Introdução: Destaca-se a importância dos organismos do gênero Legionella como agentes causadores de doenças do aparelho respiratório como a legionelose ou doença do Legionário, e ainda de um controle na manutenção adequada de sistemas de ar condicionado, uma vez que estes foram identificados como ambientes favoráveis à proliferação desses microrganismos. Objetivo: Devido à importância de bactérias do gênero Legionella como agentes causadores de doenças, e um crescente número de casos de legionelose relacionados à permanência em ambientes fechados, este estudo teve como objetivo determinar a presença de Legionella em águas de bandejas de sistemas de ar condicionado. Métodos: Foram realizadas análises de amostras de água provenientes de bandejas de ar condicionado de dois hospitais, uma instituição de ensino superior e um centro comercial, totalizando 41 amostras. Volumes de 1L das amostras coletadas foram concentrados em membrana, seguida de tratamento ácido. Alíquotas das amostras tratadas e não tratadas com ácido, foram então inoculadas em meio Agar BCYE-, com e sem adição de antibióticos. As cepas isoladas foram submetidas a teste sorológico para identificação. Resultados: Das amostras analisadas, 4 (9,5%) apresentaram resultado positivo para Legionella sp.; uma no centro comercial e três em um dos hospitais estudados. Os isolados foram submetidos a teste sorológico, sendo um identificado como Legionella pneumophila sorogrupo 1 e três como seis prováveis espécies:L. longbeachae, L. bozemanni, L. dumoffi, L. gormanii, L. jordanis, L. micdadei, L. anisa . Conclusão: Os resultados deste estudo comprovam a presença de Legionella sp. nos sistemas de ar condicionado pesquisados, sendo observada maior freqüência dos isolados em amostras analisadas oriundas dos hospitais.Os resultados demonstraram a importância e a necessidade de planos de monitoramento em sistemas de 7 ar condicionado como medida preventiva à colonização por organismos patogênicos nesses sistemas e dessa forma proteger a saúde dos ocupantes e usuários de ambientes climatizados artificialmente. / Introduction: It highlights the importance of organisms genus Legionella as responsible for diseases of the respiratory system such as legionellosis or Legionnaires´disease, and a control on proper maintenance of air conditioning systems, as these were identified as favorable environment for the proliferation of these microorganisms. Objective: Due to the importance of Legionella as causative agents of disease and a growing number of cases of legionellosis related to stay indoors, this study aimed to determine the presence of Legionella in water samples from air-conditioning systems. Methods: We performed analysis of samples of water from trays of airconditioning systems in two hospitals, a university and a trade center, totalizing 41 samples. One liter volumes of water samples were concentrated on membrane, followed by acid treatment. Aliquots of the sample treated and not treated with acid, were inoculated on BCYE- agar medium, with and without added antibiotics. The strains were subjected to serological identification. Results: Out of the samples analyzed, 4 (9.5%) were positive for Legionella sp.; one in a trade center and three in one of the hospitals. Isolates were submitted to serological testing, one identified as Legionella pneumophila serogroup 1 and three as probable six species: L. longbeachae, L. bozemanni, L. dumoffi, L. gormanii, L. jordan, L. micdadei, L. anise. Conclusion: The results confirm the presence of Legionella sp. in air conditioning systems studied. It was observed a higher frequency in isolates from hospitals. The results demonstrated the importance and necessity of monitoring plans in air-conditioning systems as a preventive measure to colonization by pathogens in these systems and protect the health of occupants and users of these buildings.

