Academic literature on the topic 'Legionella longbeachae'

Although known as causes of community-acquired pneumonia and Pontiac fever, the global burden of infection caused by Legionella species other than Legionella pneumophila is under-recognised. Non-L. pneumophila legionellae have a worldwide distribution, although common testing strategies for legionellosis favour detection of L. pneumophila over other Legionella species, leading to an inherent diagnostic bias and under-detection of cases. When systematically tested for in Australia and New Zealand, L. longbeachae was shown to be a leading cause of community-acquired pneumonia. Exposure to potting soils and compost is a particular risk for infection from L. longbeachae, and L. longbeachae may be better adapted to soil and composting plant material than other Legionella species. It is possible that the high rate of L. longbeachae reported in Australia and New Zealand is related to the composition of commercial potting soils which, unlike European products, contain pine bark and sawdust. Genetic studies have demonstrated that the Legionella genomes are highly plastic, with areas of the chromosome showing high levels of recombination as well as horizontal gene transfer both within and between species via plasmids. This, combined with various secretion systems and extensive effector repertoires that enable the bacterium to hijack host cell functions and resources, is instrumental in shaping its pathogenesis, survival and growth. Prevention of legionellosis is hampered by surveillance systems that are compromised by ascertainment bias, which limits commitment to an effective public health response. Current prevention strategies in Australia and New Zealand are directed at individual gardeners who use potting soils and compost. This consists of advice to avoid aerosols generated by the use of potting soils and use masks and gloves, but there is little evidence that this is effective. There is a need to better understand the epidemiology of L. longbeachae and other Legionella species in order to develop effective treatment and preventative strategies globally.

ABSTRACT Legionella longbeachae causes most cases of legionellosis in Australia and may be underreported worldwide due to the lack of L. longbeachae-specific diagnostic tests. L. longbeachae displays distinctive differences in intracellular trafficking, caspase 1 activation, and infection in mouse models compared to Legionella pneumophila, yet these two species have indistinguishable clinical presentations in humans. Unlike other legionellae, which inhabit freshwater systems, L. longbeachae is found predominantly in moist soil. In this study, we sequenced and annotated the genome of an L. longbeachae clinical isolate from Oregon, isolate D-4968, and compared it to the previously published genomes of L. pneumophila. The results revealed that the D-4968 genome is larger than the L. pneumophila genome and has a gene order that is different from that of the L. pneumophila genome. Genes encoding structural components of type II, type IV Lvh, and type IV Icm/Dot secretion systems are conserved. In contrast, only 42/140 homologs of genes encoding L. pneumophila Icm/Dot substrates have been found in the D-4968 genome. L. longbeachae encodes numerous proteins with eukaryotic motifs and eukaryote-like proteins unique to this species, including 16 ankyrin repeat-containing proteins and a novel U-box protein. We predict that these proteins are secreted by the L. longbeachae Icm/Dot secretion system. In contrast to the L. pneumophila genome, the L. longbeachae D-4968 genome does not contain flagellar biosynthesis genes, yet it contains a chemotaxis operon. The lack of a flagellum explains the failure of L. longbeachae to activate caspase 1 and trigger pyroptosis in murine macrophages. These unique features of L. longbeachae may reflect adaptation of this species to life in soil.

In an observational study we measured the Legionella longbeachae antibody titre rise in patients mechanically ventilated for more than eight days during a two-month period. The patients were divided into two groups on the basis of the presence or absence of the adult respiratory distress syndrome (ARDS). In nine patients with ARDS all showed an antibody rise consistent with recent infection with Legionella long-beachae with a rise in titre (six patients) or a high titre after eight to ten days of ventilation (three patients). Three patients without ARDS did not show a rise in titre. Culture of the environment, ventilator circuits, humidifiers and humidification water did not reveal an environmental source of Legionella longbeachae in the Intensive Care Unit. Legionella longbeachae may be implicated as a pathogenic organism in ARDS, or as a secondary nosocomial infection. Alternatively the antibody titre rise may represent an epiphenomenon and may not be related to Legionella longbeachae infection.

