Investigation of lantibiotic regulation, immunity and synergy

Due to the increasing incidence of antibiotic resistant strains, the use of novel antimicrobials, such as bacteriocins, has become an ever more likely prospect. Lacticin 3147 (of which there are two components, Ltnα and Ltnβ) and nisin belong to the subgroup of bacteriocins called the lantibiotics, which has attracted much attention in recent years. The lantibiotics are antimicrobial peptides that contain unusual amino acids resulting from a series of enzyme-mediated post translational modifications. Given that there have been relatively few examples of lantibiotic-specific resistance; these antimicrobials appear to represent valid alternatives to classical antibiotics. However, the fact that lantibiotics are naturally only produced in small amounts often hinders their commercialisation. In order to overcome this bottleneck, several approaches can be employed. For example, we can create a situation that reduces the quantity of a lantibiotic required to inhibit a target by combining it with other antimicrobials. Here, following an initial screen involving lacticin 3147 and several classical antibiotics, it was observed between lacticin 3147 and the commercial antibiotics polymyxin B/E function synergistically. This reduced the amounts of the individual antimicrobials required for kill and broadened the spectrum of inhibition of both agents. Upon combination with polymyxins, lacticin 3147, which has been associated with Gram positive targets only, actively targeted Gram negative species such as Escherichia coli and Cronobacter sp. An alternative means of addressing problems associated with lantibiotic yield is to better understand how production is regulated, and ultimately use this information to enhance peptide levels. With this in mind the regulation of lacticin 3147 production from the promoter Pbac was investigated using a green fluorescent protein (GFP) expression reporter system. This revealed that elements within both of the divergent operons of the lacticin 3147 gene cluster are involved in Pbac regulation. That is, LtnR, already established as a negative regulator of itself and the lacticin 3147 associated immunity genes, also acts as an activator of Pbac transcription. In contrast, an enhanced level of expression is observed in the absence of the lacticin 3147 structural genes, ltnA1 and ltnA2, indicating that these genes/gene products are involved in Pbac repression. In fact, through complementation of the ltnA2 gene, it was revealed that this regulation is more likely to be dependent on the presence of the gene transcript rather that the corresponding prepropeptide or modified Ltnβ. It may be that if lacticin 3147 production is successfully enhanced, the ability of the producing cell to protect itself may become an issue. To prepare for such a possibility a bioengineered derivative of the lacticin 3147 immunity protein LtnI (LtnI I81V) which provides enhanced protection was discovered through an in depth investigation involving the site and saturation mutagenesis of this protein. In addition, the creation of truncated forms of LtnI allowed the identification of important and essential regions of this immunity protein. Finally, as mentioned, self-immunity is essential to prevent self-killing. However the discovery of nisin U immunity and regulatory gene homologues (spiFEGRR’K) within the pathogenic strain S. infantarius subsp. infantarius is a cause for concern as it represents an example of immune mimicry, a form of lantibiotic-specific resistance. The ability of spiFEG to confer protection was apparent when they successfully provided protection to nisin A, F, Z, Q and U when expressed heterologously in the nisin sensitive L. lactis HP host. As a consequence of the studies presented in this thesis, it is likely that strategies will emerge that will facilitate the production of greater levels of lacticin 3147 production and lead to enhanced immunity in lactococcal backgrounds. Alternatively the need for enhanced production could be avoided through the use of antimicrobial combinations. In addition, providing awareness of the threats of the emergence of resistance through immune mimicry can allow researchers to develop strategies to prevent this phenomenon from leading to the dissemination of lantibiotic resistance.

The biodiversity, control and genetic characterisation of the lactococcal 936 group phages

Phages belonging to the 936 group represent one of the most prevalent and frequently isolated phages in dairy fermentation processes using Lactococcus lactis as the primary starter culture. In recent years extensive research has been carried out to characterise this phage group at a genomic level in an effort to understand how the 936 group phages dominate this particular niche and cause regular problems during large scale milk fermentations. This thesis describes a large scale screening of industrial whey samples, leading to the isolation of forty three genetically different lactococcal phages. Using multiplex PCR, all phages were identified as members of the 936 group. The complete genome of thirty eight of these phages was determined using next generation sequencing technologies which identified several regions of divergence. These included the structural region surrounding the major tail protein, the replication region as well as the genes involved in phage DNA packing. For a number of phages the latter genomic region was found to harbour genes encoding putative orphan methyltransferases. Using small molecule real time (SMRT) sequencing and heterologous gene expression, the target motifs for several of these MTases were determined and subsequently shown to actively protect phage DNA from restriction endonuclease activity. Comparative analysis of the thirty eight phages with fifty two previously sequenced members of this group showed that the core genome consists of 28 genes, while the non-core genome was found to fluctuate irrespective of geographical location or time of isolation. This study highlights the continued need to perform large scale characterisation of the bacteriophage populations infecting industrial fermentation facilities in effort to further our understanding dairy phages and ways to control their proliferation.

