Identification and analysis of a Scophthalmus maximus ferritin that is regulated at transcription level by oxidative stress and bacterial infection

Ferritins are conserved Iron storage proteins that exist in most living organisms and play an essential role in Iron homeostasis. In this study, we reported the identification and analysis a ferritin M subunit, SmFerM, from turbot Scophthalmus maximus. The full length cDNA of SmFerM contains a 5'-untranslated region (UTR) of 232 bp, an open reading frame (ORF) of 531 bp, and a 3'-UTR of 196 bp The ORF encodes a putative protein of 176 amino acids, which shares extensive sequence identities with the M terrains of several fish species. In silico analysis identified in SmFerM both the ferroxidase center of mammalian H ferritins and the iron nucleation site of mammalian L ferritins. Quantitative real time reverse transcriptase-PCR analysis indicated that SmFerM expression was highest in muscle and lowest in heart and responded positively to experimental challenges with bacterial pathogens and poly(I center dot C) Exposure of cultured turbot hepatocytes to treatment of stress inducers (iron, copper, and H2O2) significantly upregulated the expression of SmFerM in a dose dependent manner. Iron chelating analysis showed that recombinant SmFerM purified from Escherichia coli exhibited apparent iron binding activity. These results suggest that SmFerM is a functional M ferritin and is likely to play a role in iron sequestration and protection against oxidative stress and microbial infection (C) 2010 Elsevier Inc All rights reserved

Dominant chloramphenicol-resistant bacteria and resistance genes in coastal marine waters of Jiaozhou Bay, China

Studies of abundance, diversity and distribution of antibiotic-resistant bacteria and their resistance determinants are necessary for effective prevention and control of antibiotic resistance and its dissemination, critically important for public health and environment management. In order to gain an understanding of the persistence of resistance in the absence of a specific antibiotic selective pressure, microbiological surveys were carried out to investigate chloramphenicol-resistant bacteria and the chloramphenicol acetyltransferase resistance genes in Jiaozhou Bay after chloramphenicol was banned since 1999 in China. About 0.15-6.70% cultivable bacteria were chloramphenicol resistant, and the highest abundances occurred mainly in the areas near river mouths or sewage processing plants. For the dominant resistant isolates, 14 genera and 25 species were identified, mostly being indigenous estuarine or marine bacteria. Antibiotic-resistant potential human or marine animal pathogens, such as Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Shewanella algae, were also identified. For the molecular resistance determinants, the cat I and cat III genes could be detected in some of the resistant strains, and they might have the same origins as those from clinical strains as determined via gene sequence analysis. Further investigation about the biological, environmental and anthropogenic mechanisms and their interactions that may contribute to the persistence of antibiotic-resistance in coastal marine waters in the absence of specific antibiotic selective pressure is necessary for tackling this complicated environmental issue.

