Legionella spp. can cause a life threatening form of pneumonia, which is observed world-wide. Outbreaks of the disease are, unfortunately, not a rare event, despite the introduction of government regulations which enforce the mandatory testing of cooling towers to ensure that they contain levels of the organism which are regarded as being within safe limits. Therefore, cooling towers should be monitored for Legionella spp. by using a biosensor. These could potentially save the community from a great deal of morbidity and mortality due to legionellosis. This study identified and investigated novel outer membrane proteins in L. pneumophila, and analysed their potential for use in a Legionella biosensor. A combination of bioinformatics and laboratory investigations was used to identify the Omp87, an outer membrane protein of L. pneumophila which had not been previously described in this organism. Sequence analysis of the protein showed that it shares similarity with various other members of the Omp85 protein family, including the D15 antigen of Haemophilus influenzae and the Oma87 of Pseudomonas aeruginosa. The omp87 gene of L. pneumophila was amplified and cloned, and was found to encode a protein of 786 amino acids, with a molecular weight of 87 kDa. Distribution studies revealed that the gene is present in most, but not all species and serogroups of Legionella. To investigate the function of the Omp87 protein in L. pneumophila, the omp87 gene was insertionally inactivated with the use of a kanamycin resistance gene. Amplicons of this disrupted gene were then introduced into L. pneumophila, and a double-cross over event occurred, integrating the inactivated gene into the genome of the organism. This resulted in non-viable cells, indicating that the gene is essential in L. pneumophila. The expression vector pRSETA was used to express the Omp87 protein in E. coli, and four truncates of varying sizes were designed, through the use of different PCR primers. Two of the protein truncates were then expressed and purified by gravity flow chromatography using columns packed with Ni-NTA sepharose resin. Following analysis of the proteins by SDS-PAGE and Western blotting, polyclonal antibodies were raised against the truncates. Distribution studies were then performed using the antiserum with different strains and species of Legionella. This study demonstrated that most serogroups of L. pneumophila, and most other Legionella species reacted with the polyclonal anti-Omp87 L. pneumophila antisera. Cross-reactivity was also observed with most other Legionella related organisms tested. The results presented in this thesis demonstrated that the Omp87 protein or the omp87 gene can be used to construct a biosensor. In addition other novel outer membrane proteins were identified which could also serve as potential targets for a biosensor. These biosensors will be able to identify Legionella spp. in water reservoirs and in clinical samples and hopefully reduce the number of infections and deaths caused by this organism.

Zugl.: Berlin, Freie Universiẗat, Diss., 2008.

Würzburg, Univ., Diss., 2005. / Erscheinungsjahr an der Haupttitelstelle: 2004

No presente trabalho faz-se uma revisão da literatura sobre pneumonia por Legionella pneumophila e se comparam estes dados com a série da autora, que se compõe de 10 casos esporádicos desta pneumonia, adquiridos na comunidade, ocorridos no perÍodo entre outubro de 1983 e maio de 1989. Todos os pacientes desta série eram do sexo masculino e de cor branca, com idade variando entre 36 e 71 anos. Os sintomas mais freqüentes foram febre alta, calafrios, cefaléia, tosse seca e mialgias. A hipótese diagnóstica baseou-se nos dados clínicos, radiológicos e laboratoriais. Em todos os casos o critério de comprovaçao diagnóstica foi a imunofluorescência indireta para Legionella. Salienta-se a importância do reconhecimento desta doença, que ainda apresenta um baixo Índice de suspeição em nosso meio. Procura-se tanto ressaltar os principais achados clínicos e radiológicos como também contribuir com orientações diagnósticas e terapêuticas. ApÓs a análise dos dados obtidos no trabalho, a autora concluiu que: 1. Os achados da série nao diferem daqueles descritos na literatura. 2. A casuística é restrita para o traçado de um perfil da doença no Rio Grande do Sul. 3. Quadros pneumônicos com má resposta clÍnica à penicilina ou derivados, associados a lesões radiolÓgicas com rápida mutabilidade, devem chamar a atenção para este diagnóstico. 