The Legionella pneumophilaDot/Icm T4SS injects similar to 300 protein effector proteins into host cells. Dot/Icm substrates have been proposed to contain a carboxy-terminal signal sequence that is ...necessary and sufficient for export, although both traits have been demonstrated for only a small fraction of these proteins. In this study, we discovered that export of the substrate SidJ is mediated by dual signal sequences that include a conventional C-terminal domain and a novel internal motif. The C-terminal signal sequence facilitates secretion of SidJ into host cells at early points of infection, whereas the internal signal sequence mediates secretion at later time points. Interestingly, only the internal signal sequence is necessary for complementation of the intracellular growth defect of a Delta sidJ mutant. Although this is the first report of a Dot/Icm substrate being secreted by an internal signal sequence, many other substrates may be exported in a similar manner. In addition, efficient translocation of SidJ is dependent on the chaperone-like type IV adaptors IcmS/IcmW. Five IcmS/IcmW binding domains that are distinct from both signal sequences were elucidated and, interestingly, only secretion mediated by the internal signal sequence requires IcmS/IcmW. Thus, Legionella employs multiple sophisticated molecular mechanisms to regulate the export of SidJ. Export of the protein SidJ, a Legionella Dot/Icm type IV secretion substrate, is mediated by a c-terminal and internal signal sequence that share homology. In addition, optimal secretion of SidJ requires multiple binding sites for the chaperone IcmS/IcmW.
species are environmental gram-negative bacteria able to cause a severe form of pneumonia in humans known as Legionnaires' disease. Since the identification of
in 1977, four decades of research on
...biology and Legionnaires' disease have brought important insights into the biology of the bacteria and the molecular mechanisms that these intracellular pathogens use to cause disease in humans. Nowadays,
species constitute a remarkable model of bacterial adaptation, with a genus genome shaped by their close coevolution with amoebae and an ability to exploit many hosts and signaling pathways through the secretion of a myriad of effector proteins, many of which have a eukaryotic origin. This review aims to discuss current knowledge of
infection mechanisms and future research directions to be taken that might answer the many remaining open questions. This research will without a doubt be a terrific scientific journey worth taking.
Genomics can provide the basis for understanding the evolution of emerging, lethal human pathogens such as Legionella pneumophila, the causative agent of Legionnaires' disease. This bacterium ...replicates within amoebae and persists in the environment as a free-living microbe. Among the many Legionella species described, L. pneumophila is associated with 90% of human disease and within the 15 serogroups (Sg), L. pneumophila Sg1 causes over 84% of Legionnaires' disease worldwide. Why L. pneumophila Sg1 is so predominant is unknown. Here, we report the first comprehensive screen of the gene content of 217 L. pneumophila and 32 non-L. pneumophila strains isolated from humans and the environment using a Legionella DNA-array. Strikingly, we uncovered a high conservation of virulence- and eukaryotic-like genes, indicating strong environmental selection pressures for their preservation. No specific hybridization profile differentiated clinical and environmental strains or strains of different serogroups. Surprisingly, the gene cluster coding the determinants of the core and the O side-chain synthesis of the lipopolysaccaride (LPS cluster) determining Sg1 was present in diverse genomic backgrounds, strongly implicating the LPS of Sg1 itself as a principal cause of the high prevalence of Sg1 strains in human disease and suggesting that the LPS cluster can be transferred horizontally. Genomic analysis also revealed that L. pneumophila is a genetically diverse species, in part due to horizontal gene transfer of mobile genetic elements among L. pneumophila strains, but also between different Legionella species. However, the genomic background also plays a role in disease causation as demonstrated by the identification of a globally distributed epidemic strain exhibiting the genotype of the sequenced L. pneumophila strain Paris.
Signalling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalysed by the E1, E2 and E3 three-enzyme cascade, which ...links the carboxy terminus of ubiquitin to the ε-amino group of, in most cases, a lysine of the substrate via an isopeptide bond. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents. For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases or as enzymes that directly attack ubiquitin. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes. Here we demonstrate that members of the SidE effector family of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum. Moreover, we show that these proteins are capable of catalysing ubiquitination without the need for the E1 and E2 enzymes. A putative mono-ADP-ribosyltransferase motif critical for the ubiquitination activity is also essential for the role of the SidE family in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalysed by these enzymes is energized by nicotinamide adenine dinucleotide, which activates ubiquitin by the formation of ADP-ribosylated ubiquitin. These results establish that ubiquitination can be catalysed by a single enzyme, the activity of which does not require ATP.
The genus Legionella comprises 65 species, among which Legionella pneumophila is a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human ...pathogen, we have functionally analyzed 80 Legionella genomes spanning 58 species. Uniquely, an immense repository of 18,000 secreted proteins encoding 137 different eukaryotic-like domains and over 200 eukaryotic-like proteins is paired with a highly conserved type IV secretion system (T4SS). Specifically, we show that eukaryotic Rho- and Rab-GTPase domains are found nearly exclusively in eukaryotes and Legionella. Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F-box, U-box, and SET domains were present in >70% of all species, suggesting that manipulation of host signal transduction, protein turnover, and chromatin modification pathways are fundamental intracellular replication strategies for legionellae. In contrast, the Sec-7 domain was restricted to L. pneumophila and seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly, this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis, indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by interdomain gene transfer. Furthermore, we revealed the surprising extent to which legionellae have coopted genes and thus cellular functions from their eukaryotic hosts, providing an understanding of how dynamic reshuffling and gene acquisition have led to the emergence of major human pathogens.