鱼类疫苗的研究：I: 海水鱼类病原菌乳化口服疫苗的研究；II：迟缓爱德华氏菌和嗜水气单胞菌外膜蛋白疫苗的研究

本论文的目的是研究几种病原菌口服疫苗接种鱼类的免疫效果，并从常见病原菌株中筛选几个具有较好保护效果的蛋白抗原，利用口服免疫的方式，接种养殖动物，并检测免疫效果。 以10号白油为有机溶剂，采用搅拌与均浆方法制备鳗弧菌M3和SMP1的油乳化二价疫苗，用饵料包埋后以口服途径免疫养殖大菱鲆，评价免疫大菱鲆的免疫应答和疫苗的保护效果。结果显示，以油乳化和未油乳化疫苗分别连续口服免疫大菱鲆一周后，在后肠组织，乳化疫苗刺激产生的非特异性活力、特异性抗体水平均高于未乳化疫苗；而在血清，两种疫苗引起的两种酶的活力、SMP1抗体水平没有变化，但在乳化疫苗免疫的大菱鲆检测到明显高于未免疫对照大菱鲆的M3抗体水平。大菱鲆后肠组织原位杂交结果显示，口服免疫的大菱鲆后肠褶皱有IgM抗体的产生和分布。其中，乳化疫苗免疫大菱鲆的IgM抗体的产生和分布水平高于未乳化疫苗免疫的大菱鲆。攻毒实验显示，乳化疫苗免疫的大菱鲆对M3和SMP1的感染分别获得100%和50%的免疫保护率，而未乳化疫苗获得的免疫保护率分别为57.9%和0%，表明乳化疫苗比未乳化疫苗更有效地保护大菱鲆、抵抗病原的感染。在乳化疫苗免疫持续期的研究中，免疫的大菱鲆后肠在免疫后120天仍能检测到抗体效价，在免疫后90天还能观察到一定的免疫保护效果。免疫30天、60天、90天和120天的大菱鲆分别获得100%、66.7%、36.7%和13.3%的免疫保护力。 以鳗弧菌M3和SMP1、链球菌CF、迟缓爱德华菌SMW7作为细菌抗原制备油乳化多价口服疫苗和轮虫携带疫苗，口服途径免疫养殖大菱鲆与大菱鲆初孵仔鱼。结果显示，在免疫大菱鲆后肠可检测到抗M3抗体水平的提高（P<0.05），而在其胆汁、鳃、中肠、体表黏液、前肠与血清中抗体效价变化与对照组没有显示出差异；没有检测到免疫大菱鲆后肠抗SMP1、SMW7、CF抗体效价。M3浸泡攻毒实验显示，口服免疫的大菱鲆获得了100%的免疫保护力；在M3注射攻毒和SMP1、CF、SMW7浸泡攻毒大菱鲆的实验中，在每个攻毒组中，免疫组大菱鲆开始死亡的时间都要比对照组有不同程度的延迟，但攻毒大菱鲆都发生死亡，不能显示出与对照组的差异。轮虫携带免疫的结果显示，免疫的大菱鲆初孵仔鱼并未获得较好的保护效果，与对照大菱鲆没有体现出差异。 从致病性病嗜水气单胞菌（Aeromonas hydrophila）LSA34克隆并表达ahaI基因和gapA基因，从迟缓爱德华菌（Edwardsiella tarda）LSE40克隆并表达eseB，将所得蛋白分别通过腹腔注射途径免疫大菱鲆，检测蛋白的免疫原性和免疫保护。结果在免疫后7天就可以检测到AhaI、GapA蛋白免疫组大菱鲆产生的抗体，至第40天可以检测到明显的保护性抗体，之后抗体效价增加明显，直至第60天时达到最高值。EseB免疫的大菱鲆第一次免疫后15天就有较高的抗体效价产生，明显高于对照组大菱鲆血清抗体效价，到距第一次免疫60天时，抗体效价达到最高值。攻毒实验显示，与对照组相比，AhaI免疫组和GapA免疫组对LSA34感染的免疫保护力分别为80%和100%；AhaI免疫组和GapA免疫组对LSE40感染的免疫保护力分别为30%和10%。，而对照组牙鲆对人工攻毒不具有保护力。以AhaI和GapA作为疫苗免疫大菱鲆，使大菱鲆获得了对嗜水气单胞菌LSA34较高的免疫保护；而对迟缓爱德华氏菌LSE40的交叉保护能力没有明显提高。EseB免疫的大菱鲆在攻毒实验中并没有显示出较好的保护效果，与对照组相比，只是在死亡时间上有所延迟。 以从致病性嗜水气单胞菌中克隆的ahaI和gapA基因表达出的蛋白为蛋白抗原制备油乳化疫苗，用饵料包埋后以口服途径免疫养殖牙鲆，评价免疫牙鲆的免疫应答和疫苗的保护效果。结果显示，以油乳化和未油乳化疫苗分别免疫牙鲆一周后，在后肠组织，AhaI和GapA乳化疫苗免疫组牙鲆检测到抗体，且分别高于AhaI和GapA未乳化疫苗免疫的牙鲆；而在血清，GapA的两种疫苗引起的GapA抗体水平没有变化；但在AhaI乳化疫苗免疫的牙鲆第21天和第35天的血清中检测到高于未免疫对照牙鲆的AhaI抗体水平，AhaI未乳化疫苗免疫牙鲆血清对照组相比没有检测到AhaI抗体水平的变化。

