Agent Of Legionnaires Research Articles

Assessing the impact, genomics and evolution of type II secretion across a large, medically important genus: the Legionella type II secretion paradigm.

The type II secretion system (T2SS) plays a major role in promoting bacterial survival in the environment and in human hosts. One of the best characterized T2SS is that of Legionella pneumophila, the agent of Legionnaires’ disease. Secreting at least 25 proteins, including degradative enzymes, eukaryotic-like proteins and novel effectors, this T2SS contributes to the ability of L. pneumophila to grow at low temperatures, infect amoebal and macrophage hosts, damage lung tissue, evade the immune system, and undergo sliding motility. The genes encoding the T2SS are conserved across the genus Legionella, which includes 62 species and >30 pathogens in addition to L. pneumophila. The vast majority of effectors associated with L. pneumophila are shared by a large number of Legionella species, hinting at a critical role for them in the ecology of Legionella as a whole. However, no other species has the same repertoire as L. pneumophila, with, as a general rule, phylogenetically more closely related species sharing similar sets of effectors. T2SS effectors that are involved in infection of a eukaryotic host(s) are more prevalent throughout Legionella, indicating that they are under stronger selective pressure. The Legionella T2SS apparatus is closest to that of Aquicella (another parasite of amoebae), and a significant number of L. pneumophila effectors have their closest homologues in Aquicella. Thus, the T2SS of L. pneumophila probably originated within the order Legionellales, with some of its effectors having arisen within that Aquicella-like progenitor, while other effectors derived from the amoebal host, mimiviruses, fungi and less closely related bacteria.

Search for <i>Legionella pneumophila</i> in domestic water system in Benin Metropolis

Legionella species, a Gram negative bacterium is the causative agent of Legionnaires’ disease, a potentially fatal pneumonic syndrome of widely recognized public health importance. The aim of the study was to determine the presence of Legionella pneumophila in domestic water (borehole) in Benin metropolis by cultural method. One hundred and ninety-eight (198) water samples from the eight-two facilities (grouped into three: public apartments/hotels, private apartments and eateries) were cultured on BCYE made selective with the addition of legionella supplement IV and growth supplement after concentration and heat treatment at 50 0 C for 30 minutes and incubated at 37 0 C. Isolates were identified by doing gram stain, oxidase, catalase and hippurate test, final identification was done by using PCR, sequence and blast search using National Center for Biotechnology Information (NCBI). The results obtained showed that Legionella pneumophila or Legionella species was not isolated though other bacteria (such as Burkholderia bacterium MSMB7 (32%), Pseudomonas antarctica (28%), Cupriavidus gilaridii (14%), Microbacterium paraoxydans (14%), Bacillus thuringensis (4%), Bacillus cereus (2%), Acinetobacter johnsonii (6%)) were detected. In conclusion zero percent (0%) prevalence of Legionella species in the water systems investigated though other bacteria with pathogenic potential were recovered. This finding suggests that water systems in Benin metropolis may not present vehicles for the transmission of diseases associated with Legionella species. Keywords: Legionella pneumophila , domestic water, search, BCYE, Benin metropolis

The large GTPase atlastin controls ER remodeling around a pathogen vacuole

ABSTRACTThe ubiquitous environmental bacterium Legionella pneumophila is the causative agent of Legionnaires' pneumonia and replicates in free-living protozoa and mammalian macrophages in a specific compartment, the Legionella-containing vacuole (LCV). LCVs communicate with the endosomal, retrograde and secretory vesicle trafficking pathway, and eventually tightly interact with the endoplasmic reticulum (ER). In Dictyostelium discoideum amoebae and macrophages, the ER tubule-resident large GTPase Sey1/atlastin3 (Atl3) accumulates on LCVs and promotes LCV expansion and intracellular replication of L. pneumophila. Fluorescence microscopy of D. discoideum infected with L. pneumophila indicated that Sey1 is involved in extensive ER remodeling around LCVs. An ultrastructural analysis confirmed these findings. Moreover, dominant negative Sey1_K154A compromises ER accumulation on LCVs and causes an aberrant ER morphology in uninfected D. discoideum as well as in amoebae infected with avirulent L. pneumophila that lack a functional type IV secretion system. Thus, the large, dynamin-like GTPase Sey1/Atl3 controls circumferential ER remodeling during LCV maturation.

