Exploring Anti-Bacterial Compounds against Intracellular Legionella

Harrison, Christopher F.; Kicka, Sébastien; Trofimov, Valentin; Berschl, Kathrin; Ouertatani-Sakouhi, Hajer; Ackermann, Nikolaus; Hedberg, Christian; Cosson, Pierre; Soldati, Thierry; Hilbi, Hubert

doi:10.1371/journal.pone.0074813

Cited by 30 publications

(49 citation statements)

References 53 publications

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“…Intracellular replication of L. pneumophila was assessed as previously published (46). In brief, A. castellanii or D. discoideum amoebae were grown in PYG or HL5 medium, respectively, to ϳ80% confluence, and 4 ϫ 10 4 cells per well were seeded in 96-well plates.…”

Section: Methodsmentioning

confidence: 99%

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

Manske

Schell

Hilbi

2016

Appl Environ Microbiol

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Legionella pneumophila is 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. The L. pneumophila genome harbors the cluster lpg1653 to lpg1649 putatively involved in the metabolism of the abundant carbohydrate myo-inositol (here termed inositol). To assess inositol metabolism by L. pneumophila, we constructed defined mutant strains lacking lpg1653 or lpg1652, 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 of Acanthamoeba 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 to L. pneumophila-infected amoebae or macrophages promoted intracellular growth of the parental strain, but not of the ⌬iolT or ⌬iolG mutant, and growth stimulation by inositol was restored by complementation of the mutant strains. The expression of the P iol promoter and bacterial uptake of inositol required the alternative sigma factor RpoS, a key virulence regulator of L. pneumophila. Finally, the parental strain and ⌬iolG mutant bacteria but not the ⌬iolT mutant strain accumulated [U-14 C 6 ]inositol, indicating that IolT indeed functions as an inositol transporter. Taken together, intracellular L. pneumophila metabolizes inositol through the iol gene products, thus promoting the growth and virulence of the pathogen. IMPORTANCEThe environmental bacterium Legionella pneumophila is 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 for L. 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 growing L. pneumophila. By using genetic, biochemical, and cell biological approaches, we found that L. pneumophila accumulates and metabolizes inositol through the iol gene 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. L egionella pneumophila is a Gram-negative ubiquitous environmental bacterium that survives in complex multispecies biofilms in natural or manmade water sources (1-3). Predominantly, however, Legionella spp. parasitize free-living protozoa and grow within these u...

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Section: Methodsmentioning

confidence: 99%

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

Manske

Schell

Hilbi

2016

Appl Environ Microbiol

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“…This was not due to overall differences in the model systems, as antibiotic treatment continued to reduce replication levels (Fig. 4a), and we had previously shown correlation between antibiotic effects in A. castellanii and RAW 264.7 macrophages (Harrison et al, 2013). The increased bacterial replication in macrophages correlated with an extended lag phase prior to the onset of growth, as seen in time-course measurements of replication in the presence of different concentrations of LED209 (Fig.…”

Section: Led209 and Naftopidil Promote Growth Of L Pneumophila In Mamentioning

confidence: 51%

“…Extracellular replication of L. pneumophila in the presence of compounds was determined using previously published protocols (Harrison et al, 2013). Briefly, L. pneumophila were taken from CYE plates after 3 days of growth, resuspended in 3 ml AYE at a final OD 600 of 0.02 (matching the bacterial concentration used for intracellular assays; see below) and incubated in test tubes on a rotating wheel overnight at 37 uC.…”