Legionella longbeachae é uma espécie da família Legionellaceae que é comumente presente no solo em diversas regiões do globo. Uma infecção por L. longbeachae em indivíduos imunocomprometidos causa uma pneumonia severa, frequentemente levando a hospitalização e à morte. A prevalência destas bactérias como causa de pneumonia é grande, e certamente sub-estimada, uma vez que os métodos de diagnóstico convencionais detectam apenas as espécies de Legionella pneumophila. A anafilatoxina C5a è uma proteína inflamatória ativada pelo complemento, a qual é envolvida no recrutamento de células inflamatórias, um processo induzido pelas células da imunidade inata que leva a dano tecidual. Dados recentes gerados no nosso laboratório sugerem que a mortalidade de camundongos após a infecção por L. longbeachae é causada por uma falência pulmonar, associada a indução de um intenso processo inflamatório nos pulmões dos animais infectados. Nesse trabalho, nós investigamos papel do receptor de C5a (C5aR) na replicação bacteriana e na resistência de camundongos diante de uma infecção letal por L. longbeachae. Experimentos realizados com animais deficientes no receptor C5a indicam que os animais são protegidos durante uma infecção letal por L. longbeachae em comparação com animais selvagens, da linhagem BALB/c. De acordo com esses resultados, foi detectada uma menor carga bacteriana nos pulmões dos animais C5a-/- em comparação com animais selvagens. Experimentos realizados com animais controles da mesma linhagem demonstraram que C5a-/- diferem de animais C5a+/-, o que suporta o papel desse receptor durante a infecção por L. longbeachae. Dessa forma, nossos dados sugerem que a sinalização via C5aR contribui para a patogênese da doença em modelo murino da infecção por L. longbeachae. Os mecanismos envolvidos na patogênese mediada pelo receptor C5a encontram-se sob investigação. / Legionella longbeachae is a species of the Legionellaceae family that is commonly present in the soil in various regions of the globe. Infections by L. longbeachae in immunocompromised individuals cause severe pneumonia, often leading to hospitalization and death. The prevalence of L. longbeachae as a cause of pneumonia is large, and certainly under-estimated, mainly because the conventional diagnostic methods only detect Legionella pneumophila species. The anaphylatoxin C5a is an inflammatory protein activated by the complement system, which is involved in the recruitment of inflammatory cells, a process induced by cells of the innate immunity, which leads to tissue damage. Recent data generated in our laboratory suggest that the mortality of mice after infection with L. longbeachae is caused by a lung failure, associated with the induction of an intense inflammatory process in the lungs of infected animals. In this study, we investigated the role of C5a receptor (C5aR) in bacterial replication and mice resistance on a lethal infection by L. longbeachae. Experiments with animals deficient in the C5a receptor indicate that the animals are protected during a lethal infection by L. longbeachae as compared with-wild type strain, BALB/c. According to these results, a lower bacterial load was detected in the lungs of C5a-/- animals compared with BALB/c animals. Experiments performed with control animals of the same strain demonstrated that C5a-/- differ from C5a+/- animals, which supports the role of this receptor during infection by L. longbeachae. Thus, our data suggest that C5aR signalling pathway contributes to the pathogenesis of the disease in a murine model of infection by L. longbeachae. The mechanisms involved in the pathogenesis mediated by C5a receptor are under investigation.

Introdução: Destaca-se a importância dos organismos do gênero Legionella como agentes causadores de doenças do aparelho respiratório como a legionelose ou doença do Legionário, e ainda de um controle na manutenção adequada de sistemas de ar condicionado, uma vez que estes foram identificados como ambientes favoráveis à proliferação desses microrganismos. Objetivo: Devido à importância de bactérias do gênero Legionella como agentes causadores de doenças, e um crescente número de casos de legionelose relacionados à permanência em ambientes fechados, este estudo teve como objetivo determinar a presença de Legionella em águas de bandejas de sistemas de ar condicionado. Métodos: Foram realizadas análises de amostras de água provenientes de bandejas de ar condicionado de dois hospitais, uma instituição de ensino superior e um centro comercial, totalizando 41 amostras. Volumes de 1L das amostras coletadas foram concentrados em membrana, seguida de tratamento ácido. Alíquotas das amostras tratadas e não tratadas com ácido, foram então inoculadas em meio Agar BCYE-, com e sem adição de antibióticos. As cepas isoladas foram submetidas a teste sorológico