During infection, intracellular bacterial pathogens translocate a variety of effectors into host cells that modify host membrane trafficking for their benefit. We found a self-organizing system ...consisting of a bacterial phosphoinositide kinase and its opposing phosphatase that formed spatiotemporal patterns, including traveling waves, to remodel host cellular membranes. The
effector MavQ, a phosphatidylinositol (PI) 3-kinase, was targeted to the endoplasmic reticulum (ER). MavQ and the
PI 3-phosphatase SidP, even in the absence of other bacterial components, drove rapid PI 3-phosphate turnover on the ER and spontaneously formed traveling waves that spread along ER subdomains inducing vesicle and tubule budding. Thus, bacteria can exploit a self-organizing membrane-targeting mechanism to hijack host cellular structures for survival.
The Dot/Icm type IVB secretion system of Legionella pneumophila is essential for its pathogenesis by delivering >300 effector proteins into the host cell. However, their precise secretion mechanism ...and which components interact with the host cell is only partly understood. Here, we undertook evolutionary analyses of the Dot/Icm system of 58 Legionella species to identify those components that interact with the host and/or the substrates. We show that high recombination rates are acting on DotA, DotG, and IcmX, supporting exposure of these proteins to the host. Specific amino acids under positive selection on the periplasmic region of DotF, and the cytoplasmic domain of DotM, support a role of these regions in substrate binding. Diversifying selection acting on the signal peptide of DotC suggests its interaction with the host after cleavage. Positive selection acts on IcmR, IcmQ, and DotL revealing that these components are probably participating in effector recognition and/or translocation. Furthermore, our results predict the participation in host/effector interaction of DotV and IcmF. In contrast, DotB, DotO, most of the core subcomplex elements, and the chaperones IcmS-W show a high degree of conservation and not signs of recombination or positive selection suggesting that these proteins are under strong structural constraints and have an important role in maintaining the architecture/function of the system. Thus, our analyses of recombination and positive selection acting on the Dot/Icm secretion system predicted specific Dot/Icm components and regions implicated in host interaction and/or substrate recognition and translocation, which will guide further functional analyses.
Increasing numbers of legionellosis outbreaks within the last years have shown that Legionella are a growing challenge for public health. Molecular biological detection methods capable of rapidly ...identifying viable Legionella are important for the control of engineered water systems. The current gold standard based on culture methods takes up to 10 days to show positive results. For this reason, a flow-based chemiluminescence (CL) DNA microarray was developed that is able to quantify viable and non-viable Legionella spp. as well as Legionella pneumophila in one hour. An isothermal heterogeneous asymmetric recombinase polymerase amplification (haRPA) was carried out on flow-based CL DNA microarrays. Detection limits of 87 genomic units (GU) µL−1 and 26GUµL−1 for Legionella spp. and Legionella pneumophila, respectively, were achieved. In this work, it was shown for the first time that the combination of a propidium monoazide (PMA) treatment with haRPA, the so-called viability haRPA, is able to identify viable Legionella on DNA microarrays. Different proportions of viable and non-viable Legionella, shown with the example of L. pneumophila, ranging in a total concentration between 101 to 105GUµL−1 were analyzed on the microarray analysis platform MCR 3. Recovery values for viable Legionella spp. were found between 81% and 133%. With the combination of these two methods, there is a chance to replace culture-based methods in the future for the monitoring of engineered water systems like condensation recooling plants.
•In-house RPA primer design for the detection of Legionella spp. and L. pneumophila.•Quantification by haRPA on flow-based chemiluminescence DNA microarrays.•Differentiation of Legionella spp. and L. pneumophila on one DNA microarray.•Quantification of viable Legionella spp. by combination of PMA treatment and haRPA on flow-based chemiluminescence DNA microarrays.
Enzymes with a protein kinase fold transfer phosphate from adenosine 5'-triphosphate (ATP) to substrates in a process known as phosphorylation. Here, we show that the
meta-effector SidJ adopts a ...protein kinase fold, yet unexpectedly catalyzes protein polyglutamylation. SidJ is activated by host-cell calmodulin to polyglutamylate the SidE family of ubiquitin (Ub) ligases. Crystal structures of the SidJ-calmodulin complex reveal a protein kinase fold that catalyzes ATP-dependent isopeptide bond formation between the amino group of free glutamate and the γ-carboxyl group of an active-site glutamate in SidE. We show that SidJ polyglutamylation of SidE, and the consequent inactivation of Ub ligase activity, is required for successful
replication in a viable eukaryotic host cell.
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Legionnaires disease occurs worldwide. Many authorities have guidelines and regulations to prevent and control Legionella in water systems. These regulations are based on often very ...limited field and laboratory observations and measurements. They are, therefore, very different from country to country. This article aims to map the existing regulatory framework of worldwide Legionella control to assess the feasibility of regulatory unification.
This article gives an overview of the different standards, guidelines, and recommendations as well as how various authorities and/or countries deal with Legionella infection. A 3-step process is followed to identify current regulations.
Although Legionella is a global concern with a common scientific base, the regulatory framework is different from country to country. The current guidelines and standards are not the best possible. Despite different regulatory frameworks, there is still broad unification of underlying principles. Common principles across regulations are avoiding and monitoring critical spots, avoiding water stagnation, and maintaining sufficiently high temperature (above 60°C, below 25°C). Differences between regulations are target group and dangerous Legionella concentration levels.
The comparative analysis of the framework is a good starting point for reaching future regulatory unification based on common ground.
