The amoebae Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris are free-living amoebae found in both water and soil. They are opportunistic pathogens in humans. Acanthamoeba is the most common cause of illness, usually infecting the eyes and sometimes causing a sight-threatening keratitis. Acanthamoeba spp. and B. mandrillaris can cause granulomatous amoebic encephalitis, in addition to infections of the lungs and skin. N. fowleri causes primary amoebic meningoencephalitis . There is little known regarding the ecology and occurrence of these organisms. A total of 36 high-use recreational surface waters in Arizona were surveyed over a period of two years to assess the occurrence of N. fowleri and seasonal and environmental factors. Overall, 9.3% of the warm weather samples collected were positive for N. fowleri, whereas 16.3% of the samples were positive during cold weather. Although the presence of N. fowleri could not be significantly correlated with physical and chemical parameters such as temperature, pH, turbidity, conductivity, and the presence of heterotrophic bacteria, total coliforms, and Escherichia coli, a weak correlation (0.52) with live amoebic activity was observed. Five lakes to the north and northeast of Phoenix tested positive for the N. fowleri on more than one occasion over multiple seasons. Finished drinking water samples (n= 785) from a municipal potable distribution system were evaluated for the presence of N. fowleri, B. mandrillaris and Acanthamoeba spp. from 18 different regions during three different sampling periods. Physical and chemical parameters were also evaluated but provided no significant correlations with the occurrence of amoebae or indicator organisms. A total of 138 samples (17.9%) were positive for viable amoebae in distribution water with more than an adequate chlorine residual (average of 0.86 mg/L). Microorganisms that are typically used to monitor microbial water quality such as coliforms and E. coli would likely not be found under these circumstances. Clusters with three or more samples testing positive for viable amoebae per region were observed during all three periods. Viable amoebae may not only provide a better assessment of the microbial quality of water, but such clustering could reveal areas with potential water quality issues within the distribution system.

The metabolism of iron ions is a crucial process in all living organisms and its correct regulation is essential for basic life functions. Homeostasis of iron ions is closely regulated, it usually appears as a component of various proteins and plays role in many oxidation-reduction reactions. Naegleria gruberi is a non-pathogenic, free living protozoon, that serves as a laboratory model for closely related pathogenic Naegleria fowleri. This work focuses on the study of selected metabolites of N. gruberi, that were possible to detect and quantify by the means of modern metabolomic methods, and the influence on culture cultivated in environment with lack of iron ions was shown. The discovery of effect of this condition on the energetic metabolism of this protozoan is an important aspect of understanding the biological processes on cellular level. This method proved a significant influence on certain metabolites and modification of certain metabolic pathways as a direct effect of decreased availability of iron ions. Second part of this work was focused on the enzyme alcohol dehydrogenase, that was found in the genome of this protozoon. Unusual aspects of this enzyme include a N-terminal mitochondrial presequence, prompting about mitochondrial localization, and utilization of iron ion as a prosthetic...

abstract: The deterioration of drinking-water quality within distribution systems is a serious cause for concern. Extensive water-quality deterioration often results in violations against regulatory standards and has been linked to water-borne disease outbreaks. The causes for the deterioration of drinking water quality inside distribution systems are not yet fully understood. Mathematical models are often used to analyze how different biological, chemical, and physical phenomena interact and cause water quality deterioration inside distribution systems. In this dissertation research I developed a mathematical model, the Expanded Comprehensive Disinfection and Water Quality (CDWQ-E) model, to track water quality changes in chloraminated water. I then applied CDWQ-E to forecast water quality deterioration trends and the ability of Naegleria fowleri (N.fowleri), a protozoan pathogen, to thrive within drinking-water distribution systems. When used to assess the efficacy of substrate limitation versus disinfection in controlling bacterial growth, CDWQ-E demonstrated that bacterial growth is more effectively controlled by lowering substrate loading into distribution systems than by adding residual disinfectants. High substrate concentrations supported extensive bacterial growth even in the presence of high levels of chloramine. Model results also showed that chloramine decay and oxidation of organic matter increase the pool of available ammonia, and thus have potential to advance nitrification within distribution systems. Without exception, trends predicted by CDWQ-E matched trends observed from experimental studies. When CDWQ-E was used to evaluate the ability N. fowleri to survive in finished drinking water, the model predicted that N. fowleri can survive for extended periods of time in distribution systems. Model results also showed that N. fowleri growth depends on the availability of high bacterial densities in the 105 CFU/mL range. Since HPC levels this high are rarely reported in bulk water, it is clear that in distribution systems biofilms are the prime reservoirs N. fowleri because of their high bacterial densities. Controlled laboratory experiments also showed that drinking water can be a source of N. fowleri, and the main reservoir appeared to be biofilms dominated by bacteria. When introduced to pipe-loops N. fowleri successfully attached to biofilms and survived for 5 months. / Dissertation/Thesis / Ph.D. Civil and Environmental Engineering 2010

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.