para identificação. Resultados: Das amostras analisadas, 4 (9,5%) apresentaram resultado positivo para Legionella sp.; uma no centro comercial e três em um dos hospitais estudados. Os isolados foram submetidos a teste sorológico, sendo um identificado como Legionella pneumophila sorogrupo 1 e três como seis prováveis espécies:L. longbeachae, L. bozemanni, L. dumoffi, L. gormanii, L. jordanis, L. micdadei, L. anisa . Conclusão: Os resultados deste estudo comprovam a presença de Legionella sp. nos sistemas de ar condicionado pesquisados, sendo observada maior freqüência dos isolados em amostras analisadas oriundas dos hospitais.Os resultados demonstraram a importância e a necessidade de planos de monitoramento em sistemas de 7 ar condicionado como medida preventiva à colonização por organismos patogênicos nesses sistemas e dessa forma proteger a saúde dos ocupantes e usuários de ambientes climatizados artificialmente. / Introduction: It highlights the importance of organisms genus Legionella as responsible for diseases of the respiratory system such as legionellosis or Legionnaires´disease, and a control on proper maintenance of air conditioning systems, as these were identified as favorable environment for the proliferation of these microorganisms. Objective: Due to the importance of Legionella as causative agents of disease and a growing number of cases of legionellosis related to stay indoors, this study aimed to determine the presence of Legionella in water samples from air-conditioning systems. Methods: We performed analysis of samples of water from trays of airconditioning systems in two hospitals, a university and a trade center, totalizing 41 samples. One liter volumes of water samples were concentrated on membrane, followed by acid treatment. Aliquots of the sample treated and not treated with acid, were inoculated on BCYE- agar medium, with and without added antibiotics. The strains were subjected to serological identification. Results: Out of the samples analyzed, 4 (9.5%) were positive for Legionella sp.; one in a trade center and three in one of the hospitals. Isolates were submitted to serological testing, one identified as Legionella pneumophila serogroup 1 and three as probable six species: L. longbeachae, L. bozemanni, L. dumoffi, L. gormanii, L. jordan, L. micdadei, L. anise. Conclusion: The results confirm the presence of Legionella sp. in air conditioning systems studied. It was observed a higher frequency in isolates from hospitals. The results demonstrated the importance and necessity of monitoring plans in air-conditioning systems as a preventive measure to colonization by pathogens in these systems and protect the health of occupants and users of these buildings.

L. pneumophila est l'agent responsable de la légionellose, une pneumonie sévère associée à 10% de mortalité. Cette bactérie intracellulaire a acquis la capacité de survivre et de se répliquer dans des cellules humaines. Notamment, L. pneumophila sécrète un grand nombre d'effecteurs par son système de sécrétion de type IV, qui interagissent avec différentes voies cellulaires, dont l'autophagie. L'autophagie est une voie de dégradation conservée qui permet aux cellules eucaryotes de réguler l'homéostasie cellulaire et d'éliminer les agents pathogènes intracellulaires. Néanmoins, nombre d'entre eux ont évolué pour manipuler cette voie à leur propre avantage. Même si l'interaction entre L. pneumophila et l'autophagie a été rapportée, aucun modèle clair n'est déterminé. Dans cette étude, nous montrons qu'une infection à L. pneumophila induit une stimulation globale de l'autophagie, mais que ce phénotype dépend des souches utilisées, et notamment de la présence de certains effecteurs. De plus, l'inhibition de l'autophagie est liée à un défaut de réplication intracellulaire suggérant que cette voie est bénéfique à la bactérie. Afin de rechercher les déterminants génétiques impliqués dans cette interaction, nous avons identifié des effecteurs communs sécrétés par le système de sécrétion de type IV entre L. pneumophila et Coxiella burnetii, une bactérie de l'ordre des Legionellales connue pour stimuler et détourner l'autophagie. La capacité des mutants de ces effecteurs à stimuler l'autophagie chez L. pneumophila a été analysée. Si aucun d'entre eux ne semble impliqué dans la modulation de l'autophagie, cette étude suggère d'autres fonctions pour ces effecteurs conservés / Legionella pneumophila is responsible for the legionellosis disease, a severe pneumonia associated with 10% mortality rate. This intracellular bacterium has evolved the ability to survive and replicate within human cells. Notably, L. pneumophila secretes a high number of type IV secretion system effectors that interfere with many cellular pathways including autophagy. Autophagy, a highly conserved degradative pathway, allows eukaryotic