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Legionella, an important cause of pneumonia, is acquired from environmental sources such as potable water. In this article, evidence of person-to-person spread of Legionnaires' disease is reported. To the Editor: Legionnaires’ disease is an often severe form of pneumonia that is typically acquired by susceptible persons (e.g., elderly persons and smokers) through inhalation of aerosols that contain legionella species. 1 – 4 A cluster of cases of this disease occurred in Vila Franca de Xira, Portugal, in 2014. 5 One of the first cases of disease in this cluster occurred in a 48-year-old man (Patient 1), a smoker, who had been employed since October 6, 2014, as a maintenance worker at an industrial cooling tower complex in Vila Franca de Xira that was subsequently found to be contaminated with Legionella . . .

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.

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.

An unprecedented mechanism of ubiquitination that is independent of E1 and E2 enzymes, instead relying on activation of ubiquitin by ADP-ribosylation, and which is mediated by members of the SidE effector family encoded by the bacterial pathogen Legionella pneumophila , establishes that ubiquitination can be carried out by a single enzyme. Ubiquitination catalysed by a single protein Ubiquitination is an important regulatory mechanism for many cellular processes, and several bacterial pathogens have evolved to exploit and target this protein degradation pathway to facilitate their own proliferation. Ubiquitination is catalysed by a sequential multi-enzyme cascade that involves three enzymes: E1, E2 and E3. In this study, Zhao-Qing Luo and colleagues describe an unprecedented mechanism of ubiquitination that is independent of E1 and E2 enzymes, and which is mediated by members of the SidE effector family encoded by the bacterial pathogen Legionella pneumophila . They show that SidE effectors ubiquitinate multiple Rab small GTPases in a mechanism that relies on activation of ubiquitin by ADP-ribosylation, thereby circumventing the requirement for activation by the E1 and E2 enzymes. This work establishes that ubiquitination can be carried out by a single enzyme, but future work is required to resolve the functional implications of this unique mechanism of post-translational modification. 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 1 , 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 2 . For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases 3 or as enzymes that directly attack ubiquitin 4 . The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes 5 . 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.

Due to nutrient limitation and adverse conditions in the environment, bacteria mostly do not grow exponentially but adopt a resting (“dormant”) state. Bacterial dormancy usually coincides with the formation of translationally silent (“hibernating”) ribosomes; however, the role of ribosome hibernation in intracellular pathogens is poorly understood. The facultative intracellular bacterium Legionella pneumophila is virulent in the stationary but not in the exponential growth phase, and therefore, an in-depth characterization of the pathogen’s physiological states and ribosome profiles is crucial for understanding its virulence. Using bioinformatics, bacterial genetics, biochemical, and cell biological approaches, in this study, we reveal that the L. pneumophila ribosome hibernation factors LhpF, RaiA, RsfS, and HflX determine distinct ribosome subpopulations and are implicated in starvation survival and regrowth, as well as in host cell infection, intracellular replication, and phenotypic heterogeneity. Collectively, our data highlight the critical importance of ribosome hibernation for the physiology and virulence of L. pneumophila .

Redundancy has been referred to as a state of no longer being needed or useful. Microbiologists often theorize that the only case of true redundancy in a haploid organism would be a recent gene duplication event, prior to divergence through selective pressure. However, a growing number of examples exist where an organism encodes two genes that appear to perform the same function. For example, many pathogens translocate multiple effector proteins into hosts. While disruption of individual effector genes does not result in a discernable phenotype, deleting genes in combination impairs pathogenesis: this has been described as redundancy. In many cases, this apparent redundancy could be due to limitations of laboratory models of pathogenesis that do not fully recapitulate the disease process. Alternatively, it is possible that the selective advantage achieved by this perceived redundancy is too subtle to be measured in the laboratory. Moreover, there are numerous possibilities for different types of redundancy. The most common and recognized form of redundancy is functional redundancy whereby two proteins have similar biochemical activities and substrate specificities allowing each one to compensate in the absence of the other. However, redundancy can also exist between seemingly unrelated proteins that manipulate the same or complementary host cell pathways. In this article, we outline 5 types of redundancy in pathogenesis: molecular, target, pathway, cellular process, and system redundancy that incorporate the biochemical activities, the host target specificities and the impact of effector function on the pathways and cellular process they modulate. For each type of redundancy, we provide examples from pathogenesis as this organism employs over 300 secreted virulence proteins and loss of individual proteins rarely impacts intracellular growth. We also discuss selective pressures that drive the maintenance of redundant mechanisms, the current methods used to resolve redundancy and features that distinguish between redundant and non-redundant virulence mechanisms.