Opportunistic pathogens (OPs) are of special concern for immunocompromised populations and are known to grow in both drinking water and reclaimed water (i.e., non-potable recycled water) distribution systems, with aerosol inhalation and other non-ingestion exposures that are not addressed by existing regulatory frameworks. Factors enabling the growth of OPs in water distribution and premise (i.e., building) plumbing systems distributing reclaimed and other water sources systems are poorly understood especially for the emerging OP, Naegleria fowleri (i.e. brain-eating amoeba). Three phases of investigation were carried out to identify factors that facilitate the growth of OPs in main distribution and premise plumbing systems, with particular attention on reclaimed water systems, aging water mains, and private well systems. Phase one examined the role of biological treatment to remove organic carbon and disinfectant type on the occurrence of OPs during distribution of reclaimed water. Laboratory-scale simulated reclaimed water distribution systems were employed to systematically examine the effects of prior granular activated carbon (GAC) biofiltration of the water; chlorine, chloramines, or no disinfectant, and water ages ranging up to 5 days. The second and third phases of research explored the role of nitrification, iron corrosion, and disinfectant on the growth of N. fowleri both in municipal drinking water from a city grappling with aging water infrastructure and untreated private well water. Results from the simulated reclaimed water distribution systems suggested that biologically-active GAC filtration may unintentionally select for specific OPs, contrary to expectations and experiences with oligotrophic conditions in potable water systems. While GAC biofiltration was associated with lower total bacteria and Legionella spp. gene markers, there were no apparent benefits in terms of other OPs analyzed. Similarly, disinfectant treatments successful for controlling OPs in potable water were either ineffective or associated with increased levels of OPs, such as Mycobacterium spp. and Acanthamoeba spp., in the reclaimed water examined. In the potable water study, it was possible to recreate conditions associated with growth of N. fowleri in the aged main distribution system from where the water for the experiment was collected; including corroding iron mains, nitrification, and disinfectant decay. While the effects of nitrification could not be confirmed, there was a clear association of iron corrosion with N. fowleri proliferation. The role of iron was explored further in what, to the author's knowledge, was the first study of N. fowleri in private wells. Analysis of 40 wells found correlations between N. fowleri and stagnant iron levels, further supporting the hypothesis that iron corrosion or iron encourages the growth of N. fowleri, and, because wells are not routinely disinfected, not necessarily as a result of promoting disinfectant decay. As this study took place following a major flooding event, it provided insight not only into how surface water contamination may influence private well water microbial communities, but also added to the understanding that current recommendations for disinfecting private wells are inadequate and standards should be implemented to aid homeowners in the event of flooding. This exploratory research illuminated several factors influencing the OP growth in a range of water systems. Identifying key variables that control growth is crucial to improving the safety of these systems. / MS / Water borne bacteria that effect the immune systems of the sick, known as opportunistic pathogens (OPs), have become a major heath concern. These organisms are known to grow in drinking water and reclaimed water (i.e., non-potable recycled water) distribution systems yet there are no regulations aimed at prevention. There is also limited knowledge on how premise plumbing and water sources effect the growth, population, and risk of infection by OPs, especially for Naegleria fowleri (i.e. brain-eating amoeba). An investigation was carried out in three parts to determine what influences the growth of OPs in water distribution and household plumbing systems, with particular attention to reclaimed water, municipal drinking water, and private well systems. Phase one examined the role of biological treatment to remove organic carbon and disinfectant type on the occurrence of OPs during distribution of reclaimed water. Laboratory-scale simulated reclaimed water distribution systems were used to examine the effects of granular activated carbon (GAC) biofiltration of the water, disinfectants (chlorine, chloramines, or no disinfectant), and water ages ranging zero to five days. The second and third phases of research explored the role of nitrification, iron corrosion, and disinfectant on the growth of N. fowleri both in municipal drinking water from a city with aging water infrastructure and untreated private well water. Results from the simulated reclaimed water distribution systems suggested that biologically-active GAC filtration may allow for the growth of specific OPs. While GAC biofiltration was associated with lower total bacteria and Legionella spp., there were no apparent benefits in reducing the presence of other OPs. Similarly, common disinfectant treatments for preventing OPs in drinking water were either ineffective or increased viii levels of OPs, such as Mycobacterium spp. and Acanthamoeba spp., in the reclaimed water. In the drinking water study, conditions were introduced to grow N. fowleri in aged drinking water distribution systems with the additions of corroding iron, nitrification (using nitrifying bacteria), and disinfectant. While the effects of nitrification could not be confirmed, there was a clear relationship between iron corrosion and N. fowleri growth. The role of iron was explored further in what, to the author’s knowledge, was the first study of N. fowleri in private wells. Forty wells were examined and the relationships between N. fowleri and stagnant iron levels supported the hypothesis that iron corrosion or iron increases the growth of N. fowleri. As this study took place following a major flooding event, it provided data not only into how surface water contamination may influence private well water microbial communities, but also added to the understanding that current recommendations for disinfecting private wells are inadequate and standards should be implemented to aid homeowners in the event of flooding. This exploratory research highlighted several variables that may allow for the growth of OPs in a range of water systems. Identifying key variables that control growth is crucial to improving the safety of these systems.