cells to regulate cell homeostasis and fight intracellular pathogens. Nevertheless numerous microorganisms have evolved strategies to subvert this mechanism to their own advantage. The interaction between L. pneumophila and autophagy has been reported but remains unclear. In this study, we show that L. pneumophila infection induces a global stimulation of autophagy, but importantly this autophagy stimulation depends on the bacterial strain. Moreover, we also observed that inhibition of autophagy results in decreased intracellular bacterial proliferation suggesting that host cell autophagy is benificial for L. pneumophila. In order to decipher the molecular determinants involved in the interaction with autophagy, we identified common effectors secreted by the type IV secretion system between L. pneumophila and Coxiella burnetii, a bacterium from the order Legionellale responsible for Q fever and known to stimulate and hijack host cell autophagy. Mutant of these common effectors in L. pneumophila were analysed. While, none of them seems to be implicated in autophagy modulation, this study suggests other functions for these conserved effectors

Legionella longbeachae é uma espécie da família Legionellaceae que é comumente presente no solo em diversas regiões do globo. Uma infecção por L. longbeachae em indivíduos imunocomprometidos causa uma pneumonia severa, frequentemente levando a hospitalização e à morte. A prevalência destas bactérias como causa de pneumonia é grande, e certamente sub-estimada, uma vez que os métodos de diagnóstico convencionais detectam apenas as espécies de Legionella pneumophila. A anafilatoxina C5a è uma proteína inflamatória ativada pelo complemento, a qual é envolvida no recrutamento de células inflamatórias, um processo induzido pelas células da imunidade inata que leva a dano tecidual. Dados recentes gerados no nosso laboratório sugerem que a mortalidade de camundongos após a infecção por L. longbeachae é causada por uma falência pulmonar, associada a indução de um intenso processo inflamatório nos pulmões dos animais infectados. Nesse trabalho, nós investigamos papel do receptor de C5a (C5aR) na replicação bacteriana e na resistência de camundongos diante de uma infecção letal por L. longbeachae. Experimentos realizados com animais deficientes no receptor C5a indicam que os animais são protegidos durante uma infecção letal por L. longbeachae em comparação com animais selvagens, da linhagem BALB/c. De acordo com esses resultados, foi detectada uma menor carga bacteriana nos pulmões dos animais C5a-/- em comparação com animais selvagens. Experimentos realizados com animais controles da mesma linhagem demonstraram que C5a-/- diferem de animais C5a+/-, o que suporta o papel desse receptor durante a infecção por L. longbeachae. Dessa forma, nossos dados sugerem que a sinalização via C5aR contribui para a patogênese da doença em modelo murino da infecção por L. longbeachae. Os mecanismos envolvidos na patogênese mediada pelo receptor C5a encontram-se sob investigação. / Legionella longbeachae is a species of the Legionellaceae family that is commonly present in the soil in various regions of the globe. Infections by L. longbeachae in immunocompromised individuals cause severe pneumonia, often leading to hospitalization and death. The prevalence of L. longbeachae as a cause of pneumonia is large, and certainly under-estimated, mainly because the conventional diagnostic methods only detect Legionella pneumophila species. The anaphylatoxin C5a is an inflammatory protein activated by the complement system, which is involved in the recruitment of inflammatory cells, a process induced by cells of the innate immunity, which leads to tissue damage. Recent data generated in our laboratory suggest that the mortality of mice after infection with L. longbeachae is caused by a lung failure, associated with the induction of an intense inflammatory process in the lungs of infected animals. In this study, we investigated the role of C5a receptor (C5aR) in bacterial replication and mice resistance on a lethal infection by L. longbeachae. Experiments with animals deficient in the C5a receptor indicate that the animals are protected during a lethal infection by L. longbeachae as compared with-wild type strain, BALB/c. According to these results, a lower bacterial load was detected in the lungs of C5a-/- animals compared with BALB/c animals. Experiments performed with control animals of the same strain demonstrated that C5a-/- differ from C5a+/- animals, which supports the role of this receptor during infection by L. longbeachae. Thus, our data suggest that C5aR signalling pathway contributes to the pathogenesis of the disease in a murine model of infection by L. longbeachae. The mechanisms involved in the pathogenesis mediated by C5a receptor are under investigation.