Oxidation-reduction potential and its influence on Cheddar cheese quality

Oxidation-reduction (redox) potential is a fundamental physicochemical parameter that affects the growth of microorganisms in dairy products and contributes to a balanced flavour development in cheese. Even though redox potential has an important impact on the quality of dairy products, it is not usually monitored in dairy industry. The aims of this thesis were to develop practical methods for measuring redox potential in cheese, to provide detailed information on changes in redox potential during the cheesemaking and cheese ripening and how this parameter is influenced by starter systems and to understand the relationship between redox potential and cheese quality. Methods were developed for monitoring redox potential during cheesemaking and early in ripening. Changes in redox potential during laboratory scale manufacture of Cheddar, Gouda, Emmental, and Camembert cheeses were determined. Distinctive kinetics of reduction in redox potential during cheesemakings were observed, and depended on the cheese technology and starter culture utilised. Redox potential was also measured early in ripening by embedding electrodes into Cheddar cheese at moulding together with the salted curd pieces. Using this approach it was possible to monitor redox potential during the pressing stage. The redox potential of Emmental cheese was also monitored during ripening. Moreover, since bacterial growth drives the reduction in redox potential during cheese manufacture and ripening, the ability of Lactococcus lactis strains to affect redox potential was studied. Redox potential of a Cheddar cheese extract was altered by bacterial growth and there were strain-specific differences in the nature of the redox potential/time curves obtained. Besides, strategies to control redox potential during cheesemaking and ripening were developed. Oxidizing or reducing agents were added to the salted curd before pressing and results confirmed that a negative redox potential is essential for the development of sulfur compounds in Cheddar cheese. Overall, the studies described in this thesis gave an evidence of the importance of the redox potential on the quality of dairy products. Redox potential could become an additional parameter used to select microorganisms candidate as starters in fermented dairy products. Moreover, it has been demonstrated that the redox potential influences the development of flavour component. Thus, measuring continuously changes in redox potential of a product and controlling, and adjusting if necessary, the redox potential values during manufacture and ripening could be important in the future of the dairy industry.

Galactokinases: potential biotechnological applications as biocatalysts

Galactokinase, a member of the GHMP (galactokinase, homoserine kinase, mevalonate kinase, phosphomevalonate kinase) family of kinases, catalyses the ATP-dependent phosphorylation of galactose at position 1 on the sugar. This reaction is important in the Leloir pathway of galactose catabolism. The need to produce monosaccharides phosphorylated at position 1 for the synthesis of complex molecules, including aminoglycoside antibiotics, has stimulated interest in exploiting the catalytic potential of galactokinases. However, the enzyme is quite specific, generally only catalysing the phosphorylation of D-galactose and closely related molecules. Directed evolution strategies have identified a key tyrosine residue (Tyr-371 in the Escherichia coli enzyme) which, although distant from the active site, influences the specificity of the enzyme. Alteration of this residue to histidine in E. coli and Lactococcus lactis galactokinases dramatically expanded the substrate range to include both D- and L-sugars. Similar experiments with the human enzyme demonstrated that alteration of the equivalent tyrosine (Tyr-379) to cysteine, lysine, arginine, serine or tryptophan increased the catalytic promiscuity of the enzyme. It has been hypothesised that these specificity changes arise because of alterations in the flexibility of the polypeptide chain. This hypothesis has yet to be tested experimentally. The biotechnological potential of galactokinases is clearly considerable and exploitation of closely related enzymes such as N-acetylgalactosamine kinase and arabinose kinase would expand that potential still further.

High-speed conversion of xylose to l-lactate by electrodialysis bioprocess

Conversion of xylose to l-lactate was carried out by Lactococcus lactis IO-1 using an electrodialysis bioprocess (ED-BP). At 50 g l -1 xylose, the ED-BP was already complete in half the time (32 h) taken by the control culture without electrodialysis (>60 h). At 80 g l -1 xylose, the control culture was unable to consume >50 g l -1 xylose, whereas the ED-BP consumed 75 g l -1 xylose in 45 h. Thus, the simultaneous removal of lactate and acetate by ED-BP was associated with high-speed l-lactate production, increased xylose consumption and an increased l-lactate production. Copyright (C) 1998 Elsevier Science B.V.

Fracionamento por processos de membranas do soro de queijo com vista ao uso na produção de bioetanol

Dissertação de mestrado, Engenharia Biológica, Faculdade de Ciências e da Tecnologia, Universidade do Algarve, 2015

Definição do modo de operação, descontínuo ou semi-descontínuo, para produção de bioetanol utilizando um novo substrato misto pela levedura Kluyveromyces sp.

