Autorepressor PsrA is required for optimal Legionella pneumophila growth in Acanthamoeba castellanii protozoa.

Legionella pneumophila possesses a unique intracellular lifecycle featuring distinct morphological stages that include replicative forms and transmissive cyst forms. Expression of genes associated with virulence traits and cyst morphogenesis is concomitant, and governed by a complex stringent response based-regulatory network and the stationary phase sigma factor RpoS. In Pseudomonas spp., rpoS expression is controlled by the autorepressor PsrA, and orthologs of PsrA and RpoS are required for cyst formation in Azotobacter. Here we report that the L. pneumophila psrA ortholog, expressed as a leaderless monocistronic transcript, is also an autorepressor, but is not a regulator of rpoS expression. Further, the binding site sequence recognized by L. pneumophila PsrA is different from that of Pseudomonas PsrA, suggesting a repertoire of target genes unique to L. pneumophila. While PsrA was dispensable for growth in human U937-derived macrophages, lack of PsrA affected bacterial intracellular growth in Acanthamoeba castellanii protozoa, but also increased the quantity of poly-3-hydroxybutyrate (PHB) inclusions in matured transmissive cysts. Interestingly, overexpression of PsrA increased the size and bacterial load of the replicative vacuole in both host cell types. Taken together, we report that PsrA is a host-specific requirement for optimal temporal progression of L. pneumophila intracellular lifecycle in A. castellanii.

Dictyostelium lacking the single atlastin homolog Sey1 shows aberrant ER architecture, proteolytic processes and expansion of the Legionella-containing vacuole.

Dictyostelium discoideum Sey1 is the single ortholog of mammalian atlastin 1-3 (ATL1-3), which are large homodimeric GTPases mediating homotypic fusion of endoplasmic reticulum (ER) tubules. In this study, we generated a D. discoideum mutant strain lacking the sey1 gene and found that amoebae deleted for sey1 are enlarged, but grow and develop similarly to the parental strain. The ∆sey1 mutant amoebae showed an altered ER architecture, and the tubular ER network was partially disrupted without any major consequences for other organelles or the architecture of the secretory and endocytic pathways. Macropinocytic and phagocytic functions were preserved; however, the mutant amoebae exhibited cumulative defects in lysosomal enzymes exocytosis, intracellular proteolysis, and cell motility, resulting in impaired growth on bacterial lawns. Moreover, ∆sey1 mutant cells showed a constitutive activation of the unfolded protein response pathway (UPR), but they still readily adapted to moderate levels of ER stress, while unable to cope with prolonged stress. In D. discoideum ∆sey1 the formation of the ER-associated compartment harbouring the bacterial pathogen Legionella pneumophila was also impaired. In the mutant amoebae, the ER was less efficiently recruited to the "Legionella-containing vacuole" (LCV), the expansion of the pathogen vacuole was inhibited at early stages of infection and intracellular bacterial growth was reduced. In summary, our study establishes a role of D. discoideum Sey1 in ER architecture, proteolysis, cell motility and intracellular replication of L. pneumophila.

Zinc metalloprotease ProA of Legionella pneumophila increases alveolar septal thickness in human lung tissue explants by collagen IV degradation.

ProA is a secreted zinc metalloprotease of Legionella pneumophila causing lung damage in animal models of Legionnaires' disease. Here we demonstrate that ProA promotes infection of human lung tissue explants (HLTEs) and dissect the contribution to cell type specific replication and extracellular virulence mechanisms. For the first time, we reveal that co-incubation of HLTEs with purified ProA causes a significant increase of the alveolar septal thickness. This destruction of connective tissue fibres was further substantiated by collagen IV degradation assays. The moderate attenuation of a proA-negative mutant in A549 epithelial cells and THP-1 macrophages suggests that effects of ProA in tissue mainly result from extracellular activity. Correspondingly, ProA contributes to dissemination and serum resistance of the pathogen, which further expands the versatile substrate spectrum of this thermolysin-like protease. The crystal structure of ProA at 1.48 Å resolution showed high congruence to pseudolysin of Pseudomonas aeruginosa, but revealed deviations in flexible loops, the substrate binding pocket S1 ' and the repertoire of cofactors, by which ProA can be distinguished from respective homologues. In sum, this work specified virulence features of ProA at different organisational levels by zooming in from histopathological effects in human lung tissue to atomic details of the protease substrate determination.