Outer membrane proteins (OMPs) were recovered from eleven strains (eight serogroups) of Legionella pneumophila by sequential treatment with Tris buffer (pH 8), citrate buffer(pH 2.75) and Tris buffer (pH 8). Transmission electron microscopy revealed clearly the separation of the outer membrane from the bacteria. The development of delayed hypersensitivity was also noted by measuring the area of arythema and induration produced by intradermal injections of the MPSs from Chicago 8 strain. The adjuvants enhanced greatly both active and cell-meditated immunity (CMI). Transient lymphocytopenia with a slight rise in neutrophils was noted in each of the immunized groups. Intraperitoneal challenge, seven days after the OMP booster, of one LD (1.5 x10^6) of legionellae resulted in lymphocytopenia with elevated neutrophils. All immunized rats survived the challenge, although those in the saline-OMP group were clearly the sickest. Post-challenge, legionella antibody titers rose greatly and CMI was heightened. Passive immunization (homologous and heterologous) was found to protect the rats from a challenge of on LD. Actively-immunized rats retained their immunity for at least six months as determined by their resistance to a second challenge.

The bacterial pathogen Legionella pneumophila replicates intracellularly in protozoa, but can also cause severe pneumonia, called Legionnaires' disease. The bacteria invade and proliferate in the alveolar macrophages of the human lung. L. pneumophila bacteria exhibit a biphasic life cycle: replicative bacteria are avirulent; in contrast, transmissive bacteria express virulence traits and flagella. Primarily aim of this thesis was to evaluate the impact of the regulatory proteins FleQ, FleR, and RpoN in flagellar gene regulation. Phenotypic analysis, Western blot and electron microscopy of regulatory mutants in the genes coding for FleQ, RpoN and FleR demonstrated that flagellin expression is strongly repressed and that these mutants are non-flagellated in transmissive phase. Transcriptomic studies of these putative flagellar gene expression regulators demonstrated that fleQ controls the expression of numerous flagellar biosynthetic genes. Together with RpoN, FleQ controls transcription of 14 out of 31 flagellar class II genes, coding for the basal body, hook, and regulatory proteins. Unexpectedly, 7 out of 15 late flagellar genes class III and IV) are expressed dependent on FleQ but independent of RpoN. Thus, in contrast to the commonly accepted view that enhancer binding proteins as FleQ always interact with RpoN to initiate transcription, our results strongly indicate that FleQ of L. pneumophila regulates gene expression RpoN-dependent as well as RpoN-independent. Moreover, transcriptome analysis of a fleR mutant strain elucidated that FleR does not regulate the flagellar class III genes as previously suggested. Instead FleR regulates together with RpoN numerous protein biosynthesis and metabolic genes. Based on these experimental results our modified model for the transcriptional regulation of flagellar genes in L. pneumophila is that flagellar class II genes are controlled by FleQ and RpoN, while flagellar class III and IV genes are controlled in a fleQ-dependent but rpoN-independent manner. Although all L. pneumophila strains share the same complex life style, various pathotypes have evolved. This is reflected by the genomes, which contain e.g. genomic islands. The genomic island Trb-1 of L. pneumophila Corby, carries all genes necessary for a type-IV conjugation system, an integrase gene and a putative oriT site. The second aim of this thesis was to investigate the implication of this genomic island in conjugative DNA transfer. Using conjugation assays we showed that the oriT site located on Trb-1 is functional and contributes to conjugation between different L. pneumophila strains. As this is the first oriT site of L. pneumophila known to be functional our results provide evidence that conjugation is a major mechanism for the evolution of new pathotypes in L. pneumophila. / Das pathogene Bakterium Legionella pneumophila repliziert sich in der Natur intrazellulär in Protozoen. Beim Menschen kann das Bakterium eine schwere Pneumonie, die sogenannte Legionärskrankheit auslösen. Hierbei vermehren sich die Bakterien in Alveolarmakrophagen der Lunge. Der Lebenszyklus von L. pneumophila Bakterien ist gekennzeichnet durch zwei Phase: replikative Bakterien sind avirulent; im Gegensatz dazu sind transmissive Bakterien virulent