BackgroundLegionella pneumonia (LP) has been reported in a number of outbreaks in the UK. It has significant implications for public health as outbreaks require investigation to identify a responsible source. An outbreak of LP occurred in the Stoke-on-Trent area of North Staffordshire in July 2012. This is an analysis of the clinical cases reported and subsequent public health enquiry.MethodRetrospective review of case records, pathology and radiology. Data was collated on clinical and biochemical features, microbiology and clinical outcome. Results of the public health enquiry were sourced from the local Health Protection Agency (HPA).Results20 patients were confirmed to have LP. 13 male, 7 female. Mean age was 65 years. 50% were ex-smokers. 70% had at least two co-morbidities requiring treatment. 3 were immunosuppressed on longterm steroids. Only 1 patient reported foreign travel in the preceding month. Clinical, biochemical and radiological findings are shown in Table 1. Urinary legionella antigen was detected in all patients. Sputum PCR for legionella pneumophilia was positive in 8 cases and identified a single strain present in all samples (ST1268). Mean length of stay was 8.9 days. 6 patients (30%) required admission to intensive care for respiratory support; 2 were invasively ventilated. 18 patients were discharged home, 2 died. 65% had follow-up chest xrays showing improvement or resolution of consolidation. All cases were from the Stoke area. The HPA investigation detected the same legionella pneumophilia strain in a display spa pool at a retail unit, as found in the sputum. As no other tested sites were found to have this strain, it is very likely this was the origin of the local outbreak. All cases had visited the retail unit prior to their hospital admission.ConclusionLP remains a possible diagnosis in any case of community acquired pneumonia. This outbreak is a reminder that patients do not always present following the classic travel history. The diagnosis needs to be considered and vigilance in microbiological testing is necessary to identify potential cases.Abstract P248 Table 1.Clinical, biochemical and radiology findings.Fever > 38 degree C11 (78%)Type 1 respiratory failure Type 2 respiratory failure9 (45%) 1 (0.05%)CRP > 3009 (45%)Hyponatraemia (Na < 130)7 (35%)LFT derangement (ALT > 40)16 (80%)Hypoalbuminaemia (< 25)10 (50%)Consolidation on CXR19 (95%)

Key Points Legionella pneumophila and Coxiella burnetii are two evolutionarily related intracellular bacterial pathogens that reside in distinct compartments in host cells during infection. Successful infection by both pathogens requires a functionally exchangeable type IV secretion system called Dot/Icm, which translocates hundreds of virulence factors, termed effectors, into host cells. The majority of Legionella spp. and Coxiella spp. effectors are unique to these pathogens, and functional redundancy exists among many of them. Functional domains that are associated with most of these effectors are enigmatic and cannot be readily predicted by currently available bioinformatics tools. Legionella spp. and Coxiella spp. promote intracellular bacterial replication by interfering with host gene expression through effectors that impose epigenetic modifications on host chromatin by different mechanisms. L. pneumophila extensively manipulates the early phases of the secretory branch of the host vesicle trafficking pathway by hijacking the activity of key regulatory proteins such as RAB small GTPases via multiple effectors. L. pneumophila effectors function coordinately to alter the composition of lipids, such as phosphoinositides, on the vacuole that contains the bacterium and other organelles to facilitate its intracellular growth. L. pneumophila co-opts the ubiquitin network of host cells by effectors that function through diverse biochemical mechanisms, including the SidE family effectors, which catalyse ubiquitylation by an E1 enzyme and E2 enzyme-independent mechanism, which represents a paradigm shift in our understanding of this important post-translational modification. The intracellular pathogens Legionella pneumophila and Coxiella burnetii use the Dot/Icm type IV secretion system to translocate effectors into host cells. Qiu and Luo explore the biochemical and cell biological functions of these effectors and their roles in our understanding of bacterial virulence. 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.

IFN receptor signaling induces cell-autonomous immunity to infections with intracellular bacterial pathogens. Here, we demonstrate that IFN-inducible guanylate binding protein (Gbp) proteins stimulate caspase-11–dependent, cell-autonomous immunity in response to cytoplasmic LPS. Caspase-11–dependent pyroptosis is triggered in IFN-activated macrophages infected with the Gram-negative bacterial pathogen Legionella pneumophila. The rapid induction of pyroptosis in IFN-activated macrophages required a cluster of IFN-inducible Gbp proteins encoded on mouse chromosome 3 (Gbp ᶜʰʳ³). Induction of pyroptosis in naive macrophages by infections with the cytosol-invading Δ sdhA L. pneumophila mutant was similarly dependent on Gbp ᶜʰʳ³, suggesting that these Gbp proteins play a role in the detection of bacteria accessing the cytosol. Cytoplasmic LPS derived from Salmonella ssp. or Escherichia coli has recently been shown to trigger caspase-11 activation and pyroptosis, but the cytoplasmic sensor for LPS and components of the caspase-11 inflammasome are not yet defined. We found that the induction of caspase-11–dependent pyroptosis by cytoplasmic L. pneumophila -derived LPS required Gbp ᶜʰʳ³ proteins. Similarly, pyroptosis induced by cytoplasmic LPS isolated from Salmonella was diminished in Gbp ᶜʰʳ³-deficient macrophages. These data suggest a role for Gbp ᶜʰʳ³ proteins in the detection of cytoplasmic LPS and the activation of the noncanonical inflammasome.

Understanding how bacteria interact with the immune system is crucial for developing better treatments and vaccines. This study reveals that a bacterial secretion system, the type IV secretion system (T4SS) of Legionella pneumophila , triggers a targeted immune response but also enhances broader, nonspecific immunity. Using advanced infection models, the research shows that T4SS-driven immunity protects against multiple pathogens, including bacteria and fungi, even in the absence of traditional immune signaling pathways. These findings suggest that bacterial secretion systems can serve as novel tools for training the immune system, with potential applications in vaccine development and immunotherapy. By uncovering new ways bacteria influence immune memory, this work advances our understanding of host defense mechanisms and opens new avenues for designing strategies to enhance protection against infectious diseases.