LAMP-based method for a rapid identification of Legionella spp. and Legionella pneumophila

Legionella pneumophila is accounted for more than 80% of Legionella infection. However it is difficult to discriminate between the L. pneumophila and non-L. pneumophila species rapidly. In order to detect the Legionella spp. and distinguish L. pneumophila from Legionella spp., a real-time loop-mediated isothermal amplification (LAMP) platform that targets a specific sequence of the 16S rRNA gene was developed. LS-LAMP amplifies the fragment of the 16S rRNA gene to detect all species of Legionella genus. A specific sequence appears at the 16S rRNA gene of L. pneumophila, while non-L. pneumophila strains have a variable sequence in this site, which can be recognized by the primer of LP-LAMP. In the present study, 61 reference strains were used for the method verification. We found that the specificity was 100% for both LS-LAMP and LP-LAMP, and the sensitivity of LAMP assay for L. pneumophila detection was between 52 and 5.2 copies per reaction. In the environmental water samples detection, a total of 107 water samples were identified by the method. The culture and serological test were used as reference methods. The specificity of LS-LAMP and LP-LAMP for the samples detection were 91.59% (98/107) and 93.33% (56/60), respectively. The sensitivity of LS-LAMP and LP-LAMP were 100% (51/51) and 100% (18/18). The results suggest that real-time LAMP, as a new assay, provides a specific and sensitive method for rapid detection and differentiation of Legionella spp. and L. pneumophila and should be utilized to test environmental water samples for increased rates of detection.     

Entry of poliovirus into cells does not require a low-pH step.

The requirement of a low-pH step during poliovirus entry was investigated by using the macrolide antibiotic bafilomycin A1, which is a powerful and selective inhibitor of the vacuolar proton-ATPases. Thus, viruses such as Semliki Forest virus and vesicular stomatitis virus that enter cells through endosomes and need their acidification, are potently inhibited by bafilomycin A1, whereas poliovirus infection is not affected by the antibiotic. The presence of lysosomotropic agents such as chloroquine, amantadine, dansylcadaverine, and monensin during poliovirus entry did not inhibit infection, further supporting the idea that poliovirus does not depend on a low-pH step to enter the cytoplasm. The effect of bafilomycin A1 on other members of the Picornaviridae family was also assayed. Encephalomyocarditis virus entry into HeLa cells was not affected by the macrolide antibiotic, whereas rhinovirus was sensitive. Coentry of toxins, such as alpha-sarcin, with viral particles was potently inhibited by bafilomycin A1, indicating that an active vacuolar proton-ATPase is necessary for the early membrane permeabilization (coentry of alpha-sarcin) induced by poliovirus to take place.     

Dynamics of Legionella spp. and bacterial populations during the proliferation of L. pneumophila in a cooling tower facility

The dynamics of Legionella spp. and of dominant bacteria were investigated in water from a cooling tower plant over a 9-month period which included several weeks when Legionella pneumophila proliferated. The structural diversity of both the bacteria and the Legionella spp. was monitored by a fingerprint technique, single-strand conformation polymorphism, and Legionella spp. and L. pneumophila were quantified by real-time quantitative PCR. The structure of the bacterial community did not change over time, but it was perturbed periodically by chemical treatment or biofilm detachment. In contrast, the structure of the Legionella sp. population changed in different periods, its dynamics at times showing stability but also a rapid major shift during the proliferation of L. pneumophila in July. The dynamics of the Legionella spp. and of dominant bacteria were not correlated. In particular, no change in the bacterial community structure was observed during the proliferation of L. pneumophila. Legionella spp. present in the cooling tower system were identified by cloning and sequencing of 16S rRNA genes. A high diversity of Legionella spp. was observed before proliferation, including L. lytica, L. fallonii, and other Legionella-like amoebal pathogen types, along with as-yet-undescribed species. During the proliferation of L. pneumophila, Legionella sp. diversity decreased significantly, L. fallonii and L. pneumophila being the main species recovered.     

Detection of Legionella spp. and Legionella pneumophila in water samples of Spain by specific real-time PCR

Legionella pneumophila is the primary cause of the legionellosis diseases (90 %) (Yu et al. in J Infect Dis 186:127–128, 2002; Doleans et al. in J Clin Microbiol 42:458–460, 2004; Den Boer et al. in Clin Microbiol Infect 14:459–466, 2008). In this study, methodologies based on molecular biology were developed in order to provide a quick diagnosis of the bacterial presence in water samples of Spain. Multiplex real-time polymerase chain reaction assays were realized to target the 16S rRNA and macrophage infectivity potentiator (mip) genes of, respectively, Legionella spp. and L. pneumophila including in the design of an internal control. The results obtained by the culture and the gene amplification methods agreed in 94.44 % for the 16S rRNA gene, and a concordance of 66.67 % of the cases was obtained for the mip gene.     