Studies on mass summer mortality of cultured zhikong scallops (Chlamys farreri Jones et Preston) in China

Mass mortalities of cultured zhikong scallops (Chlamys farreri) have occurred each summer in most culture areas of northern China since 1996. Among the hypothesized causes are high culture density, infectious disease and genetic inbreeding. To investigate these potential agents, C. farreri were deployed at three densities (low, medium and high) at three sites (Jiaonan, Penglai and Yantai) in the summer of 2000. Scallops were sampled for survival, growth and histopathology before, during and after a mortality episode. Most of the mortality occurred in July and August, during and toward the later part of the spawning season, when water temperature reached 23-26 degrees C. Final cumulative mortalities reached 85% to 90% at all three sites. Scallops in the medium and high densities had higher initial death rates than did those at the low density. High densities also inhibited growth. Ciliates from the genus Trichodina, larvae of various organisms and anomalous secretions were observed in sections of the gill cavity, with highest prevalence during and at the end of the mortality period. Prokaryotic inclusion bodies were found in the soft tissues, but their prevalence was low and apparently without correlation with mortalities. Genetic analysis with random amplified polymorphic DNA markers showed slightly lower heterozygosity in the cultured stocks (0.301) than in the wild stocks (0.331). It is possible that the mortalities are caused by a combination of several factors such as stress associated with reproduction, high temperature, overcrowding and poor circulation in the growout cages, opportunistic invaders or pathogens, and possibly inbreeding. (c) 2005 Elsevier B.V. All rights reserved.

An unidentified haplosporidian parasite of bay scallop Argopecten irradians cultured in the Shandong and Liaoning provinces of China

Since 1988 growers of bay scallop Argopecten irradians in China have been experiencing mortality in their cultured stocks. Although poorly documented, mortality apparently began near Qingdao and has since spread to other areas of Shandong and Liaoning provinces. Samples of cultured scallops were collected from several growing areas in these provinces and analyzed by histological methods for pathogens. An unidentified haplosporidian parasite was observed in a high proportion of scallops from two of the stocks examined. Most infections were of low intensity, but one heavy infection was also observed. Only plasmodia stages were observed; they occurred intercellularly in connective tissues throughout the scallops. Plasmodia were spherical to oval, varied from 4.0 to 17.0 mu m in diameter and contained from 2 to 18 nuclei. Absence of spores prevented generic assignment of the parasite. The source and pathogenicity of the haplosporidian could not be assessed without additional research. No other microbial parasites (i.e. rickettsia-like, chlamydia-like or kidney coccidia) were observed in any of the scallops examined.

Exploitation of Ebola Virus VP35 Protein to identify new drugs counteracting its type I IFN antagonism