Development of ADPribosyl Ubiquitin Analogues to Study Enzymes Involved in Legionella Infection.

Legionnaires' disease is caused by infection with the intracellularly replicating Gram-negative bacterium Legionella pneumophila. This pathogen uses an unconventional way of ubiquitinating host proteins by generating a phosphoribosyl linkage between substrate proteins and ubiquitin by making use of an ADPribosylated ubiquitin (UbADPr ) intermediate. The family of SidE effector enzymes that catalyze this reaction is counteracted by Legionella hydrolases, which are called Dups. This unusual ubiquitination process is important for Legionella proliferation and understanding these processes on a molecular level might prove invaluable in finding new treatments. Herein, a modular approach is used for the synthesis of triazole-linked UbADPr , and analogues thereof, and their affinity towards the hydrolase DupA is determined and hydrolysis rates are compared to natively linked UbADPr . The inhibitory effects of modified Ub on the canonical eukaryotic E1-enzyme Uba1 are investigated and rationalized in the context of a high-resolution crystal structure reported herein. Finally, it is shown that synthetic UbADPr analogues can be used to effectively pull-down overexpressed DupA from cell lysate.

Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella-containing vacuole.

Legionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high-throughput RNAi screens against host membrane trafficking genes to identify factors that antagonise vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the ΔsdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilise the replication vacuole, EEA1 and Rab11FIP1 showed increased density about the sdhA mutant vacuole compared with the wild type (WT) vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant redistribution. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilise vacuole integrity during infection.

Cationic Porphyrins as Effective Agents in Photodynamic Inactivation of Opportunistic Plumbing Pathogen Legionella pneumophila.

Legionella pneumophila is an environmental bacterium, an opportunistic premise plumbing pathogen that causes the Legionnaires' disease. L. pneumophila presents a serious health hazard in building water systems, due to its high resistance to standard water disinfection methods. Our aim was to study the use of photodynamic inactivation (PDI) against Legionella. We investigated and compared the photobactericidal potential of five cationic dyes. We tested toluidine blue (TBO) and methylene blue (MB), and three 3-N-methylpyridylporphyrins, one tetra-cationic and two tri-cationic, one with a short (CH3) and the other with a long (C17H35) alkyl chain, against L. pneumophila in tap water and after irradiation with violet light. All tested dyes demonstrated a certain dark toxicity against L. pneumophila; porphyrins with lower minimal effective concentration (MEC) values than TBO and MB. Nanomolar MEC values, significantly lower than with TBO and MB, were obtained with all three porphyrins in PDI experiments, with amphiphilic porphyrin demonstrating the highest PDI activity. All tested dyes showed increasing PDI with longer irradiation (0-108 J/cm2), especially the two hydrophilic porphyrins. All three porphyrins caused significant changes in cell membrane permeability after irradiation and L. pneumophila, co-cultivated with Acanthamoeba castellanii after treatment with all three porphyrins and irradiation, did not recover in amoeba. We believe our results indicate the considerable potential of cationic porphyrins as effective anti-Legionella agents.

Chlorine and Monochloramine Disinfection of Legionella pneumophila Colonizing Copper and Polyvinyl Chloride Drinking Water Biofilms.

