Therapeutic Antibodies Against Shiga Toxins: Trends and Perspectives.

Shiga toxins (Stx) are AB5-type toxins, composed of five B subunits which bind to Gb3 host cell receptors and an active A subunit, whose action on the ribosome leads to protein synthesis suppression. The two Stx types (Stx1 and Stx2) and their subtypes can be produced by Shiga toxin-producing Escherichia coli strains and some Shigella spp. These bacteria colonize the colon and induce diarrhea that may progress to hemorrhagic colitis and in the most severe cases, to hemolytic uremic syndrome, which could lead to death. Since the use of antibiotics in these infections is a topic of great controversy, the treatment remains supportive and there are no specific therapies to ameliorate the course. Therefore, there is an open window for Stx neutralization employing antibodies, which are versatile molecules. Indeed, polyclonal, monoclonal, and recombinant antibodies have been raised and tested in vitro and in vivo assays, showing differences in their neutralizing ability against deleterious effects of Stx. These molecules are in different phases of development for which we decide to present herein an updated report of these antibody molecules, their source, advantages, and disadvantages of the promising ones, as well as the challenges faced until reaching their applicability.

Recombinant PilS: cloning, expression and biochemical characterization of a Pil-Fimbriae subunit

Pil-fimbriae is a type IV pili member, which is a remarkably versatile component with a wide variety of functions, including motility, attachment to different surfaces, electrical conductance, DNA acquisition, and secretion of a broad range of structurally distinct protein substrates. Despite the previous functional characterization of Pil, more studies are required to understand the regulation of Pil expression and production, since the exact mechanisms involved in these steps are still unknown. Therefore it is extremely important to have a protein with the correct secondary and tertiary structure that will enable an accurate characterization and a specific antisera generation. For this reason, the aim of this work was to generate potential tools for further investigations to comprehend the mechanisms involved in Pil regulation and its role in pathogenic E. coli infections with the obtaining of a precise native-like recombinant PilS and the corresponding antisera. The pilS gene was successfully cloned into an expression vector, and recombinant PilS (rPilS) was efficiently solubilized and purified by metal affinity chromatography. Protein characterization analyses indicated that rPilS presented native-like secondary and tertiary structures after the refolding process. The generated anti-rPilS sera efficiently recognized recombinant and native proteins from atypical enteropathogenic E. coli strains.

Therapeutic antibodies against shiga toxins: trends and perspectives

Shiga toxins (Stx) are AB5-type toxins, composed of five B subunits which bind to Gb3 host cell receptors and an active A subunit, whose action on the ribosome leads to protein synthesis suppression. The two Stx types (Stx1 and Stx2) and their subtypes can be produced by Shiga toxin-producing Escherichia coli strains and some Shigella spp. These bacteria colonize the colon and induce diarrhea that may progress to hemorrhagic colitis and in the most severe cases, to hemolytic uremic syndrome, which could lead to death. Since the use of antibiotics in these infections is a topic of great controversy, the treatment remains supportive and there are no specific therapies to ameliorate the course. Therefore, there is an open window for Stx neutralization employing antibodies, which are versatile molecules. Indeed, polyclonal, monoclonal, and recombinant antibodies have been raised and tested in vitro and in vivo assays, showing differences in their neutralizing ability against deleterious effects of Stx. These molecules are in different phases of development for which we decide to present herein an updated report of these antibody molecules, their source, advantages, and disadvantages of the promising ones, as well as the challenges faced until reaching their applicability.

Cultivos em meios complexos para produção de L-asparaginase em Escherichia coli BL21(DE3)