In addition to Legionella pneumophila, about 20 Legionella species have been documented as human pathogens. The majority of infections by non-pneumophila Legionella species occur in immunocompromised and splenectomized patients. Here, we report a case of ‘classical’ lobar pneumonia caused by Legionella longbeachae in a splenectomized patient receiving corticosteroids for chronic immune thrombocytopenia. Tests for Legionella antigen were negative. L. longbeachae was immediately detected in bronchoalveolar fluid by PCR and subsequently confirmed by culture on legionella-selective media. The features of Legionnaires' disease in immunocompromised patients with special emphasis on significance and detection of non-pneumophila species are reviewed.

This study established an experimental model of replicative Legionella longbeachae infection in A/J mice. The animals were infected by intratracheal inoculation of 103–109 c.f.u. L. longbeachae serogroup 1 (USA clinical isolates D4968, D4969 and D4973). The inocula of 109, 108, 107 and 106 c.f.u. of all tested L. longbeachae serogroup 1 isolates were lethal for A/J mice. Inoculation of 105 c.f.u. L. longbeachae caused death in 90 % of the animals within 5 days, whilst inoculation of 104 c.f.u. caused sporadic death of mice. All animals that received 103 c.f.u. bacteria developed acute lower respiratory disease, but were able to clear Legionella from the lungs within 3 weeks. The kinetics of bacterial growth in the lungs was independent of inoculum size and reached a growth peak about 3 logarithms above the initial inoculum at 72 h after inoculation. The most prominent histological changes in the lungs were observed at 48–72 h after inoculation in the form of a focal, neutrophil-dominant, peribronchiolar infiltration. The inflammatory process did not progress towards the interstitial or alveolar spaces. Immunohistological analyses revealed L. longbeachae serogroup 1 during the early phase of infection near the bronchiolar epithelia and later co-localized with inflammatory cells. BALB/c and C57BL/6 mice strains were also susceptible to infection with all L. longbeachae serogroup 1 strains tested and very similar changes were observed in the lungs of infected animals. These results underline the infection potential of L. longbeachae serogroup 1, which is associated with high morbidity and lethality in mice.

Legionella longbeachae is almost as frequent a cause of legionellosis in Australia as Legionella pneumophila, but epidemiological investigation of possible environmental sources and clinical cases has been limited by the lack of a discriminatory subtyping method. The purpose of this study was to examine the genetic variability among Australian isolates of L. longbeachaeserogroup 1. Pulsed-field gel electrophoresis (PFGE) ofSfiI fragments revealed three distinct pulsotypes among 57 clinical and 11 environmental isolates and the ATCC control strains of L. longbeachae serogroups 1 and 2. Each pulsotype differed by four bands, corresponding to <65% similarity. A clonal subgroup within each pulsotype was characterized by >88% similarity. The largest major cluster was pulsotype A, which included 43 clinical isolates and 9 environmental isolates and was divided into five subgroups. Pulsotypes B and C comprised smaller numbers of clinical and environmental isolates, which could each be further divided into three subgroups. The ATCC type strain of L. longbeachae serogroup 1 was classified as pulsotype B, subtype B3, while the ATCC type strain of L. longbeachae serogroup 2 was identified as a different pulsotype, LL2. SfiI macrorestriction analysis followed by PFGE showed that the AustralianL. longbeachae strains are not a single clonal population as previously reported.

Bibliography: leaves 246-304. Describes experiments aimed at characterising the potential virilant factors of Legionella longbeachae sg 1, an important human pathogen which is responsible for nearly half of all clinical cases of Legionella related pneumonia reported each year.

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.