Building water systems promote the regrowth and survival of opportunistic pathogens, such as Legionella pneumophila, especially within biofilms, where most drinking water microbes reside. However, compared to their planktonic form, disinfection efficacy for the biofilm-associated forms of water-based pathogens is unclear. The aim of this study was to determine the effectiveness of free chlorine and monochloramine in the inactivation of biofilm-associated L. pneumophila strain Philadelphia-1 serogroup 1 (LpP1s1). Mature (1.5- to 2-year-old) drinking water biofilms were developed on copper (Cu) and polyvinyl chloride (PVC) slides within biofilm annular reactors, then colonized with LpP1s1 at approximately 4 log10 CFU cm-2 and exposed to 2 mg liter-1 of free chlorine or monochloramine. Ct (disinfectant concentration × time, expressed as mg min liter-1) inactivation values for 2-, 3-, and 4-log10 reductions of planktonic and biofilm LpP1s1 were determined. For planktonic LpP1s1, free chlorine was more effective at inactivation than was monochloramine treatment, and for biofilm-associated LpP1s1, monochloramine was more effective on Cu biofilms while free chlorine was more effective on PVC biofilms. In contrast to monochloramine, free chlorine treatment of Cu and PVC biofilms, negatively impacted LpP1s1 16S rRNA gene transcript levels and may act synergistically with Cu surfaces to further reduce transcript levels. Moreover, LpP1s1 cells shed from biofilms into the bulk water were more resistant to disinfection than were prepared planktonic LpP1s1 cells. Results from this study indicate that biofilm association, disinfectant type, and substratum play an important role in the survival of Legionella pneumophila in building water systems.IMPORTANCE Microbial regrowth within building water systems are promoted by water stagnation, low disinfectant residual, high surface-to-volume ratio, amenable growth temperatures, and colonization of drinking water biofilms. Moreover, biofilms provide protection from environmental stresses, access to higher levels of nutrients, and opportunities for symbiotic interactions with other microbes. Disinfectant efficacy information is historically based on inactivation of pathogens in their planktonic, free-floating forms. However, due to the ecological importance of drinking water biofilms for pathogen survival, this study evaluated the efficacy of two common disinfectants, free chlorine and monochloramine, on Legionella pneumophila colonizing mature, drinking water biofilms established on copper and PVC surfaces. Results showed that inactivation was dependent on the disinfectant type and biofilm substratum. Overall, this, and other related research, will provide a better understanding of Legionella ecological stability and survival and aid policy makers in the management of exposure risks to water-based pathogens within building water systems.

LotA, a Legionella deubiquitinase, has dual catalytic activity and contributes to intracellular growth.

The intracellular bacterial pathogen, Legionella pneumophila, establishes the replicative niche as a result of the actions of a large array of effector proteins delivered via the Legionella Type 4 secretion system. Many effector proteins are expected to be involved in biogenesis and regulation of the Legionella-containing vacuole (LCV) that is highly decorated with ubiquitin. Here, we identified a Legionella deubiquitinase, designated LotA, by carrying out a genome analysis to find proteins resembling the eukaryotic ovarian tumour superfamily of cysteine proteases. LotA exhibits a dual ability to cleave ubiquitin chains that is dependent on 2 distinctive catalytic cysteine residues in the eukaryotic ovarian tumour domains. One cysteine dominantly contributes to the removal of ubiquitin from the LCVs by its polyubiquitin cleavage activity. The other specifically cleaves conjugated Lys6-linked ubiquitin. After delivered by the Type 4 secretion system, LotA localises on the LCVs via its PI(3)P-binding domain. The lipid-binding ability of LotA is crucial for ubiquitin removal from the vacuoles. We further analysed the functional interaction of the protein with the recently reported noncanonical ubiquitin ligases of L. pneumophila, revealing that the effector proteins are involved in coordinated regulation that contributes to bacterial growth in the host cells.

ER remodeling by the large GTPase atlastin promotes vacuolar growth of Legionella pneumophila.

The pathogenic bacterium Legionella pneumophila replicates in host cells within a distinct ER-associated compartment termed the Legionella-containing vacuole (LCV). How the dynamic ER network contributes to pathogen proliferation within the nascent LCV remains elusive. A proteomic analysis of purified LCVs identified the ER tubule-resident large GTPase atlastin3 (Atl3, yeast Sey1p) and the reticulon protein Rtn4 as conserved LCV host components. Here, we report that Sey1/Atl3 and Rtn4 localize to early LCVs and are critical for pathogen vacuole formation. Sey1 overproduction promotes intracellular growth of L. pneumophila, whereas a catalytically inactive, dominant-negative GTPase mutant protein, or Atl3 depletion, restricts pathogen replication and impairs LCV maturation. Sey1 is not required for initial recruitment of ER to PtdIns(4)P-positive LCVs but for subsequent pathogen vacuole expansion. GTP (but not GDP) catalyzes the Sey1-dependent aggregation of purified, ER-positive LCVs in vitro Thus, Sey1/Atl3-dependent ER remodeling contributes to LCV maturation and intracellular replication of L. pneumophila.