Dissertação de mestrado, Engenharia Biológica, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Efeitos da ingestão de simbiótico e indol-3-carbinol sobre o processo de carcinogênese química de cólon em ratos Wistar alimentados com dieta contendo heme

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Formulação de um biofilme para controlo da listéria em queijos

Dissertação de Mestrado, Tecnologia e Segurança Alimentar, 12 Fevereiro de 2016, Universidade dos Açores.

Use of a human-like low-grade bacteremia model of experimental endocarditis to study the role of Staphylococcus aureus adhesins and platelet aggregation in early endocarditis.

Animal models of infective endocarditis (IE) induced by high-grade bacteremia revealed the pathogenic roles of Staphylococcus aureus surface adhesins and platelet aggregation in the infection process. In humans, however, S. aureus IE possibly occurs through repeated bouts of low-grade bacteremia from a colonized site or intravenous device. Here we used a rat model of IE induced by continuous low-grade bacteremia to explore further the contributions of S. aureus virulence factors to the initiation of IE. Rats with aortic vegetations were inoculated by continuous intravenous infusion (0.0017 ml/min over 10 h) with 10(6) CFU of Lactococcus lactis pIL253 or a recombinant L. lactis strain expressing an individual S. aureus surface protein (ClfA, FnbpA, BCD, or SdrE) conferring a particular adhesive or platelet aggregation property. Vegetation infection was assessed 24 h later. Plasma was collected at 0, 2, and 6 h postinoculation to quantify the expression of tumor necrosis factor (TNF), interleukin 1α (IL-1α), IL-1β, IL-6, and IL-10. The percentage of vegetation infection relative to that with strain pIL253 (11%) increased when binding to fibrinogen was conferred on L. lactis (ClfA strain) (52%; P = 0.007) and increased further with adhesion to fibronectin (FnbpA strain) (75%; P < 0.001). Expression of fibronectin binding alone was not sufficient to induce IE (BCD strain) (10% of infection). Platelet aggregation increased the risk of vegetation infection (SdrE strain) (30%). Conferring adhesion to fibrinogen and fibronectin favored IL-1β and IL-6 production. Our results, with a model of IE induced by low-grade bacteremia, resembling human disease, extend the essential role of fibrinogen binding in the initiation of S. aureus IE. Triggering of platelet aggregation or an inflammatory response may contribute to or promote the development of IE.

Functional and structural basis for a bacteriophage homolog of human RAD52.

In eukaryotes, homologous recombination proteins such as RAD51 and RAD52 play crucial roles in DNA repair and genome stability. Human RAD52 is a member of a large single-strand annealing protein (SSAP) family [1] and stimulates Rad51-dependent recombination [2, 3]. In prokaryotes and phages, it has been difficult to establish the presence of RAD52 homologs with conserved sequences. Putative SSAPs were recently found in several phages that infect strains of Lactococcus lactis[4]. One of these SSAPs was identified as Sak and was found in the virulent L. lactis phage ul36, which belongs to the Siphoviridae family [4, 5]. In this study, we show that Sak is homologous to the N terminus of human RAD52. Purified Sak binds single-stranded DNA (ssDNA) preferentially over double-stranded DNA (dsDNA) and promotes the renaturation of long complementary ssDNAs. Sak also binds RecA and stimulates homologous recombination reactions. Mutations shown to modulate RAD52 DNA binding [6] affect Sak similarly. Remarkably, electron-microscopic reconstruction of Sak reveals an undecameric (11) subunit ring, similar to the crystal structure of the N-terminal fragment of human RAD52 [7, 8]. For the first time, we propose a viral homolog of RAD52 at the amino acid, phylogenic, functional, and structural levels.

Iron-regulated surface determinant protein a mediates adhesion of staphylococcus aureus to human corneocyte envelope proteins

The ability of Staphylococcus aureus to colonize the human nares is a crucial prerequisite for disease. IsdA is a major S. aureus surface protein that is expressed during human infection and required for nasal colonization and survival on human skin. In this work, we show that IsdA binds to involucrin, loricrin, and cytokeratin K10, proteins that are present in the cornified envelope of human desquamated epithelial cells. To measure the forces and dynamics of the interaction between IsdA and loricrin (the most abundant protein of the cornified envelope), single-molecule force spectroscopy was used, demonstrating high-specificity binding. IsdA acts as a cellular adhesin to the human ligands, promoting whole-cell binding to immobilized proteins, even in the absence of other S. aureus components (as shown by heterologous expression in Lactococcus lactis). Inhibition experiments revealed the binding of the human ligands to the same IsdA region. This region was mapped to the NEAT domain of IsdA. The NEAT domain also was found to be required for S. aureus whole-cell binding to the ligands as well as to human nasal cells. Thus, IsdA is an important adhesin to human ligands, which predominate in its primary ecological niche.