4. A infreqüência deste diagnóstico em nosso meio deve-se ao baixo Índice de suspeição. Logo, a divulgação de informações sobre a doença pode resultar em substancial acréscimo ao registro de casos. / In the present work a review is made on the 1iterature about pneumonia by Legione11a pneumophi1a and the data are compared with the series presenteà by the author, composed of 10 sporadic cases of this pneumonia, acquired in the community, October, 1983 and May, 1989. between A11 the patients of this group were ma1e, caucasian, age varying from 36 to 71. The most frequent symptoms were high fever, chills, headache, dry cough and myalgia. The diagnostic hypothesis was baseà on laboratory, radiological and clinicai data. For all the cases, the criterion for diagnostic comprovation was indirect immunofluorescence for Legionella. The importance of the recognition of this disease s emphasized for it still shows a very low 1evel of suspicion in Rio Grande do Sul. The author looks for to ernphazise the main clinicai and radiological findings as well as contributes with diagnostics and therapeutical orientations. After the analysis of the data obtained in the present work the author concludes that: 1. The findings of this series does no differ from those described in the 1iterature. 2. The casuistic is too restricted to draw a profile of the disease in Rio Grande do Sul. 3. Pneumonias with bad clinicai response to penicillin or its derivatives, associated with radiologic lesions with rapid mutability shall call the attention for this diagnosis. 4. The low frequency of this diagnosis in our country is due to the low index of suspicion. Thus, the divulgation of information about the disease could result in substantial increase in the record of new cases.

No presente trabalho faz-se uma revisão da literatura sobre pneumonia por Legionella pneumophila e se comparam estes dados com a série da autora, que se compõe de 10 casos esporádicos desta pneumonia, adquiridos na comunidade, ocorridos no perÍodo entre outubro de 1983 e maio de 1989. Todos os pacientes desta série eram do sexo masculino e de cor branca, com idade variando entre 36 e 71 anos. Os sintomas mais freqüentes foram febre alta, calafrios, cefaléia, tosse seca e mialgias. A hipótese diagnóstica baseou-se nos dados clínicos, radiológicos e laboratoriais. Em todos os casos o critério de comprovaçao diagnóstica foi a imunofluorescência indireta para Legionella. Salienta-se a importância do reconhecimento desta doença, que ainda apresenta um baixo Índice de suspeição em nosso meio. Procura-se tanto ressaltar os principais achados clínicos e radiológicos como também contribuir com orientações diagnósticas e terapêuticas. ApÓs a análise dos dados obtidos no trabalho, a autora concluiu que: 1. Os achados da série nao diferem daqueles descritos na literatura. 2. A casuística é restrita para o traçado de um perfil da doença no Rio Grande do Sul. 3. Quadros pneumônicos com má resposta clÍnica à penicilina ou derivados, associados a lesões radiolÓgicas com rápida mutabilidade, devem chamar a atenção para este diagnóstico. 4. A infreqüência deste diagnóstico em nosso meio deve-se ao baixo Índice de suspeição. Logo, a divulgação de informações sobre a doença pode resultar em substancial acréscimo ao registro de casos. / In the present work a review is made on the 1iterature about pneumonia by Legione11a pneumophi1a and the data are compared with the series presenteà by the author, composed of 10 sporadic cases of this pneumonia, acquired in the community, October, 1983 and May, 1989. between A11 the patients of this group were ma1e, caucasian, age varying from 36 to 71. The most frequent symptoms were high fever, chills, headache, dry cough and myalgia. The diagnostic hypothesis was baseà on laboratory, radiological and clinicai data. For all the cases, the criterion for diagnostic comprovation was indirect immunofluorescence for Legionella. The importance of the recognition of this disease s emphasized for it still shows a very low 1evel of suspicion in Rio Grande do Sul. The author looks for to ernphazise the main clinicai and radiological findings as well as contributes with diagnostics and therapeutical orientations. After the analysis of the data obtained in the present work the author concludes that: 1. The findings of this series does no differ from those described in the 1iterature. 2. The casuistic is too restricted to draw a profile of the disease in Rio Grande do Sul. 3. Pneumonias with bad clinicai response to penicillin or its derivatives, associated with radiologic lesions with rapid mutability shall call the attention for this diagnosis. 4. The low frequency of this diagnosis in our country is due to the low index of suspicion. Thus, the divulgation of information about the disease could result in substantial increase in the record of new cases.

