Recent work has shown that microorganisms are surprisingly like animals in having sophisticated behaviours such as cooperation, communication, and recognition, as well as many kinds of symbioses. Here we show first that the social amoeba Dictyostelium discoideum has a primitive farming symbiosis that includes dispersal and prudent harvesting of the crop. About one-third of wild-collected clones engage in husbandry of bacteria. Instead of consuming all bacteria in their patch, they stop feeding early and incorporate bacteria into their fruiting bodies. They then carry bacteria during spore dispersal and can seed a new food crop, which is a major advantage if edible bacteria are lacking at the new site. However, if they arrive at sites already containing appropriate bacteria, the costs of early feeding cessation are not compensated, which may account for the dichotomous nature of this farming symbiosis. We also observed farmer Dictyostelium discoideum clones carry bacteria that they do not use as food. We hypothesized that these bacteria may play a defensive role against other D. discoideum clones. In our second study, we investigated the impact of these bacteria-carrying farmers on non-farming D. discoideum clones. We found that the presence of farming clones reduces spore production in non-farmers. Furthermore, this effect increases with frequency of farming clones, demonstrating the vulnerability of non-farming clones to farmers though in this experiment we had not separated the effects of the farmer clone and the bacteria they carry. In our third study we exposed non-farmers to a filtered supernatant from the most common non-food carried bacterium, Burkholderia xenovorans . This supernatant is likely to carry whatever the bacteria are producing. We treated Dictyostelium clones at the beginning of the social stage and found that the supernatant enhanced spore production of farming clones and hurt spore production of non-farming clones. This study shows that the effects of the bacteria can be restricted to a filtered supernatant alone. This discovery of symbiosis of D. discoideum with bacteria, and its impact on social interactions among D. discoideum clones will provide a fertile ground for further experiments on the evolution of sociality.

Thesis--University of California, Berkeley, 1959. / "Biology and Medicine" -t.p. Includes bibliographical references (p. 66-70).

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 117-136.

Marseilleviridae é uma família de vírus gigantes cujos membros infectam amebas de vida livre. Esses vírus têm sido encontrados em amostras ambientais de água, larvas de inseto, torres de resfriamento e mais recentemente em amostras humanas. Eles possuem capsídeo icosaédrico medindo entre 190-250 nm e genoma de DNA dupla fita circular ou linear. Sua replicação ocorre no citoplasma amebiano onde observam-se fábricas virais. Este trabalho tem como objetivo investigar a presença de vírus gigantes em mexilhões-dourados (Limnoperna fortunei) que habitam o Lago Guaíba, Porto Alegre, Brasil. Quarenta indivíduos foram coletados e agrupados em pools de 5 amostras (água interna e corpo, totalizando dezesseis pools). Os pools foram homogeneizados em tampão fosfato, centrifugados e o sobrenadante filtrado em membrana de 0,45μM. Foram cultivadas amebas da espécie Acanthamoeba polyphaga em meio PYG em microplacas de 24 poços, inoculadas com os sobrenadantes, incubadas a 30ºC e examinadas diariamente em busca de efeito citopático (ECP) até 72 horas após a inoculação. Quando CPE era evidente, os sobrenadantes foram coletados e ultracentrifugados através de um gradiente de sacarose de 25%. Um dos dezesseis pools induzindo CPE claro foi submetido à extração de DNA e sequenciamento do genoma viral completo um sequenciador de nova geração (Illumina MiSeq). O genoma do vírus chamado Golden mussel marseillevirus consiste de uma única molécula de DNA de 360610 pb, com um teor de G+C de 43,1%. A análise da sequência de nucleotídeos traduzida revelou a presença de proteínas virais que apresentam homologia com proteínas de outros membros da família Marseilleviridae, como Lausanevirus e vírus Insectomime, porém grande parte do seu genoma não apresenta identidade com proteínas depositadas no banco de dados. A análise filogenética do gene D6/D11 Helicase sugere que este vírus faça parte de uma nova linhagem de marseillevirus. Este é o primeiro estudo que demonstra o isolamento de um vírus gigante a partir de tecidos de mexilhão-dourado e infere que estes vírus estão distribuídos amplamente no meio ambiente. / Marseilleviridae is a family of giant viruses whose members infect free living amoebae. These viruses have been found in environmental samples of water, insect larvae, cooling towers and, more recently, in human samples. They have icosahedral capsids measuring between 190-250 nm and their genome is double-stranded circular or linear DNA. Replication occurs in the host cell cytoplasm, inside the viral factories. This study aims to investigate the presence of giant viruses in tissues of golden mussels (Limnoperna fortunei) that inhabit the Guaiba Lake, Porto Alegre, Brazil. Forty specimens were pooled in groups of 5 specimens (internal water and body, totalizing sixteen pools). The pools were homogenized with phosphate buffer, centrifuged and the supernatant was filtered in 0,45μM. Monolayers of Acanthamoeba polyphaga were cultivated with PYG medium in 24-well microplates, inoculated with the pooled samples, incubated at 30 ºC and examined daily in search for cytopathic effect (CPE) up to 72 hours after inoculation. When CPE was evident, the supernatants were collected, clarified and ultra centrifuged through a 25% sucrose cushion. One out of the sixteen CPE positive pools was submitted to DNA extraction and complete sequencing of the viral genome in a NGS apparatus (Illumina MiSeq). The genome of the virus named Golden mussel marseillevirus consists of a single DNA molecule of 360,610 bp, with a G+C content of 43.1%. The analysis of the translated nucleotide sequence reveals the presence of proteins which are homologs to proteins predicted in other members of the family Marseilleviridae like, e.g. Lausannevirus and Isectomime virus. However, part of the viral genome has no identity with the nucleotide sequences available at the database. The phylogenetic analysis of the D6/D11 Helicase gene suggests that this virus is part of a new lineage of marseillevirus. This is the first study where the isolation of a giant virus from golden mussel tissues is achieved, suggesting that these viruses are widely distributed in the environment.