KKL-35 Exhibits Potent Antibiotic Activity against Legionella Species Independently of trans-Translation Inhibition.

trans-Translation is a ribosome-rescue system that is ubiquitous in bacteria. Small molecules defining a new family of oxadiazole compounds that inhibit trans-translation have been found to have broad-spectrum antibiotic activity. We sought to determine the activity of KKL-35, a potent member of the oxadiazole family, against the human pathogen Legionella pneumophila and other related species that can also cause Legionnaires' disease (LD). Consistent with the essential nature of trans-translation in L. pneumophila, KKL-35 inhibited the growth of all tested strains at submicromolar concentrations. KKL-35 was also active against other LD-causing Legionella species. KKL-35 remained equally active against L. pneumophila mutants that have evolved resistance to macrolides. KKL-35 inhibited the multiplication of L. pneumophila in human macrophages at several stages of infection. No resistant mutants could be obtained, even during extended and chronic exposure. Surprisingly, KKL-35 was not synergistic with other ribosome-targeting antibiotics and did not induce the filamentation phenotype observed in cells defective for trans-translation. Importantly, KKL-35 remained active against L. pneumophila mutants expressing an alternate ribosome-rescue system and lacking transfer-messenger RNA, the essential component of trans-translation. These results indicate that the antibiotic activity of KKL-35 is not related to the specific inhibition of trans-translation and its mode of action remains to be identified. In conclusion, KKL-35 is an effective antibacterial agent against the intracellular pathogen L. pneumophila with no detectable resistance development. However, further studies are needed to better understand its mechanism of action and to assess further the potential of oxadiazoles in treatment.

Secreted phospholipases of the lung pathogen Legionella pneumophila.

Legionella pneumophila is an intracellular pathogen and the main causative agent of Legionnaires' disease, a potentially fatal pneumonia. The bacteria infect both mammalian cells and environmental hosts, such as amoeba. Inside host cells, the bacteria withstand the multifaceted defenses of the phagocyte and replicate within a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). For establishment and maintenance of the infection, L. pneumophila secretes many proteins including effector proteins by means of different secretion systems and outer membrane vesicles. Among these are a large variety of lipolytic enzymes which possess phospholipase/lysophospholipase and/or glycerophospholipid:cholesterol acyltransferase activities. Secreted lipolytic activities may contribute to bacterial virulence, for example via modification of eukaryotic membranes, such as the LCV. In this review, we describe the secretion systems of L. pneumophila, introduce the classification of phospholipases, and summarize the state of the art on secreted L. pneumophila phospholipases. We especially highlight those enzymes secreted via the type II secretion system Lsp, via the type IVB secretion system Dot/Icm, via outer membrane vesicles, and such where the mode of secretion has not yet been defined. We also give an overview on the complexity of their activities, activation mechanisms, localization, growth-phase dependent abundance, and their role in infection.

Primary Colonizing Betaproteobacteriales Play a Key Role in the Growth of Legionella pneumophila in Biofilms on Surfaces Exposed to Drinking Water Treated by Slow Sand Filtration.

