Legionella pneumophila and Coxiella burnetii are two evolutionarily related intracellular pathogens that use the Dot/Icm type IV secretion system to translocate effectors into host cells. These ...effectors are essential for the establishment of membrane-bound compartments known as replication vacuoles, which enable the survival and replication of bacteria inside host cells. The effectors interfere with diverse signalling pathways to co-opt host processes, such as vesicle trafficking, ubiquitylation, gene expression and lipid metabolism, to promote pathogen survival. In this Review, we explore Dot/Icm effectors from L. pneumophila and C. burnetii as key virulence factors, and we examine the biochemical and cell biological functions of these effectors and their roles in our understanding of bacterial virulence.
While it is well-established that
Legionella are able to colonize engineered water systems, the number of interacting factors contributing to their occurrence, proliferation, and persistence are ...unclear. This review summarizes current methods used to detect and quantify legionellae as well as the current knowledge of engineered water system characteristics that both favour and promote legionellae growth. Furthermore, the use of quantitative microbial risk assessment (QMRA) models to predict potentially critical human exposures to legionellae are also discussed. Understanding the conditions favouring
Legionella occurrence in engineered systems and their overall ecology (growth in these systems/biofilms, biotic interactions and release) will aid in developing new treatment technologies and/or systems that minimize or eliminate human exposure to potentially pathogenic legionellae.
► We review current legionellae detection and quantification methods. ► We discuss conditions favouring Legionellae occurrence in engineered systems. ► Quantitative microbial risk assessment is a tool to predict
legionella exposure risk. ► We emphasize the need and rationale to better understand legionellae ecology.
Summary
Legionella pneumophila is an amoeba‐resistant opportunistic pathogen that performs cell–cell communication through the signalling molecule 3‐hydroxypentadecane‐4‐one (LAI‐1, Legionella ...autoinducer‐1). The lqs (Legionella quorum sensing) gene cluster encodes the LAI‐1 autoinducer synthase LqsA, the cognate sensor kinase LqsS and the response regulator LqsR. Here we show that the Lqs system includes an ‘orphan’ homologue of LqsS termed LqsT. Compared with wild‐type L. pneumophila, strains lacking lqsT or both lqsS and lqsT show increased salt resistance, greatly enhanced natural competence for DNA acquisition and impaired uptake by phagocytes. Sensitive novel single round growth assays and competition experiments using Acanthamoeba castellanii revealed that ΔlqsT and ΔlqsS‐ΔlqsT, as well as ΔlqsA and other lqs mutant strains are impaired for intracellular growth and cannot compete against wild‐type bacteria upon co‐infection. In contrast to the ΔlqsS strain, ΔlqsT does not produce extracellular filaments. The phenotypes of the ΔlqsS‐ΔlqsT strain are partially complemented by either lqsT or lqsS, but are not reversed by overexpression of lqsA, suggesting that LqsT and LqsS are the sole LAI‐1‐responsive sensor kinases in L. pneumophila. In agreement with the different phenotypes of the ΔlqsT and ΔlqsS strains, lqsT and lqsS are differentially expressed in the post‐exponential growth phase, and transcriptome studies indicated that 90% of the genes, which are downregulated in absence of lqsT, are upregulated in absence of lqsS. Reciprocally regulated genes encode components of a 133 kb genomic ‘fitness island’ or translocated effector proteins implicated in virulence. Together, these results reveal a unique organization of the L. pneumophila Lqs system comprising two partially antagonistic LAI‐1‐responsive sensor kinases, LqsT and LqsS, which regulate distinct pools of genes implicated in pathogen–host cell interactions, competence, expression of a genomic island or production of extracellular filaments.
Summary Objectives Urinary antigen testing for Legionella pneumophila serogroup 1 is the leading rapid diagnostic test for Legionnaires' Disease (LD); however other Legionella species and serogroups ...can also cause LD. The aim was to determine the utility of front-line L. pneumophila and Legionella species PCR in a severe respiratory infection algorithm. Methods L. pneumophila and Legionella species duplex real-time PCR was carried out on 1944 specimens from hospitalised patients over a 4 year period in Edinburgh, UK. Results L. pneumophila was detected by PCR in 49 (2.7%) specimens from 36 patients. During a LD outbreak, combined L. pneumophila respiratory PCR and urinary antigen testing had optimal sensitivity and specificity (92.6% and 98.3% respectively) for the detection of confirmed cases. Legionella species was detected by PCR in 16 (0.9%) specimens from 10 patients. The 5 confirmed and 1 probable cases of Legionella longbeachae LD were both PCR and antibody positive. Conclusions Front-line L. pneumophila and Legionella species PCR is a valuable addition to urinary antigen testing as part of a well-defined algorithm. Cases of LD due to L. longbeachae might be considered laboratory-confirmed when there is a positive Legionella species PCR result and detection of L. longbeachae specific antibody response.