Marseilleviridae é uma família de vírus gigantes cujos membros infectam amebas de vida livre. Esses vírus têm sido encontrados em amostras ambientais de água, larvas de inseto, torres de resfriamento e mais recentemente em amostras humanas. Eles possuem capsídeo icosaédrico medindo entre 190-250 nm e genoma de DNA dupla fita circular ou linear. Sua replicação ocorre no citoplasma amebiano onde observam-se fábricas virais. Este trabalho tem como objetivo investigar a presença de vírus gigantes em mexilhões-dourados (Limnoperna fortunei) que habitam o Lago Guaíba, Porto Alegre, Brasil. Quarenta indivíduos foram coletados e agrupados em pools de 5 amostras (água interna e corpo, totalizando dezesseis pools). Os pools foram homogeneizados em tampão fosfato, centrifugados e o sobrenadante filtrado em membrana de 0,45μM. Foram cultivadas amebas da espécie Acanthamoeba polyphaga em meio PYG em microplacas de 24 poços, inoculadas com os sobrenadantes, incubadas a 30ºC e examinadas diariamente em busca de efeito citopático (ECP) até 72 horas após a inoculação. Quando CPE era evidente, os sobrenadantes foram coletados e ultracentrifugados através de um gradiente de sacarose de 25%. Um dos dezesseis pools induzindo CPE claro foi submetido à extração de DNA e sequenciamento do genoma viral completo um sequenciador de nova geração (Illumina MiSeq). O genoma do vírus chamado Golden mussel marseillevirus consiste de uma única molécula de DNA de 360610 pb, com um teor de G+C de 43,1%. A análise da sequência de nucleotídeos traduzida revelou a presença de proteínas virais que apresentam homologia com proteínas de outros membros da família Marseilleviridae, como Lausanevirus e vírus Insectomime, porém grande parte do seu genoma não apresenta identidade com proteínas depositadas no banco de dados. A análise filogenética do gene D6/D11 Helicase sugere que este vírus faça parte de uma nova linhagem de marseillevirus. Este é o primeiro estudo que demonstra o isolamento de um vírus gigante a partir de tecidos de mexilhão-dourado e infere que estes vírus estão distribuídos amplamente no meio ambiente. / Marseilleviridae is a family of giant viruses whose members infect free living amoebae. These viruses have been found in environmental samples of water, insect larvae, cooling towers and, more recently, in human samples. They have icosahedral capsids measuring between 190-250 nm and their genome is double-stranded circular or linear DNA. Replication occurs in the host cell cytoplasm, inside the viral factories. This study aims to investigate the presence of giant viruses in tissues of golden mussels (Limnoperna fortunei) that inhabit the Guaiba Lake, Porto Alegre, Brazil. Forty specimens were pooled in groups of 5 specimens (internal water and body, totalizing sixteen pools). The pools were homogenized with phosphate buffer, centrifuged and the supernatant was filtered in 0,45μM. Monolayers of Acanthamoeba polyphaga were cultivated with PYG medium in 24-well microplates, inoculated with the pooled samples, incubated at 30 ºC and examined daily in search for cytopathic effect (CPE) up to 72 hours after inoculation. When CPE was evident, the supernatants were collected, clarified and ultra centrifuged through a 25% sucrose cushion. One out of the sixteen CPE positive pools was submitted to DNA extraction and complete sequencing of the viral genome in a NGS apparatus (Illumina MiSeq). The genome of the virus named Golden mussel marseillevirus consists of a single DNA molecule of 360,610 bp, with a G+C content of 43.1%. The analysis of the translated nucleotide sequence reveals the presence of proteins which are homologs to proteins predicted in other members of the family Marseilleviridae like, e.g. Lausannevirus and Isectomime virus. However, part of the viral genome has no identity with the nucleotide sequences available at the database. The phylogenetic analysis of the D6/D11 Helicase gene suggests that this virus is part of a new lineage of marseillevirus. This is the first study where the isolation of a giant virus from golden mussel tissues is achieved, suggesting that these viruses are widely distributed in the environment.

