The Legionella pneumophila Dot/Icm-secreted Effector PlcC/CegC1 Together with PlcA and PlcB Promotes Virulence and Belongs to a Novel Zinc Metallophospholipase C Family Present in Bacteria and Fungi

Auraß, Philipp; Schlegel, Maren; Metwally, Omar; Harding, Clare R.; Schroeder, Gunnar N.; Frankel, Gad; Flieger, Antje

doi:10.1074/jbc.m112.426049

Cited by 48 publications

(48 citation statements)

References 97 publications

(105 reference statements)

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“…In addition, use of this model will allow the identification and further characterization of novel virulence factors including secreted and translocated effector proteins. Recently, it has been shown that phospholipase C activity of L. pneumophila has a role in G. mellonella virulence 60 and that the Dot/Icm effector protein SdhA is required for virulence 37 . In addition, we have recently demonstrated that there is a correlation between the phenotypes observed in G. mellonella and in the A/J mouse strain 37 .…”

Section: Discussionmentioning

confidence: 99%

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Harding

Schroeder

Collins

et al. 2013

JoVE

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Citation: Harding, C.R., Schroeder, G.N., Collins, J.W., Frankel, G. Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection. J. Vis. Exp. (81), e50964, doi:10.3791/50964 (2013). AbstractLegionella pneumophila, the causative agent of a severe pneumonia named Legionnaires' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae's immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

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Section: Discussionmentioning

confidence: 99%

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Harding

Schroeder

Collins

et al. 2013

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“…The remainder was PE-up and included seven extracellular PE-on proteins (ABC transporter periplasmic-binding protein ArtJ, hypothetical protein Lpg0264, flagellar hook protein FlgK, phospholipase C PlcB, protease/elastase LasB, polysaccharide deacetylase Lpg1993, Lpg2443) (70,(92)(93)(94). The further PE-up extr majorly included 8 proteins without assigned function (Lpg2220, Lpg1647, Lpg1645, Lpg0957, Lpg0301 Lpg1318, Lpg2206, Lpg2246), 5 T2SS substrates (chitinase ChiA, phospholipase A/acyltransferase PlaC, hypothetical protein Lpg0956, Lpg0189, Lpg0264), three Dot/Icm effectors (LegP, Lpg1667, Lpg2443) and Dot/Icm apparatus core complex protein DotC, as well as further (in addition to FlgK) motility-related proteins (FliC, FliD) (26,70,(95)(96)(97).…”

Section: Overview Of L Pneumophila E and Pe Soluble Whole Cellmentioning

confidence: 99%

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors

Auraß

Gerlach

Becher

et al. 2016

Molecular & Cellular Proteomics

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Major differences in the transcriptional program underlying the phenotypic switch between exponential and post-exponential growth of Legionella pneumophila were formerly described characterizing important alterations in infection capacity. Additionally, a third state is known where the bacteria transform in a viable but nonculturable state under stress, such as starvation. We here describe phase-related proteomic changes in exponential phase (E), postexponential phase (PE) bacteria, and unculturable microcosms (UNC) containing viable but nonculturable state cells, and identify phasespecific proteins. We present data on different bacterial subproteomes of E and PE, such as soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins. In total, 1368 different proteins were identified, 922 were quantified and 397 showed differential abundance in E/PE. The quantified subproteomes of soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins; 841, 55, and 77 proteins, respectively, were visualized in Voronoi treemaps. 95 proteins were quantified exclusively in E, such as cell division proteins MreC, FtsN, FtsA, and ZipA; 33 exclusively in PE, such as motility-related proteins of flagellum biogenesis FlgE, FlgK, and FliA; and 9 exclusively in unculturable microcosms soluble whole cell proteins, such as hypothetical, as well as transport/binding-, and metabolism-related proteins. A high frequency of differentially abundant or phase-exclusive proteins was observed among the 91 quantified effectors of the major virulence-associated protein secretion system Dot/Icm (> 60%). 24 were E-exclusive, such as LepA/B, YlfA, MavG, Lpg2271, and 13 were PE-exclusive, such as RalF, VipD, Lem10. The growth phase-related specific abundance of a subset of Dot/Icm virulence effectors was confirmed by means of Western blotting. We therefore conclude that many effectors are predominantly abundant at either E or PE which suggests their phase specific function. The distinct temporal or spatial presence of such proteins might have important implications for functional assignments in the future or for use as lifestage specific markers for pathogen analysis. Molecular

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“…pneumophila produces three PLCs, designated PlcA, PlcB, and PlcC/CegC1 (Table 5), which are induced during host cell infection (275,276). plcA is found in L. pneumophila genomes, and plcB is also present in some nonpneumophila strains, whereas plcC/cegC1 is found in all Legionella genomes sequenced so far (274). PlcC/CegC1 is injected into the host cell by a Dot/Icm type IVB secretion system, whereas PlcA and likely PlcB are secreted via an Lsp type II secretion system (274,(277)(278)(279).…”

Section: Fig 11mentioning

confidence: 99%

“…L. pneumophila PLCs constitute a separate Zn 2ϩ -dependent PLC class, together with the Pseudomonas fluorescens PC-preferring PLC and not-yet-characterized homologous enzymes from fungi (274).…”

Section: Fig 11mentioning

confidence: 99%

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

Flores-Dı́az

Monturiol-Gross

Naylor

et al. 2016

Microbiol Mol Biol Rev

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143

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SUMMARYBacterial sphingomyelinases and phospholipases are a heterogeneous group of esterases which are usually surface associated or secreted by a wide variety of Gram-positive and Gram-negative bacteria. These enzymes hydrolyze sphingomyelin and glycerophospholipids, respectively, generating products identical to the ones produced by eukaryotic enzymes which play crucial roles in distinct physiological processes, including membrane dynamics, cellular signaling, migration, growth, and death. Several bacterial sphingomyelinases and phospholipases are essential for virulence of extracellular, facultative, or obligate intracellular pathogens, as these enzymes contribute to phagosomal escape or phagosomal maturation avoidance, favoring tissue colonization, infection establishment and progression, or immune response evasion. This work presents a classification proposal for bacterial sphingomyelinases and phospholipases that considers not only their enzymatic activities but also their structural aspects. An overview of the main physiopathological activities is provided for each enzyme type, as are examples in which inactivation of a sphingomyelinase- or a phospholipase-encoding gene impairs the virulence of a pathogen. The identification of sphingomyelinases and phospholipases important for bacterial pathogenesis and the development of inhibitors for these enzymes could generate candidate vaccines and therapeutic agents, which will diminish the impacts of the associated human and animal diseases.

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The Legionella pneumophila Dot/Icm-secreted Effector PlcC/CegC1 Together with PlcA and PlcB Promotes Virulence and Belongs to a Novel Zinc Metallophospholipase C Family Present in Bacteria and Fungi

Cited by 48 publications

References 97 publications

Use of <em>Galleria mellonella</em> as a Model Organism to Study <em>Legionella pneumophila</em> Infection

Use of <em>Galleria mellonella</em> as a Model Organism to Study <em>Legionella pneumophila</em> Infection

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

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