Methods for NAD-Dependent Ubiquitination Catalyzed by Legionella pneumophila Effector Proteins.

Ubiquitination is one of the most important posttranslational modifications in eukaryotic cells where it regulates the activity, cellular localization, and half-life of proteins. Ubiquitination thus affects many essential cellular processes, including vesicle trafficking, cell cycle, DNA repair, immune response, and protein homeostasis. The ubiquitin system is exclusive to eukaryotes; however, pathogenic bacteria have developed effective strategies to influence the host ubiquitin system for their own benefit. Legionella pneumophila is the causative agent of Legionnaires' disease, a severe form of pneumonia. This bacterium utilizes a type IV secretion system to translocate more than 300 effector proteins into host cells. These virulence factors modulate a wide spectrum of host processes to support its intracellular survival and replication. Hijacking of host ubiquitin system is an important theme in Legionella virulence, and a number of L. pneumophila effector proteins have been shown to possess ubiquitin ligase or deubiquitinase activity. Among these, members of the SidE family effector proteins (SidEs) catalyze ubiquitination of several ER-associated Rab small GTPases by a mechanism that bypasses the requirement of ATP and the E1, E2 enzymes. Here, we summarize the experimental details of Rab small GTPases ubiquitination catalyzed by SdeA, a member of the SidE family.

The Legionella pneumophila Siderophore Legiobactin Is a Polycarboxylate That Is Identical in Structure to Rhizoferrin.

Legionella pneumophila, the agent of Legionnaires' disease, secretes a siderophore (legiobactin) that promotes bacterial infection of the lung. In past work, we determined that cytoplasmic LbtA (from Legiobactin gene A) promotes synthesis of legiobactin, inner membrane LbtB aids in export of the siderophore, and outer membrane LbtU and inner membrane LbtC help mediate ferrilegiobactin uptake and assimilation. However, the past studies examined legiobactin contained within bacterial culture supernatants. By utilizing high-pressure liquid chromatography that incorporates hydrophilic interaction-based chemistry, we have now purified legiobactin from supernatants of virulent strain 130b that is suitable for detailed chemical analysis. High-resolution mass spectrometry (MS) revealed that the molecular mass of (protonated) legiobactin is 437.140 Da. On the basis of the results obtained from both MS analysis and various forms of nuclear magnetic resonance, we found that legiobactin is composed of two citric acid residues linked by a putrescine bridge and thus is identical in structure to rhizoferrin, a polycarboxylate-type siderophore made by many fungi and several unrelated bacteria. Both purified legiobactin and rhizoferrin obtained from the fungus Cunninghamella elegans were able to promote Fe(3+) uptake by wild-type L. pneumophila as well as enhance growth of iron-starved bacteria. These results did not occur with 130b mutants lacking lbtU or lbtC, indicating that both endogenously made legiobactin and exogenously derived rhizoferrin are assimilated by L. pneumophila in an LbtU- and LbtC-dependent manner.

Looking at protists as a source of pathogenic viruses

In the environment, protozoa are predators of bacteria and feed on them. The possibility that some protozoa could be a source of human pathogens is consistent with the discovery that free-living amoebae were the reservoir of Legionella pneumophila, the agent of Legionnaires' disease. Later, while searching for Legionella in the environment using amoeba co-culture, the first giant virus, Acanthamoeba polyphaga mimivirus, was discovered. Since then, many other giant viruses have been isolated, including Marseilleviridae, Pithovirus sibericum, Cafeteria roenbergensis virus and Pandoravirus spp. The methods used to isolate all of these viruses are herein reviewed. By analogy to Legionella, it was originally suspected that these viruses could be human pathogens. After showing by indirect evidence, such as sero-epidemiologic studies, that it was possible for these viruses to be human pathogens, the recent isolation of some of these viruses (belonging to the Mimiviridae and Marseilleviridae families) in humans in the context of pathologic conditions shows that they are opportunistic human pathogens in some instances.