Made available in DSpace on 2017-07-20T12:40:20Z (GMT). No. of bitstreams: 1 Agnes Thiane Machado.pdf: 7176772 bytes, checksum: 01a4049f5c4aed0935803a0cf3a6468d (MD5) Previous issue date: 2017-03-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Study of protein three-dimensional structures allow us to investigate the relations between amino acid sequence, structure and function, what is important chiefly for proteins from pathogenic organisms or ones that belong to the same genus of these, such that they can be used as a structural model. In this context, this work aims at the structural characterization of the enzymes alanine racemase long isoform from Trypanosoma cruzi and glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi. The long isoform of alanine racemase catalyzes the conversion between L and D-alanine which, in turn, is part of one of the metabolic pathways in Trypanosoma cruzi, the etiologic agent of Chagas disease. The heterologous expression of this enzyme in Escherichia coli BL21 (DE3) GroEL was analyzed by SDS-PAGE, which revealed that the protein is in higher proportion in the insoluble fraction, thus it was necessary to establish a recovery protocol followed by an in vitro refolding. Data from enzymatic assays and circular dichroism revealed the success of the recovery/refolding protocol, which may in the future contribute to the search for specific inhibitors. Glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi catalyzes the sixth step of the organism’s glycolytic pathway. NgGAPDH enzyme was expressed in E. coli (DE3) using the pET-15b vector, and then purified by tree chromatographic steps, two of nickel affinity and one of size exclusion. The enzymatic characterization was investigated with the enzyme without the his-tag; NgGAPDH presented higher activity at pH 8.0, 25 °C and 10 mM of arsenate, and positive cooperativity for substrates G3P and NAD+. His-tag depleted NgGAPDH crystals appeared in 3 days after drop settings, the best crystal diffracted to 1.94 A resolution and belongs to space group P21 with cell parameters a = 83.74 A, b = 94.55 A, c = 90.93 A, = 99.96 °. The final refined structure presents R = 0.1652 and Rfree = 0.2029. The catalytic domain formed by residues 134 to 313 is highly conserved, as expected, with the exception of Asn145, present only in NgGAPDH, while the other GAPDHs present either Ser or Thr on the corresponding position. Molecular dynamics analysis revealed that Asn145 has correlated motion with residues Ala123, Thr125 and Pro126 that belong to what was called "bonded loop". It should be emphasized that this is the first GAPDH from the phylum Percolozoa that has its three dimensional structure determined and kinetic parameters established, such that we expect to have contributed to the understanding of the evolution of this class of proteins. / O estudo da estrutura tridimensional de proteínas nos permite investigar as relações entre sequência de aminoácidos, estrutura e função, o que é importante principalmente para proteínas de organismos patogênicos ou mesmo pertencente ao gênero destes, que podem ser utilizadas como modelo estrutural. Neste contexto, o presente trabalho visa caracterizar estruturalmente as enzimas alanina racemase isoforma longa de Trypanosoma cruzi e gliceraldeído-3-fosfato desidrogenase de Naegleria gruberi. A alanina racemase isoforma longa catalisa a conversão entre L e D-alanina em uma das vias metabólicas do T. cruzi, que é o agente etiológico da doença de Chagas. A análise por SDS-PAGE de amostras da expressão heteróloga dessa enzima em Escherichia coli BL21(DE3) GroEL revelou que a proteína está em maior proporção na fração insolúvel, por isso, foi necessário estabelecer um protocolo de recuperação seguido de um reenovelamento in vitro. Dados de ensaios enzimáticos e dicroísmo circular revelaram o sucesso do protocolo de recuperação/reenovelamento, o que poderá no futuro contribuir para a busca de inibidores específicos. A gliceraldeído-3-fosfato desidrogenase de Naegleria gruberi catalisa a sexta etapa da via glicolítica do organismo. A enzima NgGAPDH foi expressa em E. coli (DE3) usando-se o vetor pET-15b e então purificada em três passos de cromatografia, dois por afinidade a níquel e um por exclusão por tamanho. A caracterização enzimática foi realizada com a enzima sem a ―his-tag‖; a NgGAPDH apresentou maior atividade em pH 8,0, 25 °C e 10 mM de arsenato, e cooperatividade positiva frente aos substratos G3P e NAD+. Cristais de NgGAPDH sem a ―his-tag‖ apareceram em 3 dias após montagem das gotas e o melhor difratou a 1,94 A de resolução, pertencendo ao grupo espacial P21 com parâmetros de cela a = 83,74 Å, b = 94,55 A, c = 90,93 A e = 99,96 °. A estrutura final refinada apresenta R = 0,1652 e Rfree = 0,2029. O domínio catalítico formado pelos resíduos 134 a 313 é altamente conservado, como esperado, com exceção da Asn145, presente somente em NgGAPDH, enquanto que as demais GAPDHs apresentam Ser ou Thr na posição correspondente. Análises por dinâmica molecular revelaram que a Asn145 tem correlação de movimento com os resíduos Ala123, Thr125 e Pro126, pertencentes ao que se chamou de ―bonded loop‖. Ressalte-se que esta é a primeira GAPDH do filo Percolozoa que tem sua estrutura tridimensional determinada e parâmetros cinéticos estabelecidos, tal que se espera contribuir para o entendimento da evolução dessa classe de proteínas.