The attachment and/or penetration of animal cells by two strains of Legionella pneumophila was studied in three vertebrae cell lines in vitro . The study focused on (1) differences in attachment and penetration between the two bacterial strains (an environmental isolate, Johannesburg-2, and a clinical isolate, Chicago-8) and between the cell lines (Hep-2, WI-38 and a murine line); (2) effects of L. pneumophila on cell morphology and growth; and (3) the effects of pyruvate and six sugars or sugar derivatives (D-mannose, D-Galactose, D-Glucose, L-glucose, D-fructose, and 2-deoxy-D-glucose).

Two species of cyanobacteria (Fischeralla sp. 29161 and Phormidium autumnale) and one species of green algae (Fritschiella tuberosa) were found to promote survival of Legionella pneumophila in mineral salts medium cocultures. During the early stage of incubation Fischerella sp. supported growth of Legionella pneumophila even though the bacteria would not grow in the algae-free basal medium.

The effect of copper on Legionella pneumophila in potable water plumbing systems is dependent on its dose and water chemistry. For instance, prior research demonstrated that the presence of aluminum hydroxide from anode rods in water heaters can bind copper and render high doses non-toxic. On this basis it was also hypothesized that iron hydroxide would have similar effects and that lower levels of copper may act as a nutrient encouraging Legionella growth. Here we conducted complementary experiments at bench, microcosm and pilot-scale to evaluate the effect of copper speciation and dose on L. pneumophila. At bench-scale, the addition of 5 mg/L as Fe iron hydroxide to a solution with 1 mg/L copper decreased soluble copper from > 90% down to < 20% at pH 6.5-7. The reduction in soluble copper caused ~3-logs higher L. pneumophila culturability when iron was added with copper when compared to a condition with copper alone. In a 9-month microcosm test using simulated glass water heaters with PEX pipe, a complete range of copper doses (0, 4, 30, 250 and 2000 g/L) were tested in triplicate. Over the first phase of research covered herein, the L. pneumophila levels were low at the four lowest doses of copper, and non-detectable at the highest dose. Moreover, total cell counts were highest at 250 g/L copper, lowest at 2000 g/L copper, and in between these extremes at the lower copper doses. This ongoing experiment will continue for months after this thesis was complete. Pilot-scale experiments were conducted with anode rods removed from tank water heaters, to examine the effects of unprotected corrosion of the steel on iron release and Legionella pneumophila levels in systems with 1) added copper (WH-Cu), 2) copper and phosphate corrosion inhibitor (WH-Cu+PO4-3), 3) phosphate corrosion inhibitor alone (WH-PO4-3) and 4) a control with neither copper nor inhibitor (WH-Control). While there were slight differences in iron between the conditions, the iron concentration in the water of the tanks did not dramatically increase compared to when the powered anodes were still installed and reducing corrosion. Because the iron level was usually < 0.1 mg/L, the released iron dose was not high enough to reduce soluble copper. In fact, oddly, soluble copper increased by 37-183%, mostly likely because the installed anode rods were suppressing copper release. Consequently, with the anode rods removed, the dose of 2000 g/L copper still had a strong antimicrobial effect. The levels of L. pneumophila increased in the order WH-Cu (2.6-logs CFU/L) < WH-PO4-3 (5.1-logs CFU/L) ≈ WH-Control (5.1-logs CFU/L). The addition of phosphate precipitated some of the added copper, such that the condition with copper and phosphate [WH-Cu+PO4-3 (4.2-logs CFU/L)] had L. pneumophila in between the condition with copper alone and that with no added copper. When the copper dose in the pilot rig was reduced to 1000 g/L, Legionella increased somewhat in the system with added copper compared to the control, and L. pneumophila increased in the water heaters