Relação entre a Infeção por Escherichia coli Aderente-Invasina e a Doença de Crohn

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Estudo da suscetibilidade aos antibióticos em isolados de Enterobacteriaceae provenientes de suiniculturas Portuguesas

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Nanopartículas lipídicas na promoção da absorção oral de fármacos

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Genome-scale analyses of health-promoting bacteria: probiogenomics

The human body is colonized by an enormous population of bacteria (microbiota) that provides the host with coding capacity and metabolic activities. Among the human gut microbiota are health-promoting indigenous species (probiotic bacteria) that are commonly consumed as live dietary supplements. Recent genomics-based studies (probiogenomics) are starting to provide insights into how probiotic bacteria sense and adapt to the gastrointestinal tract environment. In this Review, we discuss the application of probiogenomics in the elucidation of the molecular basis of probiosis using the well-recognized model probiotic bacteria genera Bifidobacterium and Lactobacillus as examples.

Investigation of growth and stress tolerance characteristics of Cronobacter spp.

Cronobacter spp. are opportunistic pathogens which can be isolated from a wide variety of foods and environments. They are Gram negative, motile, non-spore forming, peritrichous rods of the Enterobacteriaceae family. This food-borne pathogen is associated with the ingestion of contaminated infant milk formula (IMF), causing necrotizing enterocolitis, sepsis and meningitis in neonatal infants. The work presented in this thesis involved the investigation and characterisation of a bank of Cronobacter strains for their ability to tolerate physiologically relevant stress conditions that are commonly encountered in the gastrointestinal tract. While all strains were able to endure the suboptimal conditions tested, noteworthy variations were observed between strains. A collection of these strains were Lux-tagged to determine if their growth could be tracked in IMF by measuring bioluminescence. The resulting strains could be easily and reproducibly monitored in real time by measuring light emission. Following this a transposon mutagenesis library was created in one of the Lux-tagged strains of Cronobacter sakazakii. This library was screened for mutants with affected growth in milk. The majority of mutants identified were associated with amino acid metabolism. The final section of this thesis identified genes involved in the tolerance of C. sakazakii to the milk derived antimicrobial peptide, Lactoferricin B (Lfcin B). This was achieved by creating a transposon mutagenesis library in C. sakazakii and screening for mutants with increased susceptibility to Lfcin B. Overall this thesis demonstrates the variation between Cronobacter strains. It also identifies genes required for growth of the bacteria in milk, as well as genes needed for antimicrobial peptide tolerance.

An evaluation of reovirus as an effective therapeutic for cancer

Cancer is a global problem. Despite the significant advances made in recent years, a definitively effective therapeutic has yet to be developed. Oncolytic virology has fallen back into favour for the treatment of cancer with several viruses and viral vectors currently under investigation including vesicular stomatitis virus (VSV), adenovirus vectors and herpes simplex virus (HSV) vectors. Reovirus has an advantage over many viral vectors in that its wild-type form is non-pathogenic and will selectively infect transformed cells, particularly those mutated in the Ras pathway. These advantages make Reovirus an ideal candidate as a safe and non-toxic therapeutic. The aim of the first part of this study was to determine the effect, if any, of Reovirus on cell lines derived from cancers of the gastrointestinal tract. These cancers, particularly those of the oesophagus and stomach, have extremely poor prognoses and little improvement has been seen in survival of these patients in recent years. Reovirus as a single therapy showed promising results in cell lines of oesophageal, gastric and colorectal origin. Further study of partially resistant cell lines using a combination of Reovirus and conventional therapies, either chemotherapy or radiation, showed that a multi-modal approach to therapy is possible with Reovirus and no antagonism between Reovirus and other treatments was observed. The second part of this study focused on investigating a novel use of Reovirus in an in vivo setting. Cancer vaccination or the use of vaccines in cancer therapy is gaining momentum and success has been seen both in a prophylactic approach and a therapeutic approach. A cell-based Reovirus vaccine was used in both these approaches with encouraging success. When used as a prophylactic vaccine tumour development was subsequently inhibited even upon exposure to a tumorigenic dose of cells. The use of the cell-based Reovirus vaccine as a therapeutic for established tumours showed significant delay in tumour growth and a prolongation of survival in all models. This study has proven that Reovirus is an effective therapeutic in a range of cancers and the successful use of a cell-based Reovirus vaccine leads the way for new advancements in cancer immunotherapy.