Quantitative Measurement ofLegionella PneumophilaCounts in Routinely Maintained Commercial and Industrial Cooling Towers

Cooling towers are associated with transmission of the bacterium Legionella, the agent of Legionnaires' Disease and Pontiac Fever. This study analyzed the numbers of Legionella pneumophila collecte...

Legionella pneumophila persists within biofilms formed by Klebsiella pneumoniae, Flavobacterium sp., and Pseudomonas fluorescens under dynamic flow conditions.

Legionella pneumophila, the agent of Legionnaires' disease pneumonia, is transmitted to humans following the inhalation of contaminated water droplets. In aquatic systems, L. pneumophila survives much of time within multi-organismal biofilms. Therefore, we examined the ability of L. pneumophila (clinical isolate 130b) to persist within biofilms formed by various types of aquatic bacteria, using a bioreactor with flow, steel surfaces, and low-nutrient conditions. L. pneumophila was able to intercalate into and persist within a biofilm formed by Klebsiella pneumoniae, Flavobacterium sp. or Pseudomonas fluorescens. The levels of L. pneumophila within these biofilms were as much as 4×104 CFU per cm2 of steel coupon and lasted for at least 12 days. These data document that K. pneumoniae, Flavobacterium sp., and P. fluorescens can promote the presence of L. pneumophila in dynamic biofilms. In contrast to these results, L. pneumophila 130b did not persist within a biofilm formed by Pseudomonas aeruginosa, confirming that some bacteria are permissive for Legionella colonization whereas others are antagonistic. In addition to colonizing certain mono-species biofilms, L. pneumophila 130b persisted within a two-species biofilm formed by K. pneumoniae and Flavobacterium sp. Interestingly, the legionellae were also able to colonize a two-species biofilm formed by K. pneumoniae and P. aeruginosa, demonstrating that a species that is permissive for L. pneumophila can override the inhibitory effect(s) of a non-permissive species.

Small Regulatory RNA and Legionella pneumophila

Legionella pneumophila is a gram-negative bacterial species that is ubiquitous in almost any aqueous environment. It is the agent of Legionnaires’ disease, an acute and often under-reported form of pneumonia. In mammals, L. pneumophila replicates inside macrophages within a modified vacuole. Many protein regulators have been identified that control virulence-related properties, including RpoS, LetA/LetS, and PmrA/PmrB. In the past few years, the importance of regulation of virulence factors by small regulatory RNA (sRNAs) has been increasingly appreciated. This is also the case in L. pneumophila where three sRNAs (RsmY, RsmZ, and 6S RNA) were recently shown to be important determinants of virulence regulation and 79 actively transcribed sRNAs were identified. In this review we describe current knowledge about sRNAs and their regulatory properties and how this relates to the known regulatory systems of L. pneumophila. We also provide a model for sRNA-mediated control of gene expression that serves as a framework for understanding the regulation of virulence-related properties of L. pneumophila.

