Reclamation of Ampicillin Sensitivity for the Genetic Manipulation of Legionella pneumophila

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

Applied and Environmental Microbiology ◽

10.1128/aem.01018-16 ◽

2016 ◽

Vol 82 (16) ◽

pp. 5000-5014 ◽

Cited By ~ 20

Author(s):

Christian Manske ◽

Ursula Schell ◽

Hubert Hilbi

Keyword(s):

Amino Acids ◽

Causative Agent ◽

Legionella Pneumophila ◽

Parental Strain ◽

Severe Pneumonia ◽

Gene Products ◽

Intracellular Growth ◽

Content Type ◽

Mutant Strains ◽

Legionnaires Disease

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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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 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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Glyphosate Resistance as a Novel Select-Agent-Compliant, Non-Antibiotic-Selectable Marker in Chromosomal Mutagenesis of the Essential Genes asd and dapB of Burkholderia pseudomallei

Applied and Environmental Microbiology ◽

10.1128/aem.00820-09 ◽

2009 ◽

Vol 75 (19) ◽

pp. 6062-6075 ◽

Cited By ~ 32

Author(s):

Michael H. Norris ◽

Yun Kang ◽

Diana Lu ◽

Bruce A. Wilcox ◽

Tung T. Hoang

Keyword(s):

Burkholderia Pseudomallei ◽

Selectable Marker ◽

Genetic Manipulation ◽

Chromosome 1 ◽

Chromosome 2 ◽

Gene Encoding ◽

Selectable Markers ◽

Genetic Manipulations ◽

Semialdehyde Dehydrogenase ◽

Allelic Replacement

ABSTRACT Genetic manipulation of the category B select agents Burkholderia pseudomallei and Burkholderia mallei has been stifled due to the lack of compliant selectable markers. Hence, there is a need for additional select-agent-compliant selectable markers. We engineered a selectable marker based on the gat gene (encoding glyphosate acetyltransferase), which confers resistance to the common herbicide glyphosate (GS). To show the ability of GS to inhibit bacterial growth, we determined the effective concentrations of GS against Escherichia coli and several Burkholderia species. Plasmids based on gat, flanked by unique flip recombination target (FRT) sequences, were constructed for allelic-replacement. Both allelic-replacement approaches, one using the counterselectable marker pheS and the gat-FRT cassette and one using the DNA incubation method with the gat-FRT cassette, were successfully utilized to create deletions in the asd and dapB genes of wild-type B. pseudomallei strains. The asd and dapB genes encode an aspartate-semialdehyde dehydrogenase (BPSS1704, chromosome 2) and dihydrodipicolinate reductase (BPSL2941, chromosome 1), respectively. Mutants unable to grow on media without diaminopimelate (DAP) and other amino acids of this pathway were PCR verified. These mutants displayed cellular morphologies consistent with the inability to cross-link peptidoglycan in the absence of DAP. The B. pseudomallei 1026b Δasd::gat-FRT mutant was complemented with the B. pseudomallei asd gene on a site-specific transposon, mini-Tn7-bar, by selecting for the bar gene (encoding bialaphos/PPT resistance) with PPT. We conclude that the gat gene is one of very few appropriate, effective, and beneficial compliant markers available for Burkholderia select-agent species. Together with the bar gene, the gat cassette will facilitate various genetic manipulations of Burkholderia select-agent species.

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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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Altered Murine Tissue Colonization by Borrelia burgdorferi following Targeted Deletion of Linear Plasmid 17-Carried Genes

Infection and Immunity ◽

10.1128/iai.05984-11 ◽

2012 ◽

Vol 80 (5) ◽

pp. 1773-1782 ◽

Cited By ~ 9

Author(s):

Timothy Casselli ◽

Yvonne Tourand ◽

Troy Bankhead

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Mutant Strain ◽

Genetic Manipulation ◽

Infectious Clone ◽

Linear Plasmid ◽

Peripheral Tissues ◽

Content Type ◽

Immunodeficient Mice

ABSTRACTThe causative agent of Lyme disease,Borrelia burgdorferi, possesses a segmented genome comprised of a single linear chromosome and upwards of 23 linear and circular plasmids. Much of what is known about plasmid-borne genes comes from studying laboratory clones that have spontaneously lost one or more plasmids duringin vitropassage. Some plasmids, including the linear plasmid lp17, are never or rarely reported to be lost during routine culture; therefore, little is known about the requirement of these conserved plasmids for infectivity. In this study, the effects of deleting regions of lp17 were examined bothin vitroandin vivo. A mutant strain lacking the genesbbd16tobbd25showed no deficiency in the ability to establish infection or disseminate to the bloodstream of mice; however, colonization of peripheral tissues was delayed. Despite the ability to colonize ear, heart, and joint tissues, this mutant exhibited a defect in bladder tissue colonization for up to 56 days postinfection. This phenotype was not observed in immunodeficient mice, suggesting that bladder colonization by the mutant strain was inhibited by an adaptive immune-based mechanism. Moreover, the mutant displayed increased expression of outer surface protein Cin vitro, which was correlated with the absence of the genebbd18. To our knowledge, this is the first report involving genetic manipulation of lp17 in an infectious clone ofB. burgdorferiand reveals for the first time the effects of lp17 gene deletion during murine infection by the Lyme disease spirochete.