The role of metabolism in the pathogenesis of adherent invasive Escherichia coli (AIEC)

Crohn’s disease (CD) is a chronic, relapsing inflammatory condition affecting the gastrointestinal tract of humans, of which there is currently no cure. The precise etiology of CD is unknown, although it has become widely accepted that it is a multifactorial disease which occurs as a result of an abnormal immune response to commensal enteric bacteria in a genetically susceptible host. Recent studies have shown that a new group of Escherichia coli, called Adherent Invasive Escherichia coli (AIEC) are present in the guts of CD patients at a higher frequency than in healthy subjects, suggesting that they may play a role in the initiation and/or maintenance of the inflammation associated with CD. Two phenotypes define an AIEC and differentiate them from other groups of E. coli. Firstly, AIEC can adhere to and invade epithelial cells; and secondly, they can replicate in macrophages. In this study, we use a strain of AIEC which has been isolated from the colonic mucosa of a CD patient, called HM605, to examine the relationship between AIEC and the macrophage. We show, using a systematic mutational approach, that while the Tricarboxylic acid (TCA) cycle, the glyoxylate pathway, the Entner-Doudoroff (ED) pathway, the Pentose Phosphate (PP) pathway and gluconeogenesis are dispensable for the intramacrophagic growth of HM605, glycolysis is an absolute requirement for the ability of this organism to replicate intracellularly. We also show that HM605 activates the inflammasome, a major driver of inflammation in mammals. Specifically, we show that macrophages infected with HM605 produce significantly higher levels of the pro-inflammatory cytokine IL-1β than macrophages infected with a non-AIEC strain, and we show by immunoblotting that this is due to cleavage of caspase-1. We also show that macrophages infected with HM605 exhibit significantly higher levels of pyroptosis, a form of inflammatory cell death, than macrophages infected with a non-AIEC strain. Therefore, AIEC strains are more pro-inflammatory than non-AIEC strains and this may have important consequences in terms of CD pathology. Moreover, we show that while inflammasome activation by HM605 is independent of intracellular bacterial replication, it is dependent on an active bacterial metabolism. Through the establishment of a genetic screen aimed at identifying mutants which activate the inflammasome to lower levels than the wild-type, we confirm our observation that bacterial metabolism is essential for successful inflammasome activation by HM605 and we also uncover new systems/structures that may be important for inflammasome activation, such as the BasS/BasR two-component system, a type VI secretion system and a K1 capsule. Finally, in this study, we also identify a putative small RNA in AIEC strain LF82, which may be involved in modulating the motility of this strain. Overall this works shows that, in the absence of specialised virulence factors, the ability of AIEC to metabolise within the host cell may be a key determinant of its pathogenesis.

The gut microbiota as a contributing factor to antipsychotic-induced weight gain and metabolic dysfunction

Schizophrenia represents one of the world’s most devastating illnesses due to its often lifelong course and debilitating nature. The treatment of schizophrenia has vastly improved over recent decades with the discovery of several antipsychotic compounds; however these drugs are not without adverse effects that must be addressed to maximize their therapeutic value. Newer, atypical, antipsychotics are associated with a compilation of serious metabolic side effects including weight gain, insulin resistance, fat deposition, glucose dysregulation and ensuing co-morbidities such as type II diabetes mellitus. The mechanisms underlying these side effects remain to be fully elucidated and adequate interventions are lacking. Further understanding of the factors that contribute these side effects is therefore required in order to develop effective adjunctive therapies and to potentially design antipsychotic drugs in the future with reduced impact on the metabolic health of patients. We investigated if the gut microbiota represented a novel mechanism contributing to the metabolic dysfunction associated with atypical antipsychotics. The gut microbiota comprises the bacteria that exist symbiotically within the gastrointestinal tract, and has been shown in recent years to be involved in several aspects of energy balance and metabolism. We have demonstrated that administration of certain antipsychotics in the rat results in an altered microbiota profile and, moreover, that the microbiota is required for the full scale of metabolic dysfunction to occur. We have further shown that specific antibiotics can attenuate certain aspects of olanzapine and risperidone–induced metabolic dysfunction, in particular fat deposition and adipose tissue inflammation. Mechanisms underlying this novel link appear to involve energy utilization via expression of lipogenic genes as well as reduced inflammatory tone. Taken together, these data indicate that the gut microbiota is an important factor involved in the myriad of metabolic complications associated with antipsychotic therapy. Furthermore, these data support the future investigation of microbial-based therapeutics for not only antipsychotic-induced weight gain but also for tackling the global obesity epidemic.

