Streptococcus anginosus como agentes de infeção em Humanos: um estudo epidemiológico numa taxonomia em mudança

Dissertação para obtenção do grau de Mestre em Microbiologia MédicaEstreptococos do grupo anginosus (SAG) são um grupo de bactérias comensais da microbiota do Homem. Contudo são também isoladas de infeção invasiva e não invasiva com diferentes apresentações, tendo uma considerável morbilidade e mortalidade. O grupo apresenta uma extensa diversidade fenotípica e genética, que dificulta a sua definição taxonómica e consequentemente a sua identificação. Atualmente SAG inclui seis taxa: Streptococcus anginosus subsp. anginosus, S. anginosus subsp. whileyi, S. intermedius, S. constellatus subsp. constellatus, S. constellatus subsp. pharyngis e S. constellatus subsp. viborgensis. Este trabalho teve como objetivos avaliar e comparar os métodos de identificação existentes para o grupo com vista à escolha de um método de referência. Exploraram-se potenciais associações entre os diferentes taxa e os grupos etários, género, apresentação clínica e perfil de resistência aos antimicrobianos. Foi também analisada a clonalidade das estirpes que causam infeção no Homem. Por fim, foi estudada a diversidade genética inter-taxa e intra-taxon, a classificação e a divisão taxonómica de SAG. Neste trabalho foram analisadas 213 estirpes provenientes de laboratórios hospitalares Portugueses, das quais 98 foram recolhidas de infeção invasiva e 115 de infeção não invasiva. Todas as estirpes foram caracterizadas pelo grupo de Lancefield e pelo tipo de hemólise. A susceptibilidade aos antimicrobianos foi testada através do método de difusão em disco de Kirby-Bauer e E-test. A identificação ao nível da espécie e subespécie foi feita por PCR com iniciadores para uma porção do gene que codifica a proteína de ligação à penicilina 2B (pbp2b), para o gene 16S rRNA de S. anginosus e para os genes que codificam os fatores de virulência hialuronidase e intermedilisina. Este método foi assumido como “gold standard” e comparado com métodos de identificação não genotípicos: Enz 5 baseado na actividade enzimática (β-N-acetilgalactosaminidase, β-N-fucosidase, α-glicosidase, β-glicosidase e α-N-acetilneuraminidase), Rapid ID 32 Strep e “Matrix-assisted laser desorption/ionization time of flight mass spectrometry” (MALDI-TOF-MS). As estirpes foram submetidas a tipagem molecular por “pulsed-field gel electrophoresis” (PFGE). A coleção invasiva foi analisada por “multilocus sequence analysis” (MLSA) e “multilocus sequence typing” (MLST) através da sequenciação dos genes map, pfl, ppac, pyk, rpoB, sodA e tuf....Unidade de Microbiologia Molecular e Infeção (UMMI) do Instituto de Medicina Molecular (IMM), Faculdade de Medicina da Universidade de Lisbo

Maintaining the balance: persistent baculovirus infection in insect cells

Baculoviruses such as Autographa californica nucleopolyhedrovirus (AcMNPV) are insect-specific viruses that usually kill the host. However, many species harbour persistent, non-lethal covert baculovirus infections although these have rarely been observed in vitro. Hence, little is known about the mechanisms for establishment or maintenance of persistent infections. Re-infection with a homologous baculovirus (known as superinfection) has been shown to activate the persistent infection to an overt form. In vitro studies have shown that infected cells can be resistant to superinfection although the mechanisms involved are largely unknown. In 2011, a serendipitous event led to the creation of a persistently infected Trichoplusia ni cell line (Hi5) with an AcMNPV p10-deletion mutant (AcUW1.lacZ). From this culture, a clonal cell line (C20) was established that has been maintained to this day. This thesis aimed to use C20 to further our knowledge of the fine balance between host and virus (designated AcC20) that promotes the establishment and maintenance of a persistent infection. The cell line also enabled study of the superinfection exclusion phenomenon. C20 cells demonstrated remarkable viability and diversity of cell size/shape compared to the parental cells. The persistent AcC20 virus was maintained at low levels but budded virus (BV) titres varied considerably within and between different cultures. Presence of BV and the major membrane protein GP64 confirmed C20 harboured a persistent rather than latent infection. Analysis indicated that only a small proportion (<7%) of cells were producing BV whilst superinfection studies demonstrated that the majority of cells were resistant to a secondary infection. Microscopy studies indicated that superinfection may be blocked as early as the cell binding stage although virus uptake was also affected. Whole genome analysis of AcC20 showed four major deletions or insertions with other mutations in 34 coding regions affecting eight essential genes and a homologous region. Transcriptome analysis showed that all virus genes were expressed in C20 cells although the regulated phases of gene expression observed in a typical lytic infection were absent. Host cell transcriptome analysis also revealed differences between the cell response to a lytic and a persistent infection. In particular, endocytic pit formation and host defence pathways appeared to be compromised. The former may help explain resistance to superinfection and the latter may help maintain the persistent infection. Attempts to create further persistent cell lines proved difficult. However, using viruses with a mutation in lef2, in which BV production is severely reduced, overcame this problem. These persistent infections were also used to evaluate the possibility of continuous recombinant protein production by expressing dsred and urokinase under strong promoters. DsRed but not urokinase was produced over several cell passages. This thesis demonstrated establishment and maintenance of persistent infections in cell culture. Further research is needed to understand this process and why these cells are resistant to superinfection