Acute Lymphoblastic Leukemia (ALL) is a type of neoplasia that has L-asparaginase as a highly effective treatment. This biopharmaceutical cleaves asparagine and releases ammonium and aspartic acid; this neoplastic cells do not produce asparagine due to their still immature cellular machinery and end up dying in the absence of that amino acid. In Brazil, patients have difficulty in accessing this medicine because it is imported, has a high cost, in addition to problems in supply. Therefore, a group of researchers seeks to develop a national drug, with less allergic potential, therefrom the country will have autonomy, enabling access to the population through SUS. Escherichia coli BL21 (DE3) has been widely used for the expression of heterologous proteins and to reach a high cell density. This work consists of evaluating different complex media, micronutrients and appropriate inducer for the production of recombinant L-asparaginase. For this study was used E.coli BL21 (DE3) containing the expression vector pET28a, with T7 promoter, selection marker for kanamycin and carrying the KY305877 gene sequence. Experiments in stirred flask were performed at 300 rpm, 37 ° C in: Luria Bertani (LB), Semi Defined (SD), Super Broth (SB), Terrific Broth (TB), TB mod., TB mod. + nutrients, TB mod. in different carbon sources, TB mod. in lactose as an inducer. Bioreactor tests were also carried out with the modified TB medium. Samples were collected every hour for analysis of biomass, enzymatic activity and electrophoresis. The TB medium showed the highest DO600nm and enzymatic activity. The tryptone of the TB medium was replaced by soytone, a component of plant origin, and adjusted in its concentration (17 g / L) to achieve similar values in growth and asparaginase activity obtained with the tryptone originating the TB mod medium. In this composition there was no need for micronutrient supplementation. The increment of the TB medium mod. with glucose, glycerol and lactose, with lactose also acting as an inducer, showed promising enzymatic activity in stirred flasks of 5 IU / ml of culture through 6UI / ml of culture with IPTG. The use of IPTG is inappropriate due to the high cost, toxicity and limited monitoring during cultivation, on the other hand, lactose may be introduced in the formulation of the medium together with other components and will act as an inducer, when the glucose runs out, and as a carbon source. This type of medium is known as a self-inducing medium. In a bioreactor the DO600nm reached 39.9 AU with enzymatic activity of 29 IU / Cultivation.

A Leucemia linfoide aguda (LLA) é um tipo de neoplasia que tem como tratamento altamente eficaz a L-asparaginase. Este biofármaco cliva a asparagina e libera amônio e ácido aspártico; as células neoplásicas que não produzem asparagina devido a sua maquinaria celular ainda imatura acabam morrendo na ausência desse aminoácido. No Brasil, os pacientes têm dificuldade no acesso a esse medicamento por ser importada, de alto custo, além de problemas no fornecimento. Assim, um grupo de pesquisadores buscam desenvolver um medicamento nacional, com menor potencial alérgico, para que o país tenha autonomia, possibilitando o acesso à população através do SUS. A Escherichia coli BL21(DE3) tem sido amplamente utilizada para a expressão de proteínas heterólogas e possibilitar o aumento da densidade celular. Este trabalho consiste em avaliar diferentes meios complexos, os micronutrientes e indutor apropriado para a produção de L-asparaginase recombinante. E.coli BL21(DE3) contendo o vetor de expressão pET28a, com promotor T7, marcador de seleção para Canamicina e carregando a sequência de gene KY305877 foi utilizada. Experimentos em fracos agitados foram realizados a 300 rpm, 37°C em: Luria Bertani (LB), Semi Definido (SD), Super Broth (SB), Terrific Broth (TB), TB mod., TB mod. + nutrientes, TB mod. em diferentes fontes de carbono, TB mod. em lactose como indutor. Foram ainda realizados ensaios em biorreator com o meio TB modificado. Amostras foram coletadas a cada hora para análise de biomassa, atividade enzimática e eletroforese. O meio TB apresentou a maior DO600nm e atividade enzimática. A triptona do meio TB foi substituída por soytone, um componente de origem vegetal, e ajustada na sua quantidade (17 g/L) para atingir valores similares em crescimento e atividade da asparaginase obtida com a triptona originando o meio TB mod. Nesta composição não houve a necessidade de suplementação de micronutrientes. A incrementação do meio TB mod. com glicose, glicerol e lactose, sendo a lactose atuando também como indutor, apresentou atividade enzimática promissor em frascos agitados de 5 UI/Lcultivo mediante a 6UI/Lcultivo com IPTG. O uso IPTG é inadequado devido ao custo elevado, toxicidade e limitação no monitoramento durante o cultivo, por outro lado a lactose poderá ser introduzida na formulação do meio juntamente com demais componentes e atuará como indutor quando a glicose acabar e como fonte de carbono. Este tipo de meio é conhecido como meio de auto-indução. Em biorreator a DO600nm chegou a 39,9 UA com atividade enzimática de 29 UI/Lcultivo.