Investigations into selective metabolic aspects of bifidobacteria: carbohydrate metabolism, fatty acid biosynthesis and plasmid biology

The gastrointestinal tract (GIT) is a diverse ecosystem, and is colonised by a diverse array of bacteria, of which bifidobacteria are a significant component. Bifidobacteria are Gram-positive, saccharolytic, non-motile, non-sporulating, anaerobic, Y-shaped bacteria, which possess a high GC genome content. Certain bifidobacteria possess the ability to produce conjugated linoleic acid (CLA) from linoleic acid (LA) by a biochemical pathway that is hypothesised to be achieved via a linoleic isomerase. In Chapter two of this thesis it was found that the MCRA-specifying gene is not involved in CLA production in B. breve NCFB 2258, and that this gene specifies an oleate hydratase involved in the conversion of oleic acid into 10-hydroxystearic acid. Prebiotics are defined as non-digestible food ingredients that beneficially affect the host by selectively stimulating growth and/or activity of one or a limited number of bacteria in the colon. Key to the development of such novel prebiotics is to understand which carbohydrates support growth of bifidobacteria and how such carbohydrates are metabolised. In Chapter 3 of this thesis we describe the identification and characterisation of two neighbouring gene clusters involved in the metabolism of raffinose-containing carbohydrates (plus related carbohydrate melibiose) and melezitose by Bifidobacterium breve UCC2003. The fourth chapter of this thesis describes the analysis of transcriptional regulation of the raf and mel clusters. In the final experimental chapter two putative rep genes, designated repA7017 and repB7017, are identified on the megaplasmid pBb7017 of B. breve JCM 7017, the first bifidobacterial megaplasmid to be reported. One of these, repA7017, was subjected to an in-depth characterisation. The work described in this thesis has resulted in an improved understanding of bifidobacterial fatty acid and carbohydrate metabolism, Furthermore, attempts were made to develop novel genetic tools.

Immunomodulatory effects exerted by Lactobacillus salivarius strains on innate immune cells

Despite increased application of commensal bacteria for attempting to improve the symptoms of a variety of inflammatory conditions, including inflammatory bowel diseases, diarrhoea and irritable bowel syndrome, therapeutic approaches that involve live bacteria are hampered by a limited understanding of bacterium-host interactions. Lactobacilli are natural inhabitants of the mammalian gastrointestinal tract and many lactobacilli are regarded as probiotics meaning that they exert a beneficial influence on the health status of their consumers. Modulation of immune responses is a plausible mechanism underlying these beneficial effects. The aim of this thesis was to investigate the effect of 33 Lactobacillus salivarius strains on the production of inflammatory cytokines from a variety of human and mouse immune cells. Induction of immune responses in vitro was shown to be bacterial- and mouse strain-dependent, cell type-dependent, blood donor-dependent and bacterial cell number-dependent. Collectively, these data suggest the importance of a case-by-case selection of candidate strains for their potential therapeutic application. Toll-like receptors (TLRs) recognize microbe-associated molecular patterns (MAMPs) and play a critical role in shaping microbial-specific innate and adaptive immune responses. Following ligand engagement, TLRs trigger a complex network of signalling that culminate in the production of inflammatory mediators. The investigation of the molecular mechanisms underlying the Lb. salivarius-host interaction resulted in the identification of a novel role for TLR2 in negatively regulating TLR4 signalling originated from subcellular compartments within macrophages. Notably, sustained activation of JAK/STAT cascade and M1-signature genes in TLR2-/- macrophages was ablated by selective TLR4 and JAK inhibitors and by absence of TLR4 in TLR2/4-/- cells. In addition, other negative regulators of TLR signalling triggered by Lb. salivarius strains were found to be the adapter molecules TIRAP and TRIF. Understanding negative regulation of TLR signalling may pave the way for the development of novel therapeutics to limit inflammation in multiple diseases.