Il a été bien documenté que les différentes canalisations des unités de soins dentaires contiennent un épais biofilm. Ce biofilm est constitué entre autres de bactéries, mais aussi d’amibes. Certaines amibes ont un potentiel pathogène et peuvent causer des infections graves. Deux cas d’infections amibiennes et possiblement reliées aux unités dentaires ont retenu notre attention et sont à l’origine du présent projet. L’identification morphologique des amibes afin de déterminer si elles présentent un potentiel pathogène ou non est une tâche ardue, même pour les protozoologistes chevronnés. Nous avons donc utilisé la réaction de polymérase en chaîne (PCR) pour identifier les amibes. Des nouvelles amorces ont été élaborées pour détecter les amibes des genres Acanthamoeba ainsi que Naegleria. Des échantillons d’eau et de terre ont été prélevés dans l’environnement, et des échantillons d’eau et de biofilm ont été prélevés dans les unités dentaires. Une partie de chaque échantillon a été mise en culture selon une méthode améliorée pour une identification morphologique, et l’autre partie a été soumise à un PCR direct. Des Acanthamoebae et/ou des Naegleriae ont été détectées dans 100% des échantillons, mais les espèces varient d’un échantillon à l’autre. Des amibes à potentiel pathogènes sont détectables dans les unités dentaires ainsi que dans l’environnement, et celles-ci pourraient représenter un risque pour la santé de certains individus. / It has been well documented that the various tubing of a dental unit are covered with a thick biofilm. This biofilm mostly consists of bacteria, but amoebae can be found within the biofilm as well. Some amoebae are potential pathogens and may cause serious infections. Two cases of amoebic infections that were possibly linked with dental units drew our attention and stimulated our researches. Morphologic identification of amoebae in order to determine their possible pathogenicity requires much expertise, and is even difficult for proficient protozoologists. Therefore, the use of PCR is essential to detect potentially pathogenic amoebae with subjectivity. We elaborated new primers for the detection of Acanthamoeba spp. and Naegleria spp. Samples of water and dirt were taken in the environment, and samples of water and biofilm were taken in dental units. A part of each samples was cultivated for morphological identification, when a second part was utilized for PCR identification. Acanthamoebae and/or Naegleriae were detected in 100% of our samples, but the species varied from one sample to another. Potentially pathogenic amoebae were detected in dental units and in the environment, which could represent a health risk for some individuals.