Ebolaviruses (EBOVs) are among the most virulent and deadly pathogens ever known, causing fulminant haemorrhagic fevers in humans and non-human primates. The 2014 outbreak of Ebola virus disease (EVD) in West Africa has claimed more lives than all previous EVD outbreaks combined. The EBOV high mortality rates have been related to the virus-induced impairment of the host innate immunity reaction due to two virus-coded proteins, VP24 and VP35. EBOV VP35 is a multifunctional protein, it is essential for viral replication as a component of the viral RNA polymerase and it also participates in nucleocapsid assembly. Early during EBOV infection, alpha-beta interferon (IFN-α/β) production would be triggered upon recognition of viral dsRNA products by cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs). However, this recognition is efficiently prevented by the double-stranded RNA (dsRNA) binding activity of the EBOV VP35 protein, which hides RLRs binding sites on the dsRNA phosphate backbone as well the 5’-triphosphate (5’-ppp) dsRNA ends to RIG-I recognition. In addition to dsRNA binding and sequestration, EBOV VP35 inhibits IFN-α/β production preventing the activation of the IFN regulatory factor 3 (IRF-3) by direct interaction with cellular proteins. Previous studies demonstrated that single amino acid changes in the VP35 dsRNA binding domain reduce EBOV virulence, indicating that VP35 is an attractive target for antiviral drugs development. Within this context, here we report the establishment of a novel method to characterize the EBOV VP35 inhibitory function of the dsRNA-dependent RIG-I-mediated IFN-β signaling pathway in a BLS2 cell culture setting. In such system, a plasmid containing the promoter region of IFN-β gene linked with a luciferase reporter gene was transfected, together with a EBOV VP35 mammalian expression plasmid, into the IFN-sensitive A549 cell line, and the IFN-induction was stimulated through dsRNA transfection. Through alanine scanning mutational studies with biochemical, cellular and computational methods we highlighted the importance of some VP35 residues involved in dsRNA end-capping binding, such as R312, K282 and R322, that may serve as target for the development of small-molecule inhibitors against EBOV. Furthermore, we identified a synthetic compound that increased IFN-induction only under antiviral response stimulation and subverted VP35 inhibition, proving to be very attractive for the development of an antiviral drug. In conclusion, our results provide the establishment of a new assay as a straightforward tool for the screening of antiviral compounds that target i) dsRNA-VP35 or cellular protein-VP35 interaction and ii) dsRNA-dependent RIG-I-mediated IFN signaling pathway, in order to potentiate the IFN response against VP35 inhibition, setting the bases for further drug development.

Sinusite odontogénica

Monografia apresentada à Universidade Fernando Pessoa para obtenção do grau de Licenciada em Medicina Dentária.

Doença de Alzheimer e Periodontite

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

Controlo do tratamento endodontico não-cirúrgico de dentes com diagnóstico de necrose pulpar associada ou não a lesão apical

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

Molecular analysis of virulence mechanisms associated with adherent-invasive Escherichia coli (AIEC)

Crohn's Disease (CD) is a chronic inflammatory bowel disease of unknown etiology. Recent work has shown that a new pathotype of Escherichia coli, Adherent Invasive E. coli (AIEC) may be associated with CD. AIEC has been shown to adhere to and invade epithelial cells and to replicate within macrophages (together this is called the AIEC phenotype). In this thesis, the AIEC phenotype of 84 E. coli strains were determined in order to identify the prevalence of this phenotype within the E. coli genus. This study showed that a significant proportion of E. coli strains (approx. 5%) are capable of adhering to and invading epithelial cells and undergoing intramacrophage replication. Moreover, the results presented in this study indicate a correlation between survival in macrophage and resistance to grazing by amoeba supporting the coincidental evolution hypothesis that resistance to amoebae could be a driving force in the evolution of pathogenicity in some bacteria, such as AIEC. In addition, this study has identified an important regulatory role for the CpxA/R two component system (TCS) in the invasive abilities of AIEC HM605, a colonic mucosa-associated CD isolate. A mutation in cpxR was shown to be defective in the invasion of epithelial cells and this defect was shown to be independent of motility or the expression of Type 1 fimbriae, factors that have been shown to be involved in the invasion of another strain of AIEC, isolated from a patient with ileal CD, called LF82. The CpxA/R TCS responds to disturbances in the cell envelope and has been implicated in the virulence of a number of Gram negative pathogens. In this study it is shown that the CpxA/R TCS regulates the expression of a potentially novel invasin called SinH. SinH is found in a number of invasive strains of E. coli and Salmonella. Moreover work presented here shows that a critical mechanism underpinning AIEC persistence in macrophages is the repair of DNA bases damaged by macrophage oxidants. Together these findings provide evidence to suggest that AIEC are a diverse group of E. coli and possess diverse molecular mechanisms and virulence factors that contribute to the AIEC phenotype. In addition, AIEC may have gone through different evolutionary histories acquiring various molecular mechanisms ultimately culminating in the AIEC phenotype. The gastrointestinal (GI) tract harbors a diverse microbiota; most are symbiotic or commensal however some bacteria have the potential to cause disease (pathobiont). The work presented here provides evidence to support the model that AIEC are pathobionts. AIEC strains can be carried as commensals in healthy guts however, when the intestinal homeostasis is disrupted, such as in the compromised gut of CD patients, AIEC may behave as opportunistic pathogens and cause and/or contribute to disease by driving intestinal inflammation.