A doença dos legionários consiste em uma broncopneumonia severa e atípica, que acomete de 2 a 7% das pessoas infectadas com Legionella spp e que apresenta taxa de mortalidade que varia de 5 a 30%, sendo considerada uma importante causa de morbidade e mortalidade mundial. A patologia causada pela espécie L. pneumophila tem sido amplamente estudada em modelos experimentais e suas características clínicas foram extensivamente descritas. No entanto, este modelo não representa adequadamente a doença que acomete seres humanos, pois L. pneumophila não é letal aos camundongos como é para humanos. Recentemente, uma nova espécie de bactéria do gênero Legionella, denominada Legionella longbeachae, foi descrita como importante agente de doença dos legionários em países do hemisfério sul. A pneumonia induzida por L. longbeachae em humanos não difere da induzida por L. pneumophila. No entanto, L. longbeachae é letal para camundongos em doses baixas, o que torna esse modelo murino de doença dos legionários mais fidedigno ao que ocorre com humanos. Com a acentuada mudança dos hábitos de nossa sociedade, há o aumento do número de pessoas com fatores que predispõe a doença, como idade elevada ou tratamento imunossupressor. Assim, entender melhor a relação patógeno-hospedeiro no curso da doença dos legionários por meio da utilização de um modelo experimental adequado é importante para a descoberta de novos meios de combater este patógeno. Neste trabalho, geramos uma cepa de L. longbeachae mutante para rpsL, que se torna resistente à estreptomicina. Essa cepa pode ser utilizada para infecções in vivo nas quais a quantificação da CFU foi estimada em placas contendo antibiótico, o que culmina em maior eficiência experimental e menor quantidade de contaminações. Essa cepa foi utilizada em experimentos in vivo para avaliar os componentes do sistema imune que operam na resistência diante de uma dose letal bacteriana administrada pela via intranasal. Demonstramos que camundongos deficientes para as citocinas IFN ou TNF e para o receptor de quimiocinas CCR2 são mais susceptíveis à infecção do que os camundongos selvagens. No entanto, camundongos deficientes para o receptor de quimiocinas CCR5, para o receptor de IL-17, para a citocina IL-6 ou para o receptor citoplasmático NOD2 são mais resistentes à infecção quando comparados com animais selvagens. A descoberta destas moléculas em um modelo de infecção letal in vivo ressalta a importância de alguns componentes da imunidade para a resistência durante a doença dos legionários experimental e possíveis alvos terapêuticos para essa doença.
Legionnaires disease is a severe and atypical bronchopneumonia, which affects 2-7% people infected with Legionella spp and has a mortality rate of 5 to 30%, therefore it is considered an important cause of mortality and morbidity worldwide. Disease caused by Legionella pneumophila has been largely studied in experimental models and its clinical characteristics was extensively described. However this model does not adequately represent the disease that affects humans, because L. pneumophila is not lethal to mice, as it is to humans. Recently, a new species of bacterium from Legionella genus, called Legionella longbeachae, was described as an important agent of Legionnaires disease in the southern hemisphere. The pneumonia induced by L. longbeachae in humans is not different from pneumonia induced by L. pneumophila. However, a low dose of L. longbeachae is lethal to mice, which makes this murine infection model of Legionnaires disease more reliable than that which occurs in humans. Because our society is changing, there is an increase in the number of persons with predisposing factors, like higher age or immunosuppressive treatment. So, a better understanding of host-pathogen relationship by using a suitable experimental model is important to find new ways to fight this pathogen. Here, we generated a strain of rpsL mutant L. longbeachae, which becomes resistant to streptomycin. This strain could be used in in vivo infections, when CFU quantification was estimated in plates with antibiotic, culminating in greater experimental efficiency and lower contamination. This strain was used in in vivo experiments to evaluate components of the immune system that participates in resistance against lethal dose of bacteria administered intranasally. We showed that Tnf-/-, Ifn-/- or Ccr2-/- mice are more susceptible to infection than wild type mice. However Ccr5-/-, Il17r-/-, Il6-/- or Nod2-/- mice are more resistant to infection than wild type animals. The discovery of these molecules in a lethal infection model in vivo highlights the importance of some components of immunity to resistance during experimental Legionnaires disease and potential therapeutic targets to disease.

Legionella pneumophila, l’agent causal de la maladie du légionnaire, est responsable de nombreuses épidémies de pneumonie. Les légionelles sont des bactéries qui vivent en milieu aquatique où elles parasitent les amibes protozoaires. Elles sont également capables d’infecter l’homme par le biais d’aérosols. Cette bactérie possède trois gènes codant des protéines contenant le domaine F-box, très similaire aux ubiquitine-ligases de type SCF chez les eucaryotes. Au cours de ma thèse, j'ai montré que les protéines F-box sont des facteurs de virulence secrétés par le système de sécrétion Dot/Icm et qu’elles sont impliquées dans l'accumulation de protéines ubiquitinées associées à la vacuole contenant les légionelles. De plus, j’ai pu montrer l’importance de ces trois protéines au cours de l’infection de l’hôte protozoaire Acanthamoeba castellanii, des macrophages THP-1, des cellules épithéliales du poumon A549 et, in vivo, lors de l’infection pulmonaire chez la souris A/J. Nous avons ensuite caractérisé plus en détail la fonction de l’une de ces trois protéines (Lpp2082) et nous avons montré qu’elle est capable d’interagir avec les composantes du complexe cellulaire d’ubiquitination de type SCF, avec une deuxième protéine cellulaire, la ParvB et moduler son ubiquitination. Dans la deuxième partie de ma thèse, j’ai analysé la séquence du génome de Legionella longbeachae, deuxième agent de la légionellose, et notamment la présence de gènes codant des protéines similaires aux protéines eucaryotes. Cette analyse a montré une diversité de ces protéines parmi les deux espèces, mais les stratégies infectieuses semblent être similaires car les mêmes domaines eucaryotes sont présents.

