Metabolism ofmyo-Inositol by Legionella pneumophila Promotes Infection of Amoebae and Macrophages

ABSTRACTLegionella pneumophilais a natural parasite of environmental amoebae and the causative agent of a severe pneumonia termed Legionnaires' disease. The facultative intracellular pathogen employs a bipartite metabolism, where the amino acid serine serves as the major energy supply, while glycerol and glucose are mainly utilized for anabolic processes. TheL. pneumophilagenome harbors the clusterlpg1653tolpg1649putatively involved in the metabolism of the abundant carbohydratemyo-inositol (here termed inositol). To assess inositol metabolism byL. pneumophila, we constructed defined mutant strains lackinglpg1653orlpg1652, which are predicted to encode the inositol transporter IolT or the inositol-2-dehydrogenase IolG, respectively. The mutant strains were not impaired for growth in complex or defined minimal media, and inositol did not promote extracellular growth. However, upon coinfection ofAcanthamoeba castellanii, the mutants were outcompeted by the parental strain, indicating that the intracellular inositol metabolism confers a fitness advantage to the pathogen. Indeed, inositol added toL. pneumophila-infected amoebae or macrophages promoted intracellular growth of the parental strain, but not of the ΔiolTor ΔiolGmutant, and growth stimulation by inositol was restored by complementation of the mutant strains. The expression of the Piolpromoter and bacterial uptake of inositol required the alternative sigma factor RpoS, a key virulence regulator ofL. pneumophila. Finally, the parental strain and ΔiolGmutant bacteria but not the ΔiolTmutant strain accumulated [U-14C6]inositol, indicating that IolT indeed functions as an inositol transporter. Taken together, intracellularL. pneumophilametabolizes inositol through theiolgene products, thus promoting the growth and virulence of the pathogen.IMPORTANCEThe environmental bacteriumLegionella pneumophilais the causative agent of a severe pneumonia termed Legionnaires' disease. The opportunistic pathogen replicates in protozoan and mammalian phagocytes in a unique vacuole. Amino acids are thought to represent the prime source of carbon and energy forL. pneumophila. However, genome, transcriptome, and proteome studies indicate that the pathogen not only utilizes amino acids as carbon sources but possesses broader metabolic capacities. In this study, we analyzed the metabolism of inositol by extra- and intracellularly growingL. pneumophila. By using genetic, biochemical, and cell biological approaches, we found thatL. pneumophilaaccumulates and metabolizes inositol through theiolgene products, thus promoting the intracellular growth, virulence, and fitness of the pathogen. Our study significantly contributes to an understanding of the intracellular niche of a human pathogen.

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Cyclic Diguanylate Signaling Proteins Control Intracellular Growth of Legionella pneumophila

mBio ◽

10.1128/mbio.00316-10 ◽

2011 ◽

Vol 2 (1) ◽

Cited By ~ 31

Author(s):

Assaf Levi ◽

Marc Folcher ◽

Urs Jenal ◽

Howard A. Shuman

Keyword(s):