M.Tech. (Biomedical Technology) / Drinking water in the greater Johannesburg area is produced by Rand Water and is transported to local Johannesburg Water where it is stored in reservoirs for distribution. At any point during the production, distribution and storage of the water, contamination with free-living amoebae, potentially containing amoeba resistant bacteria, may occur. Free-living amoebae are often resistant to the biocides used by water treatment industries and may thus be transmitted to public facilities, consumers’ homes and informal settlements through water distribution systems and during storage in small containers. The aim of our study was to analyse the water quality around Johannesburg with regard to free-living amoebae and amoeba resistant bacteria. A total of 182 tap and 5 storage tank water samples, collected from Hillbrow, Bertrams, Riverlea, Braamfischerville and Hospital Hill, were analysed for amoebae, indicator organisms, Legionellae, environmental mycobacteria, Shigella, Salmonella and Vibrio species using amoebal enrichment method. Direct microscopy indicated the presence of amoebae in 96.1% of samples. Acanthamoeba cysts were present in 69.0% of the samples. In 55.0% of these samples visibly active intracellular bacteria were observed within the sample suspensions. In the 46 samples analysed by polymerase chain reaction, the presence of Acanthamoeba species was confirmed in 65.2%, and the intracellular bacteria such as Legionella pneumophila and Mycobacterium avium was confirmed in 23.9% and 73.9% respectively. All samples indicated the presence of Shigella species while one sample contained Salmonella species on xylose lysine desoxycholate agar after amoebal enrichment processing. Vibrio species was not confirmed in the samples. Our results indicated a high risk of transmission of amoeba resistant bacteria through drinking water to people living in these areas.

Naegleria fowleri is a free-living amoeba found in freshwater lakes and ponds that is the causative agent of Primary Amoebic Meningoencephalitis (PAM). Matrix metalloproteinases (MMPs) have been described in protozoa, such as Plasmodium falciparum, Trypanosoma brucei, and Balamuthia mandrillaris, and have been linked to their increased motility and invasive capability by degrading components of the extracellular matrix (ECM). In addition, MMPs are often upregulated in tumorigenic cells and have been attributed as responsible for the metastasis of certain cancers. In the present study, in vitro experiments indicated that MMPs are linked functionally to the ECM degradation process. Gelatin zymography demonstrated protease activity in N. fowleri whole cell lysates, conditioned media, and media collected from in vitro invasion assays. Western immunoblotting confirmed the presence of the metalloproteinases MMP-2, -9, and -14. The highly virulent mouse-passaged amoebae expressed higher levels of MMPs than the weakly virulent axenically grown amoebae. The functional relevance of MMPs found in media in degradation of ECM components was confirmed through the use of MMP inhibitors. The collective in vitro results suggest that MMPs may play a critical role in the invasion of the CNS. Furthermore, the expression of select metalloproteinases may serve as amenable targets for therapeutic manipulation of expansive PAM.

En faisant le parallèle avec les selles modernes, les coprolithes peuvent être appropriés à l'étude des habitudes alimentaires, de la flore intestinale et des maladies, des animaux et des hommes ayant vécu il y a des siècles. Dans le travail de thèse ici présenté, un coprolithe datant des 14-15ième siècles, provenant de Namur en Belgique, a été étudié. Dans un premier temps l'ensemble de la communauté microbienne associée au coprolithe été characteriser. Les résultats ont montré qu'une partie du microbiote est similaire à l'environnement et l’autre la flore intestinale, des parasites intestinaux et des pathogènes systémiques ont été aussi trouvés. Un second projet a visé à la purification de particules virales à partir du coprolithe et leur analyse par microscopie électronique et métagénomique virale. Des particules virales sphériques, ainsi que des bactériophages, ont ainsi été observés. Les virus associés au coprolithe correspondent à des virus d'eucaryotes, de procaryotes et d'archaea. La communauté virale était dominée par des bactériophages détectés dans le sol et les selles. Parmi les fonctions métaboliques détectées, une correspond d'ailleurs à des résistances aux antibiotiques. Dans un troisième projet, des cultures et des identifications moléculaires ont été réalisées sur des kystes d'amibes observés dans le coprolithe. Les amibes isolées appartiennent au genre Acanthamoeba et pourraient avoir été conservées sous forme de kystes pendant des siècles dans le coprolithe. Les co-cultures d'amibes ont mené à l'isolement d'une nouvelle bactérie bi-flagellée résistante aux amibes, proche des Rickettsiales. / By drawing parallels to modern stools, coprolites can be suitable specimen to study diet habits, gut microbiota and diseases of animals and humans that have lived centuries ago. During this thesis work, a 14-15th century coprolite specimen from Namur, Belgium was analyzed. At the initiation of this thesis work, it was aimed to characterize the entire microbial communities associated to the coprolite and to identify ancient pathogens. Results indicated that parts of the microbiota are similar to those coming from environment and the gut microbiota inhabitants. Further intestinal parasites and systemic pathogens – still relevant nowadays – were also found. In a second work, viral particles were purified from the Namur coprolite and analyzed by electron microscopic and viral metagenomic. Viral particles associated to spherical virions and bacteriophages were observable. Viruses infecting eukaryotes, bacteria and archaea were associated to the specimen. The viral community was dominated by bacteriophages commonly found in soil and in modern stools and antibiotic resistance was one of the metabolic functions detected. In a third project, culture and molecular identification were performed on amoebal cysts observed within the coprolite. The amoebas isolated belong to the genus Acanthamoeba and might have been conserved in form of cysts inside the Namur coprolite for centuries. Amoeba-co culturing leaded to the isolation and identification of a new bi-flagellar amoeba-resistant bacterium closely related to Rickettsiales.