The genus Legionella consists of environmental bacteria which are the causative agents of the severe pneumonia Legionnaires’ disease. L. longbeachae and L. pneumophila are able to replicate intracellularly in human alveolar macrophages and aquatic or soil amoebae. In order to replicate within host cells the bacteria establish a compartment derived from the endoplasmatic reticulum (ER) which is called “Legionella-containing vacuole” (LCV). A bacterial intracellular multiplication/defective in organelle transport (Icm/Dot) type IV secretion system (T4SS) is essential for the formation of this LCV. The Icm/Dot T4SS enables translocation of effector proteins into the host cell. More than 100 effector proteins are presumably translocated during an L. longbeachae infection whereas around 300 translocated effector proteins are known for L. pneumophila. During maturation the LCV communicates with vesicles from the endocytic vesicle trafficking pathway, avoids fusion with lysosomes and instead fuses with the ER. Phosphoinositides (PI) such as phosphatitdylinositol-4-phosphate (PtdIns(4)P) are enriched on the LCV which mediate the binding of Icm/Dot translocated effector proteins like SidCLpn (substrate of Icm/Dot transporter) as well as its paralogous protein SdcALpn. The 73 kDa effector SidM but not the 106 kDa SidCLpn was found in a previous phosphoinositide pulldown assay with L. pneumophila lysate to be the major PtdIns(4)P binding protein. Using L. longbeachae lysate we showed binding of the 111 kDa SidCLlo to PtdIns(4)P in a phosphoinositide pulldown. This result was confirmed by protein-lipid overlay assays using “PIP-strips”. In further analysis the P4C (PtdIns(4)P-binding of SidC) domain was identified as a 19 kDa domain of SidCLlo located in the amino acid region 609 to 782. This P4C domain was located in the same region as the 20 kDa SidCLpn_P4C domain of L. pneumophila. Both P4C domains can be used as LCV markers. This was shown with GST-tagged proteins binding to LCVs in a cell homogenate. The two P4C domains show a sequence identity of only 45% and the full-length protein of 40%. Circular dichroism measurements revealed that the secondary structure of the two proteins is similar. Moreover, isothermal titration calorimetric measurements indicated a 3.4 higher affinity of SidCLlo towards PtdIns(4)P compared with SidCLpn. In RAW 264.7 macrophages infected with L. longbeachae we showed that endogenous SidCLlo as well as heterologously produced SidCLpn is translocated to the LCV in an Icm/Dot-dependent manner. The deletion of the sidCLlo gene led to a reduced recruitment of calnexin to the LCV in infected Dictyostelium discoideum. This effect was complemented by adding plasmid-encoded SidCLlo, SidCLpn or SdcALpn. The same recruitment defect for a L. pneumophila strain lacking the sidCLpn and sdcALpn genes was complemented by the production of SidCLlo and SidCLpn as published before. Therefore, these effectors play a role for pathogen-host interactions by promoting the recruitment of ER to the LCV. L. longbeachae or L. pneumophila wild-type strains outcompeted their sidC deletion mutant in a competition assay in Acanthamoeba castellanii. However neither of the deletion mutants were impaired in their growth in single strain replication experiments. In summary despite of the small sequence identity and the higher binding affinity to PtdIns(4)P of SidCLlo compared to SidCLpn both effector proteins seem to have similar functions during an infection of Legionella. For the characterization of L. longbeachae-containing vacuoles through proteomic analysis, LCVs had to be isolated from infected D. discoideum or RAW 264.7 macrophages. Endogenous SidCLlo or heterologously produced SidCLpn were used as LCV markers for the isolation. Pathogen vacuoles harbouring L. longbeachae were isolated by immuno-affinity purification using antibodies specifically recognizing SidCLlo or SidCLpn. Future investigations aim at optimizing the LCV purification protocol for L. longbeachae to determine the proteome composition of the L. longbeachae-containing vacuole.