Immune and stress factors in the pathophysiology of the mdx mouse model of Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a fatal multi-system neuromuscular disease caused by loss of dystrophin. The loss of dystrophin from membranes of contractile muscle cells and the dysregulation of the DAPC, induces chronic inflammation due to tissue necrosis and eventual replacement with collagen which weakens muscular force and strength. Dystrophin deficiency may cause under-diagnosed features of DMD include mood disorders such as depression and anxiety and dysfunction of the gastrointestinal tract. The first study in the thesis examined mood in the dystrophin-deficient mdx mouse model of DMD and examined the effects of the tri-cyclic antidepressant, amitriptyline on behaviours. Amitriptyline had anti-depressant and anxiolytic effects in the mdx mice possibly through effects on stress factors such as corticotrophin-releasing factor (CRF). This antidepressant also reduced skeletal muscle inflammation and caused a reduction in circulating interleukin (IL)-6 levels. In the second and third studies, we specifically blocked IL-6 signalling and used Urocortin 2, CRFR2 agonist to investigate their potential as therapeutic targets in mdx mice pathophysiology. Isometric and isotonic contractile properties of the diaphragm, were compared in mdx mice treated with anti IL-6 receptor antibodies (anti IL-6R) and/or Urocortin 2. Deficits in force production, work and power detected in mdx mice were improved with treatment. In study three I investigated contractile properties in gastrointestinal smooth muscle. As compared to wild type mice, mdx mice had slower faecal transit times, shorter colons with thickened muscle layers and increased contractile activity in response to recombinant IL-6. Blocking IL-6 signalling resulted in an increase in colon length, normalised faecal output times and a reduction in IL-6-evoked contractile activity. The findings from these studies indicate that for both diaphragm and gastrointestinal function in a dystrophin-deficient model, targeting of IL-6 and CRFR2 signalling has beneficial therapeutic effects.

Metabolism of host-derived carbohydrates by Bifidobacterium breve UCC2003

Bifidobacteria are Gram positive, anaerobic, typically Y-shaped bacteria which are naturally found in the digestive tract of certain mammals, birds and insects. Bifidobacterium breve strains are numerically prevalent among the gut microbiota of many healthy breast-fed infants. The prototypical B. breve strain UCC2003 has previously been shown to utilise numerous carbohydrates of plant origin. Various aspects of host-derived carbohydrate metabolism occurring in this bacterium will be described in this thesis. Chapter II describes B. breve UCC2003 utilisation of sialic acid, a nine-carbon monosaccharide, which is found in human milk oligosaccharides (HMOs) and the mucin glycoprotein. B. breve UCC2003 was also shown to cross-feed on sialic acid released from 3’ sialyllactose, a prominent HMO, by the extracellular sialidase activity of Bifidobacterium bifidum PRL2010. Chapter III reports on the transcriptional regulation of sialic acid metabolism in B. breve UCC2003 by a transcriptional repressor encoded by the nanR gene. NanR belongs to the GntR-family of transcriptional regulators and represents the first bifidobacterial member of this family to be characterised. Chapter IV investigates B. breve UCC2003 utilisation of mucin. B. breve UCC2003 was shown to be incapable of degrading mucin; however when grown in co-culture with B. bifidum PRL2010 it exhibits enhanced growth and survival properties. A number of methods were used to investigate and identify the mucin components supporting this enhanced growth/viability phenotype. Chapter V describes the characterisation of two sulfatase-encoding gene clusters from B. breve UCC2003. The transcriptional regulation of both sulfatase-encoding gene clusters was also investigated. The work presented in this thesis represents new information on the metabolism of host-derived carbohydrates in bifidobacteria, thus increasing our understanding of how these gut commensals are able to colonise and persist in the gastrointestinal tract.

Postnatally-transmitted HIV-1 Envelope variants have similar neutralization-sensitivity and function to that of nontransmitted breast milk variants.

