GamA is a eukaryotic-like glucoamylase responsible for glycogen- and starch-degrading activity of Legionella pneumophila

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Abstract

Legionella pneumophila (Lp) is the causative agent of Legionnaires’ disease, an atypical pneumonia. Lp is found in freshwater habitats and replicates within different protozoa (amoebae). It is known that Lp uses amino acids as primary energy and carbon sources for replication. However, very recently it was reported that Lp is able to metabolize also carbohydrates (glucose). Here, we present for the first time experimental evidence that the lpp0489 [gamA] gene encodes a eukaryotic-like glucoamylase (GamA) responsible for the glycogen- and starch-degrading activities of Lp. Although not essential for intra- and extracellular growth, we showed that GamA is expressed and active during intracellular replication in Acanthamoeba castellanii, suggesting that Lp is degrading glycogen during intracellular replication. Altogether, these findings indicate that Lp is indeed able to degrade exogenous polysaccharides and to utilize carbohydrates (glucose).

Introduction

Legionella pneumophila (Lp) is the causative agent of Legionnaires’ disease, an atypical pneumonia. Lp is found in freshwater habitats where it replicates within different protozoa (amoebae). Lp is transmitted to humans by Lp-contaminated aerosols. After entering the human lung, Lp is phagocytosed by alveaolar macrophages wherein Lp is able to replicate leading to Legionnaires’ disease. Many virulence factors of Lp are already known, however, less is known about nutrition of Lp within the environment or during intracellular replication (Hoffman, 2008, Rowbotham, 1986, Swanson and Hammer, 2000).

Legionella species seem to have an obligate requirement for a host cell (in vivo) for replication in the environment, but nearly nothing is known about the life of legionellae present in biofilms. Lp survives within amoebae and macrophages due to its ability to establish a replication vacuole derived from the endoplasmic reticulum. At the end of the intracellular growth phase, Lp differentiates into a spore-like form (MIF), also called the transmissive form, which seems to be metabolically nearly dormant (Faulkner and Garduno, 2002, Garduno et al., 2002, Greub and Raoult, 2003, Hammer et al., 2002, Molofsky and Swanson, 2004, Swanson and Hammer, 2000). These transmissive forms of Lp may be able to persist in the environment for long periods.

It is known that Lp uses amino acids as primary energy and carbon sources (Pine et al., 1979, Reeves et al., 1981, Ristroph et al., 1981, Sauer et al., 2005, Tesh et al., 1983, Wieland et al., 2005). In line with this is the observation that amino acid transporters of the host cell and of Lp are essential for intracellular replication (Sauer et al., 2005, Wieland et al., 2005) and that the resective genes are highly upregulated (Brüggemann et al., 2006). However, recently, it was reported, that Lp secretes an endoglucanase (CelA), which is able to degrade carboxymethyl cellulose (Debroy et al., 2006). In addition, some studies have indicated that Lp is able to utilize carbohydrates (Tesh et al., 1983, Weiss et al., 1980) and only recently, it was demonstrated that the Entner–Doudoroff (ED) pathway is necessary for intracellular growth of L. peumophila (Eylert et al., 2010, Harada et al., 2010). Moreover, the available 4 genome sequences of Lp [Lp Philadelphia (Chien et al., 2004), Lp Paris, Lp Lens (Cazalet et al., 2004), and Lp Corby (Glöckner et al., 2008)] show that the Lp genome contains genes that code for all proteins of the Embden–Meyerhof–Parnas (EMP) pathway, the complete ED, and the pentose phosphate (PP) pathway (Fonseca et al., 2008, Hoffman, 2008). There are also various ABC-type transport systems, and some of them seem to be involved in sugar uptake. Very recently, we demonstrated by isotopoloque profiling studies that Lp strain Paris uses glucose as a carbon source (Eylert et al., 2010). Specifically, 13C-label from [U-13C6]glucose was found in various amino acids and in the energy and carbon storage compound poly-3-hydroxybutyrate (PHB) (Eylert et al., 2010). This leaves the question about the source for glucose in the natural environments of Lp.