Die Gattung Legionella besteht aus opportunistischen Pathogenen, die Auslöser für die schwere Lungenentzündung Legionärskrankheit sind. Die Legionella-Spezies L. longbeachae sowie L. pneumophila vermehren sich intrazellulär in humanen alveolaren Makrophagen sowie in aquatischen oder im Boden lebenden Amöben. Ein vom endoplasmatischen Retikulum (ER) abstammendes Kompartiment ist notwendig für die intrazelluläre Replikation. Diese Nische wird als „Legionella-containing vacuole“ (LCV) bezeichnet. Die Bildung der LCV benötigt ein „intracellular multiplication/defective in organelle transport“ (Icm/Dot) Typ IV Sekretionssystem (T4SS), das Effektorproteine in die Wirtszelle transportiert. Zurzeit sind über 100 vermutete Effektorproteine für L. longbeachae und etwa 300 Effektorproteine für L. pneumophila beschrieben. Im Verlauf eines Reifungsprozesses kommuniziert die LCV mit endosomalen Vesikeln, verhindert eine Fusion mit den Lysosomen und fusioniert mit dem ER. Phosphoinositide wie das PtdIns(4)P wurden auf der LCV gefunden. Diese dienen als Bindestellen für die durch das Icm/Dot translozierten Effektorproteine wie das SidCLpn und sein paraloges Protein SdcALpn. In einer früheren Studie wurde in einem Phosphoinositid-Pulldown Experiment das 73 kDa Effektorprotein SidM aber nicht das 106 kDa Protein SidCLpn als Bindepartner von PtdIns(4)P nachgewiesen. Wir konnten in einem Phosphoinositid-Pulldown Experiment mit L. longbeachae Lysat zeigen, dass das 111 kDa homologe Protein von SidCLpn SidCLlo der Bindepartner von L. longbeachae für PtdIns(4)P ist. Ein 19 kDa großes SidCLlo- Fragment im Bereich der Aminosäuren 609 bis 782 konnte identifiziert werden, das für die Bindung von SidCLlo an PtdIns(4)P notwendig ist. Interessanterweise liegt die früher beschriebene 20 kDa große P4C Domäne von SidCLpn in der gleichen Region. Durch Inkubation von GST-gekoppelten SidCLlo_P4C-Proteinen mit L. pneumophila Zellhomogenat konnten wir zeigen, dass SidCLlo_P4C die Vakuole von L. pneumophila homogen dekoriert. Daher kann SidCLlo_P4C genauso wie das SidCLpn_P4C als LCV Marker benutzt werden. Die P4C Domänen besitzen eine Sequenzhomologie von 45% und SidCLlo und SidCLpn zeigen eine Sequenzhomologie von 40%. Mittels zirkularer Dichroismus Messung konnte gezeigt werden, dass die beiden Proteine ähnliche Sekundärstrukturen besitzen. Mittels isothermer Titrationskalorimetrie konnten wir zeigen, dass SidCLlo eine 3.4-fach höhere Bindeaffinität zu PtdIns(4)P besitzt als SidCLpn. In infizierten RAW 264.7 Makrophagen konnte wir zeigen, dass L. longbeachae nicht nur sein eigenes endogen produzierten SidCLlo sondern auch ein heterolog exprimiertes SidCLpn in einer Icm/Dot abhängigen Art und Weise auf die LCV transloziert. Frühere Studien zeigten, dass in einer sidC-sdcALpn Deletionsmutante die ER Rekrutierung zu der LCV in infizierten D. discoideum Zellen beeinträchtigt ist. Wir konnten zeigen, dass die heterologe Produktion von SidCLlo diesen Rekrutierungsfehler komplementieren kann, ebenso wie Plasmid-kodiertes SidCLpn oder SdcALpn. Die Deletion vom Gen sidCLlo in L. longbeachae führt ebenfalls zu einer verminderten Rekrutierung von ER-Markern zur LCV in infizierten D. discoideum. Dieser Effekt konnte durch eine Produktion von SidCLlo, SidCLpn und SdcALpn komplementiert werden. Die SidC Deletionsstämme von L. longbeachae oder L. pneumophila replizierten in Acanthamoeba castellanii wie die entsprechenden Wildtyp-Stämme, aber in direkter Konkurrenz wurden die Deletionsmutanten von den Wildtyp-Stämmen verdrängt. Insgesamt scheinen trotz der geringen Sequenzidentität und der höheren Bindeaffinität von SidCLlo im Vergleich zu SidCLpn zu PtdIns(4)P beide Effektorproteine ähnliche Funktionen im Infektionsweg von Legionella wahr zu nehmen. Für die Charakterisierung von L. longbeachae-enthaltenden Vakuolen in einer Proteomanalyse müssen LCVs aus D. discoideum oder RAW 264.7 Makrophagen isoliert werden. Endogenes SidCLlo oder heterolog produziertes SidCLpn wurden als Vakuolen- Marker für die Isolation von L. longbeachae-enthaltenen Vakuolen verwendet. L. longbeachae-enthaltene Vakuolen wurden in einer Immunaffinitätsaufreinigung mit Hilfe spezifischer Antikörper gegen SidCLlo oder SidCLpn isoliert. Weitere Studien zielen auf die Verbesserung der Vakuolen-Isolation von L. longbeachae, um das Proteom dieser LCV zu charakterisieren.