Causative Agent ◽

Legionella Pneumophila ◽

Second Messenger ◽

Host Cells ◽

Diguanylate Cyclase ◽

Intracellular Growth ◽

Cyclic Diguanylate ◽

Content Type ◽

Legionnaires Disease

ABSTRACTProteins that metabolize or bind the nucleotide second messenger cyclic diguanylate regulate a wide variety of important processes in bacteria. These processes include motility, biofilm formation, cell division, differentiation, and virulence. The role of cyclic diguanylate signaling in the lifestyle ofLegionella pneumophila, the causative agent of Legionnaires’ disease, has not previously been examined. TheL. pneumophilagenome encodes 22 predicted proteins containing domains related to cyclic diguanylate synthesis, hydrolysis, and recognition. We refer to these genes ascdgS(cyclicdiguanylatesignaling) genes. Strains ofL. pneumophilacontaining deletions of all individualcdgSgenes were created and did not exhibit any observable growth defect in growth medium or inside host cells. However, when overexpressed, severalcdgSgenes strongly decreased the ability ofL. pneumophilato grow inside host cells. Expression of thesecdgSgenes did not affect the Dot/Icm type IVB secretion system, the major determinant of intracellular growth inL. pneumophila.L. pneumophilastrains overexpressing thesecdgSgenes were less cytotoxic to THP-1 macrophages than wild-typeL. pneumophilabut retained the ability to resist grazing by amoebae. In many cases, the intracellular-growth inhibition caused bycdgSgene overexpression was independent of diguanylate cyclase or phosphodiesterase activities. Expression of thecdgSgenes in aSalmonella entericaserovar Enteritidis strain that lacks all diguanylate cyclase activity indicated that severalcdgSgenes encode potential cyclases. These results indicate that components of the cyclic diguanylate signaling pathway play an important role in regulating the ability ofL. pneumophilato grow in host cells.IMPORTANCEAll bacteria must sense and respond to environmental cues. Intracellular bacterial pathogens must detect and respond to host functions that limit their ability to carry out a successful infection. Small-molecule second messengers play key roles in transmitting signals from environmental receptors to the proteins and other components that respond to signals. Cyclic diguanylate is a ubiquitous bacterial second messenger known to play an important role in many sensing and signaling systems in bacteria. The causative agent of Legionnaires’ disease,Legionella pneumophila, is an intracellular pathogen that grows inside environmental protists and human macrophages by subverting the normal processes that these cells use to capture and destroy bacteria. We show that the several cyclic diguanylate signaling components inLegionellaplay a role in the ability to grow inside both kinds of host cells. This work highlights the role of cyclic diguanylate signaling during intracellular growth.

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Reclamation of Ampicillin Sensitivity for the Genetic Manipulation of Legionella pneumophila

Applied and Environmental Microbiology ◽

10.1128/aem.00669-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5457-5459 ◽

Cited By ~ 1

Author(s):

Molly C. Sutherland ◽

Joseph P. Vogel

Keyword(s):

Mutant Strain ◽

Causative Agent ◽

Legionella Pneumophila ◽

Selectable Marker ◽

Genetic Manipulation ◽

Selectable Markers ◽

Content Type ◽

Enhanced Sensitivity ◽

Legionnaires Disease

ABSTRACTResearch onLegionella pneumophila, the causative agent of Legionnaires' disease, has been hampered due to the lack of selectable markers for genetic manipulation. We report the construction of a mutant strain ofL. pneumophilalackingloxA, a chromosomally encoded β-lactamase, that has enhanced sensitivity to ampicillin. Also described are a method for convertingLegionellastrains to ampicillin sensitivity and conditions for utilizingblaas a selectable marker.

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Legionella pneumophila OxyR Is a Redundant Transcriptional Regulator That Contributes to Expression Control of the Two-Component CpxRA System

Journal of Bacteriology ◽

10.1128/jb.00690-16 ◽

2016 ◽

Vol 199 (5) ◽

Cited By ~ 3

Author(s):

Jennifer R. Tanner ◽

Palak G. Patel ◽

Jacqueline R. Hellinga ◽

Lynda J. Donald ◽

Celine Jimenez ◽

...

Keyword(s):