Slow sand filtration with extensive pretreatment reduces the microbial growth potential of drinking water to a minimum level at four surface water supplies in The Netherlands. The potential of these slow sand filtrates (SSFs) to promote microbial growth in warm tap water installations was assessed by measuring biofilm formation and growth of Legionella bacteria on glass and chlorinated polyvinylchloride (CPVC) surfaces exposed to SSFs at 37 ± 2°C in a model system for up to six months. The steady-state biofilm concentration ranged from 230 to 3,980 pg ATP cm-2 on glass and 1.4 (±0.3)-times-higher levels on CPVC. These concentrations correlated significantly with the assimilable organic carbon (AOC) concentrations of the warm water (8 to 24 µg acetate-C equivalents [ac-C eq] liter-1), which were raised about 2 times by mixing cold and heated (70°C) SSFs. All biofilms supported growth of Legionella pneumophila with maximum concentrations ranging from 6 × 102 to 1.5 × 105 CFU cm-2 Biofilms after ≤50 days of exposure were predominated by Betaproteobacteriales, mainly Piscinibacter, Caldimonas, Methyloversatilis, and an uncultured Rhodocyclaceae bacterium. These rapidly growing primary colonizers most likely served as prey for the host amoebae of L. pneumophilaAlphaproteobacteria, mostly Xanthobacteraceae, e.g., Bradyrhizobium, Pseudorhodoplanes, and other amoeba-resistant bacteria, accounted for 37.5% of the clones retrieved. A conceptual model based on a quadratic relationship between the L. pneumophila colony count and the biofilm concentration under steady-state conditions is used to explain the variations in the Legionella CFU pg-1 ATP ratios in the biofilms.IMPORTANCE Proliferation of L. pneumophila in premise plumbing poses a public health threat. Extended water treatment using physicochemical and biofiltration processes, including slow sand filtration, at four surface water supplies in The Netherlands reduces the microbial growth potential of the treated water to a minimum level, and the distributed drinking water complies with high quality standards. However, heating of the water in warm tap water installations increases the concentration of easily assimilable organic compounds, thereby promoting biofilm formation and growth of L. pneumophila Prevention of biofilm formation in plumbing systems by maintenance of a disinfectant residual during distribution and/or further natural organic matter (NOM) removal is not feasible in the supplies studied. Temperature management in combination with optimized hydraulics and material selection are therefore essential to prevent growth of L. pneumophila in premise plumbing systems. Still, reducing the concentration of biodegradable compounds in drinking water by appropriate water treatment is important for limiting the Legionella growth potential.

Persistent presence of outer membrane epitopes during short- and long-term starvation of five Legionella pneumophila strains.

BACKGROUND: Legionella pneumophila, the causative agent of Legionnaire's disease, may enter a viable but non-culturable (VBNC) state triggered by environmental stress conditions. Specific outer-membrane epitopes of L. pneumophila are used in many diagnostic applications and some of them are linked to important virulence-related factors or endotoxins. However, it is not clear how the presence and status of these epitopes are influenced by environmental stress conditions. In this study, changes of outer membrane epitopes for monoclonal antibodies (mAb) from the Dresden panel and the major outer membrane protein MOMP were analysed for five L. pneumophila strains during short- and long-term starvation in ultrapure water. RESULTS: With ELISA and single cell immuno-fluorescence analysis, we could show that for most of the investigated mAb-strain combinations the total number of mAb-stained Legionella cells stayed constant for up to 400 days. Especially the epitopes of mAb 3/1, 8/5, 26/1 and 20/1, which are specific for L. pneumophila serogroup 1 subtypes, and the mAb 9/1, specific for serogroup 6, showed long-term persistence. For most mAb- stained cells, a high percentage of viable cells was observed at least until 118 days of starvation. At the same time, we observed a reduction of the fluorescence intensity of the stained cells during starvation indicating a loss of epitopes from the cell surface. However, most of the epitopes, including the virulence-associated mAb 3/1 epitope were still present with high fluorescence intensity after 400 days of starvation in up to 50% of the starved L. pneumophila population. CONCLUSIONS: The results demonstrate the continuous presence of outer membrane epitopes of L. pneumophila during short-term and long-term starvation. Thus, culture-independent mAb-based diagnostic and detection tools, such as immuno-magnetic separation and microarray techniques are applicable for both L. pneumophila in the culturable and the VBNC state even after long-term starvation but nevertheless require careful testing before application. However, the mere presence of those epitopes is not necessarily an indication of viability or infectivity.

Rapid detection of viable Legionella pneumophila in tap water by a qPCR and RT-PCR-based method.