The Legionella pneumophila effector MavC induces ubiquitination of the E2 ubiquitin‐conjugating enzyme UBE2N by transglutamination, thereby abolishing its function in the synthesis of K63‐type ...polyubiquitin chains. The inhibition of UBE2N activity creates a conundrum because this E2 enzyme is important in multiple signaling pathways, including some that are important for intracellular L. pneumophila replication. Here, we show that prolonged inhibition of UBE2N activity by MavC restricts intracellular bacterial replication and that the activity of UBE2N is restored by MvcA, an ortholog of MavC (50% identity) with ubiquitin deamidase activity. MvcA functions to deubiquitinate UBE2N‐Ub using the same catalytic triad required for its deamidase activity. Structural analysis of the MvcA‐UBE2N‐Ub complex reveals a crucial role of the insertion domain in MvcA in substrate recognition. Our study establishes a deubiquitination mechanism catalyzed by a deamidase, which, together with MavC, imposes temporal regulation of the activity of UBE2N during L. pneumophila infection.
Synopsis
Transglutaminase MavC of the bacterial pathogen Legionella pneumophila catalyzes atypical ubiquitination of the host cell E2 ubiquitin conjugation enzyme UBE2N. Here, the L. pneumophila ubiquitin deamidase MvcA is found to reverse MavC‐induced UBE2N ubiquitination and to restore its activity to promote host cell survival at later stages of L. pneumophila infection.
Expression of MvcA reduces MavC‐induced UBE2N ubiquitination.
MvcA promotes synthesis of K63‐type polyubiquitin chains by UBE2N during L. pneumophila infection.
MvcA is a UBE2N‐specific deubiquitinase that cleaves the isopeptide bond between Gln40 of ubiquitin and Lys92 of UBE2N.
Structure of the MvcA‐UBE2N‐Ub complex reveals involvement of the MvcA “insertion domain” in substrate recognition.
MvcA counteracts MavC activity at the late stage of infection to promote intracellular replication of L. pneumophila.
Dynamic regulation of activity and atypical ubiquitination of the host E2 enzyme UBE2N via bacterial effectors MavC and MvcA over the course of infection is important for pathogen replication.
Infection by the human pathogen Legionella pneumophila relies on the translocation of ∼ 300 virulence proteins, termed effectors, which manipulate host cell processes. However, almost no information ...exists regarding effectors in other Legionella pathogens. Here we sequenced, assembled and characterized the genomes of 38 Legionella species and predicted their effector repertoires using a previously validated machine learning approach. This analysis identified 5,885 predicted effectors. The effector repertoires of different Legionella species were found to be largely non-overlapping, and only seven core effectors were shared by all species studied. Species-specific effectors had atypically low GC content, suggesting exogenous acquisition, possibly from the natural protozoan hosts of these species. Furthermore, we detected numerous new conserved effector domains and discovered new domain combinations, which allowed the inference of as yet undescribed effector functions. The effector collection and network of domain architectures described here can serve as a roadmap for future studies of effector function and evolution.
Bacteria of the genus Legionella cause water-based infections, resulting in severe pneumonia. To improve our knowledge about Legionella spp. ecology, its prevalence and its relationships with ...environmental factors were studied. Seasonal samples were taken from both water and biofilm at seven sampling points of a small drinking water distribution system in Israel. Representative isolates were obtained from each sample and identified to the species level. Legionella pneumophila was further determined to the serotype and genotype level. High resolution genotyping of L. pneumophila isolates was achieved by Multiple-Locus Variable number of tandem repeat Analysis (MLVA). Within the studied water system, Legionella plate counts were higher in summer and highly variable even between adjacent sampling points. Legionella was present in six out of the seven selected sampling points, with counts ranging from 1.0 × 101 to 5.8 × 103 cfu/l. Water counts were significantly higher in points where Legionella was present in biofilms. The main fraction of the isolated Legionella was L. pneumophila serogroup 1. Serogroup 3 and Legionella sainthelensis were also isolated. Legionella counts were positively correlated with heterotrophic plate counts at 37 °C and negatively correlated with chlorine. Five MLVA-genotypes of L. pneumophila were identified at different buildings of the sampled area. The presence of a specific genotype, “MLVA-genotype 4”, consistently co-occurred with high Legionella counts and seemed to “trigger” high Legionella counts in cold water. Our hypothesis is that both the presence of L. pneumophila in biofilm and the presence of specific genotypes, may indicate and/or even lead to high Legionella concentration in water. This observation deserves further studies in a broad range of drinking water systems to assess its potential for general use in drinking water monitoring and management.
Display omitted
•Legionella was abundant through all seasons in water and biofilm.•Legionella pneumophila serogroup 1 was the predominant identified serogroup.•When Legionella was present in biofilm its counts in water were >30 cfu/l.•Five L. pneumophila MLVA-genotypes were identified at different buildings.•MLVA-genotype 4, consistently co-occurred with high Legionella counts in cold water.