Legionella Pneumophila ◽

Mutational Analysis ◽

Transcriptional Regulator ◽

Intracellular Growth ◽

Content Type ◽

Expression Control ◽

Two Component ◽

Legionnaires Disease

ABSTRACT Nominally an environmental organism, Legionella pneumophila is an intracellular parasite of protozoa but is also the causative agent of the pneumonia termed Legionnaires' disease, which results from inhalation of aerosolized bacteria by susceptible humans. Coordination of gene expression by a number of identified regulatory factors, including OxyR, assists L. pneumophila in adapting to the stresses of changing environments. L. pneumophila OxyR (OxyRLp) is an ortholog of Escherichia coli OxyR; however, OxyRLp was shown elsewhere to be functionally divergent, such that it acts as a transcription regulator independently of the oxidative stress response. In this study, the use of improved gene deletion methods has enabled us to generate an unmarked in-frame deletion of oxyR in L. pneumophila. Lack of OxyRLp did not affect in vitro growth or intracellular growth in Acanthamoeba castellanii protozoa and U937-derived macrophages. The expression of OxyRLp does not appear to be regulated by CpxR, even though purified recombinant CpxR bound a DNA sequence similar to that reported for CpxR elsewhere. Surprisingly, a lack of OxyRLp resulted in elevated activity of the promoters located upstream of icmR and the lpg1441-cpxA operon, and OxyRLp directly bound to these promoter regions, suggesting that OxyRLp is a direct repressor. Interestingly, a strain overexpressing OxyRLp demonstrated reduced intracellular growth in A. castellanii but not in U937-derived macrophages, suggesting that balanced expression control of the two-component CpxRA system is necessary for survival in protozoa. Taken together, this study suggests that OxyRLp is a functionally redundant transcriptional regulator in L. pneumophila under the conditions evaluated herein. IMPORTANCE Legionella pneumophila is an environmental pathogen, with its transmission to the human host dependent upon its ability to replicate in protozoa and survive within its aquatic niche. Understanding the genetic factors that contribute to L. pneumophila survival within each of these unique environments will be key to limiting future point-source outbreaks of Legionnaires' disease. The transcriptional regulator L. pneumophila OxyR (OxyRLp) has been previously identified as a potential regulator of virulence traits warranting further investigation. This study demonstrated that oxyR is nonessential for L. pneumophila survival in vitro and in vivo via mutational analysis. While the mechanisms of how OxyRLp expression is regulated remain elusive, this study shows that OxyRLp negatively regulates the expression of the cpxRA two-component system necessary for intracellular survival in protozoa.

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Dot/Icm Effector Translocation by Legionella longbeachae Creates a Replicative Vacuole Similar to That of Legionella pneumophila despite Translocation of Distinct Effector Repertoires

Infection and Immunity ◽

10.1128/iai.00461-15 ◽

2015 ◽

Vol 83 (10) ◽

pp. 4081-4092 ◽

Cited By ~ 3

Author(s):

Rebecca E. Wood ◽

Patrice Newton ◽

Eleanor A. Latomanski ◽

Hayley J. Newton

Keyword(s):

Legionella Pneumophila ◽

A549 Cells ◽

Severe Pneumonia ◽

Rab Gtpase ◽

Human Pathogens ◽

Immunofluorescence Analysis ◽

Content Type ◽

Extensive Analysis ◽

Legionnaires Disease ◽

Environmental Bacteria

Legionellaorganisms are environmental bacteria and accidental human pathogens that can cause severe pneumonia, termed Legionnaires' disease. These bacteria replicate within a pathogen-derived vacuole termed theLegionella-containing vacuole (LCV). Our understanding of the development and dynamics of this vacuole is based on extensive analysis ofLegionella pneumophila. Here, we have characterized theLegionella longbeachaereplicative vacuole (longbeachae-LCV) and demonstrated that, despite important genomic differences, key features of the replicative LCV are comparable to those of the LCV ofL. pneumophila(pneumophila-LCV). We constructed a Dot/Icm-deficient strain by deletingdotBand demonstrated the inability of this mutant to replicate inside THP-1 cells.L. longbeachaedoes not enter THP-1 cells as efficiently asL. pneumophila, and this is reflected in the observation that translocation of BlaM-RalFLLO(where RalFLLOis theL. longbeachaehomologue of RalF) into THP-1 cells by theL. longbeachaeDot/Icm system is less efficient than that byL. pneumophila. This difference is negated in A549 cells whereL. longbeachaeandL. pneumophilainfect with similar entry dynamics. A β-lactamase assay was employed to demonstrate the translocation of a novel family of proteins, theRab-likeeffector (Rle) proteins. Immunofluorescence analysis confirmed that these proteins enter the host cell during infection and display distinct subcellular localizations, with RleA and RleC present on thelongbeachae-LCV. We observed that the host Rab GTPase, Rab1, and the v-SNARE Sec22b are also recruited to thelongbeachae-LCV during the early stages of infection, coinciding with the LCV avoiding endocytic maturation. These studies further our understanding of theL. longbeachaereplicative vacuole, highlighting phenotypic similarities to the vacuole ofL. pneumophilaas well as unique aspects of LCV biology.

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Human Lung Tissue Explants Reveal Novel Interactions during Legionella pneumophila Infections

Infection and Immunity ◽

10.1128/iai.00703-13 ◽

2013 ◽

Vol 82 (1) ◽

pp. 275-285 ◽

Cited By ~ 35

Author(s):

Jens Jäger ◽

Sebastian Marwitz ◽

Jana Tiefenau ◽

Janine Rasch ◽

Olga Shevchuk ◽

...