AIMS: A molecular method for a rapid detection of viable Legionella pneumophila of all serogroups in tap water samples was developed as an alternative to the reference method (ISO). Legionellae are responsible for Legionnaires' disease, a severe pneumonia in humans with high lethality. METHODS AND RESULTS: The developed method is based on a nutritional stimulation and detection of an increase in precursor 16S rRNA as an indicator for viability. For quantification, DNA was detected by qPCR. This method was compared to the ISO method using water samples obtained from public sports facilities in Switzerland. The sensitivity and specificity were 91 and 97%, respectively, when testing samples for compliance with a microbiological criterion of 1000 cell equivalents per l. CONCLUSION: The new method is sensitive and specific for Leg. pneumophila and allows results to be obtained within 8 h upon arrival, compared to one week or more by the ISO method. SIGNIFICANCE AND IMPACT OF THE STUDY: The method represents a useful tool for a rapid detection of viable Leg. pneumophila of all serogroups in water by molecular biology. It can be used as an alternative to the ISO method for official water analysis for legionellae and particularly when a short test time is required.

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors.

Major differences in the transcriptional program underlying the phenotypic switch between exponential and post-exponential growth of Legionella pneumophila were formerly described characterizing important alterations in infection capacity. Additionally, a third state is known where the bacteria transform in a viable but nonculturable state under stress, such as starvation. We here describe phase-related proteomic changes in exponential phase (E), postexponential phase (PE) bacteria, and unculturable microcosms (UNC) containing viable but nonculturable state cells, and identify phase-specific proteins. We present data on different bacterial subproteomes of E and PE, such as soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins. In total, 1368 different proteins were identified, 922 were quantified and 397 showed differential abundance in E/PE. The quantified subproteomes of soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins; 841, 55, and 77 proteins, respectively, were visualized in Voronoi treemaps. 95 proteins were quantified exclusively in E, such as cell division proteins MreC, FtsN, FtsA, and ZipA; 33 exclusively in PE, such as motility-related proteins of flagellum biogenesis FlgE, FlgK, and FliA; and 9 exclusively in unculturable microcosms soluble whole cell proteins, such as hypothetical, as well as transport/binding-, and metabolism-related proteins. A high frequency of differentially abundant or phase-exclusive proteins was observed among the 91 quantified effectors of the major virulence-associated protein secretion system Dot/Icm (> 60%). 24 were E-exclusive, such as LepA/B, YlfA, MavG, Lpg2271, and 13 were PE-exclusive, such as RalF, VipD, Lem10. The growth phase-related specific abundance of a subset of Dot/Icm virulence effectors was confirmed by means of Western blotting. We therefore conclude that many effectors are predominantly abundant at either E or PE which suggests their phase specific function. The distinct temporal or spatial presence of such proteins might have important implications for functional assignments in the future or for use as life-stage specific markers for pathogen analysis.

Deletion of oxyR in Legionella pneumophila causes growth defect on agar.

The intracellular pathogen Legionella pneumophila (Lp) is a strict aerobe, surviving and replicating in environments where it frequently encounters reactive oxygen species (ROS), such as the nutrient-poor water environment and its replicative niche inside host cells. In many proteobacteria, the LysR-type regulator OxyR controls the oxidative stress response; however, the importance of the OxyR homologue in Lp is still unclear. Therefore, we undertook the characterization of phenotypes associated with the deletion of oxyR in Lp. Contrary to the wild type, the oxyR deletion mutant exhibits a severe growth defect on charcoal - yeast extract (CYE) agar lacking α-ketoglutarate supplementation. Growth in AYE broth (CYE without agar and charcoal), in amoeba and in human cultured macrophages, and survival in water is unaffected by the deletion. Supplementing CYE agar with antioxidants that neutralize ROS or introducing the oxyR gene in trans rescues the observed growth defect. Moreover, the mutant grows as well as the wild type on CYE plates made with agarose instead of agar, suggesting that a compound present in the latter is responsible for the growth defect phenotype.

MALDI-TOF MS analysis as a useful tool for an identification of Legionella pneumophila, a facultatively pathogenic bacterium interacting with free-living amoebae: A case study from water supply system of hospitals in Bratislava (Slovakia).