und flagelliert. Hauptziel dieser Arbeit war es die Beteiligung der regulatorischen Proteins FleQ, FleR, and RpoN an der Flagellengenregulation zu ermitteln. Mutanten für die Gene welche für FleQ, FleR oder RpoN codieren exprimieren in der transmissiven Phase im Genesatz zum Wildtyp nur wenig Flagellin und sind nicht flagelliert. Nachgewiesen wurde dies durch eine phänotypische Analyse, Western blot und Ektronenmikroskopie. Studien des Transkripoms dieser Mutanten zeigten, daß FleQ die Expression zahlreicher Flagellenbiosynthesegenen kontrolliert. Gemeinsam mit RpoN kontrolliert FleQ die Transkription von 14 der 31 Klasse II Flagellengene, welche für Basalkörper, Haken und regulatorische Proteine codieren. Überraschenderweise sind 7 der 15 späten Flagellengenen (Klasse III und IV) abhängig von FleQ, aber unabhängig von RpoN exprimiert. Daher und entgegen der allgemeinen Auffassung dass sogenannte ‚enhancer binding' Proteine wie FleQ zur Transkriptionsinitiation immer mit RpoN interagieren, deuten unsere Ergebnisse darauf hin, dass FleQ von L. pneumophila Genexpression sowohl RpoN-abhängig, als auch RpoN-unabhängig reguliert. Ebenso anders als zuvor vorgeschlagen, verdeutlichen Studien des Transkriptoms einer fleR Mutante, dass FleR nicht die Expression der Klasse III Flagellengene induziert. Statt dessen reguliert FleR gemeinsam mit RpoN zahlreiche Gene der Proteinbiosynthese und des Metabolismus. Basierend auf diesen experimentellen Ergebnissen sind in unserem modifizierten Modell für die transkriptionelle Regulation der L. pneumophila Flagellengene die Flagellengene der Klasse II von FleQ und RpoN kontrolliert, während die Flagellengene der Klasse III und IV in einer fleQ-abhängigen aber rpoN-unabhängigen Weise kontrolliert sind. Obwohl alle L. pneumophila Stämme den zweiphasigen Lebenszyklus aufweisen haben sich unterschiedliche Pathotypen evolviert. Das ist auch in den Genomen sichtbar, die z. B. genomische Inseln enthalten. Die genomische Insel Trb-1 von L. pneumophila Corby trägt alle Gene eines Typ-IV Konjugationssystem, ein ntegrase-Gen und einen putative oriT-Bereich. Das zweite Ziel dieser Arbeit war es also zu untersuchen, inwieweit Trb-1 an konjugativem DNA-Transfer beteiligt ist. Mit Hilfe von Konjugationsexperimenten, zeigten wir, dass der oriT-Bereich von Trb-1 funktional ist und zur Konjugation zwischen verschiedenen L. pneumophila Stämmen beiträgt. Dies ist der erste oriT Bereich von L. pneumophila, dessen Funktionalität nachgewiesen wurde. Damit bekräftigen unsere Ergebnisse, dass Konjugation eine treibende Kraft für die Evolution neuer Pathotypen in L. pneumophila ist.

The intracellular bacterial pathogen Legionella pneumophila is the etiological agent of Legionnaires’ disease, a severe pneumonia; it has also served as a valuable tool in studying host-pathogen interactions. The study of L. pneumophila pathogenesis has led to the discovery of novel biochemical and enzymatic mechanisms and a better understanding of host cell immune responses and signaling. L. pneumophila replicates within eukaryotic cells through the use of a type IV secretion system and over 330 effector proteins injected into the host cell. Only approximately 10% of these effectors have been characterized, but regardless of the small fraction, the complexity of L. pneumophila infection is clear. A good demonstration of this complexity is the large number of effector activities the bacteria uses to manipulate the small GTPase involved in ER to Golgi trafficking, Rab1. Six different effectors with eight separate activities modulate the activity of Rab1 to aid in the replication of the bacteria. We recently discovered that the protein SetA is yet another effector targeting Rab1. SetA glucosylates Rab1 using a canonical DxD motif and the glucose moiety interferes with both GTP hydrolysis and guanosine nucleotide dissociation inhibitor (GDI) binding. Based on our findings, the role of SetA is likely to aid in maintaining a pool of free Rab1, increasing availability for use by other L. pneumophila effectors. Another example of the complexity of L. pneumophila pathogenesis is the use of metaeffectors. Metaeffectors are effectors that regulate other effectors, both being produced by L. pneumophila. Three mechanisms of metaeffector regulation have been identified: 1) removal of a modification on host proteins placed by the cognate effector, 2) direct modification of the cognate effector or 3) direct binding to the cognate effector. Through the use of Size Exclusion Chromatography (SEC), binding assays with purified proteins and bacterial two-hybrid analysis, we found the mechanism of regulation for the SidI metaeffector Lpg2505 to be inactivation through direct binding. Atypical of previously identified effector characteristics, the binding of SidI by Lpg2505 occurs within the bacterial cell prior to translocation. The expression pattern of both effectors in L. pneumophila in addition to the other findings suggest a temporal role for Lpg2505 activity in which inactivation of SidI occurs after sufficient bacterial replication has occurred.<br>