Amoeba-Resistant Bacteria: Their Role in Human Infections

Free-living amoebae in water supply systems harbor bacterial endosymbionts. Legionella pneumophila, the agent of Legionnaires' disease, was one of the first agents that was demonstrated to survive and multiply within amoebae. Similar bacteria, termed Legionella-like amoebal pathogens (LLAP's), were subsequently described and implicated in human respiratory tract infections. Amoebal co-cultivation/enrichment techniques and molecular methods (e.g., PCR) have elucidated a variety of alphaproteobacteria (e.g., Afipia, Bosea, Mesorhizobium, and Rasbobacterium species) and Chlamydia-like bacteria (e.g., Parachlamydia, Simkania, Protochlamydia, and Waddlia species) within free-living amoebae in hospital water systems. These agents are also able to survive and multiply within macrophages, pneumocytes, and lung fibroblasts. Sero-epidemiological studies and application of molecular technology to clinical specimens have revealed these agents as rare causes of community- and health care-associated pneumonia, particularly among immunocompromised patients. Additional studies have implicated these agents in other infectious processes. This article reviews these newly described microorganisms and the evidence for their roles in human infections and disease.

Studies of environmental water samples for different parameters for isolation of Legionella spp.

Legionella pneumophila has frequently been isolated from patients with Legionnaires disease and in several instances also from epidemic-related environmental samples. A selective medium was developed and used successfully to isolate Legionella pneumophila and Legionella-like organisms from environmental specimens animal inoculation methods. Legionella pneumophila is a Gram-negative, facultative, intracellular bacterium that is the causative agent of Legionnaires' disease. This medium consists of charcoalyeast extract agar to which have been added cephalothin (4µg/mL), colistin (16µg/ mL), vancomycin (0.5µg/mL), and cycloheximide (80µg/mL). Pretreating of the environmental water samples with an acid buffer (pH 2.2), followed by plating on the selective medium, improved the rate of recovery of both Legionella and Legionella-like organisms relative to that with direct plating on selective media.

Mediators of Lipid A Modification, RNA Degradation, and Central Intermediary Metabolism Facilitate the Growth of Legionella pneumophila at Low Temperatures

Legionella pneumophila is an aquatic bacterium that is also the agent of Legionnaires' disease pneumonia. Since L. pneumophila is transmitted directly from the environment to the lung, it is important to understand how legionellae survive at low temperatures. To identify genes that are needed for L. pneumophila growth at low temperature, we screened a population of mutagenized legionellae for strains that are specifically impaired for growth at 17 degrees C. From the 7,400 mutants tested, 11 displayed defects ranging from ca. 10-fold to a complete inability to grow at the low temperature. PCR and sequence analysis were then utilized to identify the genes whose loss had compromised growth. The proteins thereby implicated in low-temperature growth included components of the type II secretion system (LspE, LspG, LspH), a lipid A biosynthetic enzyme (LpxP), a ribonuclease (RNAse R), an RNA helicase (CsdA/DeaD), TCA cycle enzymes (citrate synthase), enzymes linked to fatty acid (FadB) or amino acid (aspartate aminotransferase) catabolism, and two putative membrane proteins that were, based upon their sequences, unlike previously characterized proteins. Given the magnitude of their mutant's defect, the aspartate aminotransferase, RNA helicase, and one of the putative membrane proteins were the factors most critical for L. pneumophila low-temperature growth. Thus, L. pneumophila not only employs some of the same processes and factors as other bacteria do in order to survive at low temperatures (e.g., LpxP, CsdA), but it also appears to possess novel modes of cold adaptation.

Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium

Legionella pneumophila, the agent of Legionnaires’ disease, replicates intracellularly within specialized phagosomes of human macrophages and amoebae. In this study, we have developed a protocol for the isolation of Legionella-containing phagosomes from Dictyostelium discoideum. Cell fractionation, two-dimensional gel electrophoresis and MALDI-TOF MS combined with genomic data identified 157 phagosome host proteins. In addition to proteins with an evident role in phagosome maturation, we identified proteins for which a function remains to be elucidated. Possible interactions of coronin with cytosolic NADPH oxidase components and protein kinase C inhibitors which together may lead to an inhibition of phagosomal superoxide generation are discussed. Comparative proteomics of phagosomes containing highly virulent L. pneumophila Corby versus less virulent L. hackeliae revealed distinctive protein expression patterns, e.g., an abundance of RhoGDI in L. hackeliae degrading phagosomes versus little RhoGDI in L. pneumophila Corby replicative phagosomes. We present a kinetic dissection of phagosome maturation including the complex alterations of the phagosome protein composition. A reference flow chart suggests so far unrecognized consequences of infection for host cell physiology, actin degradation on phagosomes, and a putative cysteine proteinase inhibitor interference with lysosomal enzyme sorting and activation processes.