Carbohydrate preference, acid tolerance and bile tolerance in five strains of Bifidobacterium

Aims: To assess the suitability of bifidobacteria for inclusion in synbiotic products on the basis of carbohydrate preference, acid and bile tolerance. Methods and Results: Five strains of Bifidobacterium were analysed for their carbohydrate preference from 12 substrates. Maximum growth rates were used to compare substrate preferences. Galacto-oligosaccharides and isomalto-oligosaccharides were well utilized by all the test species. Most bacteria tested could also utilize at least one type of fructan molecule. To determine transit tolerance of potentially probiotic bifidobacteria, acid and bile resistance was tested. A wide range acid resistance was found. Bile tolerance also varied. Conclusions: GOS and IMO were generally well utilized by the tested species. Other substrates were used to different degrees by the different species. Most bifidobacteria are poorly resistant to strongly acidic conditions with the exception of Bifidobacterium lactis Bb12. Bile tolerances were widely variable and it was shown that caution should be exercised when using colorimetric methods to assess bile tolerance. Significance and Impact of Study: The study allows the comparison of the properties of bifidobacteria, allowing a cost effective screen for the best species for use in synbiotic products to allow better survival and efficacy.

Dietary synbiotics reduce cancer risk factors in polypectomized and colon cancer patients

Background: Animal studies suggest that prebiotics and probiotics exert protective effects against tumor development in the colon, but human data supporting this suggestion are weak. Objective: The objective was to verify whether the prebiotic concept (selective interaction with colonic flora of nondigested carbohydrates) as induced by a synbiotic preparation-oligofructose-enriched inulin (SYN1) + Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (BB12)-is able to reduce the risk of colon cancer in humans. Design: The 12-wk randomized, double-blind, placebo-controlled trial of a synbiotic food composed of the prebiotic SYN1 and probiotics LGG and BB12 was conducted in 37 colon cancer patients and 43 polypectomized patients. Fecal and blood samples were obtained before, during, and after the intervention, and colorectal biopsy samples were obtained before and after the intervention. The effect of synbiotic consumption on a battery of intermediate biomarkers for colon cancer was examined. Results: Synbiotic intervention resulted in significant changes in fecal flora: Bifidobacterium and Lactobacillus increased and Clostridium perfringens decreased. The intervention significantly reduced colorectal proliferation and the capacity of fecal water to induce necrosis in colonic cells and improve epithelial barrier function in polypectomized patients. Genotoxicity assays of colonic biopsy samples indicated a decreased exposure to genotoxins in polypectomized patients at the end of the intervention period. Synbiotic consumption prevented an increased secretion of interleukin 2 by peripheral blood mononuclear cells in the polypectomized patients and increased the production of interferon gamma in the cancer patients. Conclusions: Several colorectal cancer biomarkers can be altered favorably by synbiotic intervention.

Gut fermentation products of insulin-derived prebiotics beneficially modulate markers of tumour progression in human colon tumour cells

Insulin is a prebiotic food ingredient, which suppresses colon tumour growth and development in rats. In the gut lumen, it is fermented to lactic acid and short chain fatty acids (SCFA). Of these, butyrate has suppressing agent activities, but little is known concerning cellular responses to complex fermentation samples. To investigate the effects of fermentation products of insulin on cellular responses related to colon carcinogenesis. Fermentations were performed in anaerobic batch cultures or in a three-stage fermentation model that simulates conditions in colon-segments (proximal, transverse, distal). Substrate was insulin enriched with oligofructose (Raftilose® Synergy1), fermented with probiotics (Bifidobacterium lactis Bb12, Lactobacillus rhamnosus GG), and/or faecal inocula. HT29 or CaCo-2 cells were incubated with supernatants of the fermented samples (2.5%-25% v/v, 24-72 hours). Cellular parameters of survival, differentiation, tumour progression, and invasive growth were determined. Fermentation supernatants derived from probiotics and Synergy1 were more effective than with glucose. The additional fermentation with faecal slurries produced supernatants with lower toxicity, higher SCFA contents, and distinct cellular functions. The supernatant derived from the gut model vessel representing the distal colon, was most effective for all parameters, probably on account of higher butyrate-concentrations. Biological effects of insulin upon colon cells may be mediated not only by growth stimulation of the lactic acid-producing bacteria and/or production of butyrate, but also by other bacteria and products of the gut lumen. These newly reported properties of the supernatants to inhibit growth and metastases in colon tumour cells are important mechanisms of tumour suppression.