BACKGROUND: Breastfeeding is a leading cause of infant HIV-1 infection in the developing world, yet only a minority of infants exposed to HIV-1 via breastfeeding become infected. As a genetic bottleneck severely restricts the number of postnatally-transmitted variants, genetic or phenotypic properties of the virus Envelope (Env) could be important for the establishment of infant infection. We examined the efficiency of virologic functions required for initiation of infection in the gastrointestinal tract and the neutralization sensitivity of HIV-1 Env variants isolated from milk of three postnatally-transmitting mothers (n = 13 viruses), five clinically-matched nontransmitting mothers (n = 16 viruses), and seven postnatally-infected infants (n = 7 postnatally-transmitted/founder (T/F) viruses). RESULTS: There was no difference in the efficiency of epithelial cell interactions between Env virus variants from the breast milk of transmitting and nontransmitting mothers. Moreover, there was similar efficiency of DC-mediated trans-infection, CCR5-usage, target cell fusion, and infectivity between HIV-1 Env-pseudoviruses from nontransmitting mothers and postnatal T/F viruses. Milk Env-pseudoviruses were generally sensitive to neutralization by autologous maternal plasma and resistant to breast milk neutralization. Infant T/F Env-pseudoviruses were equally sensitive to neutralization by broadly-neutralizing monoclonal and polyclonal antibodies as compared to nontransmitted breast milk Env variants. CONCLUSION: Postnatally-T/F Env variants do not appear to possess a superior ability to interact with and cross a mucosal barrier or an exceptional resistance to neutralization that define their capability to initiate infection across the infant gastrointestinal tract in the setting of preexisting maternal antibodies.

Dietary quality and encephalization in platyrrhine primates.

The high energetic costs of building and maintaining large brains are thought to constrain encephalization. The 'expensive-tissue hypothesis' (ETH) proposes that primates (especially humans) overcame this constraint through reduction of another metabolically expensive tissue, the gastrointestinal tract. Small guts characterize animals specializing on easily digestible diets. Thus, the hypothesis may be tested via the relationship between brain size and diet quality. Platyrrhine primates present an interesting test case, as they are more variably encephalized than other extant primate clades (excluding Hominoidea). We find a high degree of phylogenetic signal in the data for diet quality, endocranial volume and body size. Controlling for phylogenetic effects, we find no significant correlation between relative diet quality and relative endocranial volume. Thus, diet quality fails to account for differences in platyrrhine encephalization. One taxon, in particular, Brachyteles, violates predictions made by ETH in having a large brain and low-quality diet. Dietary reconstructions of stem platyrrhines further indicate that a relatively high-quality diet was probably in place prior to increases in encephalization. Therefore, it is unlikely that a shift in diet quality was a primary constraint release for encephalization in platyrrhines and, by extrapolation, humans.

Kit-like immunopositive cells in sheep mesenteric lymphatic vessels.

Recent electrophysiological studies have suggested that there is a subpopulation of cells in lymphatic vessels which act as pacemakers controlling the characteristic spontaneous contractile activity in this tissue. In this study, electron microscopy and immunohistochemical techniques were used on sheep mesenteric lymphatic vessels to investigate the morphology of the cells comprising the lymphatic wall. The smooth muscle cells were not orientated in circular and longitudinal layers as is seen in the gastrointestinal tract, but were arranged in bundles which interlock and cross over in a basket-weave fashion. Antibodies to Kit and vimentin, which are widely used to label specialised pacemaking cells in the gastrointestinal tract (known as interstitial cells of Cajal), demonstrated the existence of an axially orientated subpopulation of cells lying between the endothelium and the bulk of the smooth muscle. Examination of this area using electron microscopy showed cells which were electron dense compared to the underlying smooth muscle and contained caveolae, Golgi complexes, mitochondria, 10-nm filaments, a well-developed endoplasmic reticulum and a basal lamina. The smooth muscle cells typically contained caveolae, dense bodies, mitochondria, abundant filaments, sER and basal laminae. Cells dispersed for patch-clamp studies were also stained for vimentin and myosin. Myosin-staining cells had the typical spindle appearance of smooth muscle cells whereas the vimentin-positive cells could either be branched or more closely resemble the smooth muscle cells. The present study provides the first morphological evidence that specialised cells exist within the vascular system which have the ultrastructural characteristics of pacemaker cells in other tissues and are vimentin and Kit positive.