ADDovenom: Thermostable Protein-Based ADDomer Nanoparticles as New Therapeutics for Snakebite Envenoming

Snakebite envenoming can be a life-threatening medical emergency that requires prompt medical intervention to neutralise the effects of venom toxins. Each year up to 138,000 people die from snakebites and threefold more victims suffer life-altering disabilities. The current treatment of snakebite relies solely on antivenom—polyclonal antibodies isolated from the plasma of hyperimmunised animals—which is associated with numerous deficiencies. The ADDovenom project seeks to deliver a novel snakebite therapy, through the use of an innovative protein-based scaffold as a next-generation antivenom. The ADDomer is a megadalton-sized, thermostable synthetic nanoparticle derived from the adenovirus penton base protein; it has 60 high-avidity binding sites to neutralise venom toxins. Here, we outline our experimental strategies to achieve this goal using state-of-the-art protein engineering, expression technology and mass spectrometry, as well as in vitro and in vivo venom neutralisation assays. We anticipate that the approaches described here will produce antivenom with unparalleled efficacy, safety and affordability

Comparing matrix-assisted laser desorption ionization–time of flight mass spectrometry and phenotypic and molecular methods for identification of species within the Streptococcus anginosus group

© 2015 American Society for Microbiology.The heterogeneity of members of the Streptococcus anginosus group (SAG) has traditionally hampered their correct identification. Recently, the group was subdivided into 6 taxa whose prevalence among human infections is poorly described. We evaluated the accuracy of the Rapid ID32 Strep test, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and a PCR multiplex method to identify 212 SAG isolates recovered from human infections to the species and subspecies level by using multilocus sequence analysis (MLSA) as the gold standard. We also determined the antimicrobial susceptibilities of the isolates. Representatives of all SAG taxa were found among our collection. MALDI-TOF MS and the Rapid ID32 Strep test correctly identified 92% and 68% of the isolates to the species level, respectively, but showed poor performance at the subspecies level, and the latter was responsible for major identification errors. The multiplex PCR method results were in complete agreement with the MLSA identifications but failed to distinguish the subspecies Streptococcus constellatus subsp. pharyngis and S. constellatus subsp. viborgensis. A total of 145 MLSA sequence types were present in our collection, indicating that within each taxon a number of different lineages are capable of causing infection. Significant antibiotic resistance was observed only to tetracycline, erythromycin, and clindamycin and was present in most taxa. MALDI-TOF MS is a reliable method for routine SAG species identification, while the need for identification to the subspecies level is not clearly established.info:eu-repo/semantics/publishedVersio

Optimizing Recombinant Baculovirus Vector Design for Protein Production in Insect Cells

Autographa californica nucleopolyhedrovirus is a very productive expression vector for recombinant proteins in insect cells. Most vectors are based on the polyhedrin gene promoter, which comprises a TAAG transcription initiation motif flanked by 20 base pairs upstream and 47 base pairs downstream before the native ATG. Many transfer vectors also include a short sequence downstream of the ATG, in which case this sequence is mutated to ATT to abolish translation. However, the ATT sequence, or AUU in the mRNA, is known to be leaky. If a target-coding region is placed in the frame with the AUU, then some products will comprise a chimeric molecule with part of the polyhedrin protein. In this study, we showed that if AUU is placed in the frame with a Strep tag and eGFP coding region, we could identify a protein product with both sequences present. Further work examined if alternative codons in lieu of AUG might reduce translation initiation further. We found that AUA was used slightly more efficiently than AUU, whereas AUC was the least efficient at initiating translation. The use of this latter codon suggested that there might also be a slight improvement of protein yield if this is incorporated into expression vectors

ADDovenom : Thermostable Protein-Based ADDomer Nanoparticles as New Therapeutics for Snakebite Envenoming

Snakebite envenoming can be a life-threatening medical emergency that requires prompt medical intervention to neutralise the effects of venom toxins. Each year up to 138,000 people die from snakebites and threefold more victims suffer life-altering disabilities. The current treatment of snakebite relies solely on antivenom—polyclonal antibodies isolated from the plasma of hyperimmunised animals—which is associated with numerous deficiencies. The ADDovenom project seeks to deliver a novel snakebite therapy, through the use of an innovative protein-based scaffold as a next-generation antivenom. The ADDomer is a megadalton-sized, thermostable synthetic nanoparticle derived from the adenovirus penton base protein; it has 60 high-avidity binding sites to neutralise venom toxins. Here, we outline our experimental strategies to achieve this goal using state-of-the-art protein engineering, expression technology and mass spectrometry, as well as in vitro and in vivo venom neutralisation assays. We anticipate that the approaches described here will produce antivenom with unparalleled efficacy, safety and affordability