Il a été bien documenté que les différentes canalisations des unités de soins dentaires contiennent un épais biofilm. Ce biofilm est constitué entre autres de bactéries, mais aussi d’amibes. Certaines amibes ont un potentiel pathogène et peuvent causer des infections graves. Deux cas d’infections amibiennes et possiblement reliées aux unités dentaires ont retenu notre attention et sont à l’origine du présent projet. L’identification morphologique des amibes afin de déterminer si elles présentent un potentiel pathogène ou non est une tâche ardue, même pour les protozoologistes chevronnés. Nous avons donc utilisé la réaction de polymérase en chaîne (PCR) pour identifier les amibes. Des nouvelles amorces ont été élaborées pour détecter les amibes des genres Acanthamoeba ainsi que Naegleria. Des échantillons d’eau et de terre ont été prélevés dans l’environnement, et des échantillons d’eau et de biofilm ont été prélevés dans les unités dentaires. Une partie de chaque échantillon a été mise en culture selon une méthode améliorée pour une identification morphologique, et l’autre partie a été soumise à un PCR direct. Des Acanthamoebae et/ou des Naegleriae ont été détectées dans 100% des échantillons, mais les espèces varient d’un échantillon à l’autre. Des amibes à potentiel pathogènes sont détectables dans les unités dentaires ainsi que dans l’environnement, et celles-ci pourraient représenter un risque pour la santé de certains individus. / It has been well documented that the various tubing of a dental unit are covered with a thick biofilm. This biofilm mostly consists of bacteria, but amoebae can be found within the biofilm as well. Some amoebae are potential pathogens and may cause serious infections. Two cases of amoebic infections that were possibly linked with dental units drew our attention and stimulated our researches. Morphologic identification of amoebae in order to determine their possible pathogenicity requires much expertise, and is even difficult for proficient protozoologists. Therefore, the use of PCR is essential to detect potentially pathogenic amoebae with subjectivity. We elaborated new primers for the detection of Acanthamoeba spp. and Naegleria spp. Samples of water and dirt were taken in the environment, and samples of water and biofilm were taken in dental units. A part of each samples was cultivated for morphological identification, when a second part was utilized for PCR identification. Acanthamoebae and/or Naegleriae were detected in 100% of our samples, but the species varied from one sample to another. Potentially pathogenic amoebae were detected in dental units and in the environment, which could represent a health risk for some individuals.

This thesis attempts to broaden what is known about bacterial symbionts within amoebae by the use of a number of different molecular methods. Initially a number of different amoeba strains were screened for bacterial symbionts by 16S rRNA gene PCR, then the symbionts were identified by comparative sequence analysis and phylogenetic analysis. The amoeba strains containing bacterial symbionts were characterised by cell morphology, 18S rRNA gene sequencing, internal transcribed spacer sequencing and allozyme electrophoresis. Amoebae belonging to the genera Acanthamoeba, Naegleria, Ripidomyxa and Saccamoeba were identified as containing symbionts that belonged to a wide range of different bacterial genera. Relationships between bacterial symbionts and their host amoebae were analysed by the use of transmission electron microscopy and fluorescent in situ hybridisation using symbiont specific probes. Also described are attempts that were made to isolate and grow the bacterial symbionts outside of their host amoebae as well as experiments to try to transfer bacterial symbionts from one amoeba strain to another. Lastly the results from this study are discussed as a whole to put into perspective how they contribute to the body of knowledge of symbionts within protozoa.

Type II secretion system (T2SS) is one of the secretion systems found in gram-negative bacteria that provides transport of some bacterial proteins across the outer membrane. The passage through the membrane is mediated by a pore assembled from subunits called GspD or secretin. Together with three other components of T2SS, GspD was discovered in the genome of several protists including Naegleria gruberi, Andalucia godoyi, Reclinomonas americana, Neovahlkampfia damariscottae or in s species of genus Malawimonas. Previously it was found out that these proteins localize into the mitochondria. If found functional and with analogous topology to the bacterial system, the eukaryotic T2SS would represent unique mitochondrial protein export system. Secretin is essential subunit of T2SS which is not only the passive membrane channel, but also participates in the recognition of the substrate. Therefore, the research of the eukaryotic secretin could bring a valuable knowledge about the function of the mitochondrial T2SS. The experimental part of this thesis tries to characterize the eukaryotic secretin and it focuses on (i) the assembly of the secretin channel, in both, the bacteria and in the artificial membranes, (ii) the interactions of GspD with the other subunits of T2SS and (iii) the mechanism of import...