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.

Biological wastes as plant growth substrate amendments

Globally, agriculture is being intensified with mechanization and increased use of synthetic fertilizers and pesticides. There has been a scaling up of production to satisfy the demands of supermarket distribution. Problems associated with intensification of production, trade globalisation and a larger market demand for greater volumes of fresh produce, include consumers' concern about pesticide residues and leaching of nutrients and pesticides into the environment, as well as increases in the transmission of human food-poisoning pathogens on raw vegetables and in fruit juices. The first part of this research was concerned with the evaluation of a biological control strategy for soil-borne pathogens, these are difficult to eliminate and the chemicals of which the most effective fumigants e.g. methyl bromide, are being withdrawn form use. Chitin-containing crustaceans shellfish waste was investigated as a selective growth substrate amendment in the field, in glasshouse and in storage trials against Sclerotinia disease of Helianthus tuberosus, Phytophthora fragariae disease of Fragaria vesca and Fusarium disease of Dianthus. Results showed that addition to shellfish waste stimulated substrate microbial populations and lytic activity and induced plant defense proteins, namely chitinases and cellulases. Protective effects were seen in all crop models but the results indicate that further trials are required to confirm long-term efficacy. The second part of the research investigated the persistence of enteric bacteria in raw salad vegetables using model food poisoning isolates. In clinical investigations plants are sampled for bacterial contamination but no attempt is made to differentiate between epiphytes and endophytes. Results here indicate that the mode isolates persist endophytically thereby escaping conventional chlorine washes and they may also induce host defenses, which results in their suppression and in negative results in conventional plate count screening. Finally a discussion of criteria that should be considered for a HACCP plan for safe raw salad vegetable production is presented.

Genetic analysis of bacteriophages from clinical and environmental samples

Bacteriophages, viruses infecting bacteria, are uniformly present in any location where there are high numbers of bacteria, both in the external environment and the human body. Knowledge of their diversity is limited by the difficulty to culture the host species and by the lack of the universal marker gene present in all viruses. Metagenomics is a powerful tool that can be used to analyse viral communities in their natural environments. The aim of this study was to investigate diverse populations of uncultured viruses from clinical (a sputum of patient with cystic fibrosis, CF) and environmental samples (a sludge from a dairy food wastewater treatment plant) containing rich bacterial populations using genetic and metagenomic analyses. Metagenomic sequencing of viruses obtained from these samples revealed that the majority of the metagenomic reads (97-99%) were novel when compared to the NCBI protein database using BLAST. A large proportion of assembled contigs were assignable as novel phages or uncharacterised prophages, the next largest assignable group being single-stranded eukaryotic virus genomes. Sputum from a cystic fibrosis patient contained DNA typical of phages of bacteria that are traditionally involved in CF lung infections and other bacteria that are part of the normal oral flora. The only eukaryotic virus detected in the CF sputum was Torque Teno virus (TTV). A substantial number of assigned sequences from dairy wastewater could be affiliated with phages of bacteria that are typically found in the soil and aquatic environments, including wastewater. Eukaryotic viral sequences were dominated by plant pathogens from the Geminiviridae and Nanoviridae families, and animal pathogens from the Circoviridae family. Antibiotic resistance genes were detected in both metagenomes suggesting phages could be a source for transmissible antimicrobial resistance. Overall, diversity of viruses in the CF sputum was low, with 89 distinct viral genotypes predicted, and higher (409 genotypes) in the wastewater. Function-based screening of a metagenomic library constructed from DNA extracted from dairy food wastewater viruses revealed candidate promoter sequences that have ability to drive expression of GFP in a promoter-trap vector in Escherichia coli. The majority of the cloned DNA sequences selected by the assay were related to ssDNA circular eukaryotic viruses and phages which formed a minority of the metagenome assembly, and many lacked any significant homology to known database sequences. Natural diversity of bacteriophages in wastewater samples was also examined by PCR amplification of the major capsid protein sequences, conserved within T4-type bacteriophages from Myoviridae family. Phylogenetic analysis of capsid sequences revealed that dairy wastewater contained mainly diverse and uncharacterized phages, while some showed a high level of similarity with phages from geographically distant environments.