Twitching motility inLegionella pneumophila

Twitching motility is a form of bacterial translocation over solid or semi-solid surfaces mediated by the extension, tethering, and subsequent retraction of type IV pili. These pili are also known to be involved in virulence, biofilm formation, formation of fruiting bodies, horizontal gene transfer, and protein secretion. We have characterized the presence of twitching motility on agar plates in Legionella pneumophila, the etiological agent of Legionnaires' disease. By examining twitching motility zones, we have demonstrated that twitching motility was dependent on agar thickness/concentration, the chemical composition of the media, the presence of charcoal and cysteine, proximity to other bacteria, and temperature. A knockout mutant of the pilus subunit, pilE, exhibited a total loss of twitching motility at 37 degrees C, but not at 27 degrees C, suggesting either the existence of a compensating pilus subunit or of another twitching motility system in this organism.

Discovery of Diagnostic Biomarkers for Legionnaires' Disease: Virulence Gene Expression Profiling of Legionella pneumophila serogroup 1 in A/J Mouse Model

Background: Legionella pneumophila is the causative agent of Legionnaires' disease, a severe form of pneumonia. After L. pneumophila is inhaled through contaminated aerosols, it is phagocytized by alveolar macrophages, multiplies in a specialized phagosome approximately 10 h postinfection, and event...

The Type II Secretion System ofLegionella pneumophilaElaborates Two Aminopeptidases, as Well as a Metalloprotease That Contributes to Differential Infection among Protozoan Hosts

Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular parasite of aquatic amoebae and human macrophages. A key factor for L. pneumophila in intracellular infection is its type II protein secretion system (Lsp). In order to more completely define Lsp output, we recently performed a proteomic analysis of culture supernatants. Based upon the predictions of that analysis, we found that L. pneumophila secretes two distinct aminopeptidase activities encoded by the genes lapA and lapB. Whereas lapA conferred activity against leucine, phenylalanine, and tyrosine aminopeptides, lapB was linked to the cleavage of lysine- and arginine-containing substrates. To assess the role of secreted aminopeptidases in intracellular infection, we examined the relative abilities of lapA and lapB mutants to infect human U937 cell macrophages as well as Hartmannella vermiformis and Acanthamoeba castellanii amoebae. Although these experiments identified a dispensable role for LapA and LapB, they uncovered a previously unrecognized role for the type II-dependent ProA (MspA) metalloprotease. Whereas proA mutants were not defective for macrophage or A. castellanii infection, they (but not their complemented derivatives) were impaired for growth upon coculture with H. vermiformis. Thus, ProA represents the first type II effector implicated in an intracellular infection event. Furthermore, proA represents an L. pneumophila gene that shows differential importance among protozoan infection models, suggesting that the legionellae might have evolved some of its factors to especially target certain of their protozoan hosts.

A study of Legionella pneumophilia in Tianjin, China

Legionella is a bacterium identified as the causative agent of Legionnaires' disease commonly found in water and soil in natural environments and human habitats. While most studies are focused on cooling tower water as the source of infection, few studies have compared Legionella from cooling-tower water and water in man-made environments. This cross-sectional study with a randomized cluster sampling design aims to provide information on Legionella found in cooling-tower water and water in man-made environments. Legionella was identified using a dual analytical approach: the serological and the 16SrRNA semi-nest PCR methods. Nine different types of Legionella were identified with the Lp7 type detected in China for the first time. Results indicated slight but insignificantly increased odds (OR=1.64, 95%CI=0.54-4.98) of Legionella detection in cooling tower water as compared to water obtained from fountains and parks, suggesting that the mechanism of exposure plays an important role in the causal pathway of outbreaks of Legionnaires' disease.