Abstract Legionella species and Chlamydia pneumoniae are a common cause of community-acquired pneumonia and occasional cause of hospital-acquired pneumonia. Significant mortality rates among the elderly and patients with severe underlying disease may occur as a result of infection with those pathogen. Diagnostic delay may also result in increased mortality. Therefore, nucleic acid amplification assays have been shown to be useful for the detection of Legionella.spp and Chlamydia pneumoniae. The genes that encode 16S ribosomal subunits and the macrophage infectivity potentiator (MIP) gene have been shown to contain signature sequences that are useful for the identification of L. pneumophila and a variety of other Legionella species. The pst-1 fragment is useful for identification of Chlamydia pneumoniae. Here we try to test clinical specimens by Real-time PCR assays to detect L.pneumophila and other Legionella species in the same tube, and detect Chlamydia pneumoniae by SYBR Green 1 reagent. By this method, these amplicons of 16S ribosomal subunits gene and MIP gene can be discriminated by different melting curve dependent on different Tm values. In this study, we detected more 5 and 6 patients in Legionella species and Chlamydia pneumoniae than conventional diagnostic tools. Hence, the Real-time PCR also demonstrated that it¡¦s a rapid and high sensitivity method in diagnosis of legionnaires¡¦ disease. In this study, it also demonstrated that Real-time PCR is effective in prediction of atypical pathogen infection.

The intracellular pathogen Legionella pneumophila thrives in both natural and man-made water habitats where it replicates inside freshwater amoebae. L. pneumophila follows a developmental cycle as it grows in amoebae. The actively-multiplying intracellular replicative forms (RFs) differentiate into highly virulent mature infectious forms (MIFs) late in the amoeba infection, and are then released extracellularly. L. pneumophila accidentally infects susceptible humans causing the non-communicable Legionnaires’ disease (LD). MIFs play a central role in the life cycle of L. pneumophila and are thought to be responsible for the transmission of LD. Early reports demonstrated that MIFs were poorly produced inside human macrophages, suggesting that the L. pneumophila progeny from human macrophages has fitness and infectivity disadvantages. Direct comparisons of the L. pneumophila progenies from amoebae and human macrophages have demonstrated that the progeny from amoebae is more morphologically differentiated, resistant to antibiotic challenges, and able to adhere to and initiate infections in host cells than the progeny from macrophages. Analysis of the transcriptomic and proteomic profiles of L. pneumophila inside different hosts has revealed a specific set of genes that are upregulated during differentiation of L. pneumophila into MIFs inside freshwater protozoa but not inside human macrophages, suggesting that these genes may be required for the full differentiation of L. pneumophila and, therefore, for the transmission of LD to susceptible humans. Since the expression of the gene lpg1669, which encodes a putative α-amylase, was upregulated in amoebae (highest level of upregulation among the tested genes) and inside Tetrahymena ciliates, but not inside human macrophages, the role of lpg1669 in the differentiation of L. pneumophila into MIFs was investigated. An isogenic lpg1669 deletion mutant did not display defects in morphological differentiation, in vitro (BYE broth) or in vivo (A. castellanii or U937 human macrophages) growth when compared to its parent strain, suggesting that the gene lpg1669 is not essential for the intracellular differentiation of L. pneumophila. Collectively, these findings demonstrate that L. pneumophila can reach different developmental end points in different hosts and could also provide a clue for the lack of transmission of LD among humans.