Globally disseminated bacterial pathogens frequently cause epidemics that are of major importance in public health. Of particular significance is the capacity for some of these bacteria to switch into a new environment leading to the emergence of pathogenic clones. Understanding the evolution and epidemiology of such pathogens is essential for designing rational ways for prevention, diagnosis and treatment of the diseases they cause. Whole-genome sequencing of multiple isolates facilitating comparative genomics and phylogenomic analyses provides high-resolution insights, which are revolutionizing our understanding of infectious diseases. In this thesis, a range of population genomic analyses are employed to study the molecular mechanisms and the evolutionary dynamics of bacterial pathogen niche adaptation, specifically between humans, animals and the environment. A large-scale population genomic approach was used to provide a global perspective of the host-switching events that have defined the evolution of Staphylococcus aureus in the context of its host-species. To investigate the genetic basis of host-adaptation, we performed genome-wide association analysis, revealing an array of accessory genes linked to S. aureus host-specificity. In addition, positive selection analysis identified biological pathways encoded in the core genome that are under diversifying selection in different host-species, suggesting a role in host-adaptation. These findings provide a high-resolution view of the evolutionary landscape of a model multi-host pathogen and its capacity to undergo changes in host ecology by genetic adaptation. To further explore S. aureus host-adaptive evolution, we examined the population dynamics of this pathogen after a simulated host-switch event. S. aureus strains of human origin were used to infect the mammary glands of sheep, and bacteria were passaged in multiple animals to simulate onward transmission events. Comparative genomics of passaged isolates allowed us to characterize the genetic changes acquired during the early stages of evolution in a novel host-species. Co-infection experiments using progenitor and passaged strains indicated that accumulated mutations contributed to enhanced fitness, indicating adaptation. Within-host population genomic analysis revealed the existence of population bottlenecks associated with transmission and establishment of infection in new hosts. Computational simulations of evolving genomes under regular bottlenecks supported that the fitness gain of beneficial mutations is high enough to overcome genetic drift and sweep through the population. Overall, these data provide new information relating to the critical early events associated with adaptation to novel host-species. Finally, population genomics was used to study the total diversity of Legionella longbeachae from patient and environmental sources and to investigate the epidemiology of a L. longbeachae outbreak in Scotland. We analysed the genomes of isolates from a cluster of legionellosis cases linked to commercial growing media in Scotland and of non-outbreak-associated strains from this and other countries. Extensive genetic diversity across the L. longbeachae species was identified, associated with intraspecies and interspecies gene flow, and a wide geographic distribution of closely related genotypes. Of note, a highly diverse pool of L. longbeachae genotypes within compost samples that precluded the genetic establishment of an infection source was observed. These data represent a view of the genomic diversity of this pathogen that will inform strategies for investigating future outbreaks. Overall, our findings demonstrate the application of population genomics to understand the molecular mechanisms and the evolutionary dynamics of bacterial adaptation to different ecological niches, and provide new insights relevant to other major bacterial pathogens with the capacity to spread between environments.