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Utility of PCR, Culture, and Antigen Detection Methods for Diagnosis of Legionellosis

Journal of Clinical Microbiology ◽

10.1128/jcm.01808-15 ◽

2015 ◽

Vol 53 (11) ◽

pp. 3474-3477 ◽

Cited By ~ 17

Author(s):

Derrick J. Chen ◽

Gary W. Procop ◽

Sherilynn Vogel ◽

Belinda Yen-Lieberman ◽

Sandra S. Richter

Keyword(s):

Electronic Medical Records ◽

Legionella Pneumophila ◽

Medical Records ◽

Cleveland Clinic ◽

Test Methods ◽

Detection Methods ◽

Content Type ◽

Epidemiologic Factors ◽

Legionnaires Disease ◽

Positive Results

The goal of this retrospective study was to evaluate the performance of different diagnostic tests for Legionnaires' disease in a clinical setting whereLegionella pneumophilaPCR had been introduced. Electronic medical records at the Cleveland Clinic were searched forLegionellaurinary antigen (UAG), culture, and PCR tests ordered from March 2010 through December 2013. For cases where two or more test methods were performed and at least one was positive, the medical record was reviewed for relevant clinical and epidemiologic factors. Excluding repeat testing on a given patient, 19,912 tests were ordered (12,569 UAG, 3,747 cultures, and 3,596 PCR) with 378 positive results. The positivity rate for each method was 0.4% for culture, 0.8% for PCR, and 2.7% for UAG. For 37 patients, at least two test methods were performed with at least one positive result: 10 (27%) cases were positive by all three methods, 16 (43%) were positive by two methods, and 11 (30%) were positive by one method only. For the 32 patients with medical records available, clinical presentation was consistent with proven or probableLegionellainfection in 84% of the cases. For those cases, the sensitivities of culture, PCR, and UAG were 50%, 92%, and 96%, respectively. The specificities were 100% for culture and 99.9% for PCR and UAG.

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Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) Acinetobacter baumannii HUMC1

mSphere ◽

10.1128/msphere.00140-17 ◽

2017 ◽

Vol 2 (2) ◽

Cited By ~ 6

Author(s):

Brian M. Luna ◽

Amber Ulhaq ◽

Jun Yan ◽

Paul Pantapalangkoor ◽

Travis B. Nielsen ◽

...

Keyword(s):

Molecular Biology ◽

Acinetobacter Baumannii ◽

Genetic Manipulation ◽

Multidrug Resistant ◽

Molecular Techniques ◽

Drug Resistant ◽

Selectable Markers ◽

Content Type ◽

Extensively Drug Resistant ◽

Selection Of

ABSTRACT Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of Acinetobacter baumannii have frequently been characterized. The ability of A. baumannii to develop resistance to antibiotics is a key reason this organism has been difficult to study using genetic and molecular biology approaches. Here we report selectable markers that are not only useful but necessary for the selection of drug-resistant transformants in the setting of drug-resistant backgrounds. Use of these selectable markers can be applied to a variety of genetic and molecular techniques such as mutagenesis and transformation. These selectable markers will help promote genetic and molecular biology studies of otherwise onerous drug-resistant strains, while avoiding the generation of pathogenic organisms that are resistant to clinically relevant antibiotics. Acinetobacter baumannii is one of the most antibiotic-resistant pathogens in clinical medicine, and extensively drug-resistant (XDR) strains are commonly isolated from infected patients. Such XDR strains are already resistant to traditional selectable genetic markers, limiting the ability to conduct pathogenesis research by genetic disruption. Optimization of selectable markers is therefore critical for the advancement of fundamental molecular biology techniques to use in these strains. We screened 23 drugs that constitute a broad array of antibiotics spanning multiple drug classes against HUMC1, a highly virulent and XDR A. baumannii clinical blood and lung isolate. HUMC1 is resistant to all clinically useful antibiotics that are reported by the clinical microbiology laboratory, except for colistin. Ethical concerns about intentionally establishing pan-resistance, including to the last-line agent, colistin, in a clinical isolate made identification of other markers desirable. We screened additional antibiotics that are in clinical use and those that are useful only in a lab setting to identify selectable markers that were effective at selecting for transformants in vitro. We show that supraphysiological levels of tetracycline can overcome innate drug resistance displayed by this XDR strain. Last, we demonstrate that transformation of the tetA (tetracycline resistance) and Sh ble (zeocin resistance), but not pac (puromycin resistance), resistance cassettes allow for selection of drug-resistant transformants. These results make the genetic manipulation of XDR A. baumannii strains easily achieved. IMPORTANCE Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of Acinetobacter baumannii have frequently been characterized. The ability of A. baumannii to develop resistance to antibiotics is a key reason this organism has been difficult to study using genetic and molecular biology approaches. Here we report selectable markers that are not only useful but necessary for the selection of drug-resistant transformants in the setting of drug-resistant backgrounds. Use of these selectable markers can be applied to a variety of genetic and molecular techniques such as mutagenesis and transformation. These selectable markers will help promote genetic and molecular biology studies of otherwise onerous drug-resistant strains, while avoiding the generation of pathogenic organisms that are resistant to clinically relevant antibiotics.

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