Garden soils were investigated as reservoirs and potential sources of pathogenic Legionella bacteria. Legionella bacteria were detected in 22 of 177 garden soil samples (12%) by amoebal coculture. Of ...these 22 Legionella-positive soil samples, seven contained Legionella pneumophila Several other species were found, including the pathogenic Legionella longbeachae (4 gardens) and Legionella sainthelensi (9 gardens). The L. pneumophila isolates comprised 15 different sequence types (STs), and eight of these STs were previously isolated from patients according to the European Working Group for Legionella Infections (EWGLI) database. Six gardens that were found to be positive for L. pneumophila were resampled after several months, and in three gardens, L. pneumophila was again isolated. One of these gardens was resampled four times throughout the year and was found to be positive for L. pneumophila on all occasions.
Tracking the source of infection for sporadic cases of Legionnaires' disease (LD) has proven to be hard. L. pneumophila ST47, the sequence type that is most frequently isolated from LD patients in the Netherlands, is rarely found in potential environmental sources. As L. pneumophila ST47 was previously isolated from a garden soil sample during an outbreak investigation, garden soils were investigated as reservoirs and potential sources of pathogenic Legionella bacteria. The detection of viable, clinically relevant Legionella strains indicates that garden soil is a potential source of Legionella bacteria, and future research should assess the public health implication of the presence of L. pneumophila in garden soil.
AIMS: This study investigated waterborne opportunistic pathogens (OPs) including potential hosts, and evaluated the use of Legionella spp. for indicating microbial water quality for OPs within a ...full‐scale operating drinking water distribution system (DWDS). METHODS AND RESULTS: To investigate the occurrence of specific microbial pathogens within a major city DWDS we examined large volume (90 l drinking water) ultrafiltration (UF) concentrates collected from six sites between February, 2012 and June, 2013. The detection frequency and concentration estimates by qPCR were: Legionella spp. (57%/85 cell equivalent, CE l⁻¹), Mycobacterium spp. (88%/324 CE l⁻¹), Pseudomonas aeruginosa (24%/2 CE l⁻¹), Vermamoeba vermiformis (24%/2 CE l⁻¹) and Acanthamoeba spp. (42%/5 cyst equivalent, CE l⁻¹). There was no detection of the following microorganisms: human faecal indicator Bacteroides (HF183), Salmonella enterica, Campylobacter spp., Escherichia coli O157:H7, Giardia intestinalis, Cryptosporidium spp. or Naegleria fowleri. There were significant correlations between the qPCR signals of Legionella spp. and Mycobacterium spp., and their potential hosts V. vermiformis and Acanthamoeba spp. Sequencing of Legionella spp. demonstrated limited diversity, with most sequences coming from two dominant groups, of which the larger dominant group was an unidentified species. Other known species including Legionella pneumophila were detected, but at low frequency. The densities of Legionella spp. and Mycobacterium spp. were generally higher (17 and 324 folds, respectively) for distal sites relative to the entry point to the DWDS. CONCLUSIONS: Legionella spp. occurred, had significant growth and were strongly associated with free‐living amoebae (FLA) and Mycobacterium spp., suggesting that Legionella spp. could provide a useful DWDS monitoring role to indicate potential conditions for non‐faecal OPs. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide insight into microbial pathogen detection that may aid in the monitoring of microbial water quality within DWDS prior to customer exposures.
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.
Eukaryotic cells can use the autophagy pathway to defend against microbes that gain access to the cytosol or reside in pathogen-modified vacuoles. It remains unclear if pathogens have evolved ...specific mechanisms to manipulate autophagy. Here, we found that the intracellular pathogen Legionella pneumophila could interfere with autophagy by using the bacterial effector protein RavZ to directly uncouple Atg8 proteins attached to phosphatidylethanolamine on autophagosome membranes. RavZ hydrolyzed the amide bond between the carboxyl-terminal glycine residue and an adjacent aromatic residue in Atg8 proteins, producing an Atg8 protein that could not be reconjugated by Atg7 and Atg3. Thus, intracellular pathogens can inhibit autophagy by irreversibly inactivating Atg8 proteins during infection.
The family of bacterial SidE enzymes catalyses phosphoribosyl-linked serine ubiquitination and promotes infectivity of Legionella pneumophila, a pathogenic bacteria that causes Legionnaires' disease
.... SidE enzymes share the genetic locus with the Legionella effector SidJ that spatiotemporally opposes the toxicity of these enzymes in yeast and mammalian cells, through a mechanism that is currently unknown
. Deletion of SidJ leads to a substantial defect in the growth of Legionella in both its natural hosts (amoebae) and in mouse macrophages
. Here we demonstrate that SidJ is a glutamylase that modifies the catalytic glutamate in the mono-ADP ribosyl transferase domain of the SdeA, thus blocking the ubiquitin ligase activity of SdeA. The glutamylation activity of SidJ requires interaction with the eukaryotic-specific co-factor calmodulin, and can be regulated by intracellular changes in Ca
concentrations. The cryo-electron microscopy structure of SidJ in complex with human apo-calmodulin revealed the architecture of this heterodimeric glutamylase. We show that, in cells infected with L. pneumophila, SidJ mediates the glutamylation of SidE enzymes on the surface of vacuoles that contain Legionella. We used quantitative proteomics to uncover multiple host proteins as putative targets of SidJ-mediated glutamylation. Our study reveals the mechanism by which SidE ligases are inhibited by a SidJ-calmodulin glutamylase, and opens avenues for exploring an understudied protein modification (glutamylation) in eukaryotes.
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