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.

Legionella pneumophila (Lp) and nontuberculous mycobacteria (NTM) are opportunistic pathogens that can be transmitted via drinking water, when tiny droplets containing the bacteria are aerosolized and inhaled during activities such as showering. The resulting respiratory illnesses, Legionnaires' Disease and NTM lung disease, are among the leading sources of drinking water associated disease in the United States and other parts of the world. Lp and NTM are both difficult to control, because they establish as part of natural biofilms that form within the interiors of pipes and fixtures that deliver drinking water to the point of use. These pathogens are especially problematic within premise (i.e., building) plumbing, where intermittent use throughout the day leads to long periods of stagnation, increased water age, warmer temperatures, and depleted disinfectant residuals that exacerbate bacterial growth. The recent advent of high throughput DNA sequencing has led to the discovery that drinking water microbiomes are diverse, complex, and largely comprised of non-pathogenic microbes. This has further led researchers to hypothesize that the microbial ecology of this diverse microbiome could be harnessed as a natural means to control Lp and NTM, i.e., a "probiotic" approach, but such an approach has not yet been demonstrated. The objective of this study was to assess this hypothesis by utilizing biologically active granular activated carbon (GAC) filters, which are already a widely used drinking water treatment both at the municipal and household scale, as a means to naturally shape the microbial ecology of downstream premise plumbing and inhibit Lp and NTM proliferation. GAC has an extremely high surface area that aids removal of organic carbon via adsorption but also provides an ideal habitat for establishment of biofilms, which removes organic carbon from the water via biodegradation. Convectively-mixed pipe reactors (CMPRs) were used for replicable simulation of premise plumbing distal taps. The CMPRs consisted of four-foot-long closed polyvinyl chloride (PVC) pipe segments with the sealed bottom portion resting in a ~48 °C water bath and with the top portion plugged and exposed to the cooler, ambient atmosphere (25 °C in this study), inducing convective mixing and resulting in an internal water temperature of 37 °C. PVC was chosen because it is common in premise plumbing and generally leaches the least organic carbon among the different types of plastic pipe. Four different influent water conditions were implemented in the experimental design: 1) Untreated, dechlorinated municipal tap water with high organic carbon and low biomass; 2) GAC-treated tap water with low organic carbon and elevated, viable biomass; 3) GAC-treated + 0.22-m pore size membrane-filtered tap water to remove both nutrients and biomass; 4) GAC-treated tap water pasteurized at 70 °C with low nutrients and elevated, killed biomass. The 0.22-m pore size membrane filter simulated the use of a building scale particle filter, while pasteurization simulated water passing through a hot water heater at an elevated temperature recommended for pathogen thermal disinfection. To understand the influence of these experimental conditions on older pipes containing mature biofilms versus new pipes that leach more organics and are being freshly colonized, a set of older pipes colonized with mature ~4-year-old biofilms were compared to newly purchased pipes. Each set of pipes was tested in triplicate for the four different experimental conditions with the full volume replaced three times a week for eight months, simulating infrequently used taps containing warm, continuously mixing water thought to create conditions at a very high risk for opportunistic pathogen growth. In the aged CMPR bulk water effluents, droplet-digital-polymerase-chain-reaction measurements showed a one-log reduction of Lp and NTM when receiving GAC-treated or GAC-treated + particle-filtered influent water versus receiving dechlorinated municipal tap water or GAC-treated + pasteurized water. These findings suggest that decreased biodegradable dissolved organic carbon achieved by GAC filtration acted to suppress Lp and NTM growth, while the additional step of biomass removal by particle filtration provided a more modest benefit. In the CMPRs consisting of new pipes, concentrations of Lp and NTMs in the effluent bulk water were similar among the experimental conditions, except that the CMPRs receiving the GAC-treated + particle-filtered influent water experienced a two-log reduction in NTMs. These results demonstrate that the colonization and proliferation of NTM within premise plumbing can be significantly controlled by limiting nutrients and biomass in the influent water. This work demonstrates the potential of harnessing GAC-treatment as a means to Control Lp and NTM in premise plumbing via nutrient removal. In scenarios where chemical disinfectants have been depleted, off-the-shelf GAC-treatment used as point-of-entry treatment to large buildings with recirculating plumbing could provide benefits that have previously been unrecognized. Alternatively, pasteurization in very hot water heaters could provide a short-term disinfection benefit, but eventually the nutrients embodied in the dead biomass undermine the positive influence of the nutrient removal provided by the GAC-treatment. Improved