In the 1980s, it was shown that Legionella exhibits a weak starch hydrolysis activity (Hébert et al., 1980, Morris et al., 1980, Thorpe and Miller, 1981). Indeed, analysis of the genome sequences showed that Lp possesses putative systems for degradation of trehalose, cellulose and chitin as well as a eukaryotic-like glucoamylase (lpp0489 [gamA]) putatively involved in the degradation of starch or glycogen (Brüggemann et al., 2006). It was also demonstrated by microarray analysis that gamA and other genes required for carbohydrate metabolism are induced during the intracellular replication in A. castellanii (Brüggemann et al., 2006). In this study, we thus analyzed the function of GamA further.

Section snippets

Strains, mutant construction, plasmids, and oligonucleotides

Escherichia coli DH5α was used for cloning of recombinant plasmid DNA. Experiments were done with Lp Paris [CIP 107629 (Cazalet et al., 2004)], Lp Corby (Jepras et al., 1985), and the ΔlspDE mutant strain of Lp Corby (Schunder et al., 2010). The lpp0489gamA) single mutant strain of Lp Paris was constructed as described previously (Brüggemann et al., 2006, Heuner et al., 2002). In brief, the gene lpp0489 was inactivated by insertion/deletion of a kanamycin resistance (kanR) cassette into the

L. pneumophila exhibits glycoamylase activity and degrades glycogen, starch, and cellulose

We first investigated whether Lp strain Paris is able to degrade the glucose-containing polymers starch, glycogen, and cellulose, widely found in nature. To assess glucoamylase activity, bacteria were grown on starch, glycogen, or CMC containing agar plates and activity was recorded as zones of hydrolysis after incubation at 37 °C for 2–3 days and staining with Lugol's solution (see ‘Materials and methods’) (Fig. 1A). In addition, cell-free culture supernatants of Lp Paris cultures grown to

Conclusion

The analysis of the gamA gene of Lp Paris showed that it encodes a functional glucoamylase responsible for the starch- and glycogen-degrading activity and that this protein is mainly secreted via the T2SS. This corroborates previous reports in which GamA was detected in the secreted protein fraction of Lp (De Buck et al., 2008, Galka et al., 2008). Directly upstream of the glucoamylase, gene lpp0490 encodes a putative regulatory protein (YozG). Further analyses under which environmental

Acknowledgements

This work was financed by grants from the Deutsche Forschungsgemeinschaft DFG (Bonn, Germany) awarded to W.E. and K.H. (EI 384/4-1 and HE 2845/6-1, respectively), from the Institut Pasteur, and from the Network of Excellence ‘Europathogenomics’ LSHB-CT-2005-512061 awarded to C.B. M.J. received support from PTR 185 (Institut Pasteur – Biorad).

References (40)

  • M. Chien et al.

    The genomic sequence of the accidental pathogen Legionella pneumophila

    Science

    (2004)
  • S. Debroy et al.

    Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung

    Proc. Natl. Acad. Sci. U.S.A.

    (2006)
  • G. Faulkner et al.

    Ultrastructural analysis of differentiation in Legionella pneumophila

    J. Bacteriol.

    (2002)
  • M.V. Fonseca et al.

    Nutrient acquisition and assimilation strategies of Legionella pneumophila

  • F. Galka et al.

    Proteomic characterization of the whole secretome of Legionella pneumophila and functional analysis of outer membrane vesicles

    Infect. Immun.

    (2008)
  • R.A. Garduno et al.

    Intracellular growth of Legionella pneumophila gives rise to a differentiated form dissimilar to stationary-phase forms

    Infect. Immun.

    (2002)
  • B.K. Hammer et al.

    A two-component regulator induces the transmission phenotype of stationary-phase Legionella pneumophila

    Mol. Microbiol.

    (2002)
  • E. Harada et al.

    Glucose metabolism in Legionella pneumophila: dependence on the Entner-Doudoroff pathway and connection with intracellular bacterial growth

    J. Bacteriol.

    (2010)
  • G.A. Hébert et al.

    The rickettsia-like organisms TATLOCK (1943) and HEBA (1959): bacteria phenotypically similar to but genetically distinct from Legionella pneumophila and the WIGA bacterium

    Ann. Intern. Med.

    (1980)
  • K. Heuner et al.

    Influence of the alternative sigma(28) factor on virulence and flagellum expression of Legionella pneumophila

    Infect. Immun.

    (2002)
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    Present address: Universitätsklinikum Münster, ZMBE Institut für Infektiologie, Von-Esmarch-Str. 56, 48149 Münster, Germany.

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