Legionellae, i.e. Legionella pneumophila, are human bacterial hydrophilic facultative pathogens causing pneumonia (Legionnaires' disease). Free-living amoebae (FLA) can serve as natural hosts and thus as reservoirs of many amoebae-resistant bacteria. An encysted amoeba can contribute to the resistance of intracellular L. pneumophila to various chemical and physical treatments. Humans can be infected by droplets containing bacteria from an environmental source or human-made devices such as shower heads, bathtubs, air-conditioning units or whirlpools. In this study, we were investigating the presence of FLA and L. pneumophila in plumbing systems of healthcare facilities in Bratislava (Slovakia) by standard diagnostic methods, while the presence of L. pneumophila was verified also by MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis. The results showed the occurrence of L. pneumophila and FLA in 62.26% and 66.4% of samples taken from four paediatric clinics, respectively. Both standard methods and MALDI-TOF MS showed comparable results and they can be successfully applied for the identification of L. pneumophila strains in environmental samples. Our approach could be useful for further monitoring, prevention and decreasing risk of Legionella infection also in other hospitals.

Growth-related Metabolism of the Carbon Storage Poly-3-hydroxybutyrate in Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaires disease, has a biphasic life cycle with a switch from a replicative to a transmissive phenotype. During the replicative phase, the bacteria grow within host cells in Legionella-containing vacuoles. During the transmissive phenotype and the postexponential (PE) growth phase, the pathogens express virulence factors, become flagellated, and leave the Legionella-containing vacuoles. Using (13)C labeling experiments, we now show that, under in vitro conditions, serine is mainly metabolized during the replicative phase for the biosynthesis of some amino acids and for energy generation. During the PE phase, these carbon fluxes are reduced, and glucose also serves as an additional carbon substrate to feed the biosynthesis of poly-3-hydroxybuyrate (PHB), an essential carbon source for transmissive L. pneumophila. Whole-cell FTIR analysis and comparative isotopologue profiling further reveal that a putative 3-ketothiolase (Lpp1788) and a PHB polymerase (Lpp0650), but not enzymes of the crotonyl-CoA pathway (Lpp0931-0933) are involved in PHB metabolism during the PE phase. However, the data also reflect that additional bypassing reactions for PHB synthesis exist in agreement with in vivo competition assays using Acanthamoeba castellannii or human macrophage-like U937 cells as host cells. The data suggest that substrate usage and PHB metabolism are coordinated during the life cycle of the pathogen.

Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila.

We established a new Brucella neotomaein vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus, B. melitensis, and B. suis, B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila, we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitroBrucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection.

The α-hydroxyketone LAI-1 regulates motility, Lqs-dependent phosphorylation signalling and gene expression of Legionella pneumophila.

The causative agent of Legionnaires' disease, Legionella pneumophila, employs the autoinducer compound LAI-1 (3-hydroxypentadecane-4-one) for cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, comprising the autoinducer synthase LqsA, the sensor kinases LqsS and LqsT, as well as the response regulator LqsR. Lqs-regulated processes include pathogen-host interactions, production of extracellular filaments and natural competence for DNA uptake. Here we show that synthetic LAI-1 promotes the motility of L. pneumophila by signalling through LqsS/LqsT and LqsR. Upon addition of LAI-1, autophosphorylation of LqsS/LqsT by [γ-(32) P]-ATP was inhibited in a dose-dependent manner. In contrast, the Vibrio cholerae autoinducer CAI-1 (3-hydroxytridecane-4-one) promoted the phosphorylation of LqsS (but not LqsT). LAI-1 did neither affect the stability of phospho-LqsS or phospho-LqsT, nor the dephosphorylation by LqsR. Transcriptome analysis of L. pneumophila treated with LAI-1 revealed that the compound positively regulates a number of genes, including the non-coding RNAs rsmY and rsmZ, and negatively regulates the RNA-binding global regulator crsA. Accordingly, LAI-1 controls the switch from the replicative to the transmissive growth phase of L. pneumophila. In summary, the findings indicate that LAI-1 regulates motility and the biphasic life style of L. pneumophila through LqsS- and LqsT-dependent phosphorylation signalling.