Legionella pneumophila is an environmental bacterium and the leading cause of Legionnaires' disease. Just five sequence types (ST), from more than 2000 currently described, cause nearly half of ...disease cases in northwest Europe. Here, we report the sequence and analyses of 364 L. pneumophila genomes, including 337 from the five disease-associated STs and 27 representative of the species diversity. Phylogenetic analyses revealed that the five STs have independent origins within a highly diverse species. The number of de novo mutations is extremely low with maximum pairwise single-nucleotide polymorphisms (SNPs) ranging from 19 (ST47) to 127 (ST1), which suggests emergences within the last century. Isolates sampled geographically far apart differ by only a few SNPs, demonstrating rapid dissemination. These five STs have been recombining recently, leading to a shared pool of allelic variants potentially contributing to their increased disease propensity. The oldest clone, ST1, has spread globally; between 1940 and 2000, four new clones have emerged in Europe, which show long-distance, rapid dispersal. That a large proportion of clinical cases is caused by recently emerged and internationally dispersed clones, linked by convergent evolution, is surprising for an environmental bacterium traditionally considered to be an opportunistic pathogen. To simultaneously explain recent emergence, rapid spread and increased disease association, we hypothesize that these STs have adapted to new man-made environmental niches, which may be linked by human infection and transmission.
Legionella pneumophila and L. longbeachae are two species of a large genus of bacteria that are ubiquitous in nature. L. pneumophila is mainly found in natural and artificial water circuits while L. ...longbeachae is mainly present in soil. Under the appropriate conditions both species are human pathogens, capable of causing a severe form of pneumonia termed Legionnaires' disease. Here we report the sequencing and analysis of four L. longbeachae genomes, one complete genome sequence of L. longbeachae strain NSW150 serogroup (Sg) 1, and three draft genome sequences another belonging to Sg1 and two to Sg2. The genome organization and gene content of the four L. longbeachae genomes are highly conserved, indicating strong pressure for niche adaptation. Analysis and comparison of L. longbeachae strain NSW150 with L. pneumophila revealed common but also unexpected features specific to this pathogen. The interaction with host cells shows distinct features from L. pneumophila, as L. longbeachae possesses a unique repertoire of putative Dot/Icm type IV secretion system substrates, eukaryotic-like and eukaryotic domain proteins, and encodes additional secretion systems. However, analysis of the ability of a dotA mutant of L. longbeachae NSW150 to replicate in the Acanthamoeba castellanii and in a mouse lung infection model showed that the Dot/Icm type IV secretion system is also essential for the virulence of L. longbeachae. In contrast to L. pneumophila, L. longbeachae does not encode flagella, thereby providing a possible explanation for differences in mouse susceptibility to infection between the two pathogens. Furthermore, transcriptome analysis revealed that L. longbeachae has a less pronounced biphasic life cycle as compared to L. pneumophila, and genome analysis and electron microscopy suggested that L. longbeachae is encapsulated. These species-specific differences may account for the different environmental niches and disease epidemiology of these two Legionella species.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Fourier-transform infrared (FTIR) spectroscopy using the IR Biotyper and core genome single nucleotide polymorphism (cgSNP) analysis were performed on 12
Legionella
isolates associated with an ...outbreak at a spa house in Kanagawa Prefecture, Japan, and 3 non-outbreak isolates. The discriminative power of FTIR spectroscopy for 48-h incubation conditions of
L
.
pneumophila
in this outbreak was lower than cgSNP-based typing but higher than serogroup typing. FTIR spectroscopy could screen outbreak isolates from a group of genetically related isolates and may be useful as an initial typing method in
Legionella
outbreak investigations.
Abstract Legionnaires' disease is a severe respiratory disease that is estimated to cause between 8,000 and 18,000 hospitalizations each year, though the exact burden is unknown due to ...under-utilization of diagnostic testing. Although Legionella pneumophila is the most common species detected in clinical cases (80-90%), other species have also been reported to cause disease. However, little is known about Legionnaires' disease caused by these non- pneumophila species. We designed a multiplex real-time PCR assay for detection of all Legionella spp. and simultaneous specific identification of four clinically-relevant Legionella species, L. anisa, L. bozemanii, L. longbeachae, and L. micdadei, using 5′-hydrolysis probe real-time PCR. The analytical sensitivity for detection of nucleic acid from each target species was ≤50 fg per reaction. We demonstrated the utility of this assay in spiked human sputum specimens. This assay could serve as a tool for understanding the scope and impact of non- pneumophila Legionella species in human disease.
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