in the order WH-Cu (3.4-logs CFU/L) < WH-Cu+PO4-3 (4.3-logs CFU/L) < WH-PO4-3 (4.9-logs CFU/L) ≈ WH-Control (5.0-logs CFU/L). Overall, an antimicrobial effect of copper was maintained in the water heaters even after the anodes were removed and iron in the water increased slightly. If iron corrosion and release to water were much higher without the anode rods than observed in this study, it would be predicted that the higher iron would have reduced the copper antimicrobial effect. / Master of Science / The effect of copper on Legionella pneumophila in potable water plumbing systems depends on its dose and water chemistry. For instance, prior research demonstrated that the presence of aluminum hydroxide from anode rods in water heaters can bind copper and render high doses non-toxic. It was also hypothesized that iron hydroxide would have similar effects and that lower levels of copper may act as a nutrient encouraging Legionella growth. Here we conducted complementary experiments at bench, microcosm and pilot-scale to evaluate the effect of copper's chemistry and dose on L. pneumophila. At bench-scale, the addition of a high level of iron to a solution with copper decreased the amount of copper available to Legionella from > 90% down to < 20% at pH 6.5-7. The reduction in bioavailable copper caused ~3-logs higher L. pneumophila when iron was added with copper when compared to a condition with copper alone. In a 9-month microcosm test using simulated glass water heaters with PEX pipe, a complete range of copper doses (0, 4, 30, 250 and 2000 g/L) were tested. The L. pneumophila levels were low and there were no significant differences between the five doses in the beginning phases of the experiment reported herein. However, total cell counts were highest at 250 g/L copper, lowest at 2000 g/L copper, and in between these extremes at the lower copper doses. This suggests that 250 g/L may be optimal in encouraging overall microbial growth. Pilot-scale experiments were conducted with anode rods removed from water heaters to examine the effects of unprotected corrosion of the steel tank on iron release and Legionella pneumophila levels. The four water heaters had added copper (WH-Cu), copper and phosphate corrosion inhibitor (WH-Cu+PO4-3), phosphate corrosion inhibitor alone (WH-PO4-3) and neither copper nor inhibitor (WH-Control). The iron concentration in the water of the tanks did not dramatically increase compared to when the powered anodes were used to reduce corrosion. Nevertheless, there were still slight differences in iron concentration between the replicate water heaters. When 2000 g/L copper was dosed, WH-Cu and WH-Control had statistically higher iron levels than WH-PO4-3, consistent with copper increasing corrosion of the unprotected tank. However, because the iron level was usually < 0.1 mg/L, the released iron dose was not high enough to reduce bioavailable copper. In fact, bioavailable copper went up when the anode rod was removed, most likely because the anode rods were protecting brass plumbing from corrosion. With the anode rods removed a dose of 2000 g/L copper had a strong antimicrobial effect. The levels of L. pneumophila increased in the order WH-Cu < WH-PO4-3 ≈ WH-Control. The addition of phosphate reduced some of the bioavailable copper, such that the condition with copper and phosphate (WH-Cu+PO4-3) had L. pneumophila in between the condition with copper alone and that with no added copper. When the copper dose in the water heaters was reduced to 1000 g/L, Legionella increased somewhat in the system with added copper compared to the control, and L. pneumophila increased in the order WH-Cu < WH-Cu+PO4-3 < WH-PO4-3 ≈ WH-Control. Overall, an antimicrobial effect of copper was maintained in water heaters even after the anodes were removed and even though iron in the water increased slightly. If iron corrosion and release to water was much higher than occurred in this study, it would be expected that the iron could have reduced the copper antimicrobial effect.