Legionella pneumophila est une bactérie intracellulaire facultative retrouvée aussi bien dans les environnements aqueux naturels qu'artificiels. Cette bactérie est l'agent de la légionellose, une pneumopathie sévère. Les milieux aqueux sont colonisés par des biofilms, une association de micro-organismes enveloppés d'une matrice exopolymérique. Les légionelles sont capables de coloniser et de survivre au sein de ces biofilms. Dans les réseaux d'eaux, les biofilms sont la cible de la prédation des amibes qui se retrouvent ensuite parasités par les légionelles et favorisent ainsi leur multiplication. Nous avons étudié l'impact de l'origine de la multiplication des légionelles sur la colonisation et la formation de biofilms. Notre étude a démontré que dans tous les cas les légionelles sont présentes à la surface de biofilms préformés. Notre étude a mis en évidence que les légionelles issues de la multiplication au sein d'amibes sont capables de former des agrégats compacts empaquetés dans une matrice exopolysaccharidique. Ce phénotype « biofilm » se manifeste après induction par une molécule amibienne en cours de caractérisation. De plus, notre étude a mis en évidence l'existence d'un nouveau système de communication de type Quorum Sensing permettant une communication inter et intra-espèce qui permet l'induction de ce phénotype chez les légionelles. / Legionella pneumophila is a facultative intracellular Gram-negative bacterium colonizing freshwater as well as artificial environments. This bacterium is the agent of Legionnaires' disease, a severe pneumonia. Water network are colonized by biofilms, an association of microorganisms embedded in exopolymeric matrix. Legionella are able to colonize and survive within biofilms which are the major source of human infection. In water systems, biofilms are the target of amoeba predation that can be parasitized by Legionella. Amoebae are the major multiplication site of Legionella in the environment.We studied the impact of multiplication origin on colonization and biofilm formation by Legionella. Our study demonstrated that Legionella whatever their origins are present at the surface of pre-formed biofilms. Our study showed that Legionella issued from multiplication within amoebae are able to form compact aggregates packed in an exopolysaccharidic matrix. This "biofilm" phenotype occurs after induction by an amoebic molecule that has to be characterized. In addition, our study revealed the existence of a new QS system for inter and intra-species communication that allows the induction of this biofilm phenotype to other Legionella.

Since their 'discovery' at the turn of the 21st century, giant viruses of the amoeba have captured the fascination of virologists. They have raised a plethora of questions regarding their evolution and ecological significance and have greatly defied a century's old definition of viruses. By now, it is understood that a handful of giant viruses enter the amoeba via the phagosomal pathway. This thesis chooses to focus on the giant Melbournevirus (MelV) regarding its entry and uncoating pathway. We now conclude that the initial attachment between MelV and amoeba cells is built upon glycan interactions based on evidence that mannose competitively inhibits MelV binding. This attachment likely entails an approximately 70 kDa mannose containing glycoprotein on the MelV. Mannose and other glycans induce secretion of proteins including phagosomal enzymes from amoeba. Based on these findings, it is hypothesised that the mannose-induced phagosomal enzymes could play a role in the uncoating of the MelV. The results further reveal isolated phagosomes, also to some extent the glycan-induced protein secretions, to have high levels of proteins involved in cell redox homeostasis. This implies that the highly oxidative environment of the phagosome may be an overlooked physiological factor when regarding the uncoating of the MelV. A deeper understanding of the physiological uncoating conditions can be used for studying internal structures of giant viruses, such as the enigmatic Large and Dense Body (LDB) of the MelV particle.