Exploiting the bioactive potential of marine sponge microbiota

Endospore-forming bacteria are often isolated from different marine sponges, but their abundance varies, and they are frequently missed by culture-independent studies. Within endospore-formers, Bacillus are renowned for the production of antimicrobials and other compounds of medical and industrial importance. Although this group has been well studied in many different environments, very little is known about the actual diversity and properties of sporeformers associated with marine sponges. Identification of the endospore-forming bacteria associated with the marine sponges; Haliclona simulans, Amphilectus fucorum and Cliona celata, has uncovered an abundant and diverse microbial population composed of Bacillus, Paenibacillus, Solibacillus, Halobacillus and Viridibacillus species. This diversity appears to be overlooked by other non-targeted approaches where spore-formers are masked by more dominant species within the ecosystem. In addition to the identification of two antibiotic resistant plasmids, this bank of sporeformers produce a range of bioactive compounds. New antimicrobial compounds are urgently needed to combat the spread of multidrug resistant pathogens, as few new options are entering the drug discovery pipelines for clinical trials. Based on the results of this project, endospore-formers associated with marine sponges may hold the answer. The power of coupling functional based assays with genomic approaches has enabled us to identify a novel class 1 lantibiotic, subtilomycin, which is active against several clinically relevant pathogens. Subtilomycin is encoded in the genomes of all the marine sponge B. subtilis isolates analysed. They cluster together phylogenetically and form a distinct group from other sequenced B. subtilis strains. Regardless of its potential clinical relevance, subtilomycin may be providing these strains with a specific competitive advantage(s) within the stringent confines of the marine sponge environment. This work has outlined the industrial and biotechnological potential of marine sponge endospore-formers which appear to produce a cocktail of bioactive compounds. Genome sequencing of specific marine sponge isolates highlighted the importance of mining extreme environments and habitats for new lead compounds with potential therapeutic applications.

Shining light on food microbiology; applications of luciferase-tagged microorganisms in the food industry

Bioluminescence is the production of light by living organisms as a result of a number of enzyme catalysed reactions caused by enzymes termed luciferases. The lux genes responsible for the emission of light can be cloned from one bioluminescent microorganism into one that is not bioluminescent. The light emitted can be monitored and quantified and will provide information on the metabolic activity, quantity and location of cells in a particular environment, in real-time. The primary aim of this thesis was to investigate and identify several food industry related applications of lux-tagged microorganisms. The first aim was to monitor a lux-tagged Cronobacter sakazakii in reconstituted infant milk formula, in realtime. The second aim was to investigate a bioluminescent-based early warning system for starter culture disruption by bacteriophages and antibiotic residues. The third of this thesis was to examine the use of a bioluminescent-based assay to test the activity of bioengineered Nisin derivatives M21V and S29A against foodborne pathogens in laboratory media and selected foods.