A bactéria Legionella pneumophila é um bacilo Gram-negativo, flagelado causador da doença dos legionários e febre de Pontiac. O inflamassoma mais importante no controle da replicação desta bactéria é o composto por Naip5/NLRC4, que é responsável pelo reconhecimento de flagelina. A ativação do inflamassoma de Naip5/NLRC4 pela flagelina induz a ativação de caspase-1, induzindo a formação de poros na membrana, piroptose e controle da replicação desta bactéria. A participação da proteína adaptadora ASC é essencial para a nucleação deste complexo e secreção de citocinas inflamatórias como IL-1? e IL-18 por esta via. Além do controle da replicação de L. pneumophila pelo inflamassoma NLRC4 dependente de caspase-1, foi demonstrado que existe uma via induzida por NLRC4 independente de caspase- 1/11. Dessa forma, camundongos e células Nlrc4-/- são mais susceptíveis à infecção por esta bactéria do que as células Casp1/11-/-. Neste trabalho, nós identificamos que a via independente de caspase-1/11 é composta por Naip5/NLRC4/ASC/Caspase-8 e é essencial para o controle da replicação de Legionella spp. flageladas em macrófagos e in vivo. Através da utilização de BMDMs Casp1/11-/- e Asc/Casp1/11-/- transduzidos com NLRC4-GFP ou ASC-GFP, identificamos que a formação de punctas de NLRC4 e ASC dependem do reconhecimento de flagelina e que ASC é essencial para a formação desses punctas. Também foi identificado que a infecção com L. pneumophila que expressa flagelina leva à ativação de caspase-8 de maneira dependente de ASC e Naip5, mas independente de caspase-1/11. De acordo com esses dados, o silenciamento de caspase-8 em macrófagos Casp1/11-/- aumentou a susceptibilidade dessas células à infecção com L. pneumophila flagelada. Além disso, macrófagos e camundongos Asc/Casp1/11-/- foram tão susceptíveis quanto os Nlrc4- /- e mais susceptíveis que os Casp1/11-/-. Nós observamos que o inflamassoma de NLRC4/ASC/Caspase-8 induz formação de poros e morte celular independente de gasdermina-D (GSDMD). Por meio da utilização de células de camundongos C57BL/6, foi observado que caspase-8 é recrutada para o inflamassoma de Naip5/NLRC4/ASC/Caspase-1. Entretanto, a ativação de caspase-8 só ocorre na 10 ausência de caspase-1 ou GSDMD. Nossos dados sugerem que a ativação de caspase-8 no inflamassoma composto por NLRC4/ASC/Caspase-8 representa uma via alternativa que opera para garantir o controle da replicação de bactérias flageladas em situações nas quais ou caspase-1 ou GSDMD estão inibidas. / Legionella pneumophila is a flagellated Gram-negative bacillus that is the causative agent of the legionnaire\'s disease and Pontiac fever. The most important inflammasome for the control of L. pneumophila replication is the Naip5/NLRC4, responsible for the flagellin recognition. The activation of the Naip5/NLRC4 inflammasome leads to caspase-1 activation, consequently pore formation, pyroptosis and control of bacterial replication. The participation of the adaptor molecule ASC is essential for this complex nucleation and the secretion of inflammatory cytokines like IL-1? and IL-18 by this pathway. Besides the control of L. pneumophila replication by Naip5/NLRC4/Caspase-1 inflammasome, it was demonstrated there are NLRC4 responses independent of caspase-1/11. These explain why mice and macrophages Nlrc4-/- are more susceptible than Casp1/11-/-. In this work, we identified that the caspase-1/11-independent pathway is composed of Naip5/NLRC4/ASC/Caspase-8 and it is essential for the control of flagellated Legionella spp. replication in macrophages and in vivo. Infection of Casp1/11-/- and Asc/Casp1/11-/- macrophages, transduced with NLRC4-GFP or ASC-GFP, showed that flagellin-positive bacteria triggered puncta formation that is ASC-dependent. Accordingly, Naip5 and ASC, but not caspase-1/11, were required for caspase-8 activation in response to flagellated bacteria. Silencing caspase-8 in Casp1/11-/- BMDMs increased the susceptibility to L. pneumophila infection. Furthermore, the macrophages and mice Asc/Casp1/11-/- are as susceptible as Nlrc4-/-, but more susceptible than Casp1/11-/-. We also found that the NLRC4/ASC/Caspase-8 inflammasome induces GSDMD-independent pore formation and cell death. Using C57BL/6 cells, we observed that caspase-8 is recruited to Naip5/NLRC4/ASC/Caspase-1 inflammasome. However, caspase-8 is just activated in the absence of caspase-1 or GSDMD. Our data suggest that caspase-8 activation in the NLRC4/ASC/Caspase-8 inflammasome represents an alternative pathway that operates to ensure the control of flagellated bacteria replication in situations which either caspase-1 or GSDMD are inhibited.