mechanistic understanding of probiotic strategies to opportunistic pathogen control would be needed to overcome inherent limitations to the approaches examined herein, if more effective control is desired in the absence of thermal or chemical disinfection. / Master of Science / Legionella pneumophila (Lp) and nontuberculous mycobacteria (NTM) are bacterial pathogens that are the leading source of drinking water-associated disease in the US. Unfortunately, they are not effectively controlled by protections put in place by the US Safe Drinking Water Act (SDWA). Firstly, they cause respiratory infections, which are spread when tiny droplets of water are inhaled during activities such as showering, whereas the SDWA is specifically designed to protect against ingested pathogens. Secondly, unlike fecal-derived organisms (e.g. E. coli) that are the focus of the SDWA, Lp and NTM grow naturally in drinking water distribution systems, especially in premise (i.e., building) plumbing, where water is warmer and more stagnant. Therefore, even if water leaving the treatment plant is devoid of Lp or NTM, this does not guarantee that the consumer's tap water will be Lp- or NTM-free. Also, even though chlorine or other chemical disinfectant is required by the SDWA to be added to the water leaving the treatment plant to control downstream microbial growth, the disinfectant can be depleted or absent within the premise plumbing itself. Additionally, both Lp and NTM tend to more naturally resist chemical disinfectants than fecal-derived organisms. This research is aimed at overcoming these challenges, opening the door to new approaches to controlling Lp and NTM in premise plumbing. Historically, any microbial growth occurring in drinking water has been viewed as problematic, as it usually indicates the chemical disinfectant is inadequately protecting consumers. However, this work explores whether having an abundant community of beneficial bacteria could improve microbial water quality by competing against pathogens for limited space for attachment and nutrients. Such an approach would be analogous to the use of probiotics in humans, to establish a beneficial gut flora that is less susceptible to pathogen invasion. Granular activated carbon (GAC) filters are often used at drinking water treatment plants and by consumers as a point-of-use (e.g., installed on the kitchen tap or in a refrigerator) or whole-house treatment to remove any contaminants of concern and improve the taste and odor of tap water. The granules within GAC filters have a high surface area that helps remove contaminants, but also provides an environment where microbes can live and thrive. As water enters the filter, beneficial microbes can break down any remaining nutrients in the water (e.g., organic carbon and nitrogen). Additionally, the water leaving the filter carries high levels of microbes that grow on the GAC filter that are shed as water passes through. The resulting water with reduced nutrients and higher concentrations of potentially beneficial microbes could create a competitive environment that alters growth of harmful bacteria, like Lp and NTM, in downstream portions of plumbing. The incoming cold water is also warmed by the building envelope, which increases bacterial growth rates. Thus, the underlying hypothesis of this research is that GAC treatment could provide a combination of reduced nutrients and competitive microbes as water enters downstream premise plumbing and reduce the growth of Lp and NTM. However, GAC-treated water within a building can be further altered by other treatments, like a very hot water heater, which would heat and kill the microbes flowing through it, or a particle filter, which could remove the microbes in the water. This work also seeks to understand how these additional treatments might improve or interfere the nutrient reduction and addition of competitive microbes provided by GAC treatment. This research explores how all these different scenarios affect the growth of Lp and NTM using a lab-scale simulated premise plumbing system constructed out of polyvinyl chloride (PVC) pipe that is a common plumbing material used in homes. Water that was added to the pipes was prepared in four different ways to test the probiotic control hypothesis across distinct experimental conditions that replicate the different influent water scenarios. The four conditions were implemented over the course of eight months with regular chemical and biological analyses conducted to understand the effects of the different influent waters on Lp and NTM. It was discovered that premise plumbing with mature biofilms receiving GAC-treated water or GAC-treated + particle-filtered water contained ~90% less Lp and NTM than premise plumbing receiving non-filtered municipal tap water. However, if the GAC-treated water passes through a water heater, the capacity to limit Lp or NTM growth was lost. While GAC filters are currently thought of as an instantaneous treatment that removes contaminants from water, this work demonstrates how GAC treatment might provide prolonged benefits to water, after it has passed through the filter on its journey to a shower head or faucet. Increased understanding of the exact mechanisms of limited pathogen growth gained by this research can lead to new and effective approaches to protect people from contracting diseases caused by Lp and NTM.

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.