Section: Methodsmentioning

confidence: 99%

Adrenergic antagonists restrict replication of Legionella

et al. 2015

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Legionella pneumophila is a facultative intracellular bacterium, which upon inhalation can cause a potentially fatal pneumonia termed Legionnaires' disease. The opportunistic pathogen grows in environmental amoebae and mammalian macrophages within a unique membrane-bound compartment, the 'Legionella-containing vacuole'. Bacteria are exposed to many environmental cues including small signalling molecules from eukaryotic cells. A number of pathogenic bacteria sense and respond to catecholamine hormones, such as adrenalin and noradrenalin, a process mediated via the QseBC two-component system in some bacteria. In this study, we examined the effect of adrenergic compounds on L. pneumophila, and discovered that the adrenergic receptor antagonists benoxathian, naftopidil, propranolol and labetalol, as well as the QseC sensor kinase inhibitor LED209, reduced the growth of L. pneumophila in broth or amoebae, while replication in macrophages was enhanced. Growth restriction was common to members of the genus Legionella and Mycobacterium, and was observed for L. pneumophila in the replicative but not stationary phase of the biphasic life cycle. Deletion of the L. pneumophila qseBC genes indicated that growth inhibition by adrenergics or LED209 is mediated only to a minor extent by this two-component system, implying the presence of other adrenergic sensing systems. This study identifies adrenergic molecules as novel inhibitors of extra-and intracellular growth of Legionella and reveals LED209 as a potential lead compound to combat infections with Legionella or Mycobacterium spp.

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“…23 The compounds were added at a concentration of 10 or 30 μM, and L. pneumophila replication was normalized to the range between 0 (kanamycin treatment) and 1 (vehicle controls). A replication level of 0.5 (i.e., 50% inhibition) was chosen as the cutoff value to determine any hits of interest.…”

Section: ■ Resultsmentioning

confidence: 99%

“…Previous work in our group had led to the development of a novel screening system utilizing L. pneumophila replication within a natural prokaryotic host, Acanthamoeba castellanii. 23 We used this system to screen a pathwaybased highly diverse chemical library, called the Sinergia library, for chemicals which successfully inhibited intracellular replication. Here, we report on detailed "hit finding and chemical space exploration" studies performed on hits discovered in this process, resulting in the identification and characterization of a family of advanced hits compounds derived from inhibitors of the calcineurin protein phosphatase enzyme.…”

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confidence: 99%

Amoebae-Based Screening Reveals a Novel Family of Compounds Restricting Intracellular Legionella pneumophila

et al. 2015

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The causative agent of Legionnaires' disease, Legionella pneumophila, grows in environmental amoebae and mammalian macrophages within a distinct compartment, the 'Legionella-containing vacuole' (LCV). Intracellular bacteria are protected from many antibiotics, and thus are notoriously difficult to eradicate. To identify novel compounds that restrict intracellular bacterial replication, we previously developed an assay based on a coculture of amoebae and GFP-producing L. pneumophila. This assay was used to screen a pathway-based, highly diverse chemical library, referred to as the Sinergia library. In this work, we chose to focus on a group of 11 hit compounds, the majority of which originated from the query molecule CN585, a compound that targets the protein phosphatase calcineurin. Further studies on 78 related compound variants revealed crucial structural attributes, namely a triple-ring scaffold with a central triazine moiety, substituted in positions 3 and 5 by two piperidine or pyrrolidine rings, and in position 1 by an amine group bearing a single aliphatic chain moiety. The most effective compound, ZINC00615682, inhibited intracellular replication of L. pneumophila with an IC50 of approximately 20 nM in Acanthamoeba castellanii and slightly less efficiently in Dictyostelium discoideum or macrophages. Pharmacological and genetic attempts to implicate calcineurin in the intracellular replication of L. pneumophila failed. Taken together, these results show that the amoebae-based screen and structure-activity relationship analysis is suitable for the identification of novel inhibitors of the intracellular replication of L. pneumophila. The most potent compound identified in this study targets (an) as yet unidentified host factor(s).

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Exploring Anti-Bacterial Compounds against Intracellular Legionella

Cited by 30 publications

References 53 publications

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

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

Adrenergic antagonists restrict replication of Legionella

Amoebae-Based Screening Reveals a Novel Family of Compounds Restricting Intracellular Legionella pneumophila

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