Legionella pneumophila, o agente etiológico da doença dos Legionários, é conhecida por desencadear a formação de poro em membranas de macrófagos derivados de medula óssea (BMMs) por mecanismos dependentes do sistema de secreção do tipo IV conhecido como Dot/Icm. Neste trabalho, foram utillizados vários mutantes de L. pneumophila em combinação com camundongos nocautes para investigar os fatores bacterianos e do hospedeiro envolvidos na formação de poro em BMMs. Observamos que apesar da atividade do Dot/Icm, a formação de poro não ocorre em BMMs deficientes para caspase-1 e Nlrc4. A formação de poro foi temporalmente associada com a secreção de IL-1b e precedeu a lise celular e a piroptose. A formação de poro foi dependente do Dot/Icm, mas independente de várias proteínas efetoras, da multiplicação bacteriana e da síntese de novo de proteínas. A flagelina, a qual é conhecida em ativar o inflamassoma de Nlrc4, foi necessária para a formação de poro; a bactéria mutante flaA falhou em induzir a permeabilização celular. Consequentemente, a transfecção da flagelina purificada foi suficiente para desencadear a formação de poro independente da infecção. Utilizando 11 diferentes espécies de Legionella, nós observamos alta formação de poro em resposta à L. micdadei, L. bozemanii, L. gratiana, L. jordanis e L. rubrilucens, e essa resposta estava correlacionada com a expressão de flagelina por essas espécies. Além disso, verificamos que as proteínas Asc e Caspase-11 apresentam fenótipo intermediário na formação de poro, sugerindo que outras vias podem estar envolvidas no processo. Observamos também que a formação de poro desencadeada por L. pneumophila difere daquela induzida pelo ATP. Em conjunto, nossos resultados sugerem que a formação de poro não é uma resposta específica de L. pneumophila nem o resultado de dano da membrana induzido pelo Dot/Icm. Ao invés disso, a formação de poro é uma resposta do hospedeiro altamente coordenada, dependente dos componentes do inflamassoma Nlrc4 e caspase-1 e é desencadeada em resposta a bactérias que expressam o sistema de secreção do tipo IV e flagelina. / Legionella pneumophila, the etiological agent of Legionnaires disease, is known to trigger pore formation in bone marrow-derived macrophages (BMMs) by mechanisms dependent on the type IVB secretion system known as Dot/Icm. Here, we used several mutants of L. pneumophila in combination with knockout mice to assess the host and bacterial factors involved in pore formation in BMMs. We found that regardless of Dot/Icm activity, pore formation does not occur in BMMs deficient in caspase-1 and Nlrc4/Ipaf. Pore formation was temporally associated with IL-1b secretion and preceded host cell lysis and pyroptosis. Pore-forming ability was dependent on bacterial Dot/Icm but independent of several effector proteins, multiplication and de novo protein synthesis. Flagellin, which is known to trigger the Nlrc4 inflammasome, was required for pore formation as flaA mutant bacteria failed to induce cell permeabilization. Accordingly, transfection of purified flagellin was sufficient to trigger pore formation independent of infection. By using 11 different Legionella species, we found robust pore formation in response to L. micdadei, L. bozemanii, L. gratiana, L. jordanis and L. rubrilucens, and this trait correlated with flagellin expression by these species. Furthermore, we found that Asc and Caspase-11 showed an intermediate phenotype in pore formation, suggesting that other pathways may be involved in this process. We also observed that the pore formation triggered by L. pneumophila differs from the pore induced by ATP. Together, the results suggest that pore formation is neither L. pneumophilaspecific nor the result of membrane damage induced by Dot/Icm activity; instead, it is a highly coordinated host cell response dependent on host Nlrc4 and caspase-1 and on bacterial flagellin and type IV secretion system.