The Legionella pneumophila LidA protein: a translocated substrate of the Dot/Icm system associated with maintenance of bacterial integrity

Legionella pneumophila establishes a replication vacuole within phagocytes that requires the bacterial Dot/Icm apparatus for its formation. This apparatus is predicted to translocate effectors into host cells. We hypothesized that some translocated proteins also function to maintain the integrity of the Dot/Icm translocator. Mutations that destroy this function are predicted to result in a Dot/Icm complex that poisons the bacterium, resulting in reduced viability. To identify such mutants, strains were isolated (called lid-) that showed reduced viability on bacteriological medium in the presence of an intact Dot/Icm apparatus, but which had high viability in the absence of the translocator. Several such mutants were analysed in detail to identify candidate strains that may have lost the ability to synthesize a translocated substrate of Dot/Icm. Two such strains had mutations in the lidA gene. The LidA protein exhibits properties expected for a translocated substrate of Dot/Icm that is important for maintenance of bacterial cell integrity: it associates with the phagosomal surface, promotes replication vacuole formation, and is important for both efficient intracellular growth and high viability on bacteriological media after introduction of a plasmid that allows high level expression of the dotA gene.     

The DotA protein from Legionella pneumophila is secreted by a novel process that requires the Dot/Icm transporter.

Legionella pneumophila requires the dot/icm genes to create an organelle inside eukaryotic host cells that will support bacterial replication. The dot/icm genes are predicted to encode a type IV-related secretion apparatus. However, no proteins have been identified that require the dot/icm genes for secretion. In this study we show that the DotA protein, which was previously found to be a polytopic membrane protein, is secreted by the Dot/Icm transporter into culture supernatants. Secreted DotA protein was purified and N-terminal sequencing of the purified protein revealed that a 19 amino acid leader peptide is removed from DotA prior to secretion. Extracellular DotA protein did not fractionate in membrane vesicles. Structures containing secreted DotA protein were visualized by electron microscopy and were shaped like hollow rings. These data indicate that the large poly topic membrane protein DotA is secreted from L.pneumophila by a unique process. This represents the first target secreted by the dot/icm-encoded apparatus and demonstrates that this transporter is competent for protein secretion.     

Modulation of ubiquitin dynamics and suppression of DALIS formation by the Legionella pneumophila Dot/Icm system

Legionella pneumophila is an intracellular pathogen that uses effector proteins translocated by the Dot/Icm type IV secretion system to modulate host cellular processes. Here we investigate the dynamics of subcellular structures containing ubiquitin during L. pneumophila infection of phagocytic host cells. The Dot/Icm system mediated the formation of K48 and K63 poly-ubiquitin conjugates to proteins associated with L. pneumophila -containing vacuoles in macrophages and dendritic cells, suggesting that regulatory events and degradative events involving ubiquitin are regulated by bacterial effectors during infection. Stimulation of TLR2 on the surface of macrophages and dendritic cells by L. pneumophila- derived molecules resulted in the production of ubiquitin-rich dendritic cell aggresome-like structures (DALIS). Cells infected by L. pneumophila with a functional Dot/Icm system, however, failed to produce DALIS. Suppression of DALIS formation did not affect the accumulation of ubiquitinated proteins on vacuoles containing L. pneumophila. Examining other species of Legionella revealed that Legionella jordanis was unable to suppress DALIS formation after creating a ubiquitin-decorated vacuole. Thus, the L. pneumophila Dot/Icm system has the ability to modulate host processes to promote K48 and K63 ubiquitin conjugates on proteins at the vacuole membrane, and independently suppress cellular events required for the formation of DALIS.     

Initiation of adenovirus DNA replication does not occur via a hairpin mechanism.

Models have been proposed suggesting that initiation of adenovirus DNA replication might occur via a hairpin mechanism. A consequence of the models is a covalent linkage of progeny and parental DNA in newly completed molecules. Analysis of mature molecules from KB cells infected with adenovirus type 5 indicates that, if a hairpin mechanism is involved, the length of the hairpin must be shorter than 50 basepairs long. Recent nucleotide sequence analysis of the termini of adenovirus type 5 DNA (P.H.Steenbergh et al. (1977) Nucl. Acids Res. 4, 4371-4389) has shown that a hairpin of this size does not exist and that therefore a hairpin mechanism is unlikely.     