MP066 Characterization of potential virulence genes in the environmental chlamydia isolate UWE25

Free-living amoebae in water supply systems harbor bacterial endosymbionts. Legionella pneumophila, the agent of Legionnaires' disease, was one of the first agents that was demonstrated to survive and multiply within amoebae. Similar bacteria, termed Legionella-like amoebal pathogens (LLAP's), were subsequently described and implicated in human respiratory tract infections. Amoebal co-cultivation/enrichment techniques and molecular methods (e.g., PCR) have elucidated a variety of alphaproteobacteria (e.g., Afipia, Bosea, Mesorhizobium, and Rasbobacterium species) and Chlamydia-like bacteria (e.g., Parachlamydia, Simkania, Protochlamydia, and Waddlia species) within free-living amoebae in hospital water systems. These agents are also able to survive and multiply within macrophages, pneumocytes, and lung fibroblasts. Sero-epidemiological studies and application of molecular technology to clinical specimens have revealed these agents as rare causes of community- and health care-associated pneumonia, particularly among immunocompromised patients. Additional studies have implicated these agents in other infectious processes. This article reviews these newly described microorganisms and the evidence for their roles in human infections and disease.

Mosaic structure of pathogenicity islands in Legionella pneumophila.

A gene complex, dot/icm, located in two independent chromosomal loci of L. pneumophila, the causative agent of Legionnaires' disease, is related to virulence. To investigate the evolutionary pattern of these pathogenicity islands of L. pneumophila, portions of four genes in the dot/icm complex, namely, dotA, dotB, icmB, and icmT, were amplified, sequenced, and phylogenetically analyzed, in addition to rpoB, which encodes an RNA polymerase beta-subunit. The nucleotide sequences and phylogenetic analyses of these five genes of 96 L. pneumophila strains revealed that several subgroups of L. pneumophila proliferated clonally. However, incongruent gene tree topologies and the results of statistical testing (Templeton Willcoxon signed-ranked and incongruence length differences tests) indicated that the evolutionary histories of these genes within the pathogenicity islands are not uniform, and that they constitute a mosaic structure. In addition, the nonuniform grouping of some reference strains suggests that intraspecific recombination might be still occurring in nature or in the laboratory.

Purification and characterization of a UDP-glucosyltransferase produced by Legionella pneumophila.

Legionella pneumophila is the agent of Legionnaires' disease. It invades and replicates within eukaryotic cells, including aquatic protozoans, mammalian macrophages, and epithelial cells. The molecular mechanisms of the Legionella interaction with target cells are not fully defined. In an attempt to discover novel virulence factors of L. pneumophila, we searched for bacterial enzymes with transferase activity. Upon screening ultrasonic extracts of virulent legionellae, we identified a uridine diphospho (UDP)-glucosyltransferase activity, which was capable of modifying a 45-kDa substrate in host cells. An approximately 60-kDa UDP-glucosyltransferase was purified from L. pneumophila and subjected to microsequencing. An N-terminal amino acid sequence, as well as the sequence of an internal peptide, allowed us to identify the gene for the enzyme within the unfinished L. pneumophila genome database. The intact gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified and confirmed to possess an enzymatic activity similar to that of the native UDP-glucosyltransferase. We designated this gene ugt (UDP-glucosyltransferase). The Legionella enzyme did not exhibit significant homology with any known protein, suggesting that it is novel in structure and, perhaps, in function. Based on PCR data, an enzyme assay, and an immunoblot analysis, the glucosyltransferase appeared to be conserved in L. pneumophila strains but was absent from the other Legionella species. This study represents the first identification of a UDP-glucosyltransferase in an intracellular parasite, and therefore modification of a eukaryotic target(s) by this enzyme may influence host cell function and promote L. pneumophila proliferation.