Antagonistic interactions between competing species of bacteria are an important driver of bacterial community composition in the human gut microbiota. Of particular significance is the role of the type six secretion system (T6SS), which many species of Gram-negative bacteria use to kill competitor bacteria in a contact-dependent manner. T6SSs are syringe-like nanomachines that function to deliver antibacterial toxins into susceptible competitors. Many bacteria present in the human gut microbiota possess an extremely potent T6SS that is capable of rapidly eradicating nearby bacteria. Remarkably, however, species of beneficial bacteria that coexist in the gut are often resistant to T6SS attack by their neighbours. This resistance is mediated by bacterial immunity proteins that block the activity of the antibacterial toxins delivered by the T6SS. Intriguingly, past studies have shown that the widespread T6SS-mediated competition in the gut has led to the acquisition of repertoires of immunity genes across different bacterial strains. By examining available human gut metagenomes, I identified a putative immunity locus, named I2, in a species of gut bacteria. This locus is located downstream of its cognate T6SS toxin-encoding locus, E2, and I show when co-expressed with E2 in E. coli, it protects against E2 mediated-toxicity. Additionally, I show that four gut-derived I2 homologues bearing sequence identity levels to I2 ranging from 38% to 75% are equally capable of abrogating E2 toxicity. Using quantitative biophysical measurements, I also show that these I2 homologues physically bind E2 equally tightly pointing to the potential molecular mechanism of toxin neutralization. Lastly, through mutagenesis experiments, I found that the E2-I2 interaction is likely mediated by electrostatic forces between a small number of residues found in the interaction interface of the two proteins. Overall, these findings demonstrate that a human gut microbiome encoded type VI secretion system effector can be neutralized by divergent immunity proteins. / Thesis / Master of Science (MSc)

Submitted by Geyciane Santos (geyciane_thamires@hotmail.com) on 2015-08-20T14:55:07Z No. of bitstreams: 1 Dissertação - Henriette Soares Pereira Paskinn.pdf: 1749750 bytes, checksum: a27b1ad66bc6a166ea02a478efd07806 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-08-24T20:19:41Z (GMT) No. of bitstreams: 1 Dissertação - Henriette Soares Pereira Paskinn.pdf: 1749750 bytes, checksum: a27b1ad66bc6a166ea02a478efd07806 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-08-24T20:23:45Z (GMT) No. of bitstreams: 1 Dissertação - Henriette Soares Pereira Paskinn.pdf: 1749750 bytes, checksum: a27b1ad66bc6a166ea02a478efd07806 (MD5) / Made available in DSpace on 2015-08-24T20:23:45Z (GMT). No. of bitstreams: 1 Dissertação - Henriette Soares Pereira Paskinn.pdf: 1749750 bytes, checksum: a27b1ad66bc6a166ea02a478efd07806 (MD5) Previous issue date: 2013-05-10 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Evidence has shown that the integument of social bees has a primary protective barrier with antimicrobial properties. Various types of bacteria inhabit the seed coat of healthy social bees and can interact antagonistically against bacteria or not, contributing to the health and function of the nest. Despite its likely importance in maintaining the balance of the hive, few studies have focused on the characterization of the microbiota of this tissue, since the exoskeleton is considered largely responsible for the evolutionary success of insects, not only for the protection and support that gives them, but also the interface between the animal and is environment, and can also act as a carrier and vector of microorganisms. Thus, the main objective of this study was to isolate and identify bacteria with antagonistic activity in front of pathogenic bacteria (Staphylococcus aureus ATCC 25927 and ATCC 29212 Enteroccus faecalis) by biochemical tests and 16S rRNA gene amplification. In this study it was found Staphylococus coagulase negative and positive, Serratia liquefacies and Enterococcus sp. The genera and species of bacteria found in this study were able to produce substances in the course of their growth in vitro, exerting bactericidal or bacteriostatic effect against gram-positive and are largely related to social insects. This feature may suggest that interactions between stingless bees and bacteria are at a level of association such that both are essential for both insect as microorganisms. / Evidências demonstram que o tegumento de abelhas sociais apresenta uma barreira de proteção primária com propriedades antimicrobianas. Diversos tipos de bactérias habitam o tegumento de abelhas sociais saudáveis e podem interagir de forma antagônica frente a bactérias benéficas ou não, contribuindo para a saúde e funções do ninho. Apesar de sua provável importância na manutenção do equilíbrio da colméia, poucos estudos tem se voltado para a caracterização da microbiota deste tecido, visto que o exoesqueleto é considerado como um dos grandes responsáveis pelo sucesso evolutivo dos insetos, não somente pela proteção e ao suporte que lhes confere, mas também a interface que representa entre o animal e o meio ambiente, podendo também atuar como transportador e vetor de microrganismos. Sendo assim, o principal objetivo deste trabalho foi isolar e identificar bactérias com atividade antagônica contra bactérias patogênicas (Staphylococcus aureus ATCC 25927 e Enteroccus faecalis ATCC 29212) por meio de testes bioquímicos e amplificação do gene 16S rRNA. Neste trabalho encontrou-se Staphylococus coagulase negativo e positivo, Serratia liquefacies e Enterococcus sp. Os gêneros e espécies bacterianas encontrados neste estudo foram capazes de produzir substâncias, exercendo efeito bactericida ou bacteriostático contra gram- positivos, no curso de seu crescimento in vitro e são amplamente relacionados com insetos sociais. Tal característica pode sugerir que interações entre abelhas sem ferrão e bactérias estão num patamar de associação tal que sejam imprescindíveis para ambos, tanto inseto quanto microrganismos.