Induction of apoptosis by Sindbis virus occurs at cell entry and does not require virus replication

Sindbis virus (SV) is an alphavirus that causes encephalitis in mice and can lead to the apoptotic death of infected cells. To determine the step in virus replication during which apoptosis is triggered, we used UV-inactivated SV, chemicals that block virus fusion or protein synthesis, and cells that do and do not express heparan sulfate, the initial binding molecule for SV infection of many cells. In initial experiments, UV-inactivated neuroadapted SV (NSV) induced apoptosis in Chinese hamster ovary (CHO) cells lacking heparan sulfate in the presence of cycloheximide. When fusion of prebound UV-inactivated NSV was rapidly induced at the plasma membrane by exposure to acidic pH, apoptosis was induced in CHO cells with or without heparan sulfate in the presence or absence of cycloheximide in a virus dose-dependent manner. In N18 neuroblastoma cells, the relative virulence of the virus strain was an important determinant of apoptosis induced by UV-inactivated SV. Treatment of N18 cells with monensin to prevent endosomal acidification an hour before, but not 2 h after, exposure to live NSV blocked the induction of cell death, as did treatment with NH(4)Cl or bafilomycin A1. These studies indicate that SV can induce apoptosis at the time of fusion with the cell membrane and that virus replication is not required.     

A yeast genetic system for the identification and characterization of substrate proteins transferred into host cells by the Legionella pneumophila Dot/Icm system

The Dot/Icm system is a type IVb secretion system used by Legionella pneumophila to modulate vesicular transport in both protozoan and mammalian host cells. It has been shown that proteins and processes that are highly conserved in all eukaryotic cells are targets for some of the proteins injected by the Dot/Icm system. For example, the Legionella protein RalF was shown previously to be a Dot/Icm substrate that functions as a guanine nucleotide exchange factor (GEF) for the Arf family of eukaryotic small GTP-binding proteins. Here we show that ectopic production of the RalF protein in Saccharomyces cerevisiae interferes with yeast growth. Inhibition of yeast growth was found to be dependent on the ability of RalF to function as an Arf-GEF in vivo. The possibility that other Dot/Icm substrate proteins would have the capacity to interfere with yeast growth was used as a rationale to screen plasmid libraries containing random fragments of Legionella chromosomal DNA positioned downstream of a galactose-inducible promoter. This screen identified Legionella proteins that conferred a conditional growth defect when overproduced by yeast cultured in the presence of galactose. Most of the Legionella proteins identified were determined to be substrates of the Dot/Icm system. This screen led to the identification of a new Dot/Icm substrate protein that was called YlfA, for yeast lethal factor A. A paralogue of YlfA was identified on an unlinked region of the Legionella chromosome and this protein was also translocated by the Dot/Icm system. It was determined that a hydrophobic region near the N-terminus of the YlfA protein and an adjacent region predicted to form a coiled-coil domain were necessary for a biological activity that interfered with yeast growth. The YlfA protein did not decorate the Legionella-containing vacuole during the first 7 h of infection but could be observed on the endoplasmic reticulum (ER)-derived replicative vacuole and on punctate structures throughout the host cell at later stages. Ectopic production of YlfA in mammalian cells revealed that the N-terminal hydrophobic domain in YlfA was able to localize the protein to early secretory organelles, including endoplasmic reticulum. These studies show that yeast genetics can be exploited to identify and characterize proteins that are injected into host cells by bacterial pathogens that utilize type IV secretion systems for pathogenesis.     

Usefulness of real-time PCR as a complementary tool to the monitoring of Legionella spp. and Legionella pneumophila by culture in industrial cooling systems

Aims: This study was designed to evaluate the usefulness of quantification by real‐time PCR as a management tool to monitor concentrations of Legionella spp. and Legionella pneumophila in industrial cooling systems and its ability to anticipate culture trends by the French standard method (AFNOR T90‐431). Methods and Results: Quantifications of Legionella bacteria were achieved by both methods on samples from nine cooling systems with different water qualities. Proportion of positive samples for L. pneumophila quantified by PCR was clearly lower in deionized or river waters submitted to a biocide treatment than in raw river waters, while positive samples for Legionella spp. were quantified for almost all the samples. For some samples containing PCR inhibitors, high quantification limits (up to 4·80 × 10⁵ GU l^?1) did not allow us to quantify L. pneumophila, when they were quantified by culture. Finally, the monitoring of concentrations of L. pneumophila by both methods showed similar trends for 57–100% of the samples. Conclusions: These results suggest that, if some methodological steps designed to reduce inhibitory problems and thus decrease the quantification limits, could be developed to quantify Legionella in complex waters, the real‐time PCR could be a valuable complementary tool to monitor the evolution of L. pneumophila concentrations. Significance and Impact of the Study: This study shows the possibility of using real‐time PCR to monitor L. pneumophila proliferations in cooling systems and the importance to adapt nucleic acid extraction and purification protocols to raw waters.     