Keyword(s):

Lung Tissue ◽

Legionella Pneumophila ◽

Human Lung ◽

Transcriptional Response ◽

Human Infection ◽

Bacterial Invasion ◽

Human Lung Tissue ◽

Content Type ◽

Tissue Explants ◽

Legionnaires Disease

ABSTRACTHistological and clinical investigations describe late stages of Legionnaires' disease but cannot characterize early events of human infection. Cellular or rodent infection models lack the complexity of tissue or have nonhuman backgrounds. Therefore, we developed and applied a novel model forLegionella pneumophilainfection comprising living human lung tissue. We stimulated lung explants withL. pneumophilastrains and outer membrane vesicles (OMVs) to analyze tissue damage, bacterial replication, and localization as well as the transcriptional response of infected tissue. Interestingly, we found that extracellular adhesion ofL. pneumophilato the entire alveolar lining precedes bacterial invasion and replication in recruited macrophages. In contrast, OMVs predominantly bound to alveolar macrophages. Specific damage to septa and epithelia increased over 48 h and was stronger in wild-type-infected and OMV-treated samples than in samples infected with the replication-deficient, type IVB secretion-deficient DotA−strain. Transcriptome analysis of lung tissue explants revealed a differential regulation of 2,499 genes after infection. The transcriptional response included the upregulation of uteroglobin and the downregulation of the macrophage receptor with collagenous structure (MARCO). Immunohistochemistry confirmed the downregulation of MARCO at sites of pathogen-induced tissue destruction. Neither host factor has ever been described in the context ofL. pneumophilainfections. This work demonstrates that the tissue explant model reproduces realistic features of Legionnaires' disease and reveals new functions for bacterial OMVs during infection. Our model allows us to characterize early steps of human infection which otherwise are not feasible for investigations.

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Peptidyl-Prolyl-cis/trans-Isomerases Mip and PpiB ofLegionella pneumophilaContribute to Surface Translocation, Growth at Suboptimal Temperature, and Infection

Infection and Immunity ◽

10.1128/iai.00939-17 ◽

2018 ◽

Vol 87 (1) ◽

Cited By ~ 4

Author(s):

J. Rasch ◽

C. M. Ünal ◽

A. Klages ◽

Ü. Karsli ◽

N. Heinsohn ◽

...

Keyword(s):

Legionella Pneumophila ◽

Acanthamoeba Castellanii ◽

Temperature Tolerance ◽

Lung Damage ◽

Atypical Pneumonia ◽

Content Type ◽

Wide Range ◽

Legionnaires Disease ◽

Environmental Fitness ◽

Sliding Motility

ABSTRACTThe gammaproteobacteriumLegionella pneumophilais the causative agent of Legionnaires’ disease, an atypical pneumonia that manifests itself with severe lung damage.L. pneumophila, a common inhabitant of freshwater environments, replicates in free-living amoebae and persists in biofilms in natural and man-made water systems. Its environmental versatility is reflected in its ability to survive and grow within a broad temperature range as well as its capability to colonize and infect a wide range of hosts, including protozoa and humans. Peptidyl-prolyl-cis/trans-isomerases (PPIases) are multifunctional proteins that are mainly involved in protein folding and secretion in bacteria. InL. pneumophilathe surface-associated PPIase Mip was shown to facilitate the establishment of the intracellular infection cycle in its early stages. The cytoplasmic PpiB was shown to promote cold tolerance. Here, we set out to analyze the interrelationship of these two relevant PPIases in the context of environmental fitness and infection. We demonstrate that the PPIases Mip and PpiB are important for surfactant-dependent sliding motility and adaptation to suboptimal temperatures, features that contribute to the environmental fitness ofL. pneumophila. Furthermore, they contribute to infection of the natural hostAcanthamoeba castellaniias well as human macrophages and human explanted lung tissue. These effects were additive in the case of sliding motility or synergistic in the case of temperature tolerance and infection, as assessed by the behavior of the double mutant. Accordingly, we propose that Mip and PpiB are virulence modulators ofL. pneumophilawith compensatory action and pleiotropic effects.