Gram-negative bacteria from the Legionella genus are intracellular pathogens that cause a severe form of pneumonia called Legionnaires' disease. The bacteria replicate intracellularly in macrophages, ...and the restriction of bacterial replication by these cells is critical for host resistance. The activation of the NAIP5/NLRC4 inflammasome, which is readily triggered in response to bacterial flagellin, is essential for the restriction of bacterial replication in murine macrophages. Once activated, this inflammasome induces pore formation and pyroptosis and facilitates the restriction of bacterial replication in macrophages. Because investigations related to the NLRC4-mediated restriction of Legionella replication were performed using mice double deficient for caspase-1 and caspase-11, we assessed the participation of caspase-1 and caspase-11 in the functions of the NLRC4 inflammasome and the restriction of Legionella replication in macrophages and in vivo. By using several species of Legionella and mice singly deficient for caspase-1 or caspase-11, we demonstrated that caspase-1 but not caspase-11 was required for pore formation, pyroptosis, and restriction of Legionella replication in macrophages and in vivo. By generating F1 mice in a mixed 129 × C57BL/6 background deficient (129 × Casp-11(-/-) ) or sufficient (129 × C57BL/6) for caspase-11 expression, we found that caspase-11 was dispensable for the restriction of Legionella pneumophila replication in macrophages and in vivo. Thus, although caspase-11 participates in flagellin-independent noncanonical activation of the NLRP3 inflammasome, it is dispensable for the activities of the NLRC4 inflammasome. In contrast, functional caspase-1 is necessary and sufficient to trigger flagellin/NLRC4-mediated restriction of Legionella spp. infection in macrophages and in vivo.
Aims
Culture remains the gold‐standard for the enumeration of environmental Legionella. However, it has several drawbacks including long incubation and poor sensitivity, causing delays in response ...times to outbreaks of Legionnaires' disease. This study aimed to validate real‐time PCR assays to quantify Legionella species (ssrA gene), Legionella pneumophila (mip gene) and Leg. pneumophila serogroup‐1 (wzm gene) to support culture‐based detection in a frontline public health laboratory.
Methods and Results
Each qPCR assay had 100% specificity, excellent sensitivity (5 GU/reaction) and reproducibility. Comparison of the assays to culture‐based enumeration of Legionella from 200 environmental samples showed that they had a negative predictive value of 100%. Thirty eight samples were positive for Legionella species by culture and qPCR. One hundred samples were negative by both methods, whereas 62 samples were negative by culture but positive by qPCR. The average log10 increase between culture and qPCR for Legionella spp. and Leg. pneumophila was 0·72 (P = 0·0002) and 0·51 (P = 0·006), respectively.
Conclusions
The qPCR assays can be conducted on the same 1 l water sample as culture thus can be used as a supplementary technique to screen out negative samples and allow more rapid indication of positive samples.
Significance and Impact of the Study
The assay could prove informative in public health investigations to identify or rule out sources of Legionella as well as to specifically identify Leg. pneumophila serogroup 1 in a timely manner not possible with culture.
Type IV secretion systems (T4SSs) are large macromolecular machines that translocate protein and DNA and are involved in the pathogenesis of multiple human diseases. Here, using electron ...cryotomography (ECT), we report the in situ structure of the Dot/Icm type IVB secretion system (T4BSS) utilized by the human pathogen Legionella pneumophila. This is the first structure of a type IVB secretion system, and also the first structure of any T4SS in situ. While the Dot/Icm system shares almost no sequence similarity with type IVA secretion systems (T4ASSs), its overall structure is seen here to be remarkably similar to previously reported T4ASS structures (those encoded by the R388 plasmid in Escherichia coli and the cag pathogenicity island in Helicobacter pylori). This structural similarity suggests shared aspects of mechanism. However, compared to the negative‐stain reconstruction of the purified T4ASS from the R388 plasmid, the L. pneumophila Dot/Icm system is approximately twice as long and wide and exhibits several additional large densities, reflecting type‐specific elaborations and potentially better structural preservation in situ.
Synopsis
Bacterial type IV secretion systems translocate protein and DNA, and are involved in the pathogenesis of multiple human diseases. This study presents the in situ structure of the Dot/Icm type IVB secretion system from the human pathogen Legionella pneumophila.
First structure of a type IVB secretion system, and also the first structure of any T4SS in situ.
The Dot/Icm system shares almost no sequence similarity with type IVA secretion systems (T4ASSs).
The basic architecture of the type IVB secretion system is however strikingly similar to the type IVA secretion system.
Bacterial type IV secretion systems translocate protein and DNA, and are involved in the pathogenesis of multiple human diseases. This study presents the in situ structure of the Dot/Icm type IVB secretion system from the human pathogen Legionella pneumophila.
PDF