The effect of water activity on Legionella spp. growth and survival

Growth and survival of Legionella spp. at various water activity (aw) levels were determined. Compared with Escherichia coli, the growth of Legionella spp. was limited to a high aw environment (greater than or equal to 0.98).     

The effect of water activity on Legionella spp. growth and survival

Growth and survival of Legionella spp. at various water activity (a_w) levels were determined. Compared with Escherichia coli , the growth of Legionella spp. was limited to a high a_w environment (≥0.98).     

Rapid detection and identification of Legionella pneumophila by a membrane immunoassay.

Legionella pneumophila was detected and identified by an immunoblot assay using a monoclonal antibody specific to serogroups 1 to 8. Samples containing L. pneumophila were plated on buffered charcoal yeast extract agar supplemented with glycine, vancomycin, and polymyxin B. After incubation at 35 degrees C for 3 days, colonies were transferred to nitrocellulose membranes by blotting. Simultaneous detection and identification of L. pneumophila were done by treating the membrane with the monoclonal antibody and a peroxidase conjugate to mouse immunoglobulins. A diffuse cross-reaction was observed with Pseudomonas fluorescens colonies, but this was a low-level reaction that could easily be differentiated from the strong specific reactions to L. pneumophila.     

The stimulation of heart glycolysis by increased workload does not require AMP-activated protein kinase but a wortmannin-sensitive mechanism

Increasing heart workload stimulates glycolysis by enhancing glucose transport and fructose-2,6-bisphosphate (Fru-2,6-P(2)), the latter resulting from 6-phosphofructo-2-kinase (PFK-2) activation. Here, we investigated whether adenosine monophosphate (AMP)-activated protein kinase (AMPK) mediates PFK-2 activation in hearts submitted to increased workload. When heart work was increased, PFK-2 activity, Fru-2,6-P(2) content and glycolysis increased, whereas the AMP:adenosine triphosphate (ATP) and phosphocreatine/creatine (PCr:Cr) ratios, and AMPK activity remained unchanged. Wortmannin, the well-known phosphatidylinositol-3-kinase inhibitor, blocked the activation of protein kinase B and the increase in glycolysis and Fru-2,6-P(2) content induced by increased work. Therefore, the control of heart glycolysis by contraction differs from that in skeletal muscle where AMPK is involved.     

Identification and Differentiation of Legionella pneumophila and Legionella spp. with Real-Time PCR Targeting the 16S rRNA Gene and Species Identification by mip Sequencing

Fluorescent resonance energy transfer probes targeting the 16S rRNA gene were constructed for a sensitive and specific real-time PCR for identification and differentiation of Legionella pneumophila from other Legionella spp. For identification of non-L. pneumophila spp. by direct amplicon sequencing, two conventional PCR assays targeting the mip gene were established.     

RCN1-regulated phosphatase activity and EIN2 modulate hypocotyl gravitropism by a mechanism that does not require ethylene signaling

The roots curl in naphthylphthalamic acid1 (rcn1) mutant of Arabidopsis (Arabidopsis thaliana) has altered auxin transport, gravitropism, and ethylene response, providing an opportunity to analyze the interplay between ethylene and auxin in control of seedling growth. Roots of rcn1 seedlings were previously shown to have altered auxin transport, growth, and gravitropism, while rcn1 hypocotyl elongation exhibited enhanced ethylene response. We have characterized auxin transport and gravitropism phenotypes of rcn1 hypocotyls and have explored the roles of auxin and ethylene in controlling these phenotypes. As in roots, auxin transport is increased in etiolated rcn1 hypocotyls. Hypocotyl gravity response is accelerated, although overall elongation is reduced, in etiolated rcn1 hypocotyls. Etiolated, but not light grown, rcn1 seedlings also overproduce ethylene, and mutations conferring ethylene insensitivity restore normal hypocotyl elongation to rcn1. Auxin transport is unaffected by treatment with the ethylene precursor 1-aminocyclopropane carboxylic acid in etiolated hypocotyls of wild-type and rcn1 seedlings. Surprisingly, the ethylene insensitive2-1 (ein2-1) and ein2-5 mutations dramatically reduce gravitropic bending in hypocotyls. However, the ethylene resistant1-3 (etr1-3) mutation does not significantly affect hypocotyl gravity response. Furthermore, neither the etr1 nor the ein2 mutation abrogates the accelerated gravitropism observed in rcn1 hypocotyls, indicating that both wild-type gravity response and enhanced gravity response in rcn1 do not require an intact ethylene-signaling pathway. We therefore conclude that the RCN1 protein affects overall hypocotyl elongation via negative regulation of ethylene synthesis in etiolated seedlings, and that RCN1 and EIN2 modulate hypocotyl gravitropism and ethylene responses through independent pathways.