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The amoebae plate test implicates a paralogue of lpxB in the interaction of Legionella pneumophila with Acanthamoeba castellanii

Microbiology ◽

10.1099/mic.0.27563-0 ◽

2005 ◽

Vol 151 (1) ◽

pp. 167-182 ◽

Cited By ~ 36

Author(s):

Urs Albers ◽

Katrin Reus ◽

Howard A. Shuman ◽

Hubert Hilbi

Keyword(s):

Legionella Pneumophila ◽

Lipid A ◽

Sequence Similarity ◽

Acanthamoeba Castellanii ◽

Growth Defect ◽

Wild Type ◽

Intracellular Growth ◽

Plate Test ◽

Raw264.7 Macrophages ◽

Mutant Strains

Legionella pneumophila is a bacterial parasite of freshwater amoebae which also grows in alveolar macrophages and thus causes the potentially fatal pneumonia Legionnaires' disease. Intracellular growth within amoebae and macrophages is mechanistically similar and requires the Icm/Dot type IV secretion system. This paper reports the development of an assay, the amoebae plate test (APT), to analyse growth of L. pneumophila wild-type and icm/dot mutant strains spotted on agar plates in the presence of Acanthamoeba castellanii. In the APT, wild-type L. pneumophila formed robust colonies even at high dilutions, icmT, -R, -P or dotB mutants failed to grow, and icmS or -G mutants were partially growth defective. The icmS or icmG mutant strains were used to screen an L. pneumophila chromosomal library for genes that suppress the growth defect in the presence of the amoebae. An icmS suppressor plasmid was isolated that harboured the icmS and flanking icm genes, indicating that this plasmid complements the intracellular growth defect of the mutant. In contrast, different icmG suppressor plasmids rendered the icmG mutant more cytotoxic for A. castellanii without enhancing intracellular multiplication in amoebae or RAW264.7 macrophages. Deletion of individual genes in the suppressor plasmids inserts identified lcs (Legionella cytotoxic suppressor) -A, -B, -C and -D as being required for enhanced cytotoxicity of an icmG mutant strain. The corresponding proteins show sequence similarity to hydrolases, NlpD-related metalloproteases, lipid A disaccharide synthases and ABC transporters, respectively. Overexpression of LcsC, a putative paralogue of the lipid A disaccharide synthase LpxB, increased cytotoxicity of an icmG mutant but not that of other icm/dot or rpoS mutant strains against A. castellanii. Based on sequence comparison and chromosomal location, lcsB and lcsC probably encode enzymes involved in cell wall maintenance and peptidoglycan metabolism. The APT established here may prove useful to identify other bacterial factors relevant for interactions with amoeba hosts.

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The Legionella pneumophila Metaeffector Lpg2505 (MesI) Regulates SidI-Mediated Translation Inhibition and Novel Glycosyl Hydrolase Activity

Infection and Immunity ◽

10.1128/iai.00853-19 ◽

2020 ◽

Vol 88 (5) ◽

Cited By ~ 2

Author(s):

Ashley M. Joseph ◽

Adrienne E. Pohl ◽

Theodore J. Ball ◽

Troy G. Abram ◽

David K. Johnson ◽

...

Keyword(s):

Legionella Pneumophila ◽

Glycosyl Hydrolase ◽

Protein Translation ◽

Effector Proteins ◽

Hydrolase Activity ◽

Intracellular Replication ◽

Content Type ◽

Translation Inhibition ◽

Legionnaires Disease ◽

The Impact

ABSTRACT Legionella pneumophila, the etiological agent of Legionnaires’ disease, employs an arsenal of hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic phagocytes. Several effectors, called metaeffectors, function to regulate the activity of other Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated inhibition of protein translation. SidI binding to eEF1A and Lpg2505 is not mutually exclusive, and the proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-dependent glycosyl hydrolase activity and that this activity is regulated by Lpg2505. We have therefore renamed Lpg2505 MesI (metaeffector of SidI). This work reveals novel enzymatic activity for SidI and provides insight into how intracellular replication of L. pneumophila is regulated by a metaeffector.

