the influence of CD8+ T cell quantity versus phenotype. Single or multiple therapeutic vaccinations with archaeosome-OVA yielded transient melanoma tumour protection, despite an increased frequency of circulating and tumour infiltrating CD8+ T cells. This correlated with increased expression of Program death receptor-1 (PD-1) on CD8+ T cells and induction of regulatory T cells. Prophylactic archaeosome-OVA vaccination resulted in a maximal frequency of antigen-specific CD8+ T cells of ~50-60 % with just three injections, and ~50 % of the mice were of mice were afforded long-term tumour protection (> 90 days). Overall, my study shows that the choice of vaccine adjuvant and/or vector can profoundly influence CD8+ T cell quality and cancer vaccine efficacy.

Thèse de doctorat : Biologie cellulaire et moléculaire. Immunologie : Orléans : 2009. / Titre provenant de l'écran-titre.