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Involvement of Fractalkine/CX3CL1 Expression by Dendritic Cells in the Enhancement of Host Immunity against Legionella pneumophila

Infection and Immunity ◽

10.1128/iai.73.9.5350-5357.2005 ◽

2005 ◽

Vol 73 (9) ◽

pp. 5350-5357 ◽

Cited By ~ 17

Author(s):

Toshiaki Kikuchi ◽

Sita Andarini ◽

Hong Xin ◽

Kazunori Gomi ◽

Yutaka Tokue ◽

...

Keyword(s):

T Cells ◽

Immune Responses ◽

Host Defense ◽

Legionella Pneumophila ◽

Recombinant Adenovirus ◽

Severe Pneumonia ◽

Adenovirus Vector ◽

Recombinant Adenovirus Vector ◽

Legionnaires Disease

ABSTRACT Legionnaires' disease is clinically manifested as severe pneumonia caused by Legionella pneumophila. However, the dendritic cell (DC)-centered immunological framework of the host defense against L. pneumophila has not been fully delineated. For this study, we focused on a potent chemoattractant for lymphocytes, fractalkine/CX3CL1, and observed that the fractalkine expression of DCs was somewhat up-regulated when they encountered L. pneumophila. We therefore hypothesized that fractalkine expressed by Legionella-capturing DCs is involved in the induction of T-cell-mediated immune responses against Legionella, which would be enhanced by a genetic modulation of DCs to overexpress fractalkine. In vivo immunization-challenge experiments demonstrated that DCs modified with a recombinant adenovirus vector to overexpress fractalkine (AdFKN) and pulsed with heat-killed Legionella protected immunized mice from a lethal Legionella infection and that the generation of in vivo protective immunity depended on the host lymphocyte subsets, including CD4+ T cells, CD8+ T cells, and B cells. Consistent with this, immunization with AdFKN/Legionella/DC induced significantly higher levels of serum anti-Legionella antibodies of several isotypes than those induced by control immunizations. Further analysis of spleen cells from the immunized mice indicated that the AdFKN/Legionella/DC immunization elicited Th1-dominated immune responses to L. pneumophila. These observations suggest that fractalkine may play an important role in the DC-mediated host defense against intracellular pathogens such as L. pneumophila.

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Development of a DNA Microarray Method for Detection and Identification of All 15 Distinct O-Antigen Forms of Legionella pneumophila

Applied and Environmental Microbiology ◽

10.1128/aem.01957-13 ◽

2013 ◽

Vol 79 (21) ◽

pp. 6647-6654 ◽

Cited By ~ 17

Author(s):

Boyang Cao ◽

Fangfang Yao ◽

Xiangqian Liu ◽

Lu Feng ◽

Lei Wang

Keyword(s):

Dna Microarray ◽

Legionella Pneumophila ◽

Detection Sensitivity ◽

Immunological Methods ◽

Clinical Samples ◽

Content Type ◽

O Antigen ◽

Detection And Identification ◽

Legionnaires Disease ◽

Reference Strains

ABSTRACTLegionellais ubiquitous in many environments. At least 50 species and 70 serogroups of the Gram-negative bacterium have been identified. Of the 50 species, 20 are pathogenic, andLegionella pneumophilais responsible for the great majority (approximately 90%) of the Legionnaires' disease cases that occur. Furthermore, of the 15L. pneumophilaserogroups identified, O1 alone causes more than 84% of the Legionnaires' disease cases that occur worldwide. Rapid and reliable assays for the detection and identification ofL. pneumophilain water, environmental, and clinical samples are in great demand.L. pneumophilabacteria are traditionally identified by their O antigens by immunological methods. We have recently developed an O serogroup-specific DNA microarray for the detection of all 15 distinct O-antigen forms ofL. pneumophila, including serogroups O1 to O15. A total of 35 strains were used to verify the specificity of the microarray, including 15L. pneumophilaO-antigen standard reference strains and sevenL. pneumophilaclinical isolates as target strains, seven reference strains of other non-pneumophila Legionellaspecies as closely related strains, and six non-Legionellabacterial species as nonrelated strains. The detection sensitivity was 1 ng of genomic DNA or 0.4 CFU/ml in water samples with filter enrichment and plate culturing. This study demonstrated that the microarray allows specific, sensitive, and reproducible detection ofL. pneumophilaserogroups. To the best of our knowledge, this is the first report of a microarray serotyping method for all 15 distinct O-antigen forms ofL. pneumophila.

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