PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
FEMS Microbiol Lett. Author manuscript; available in PMC 2010 November 1.
Published in final edited form as:
PMCID: PMC2766432
NIHMSID: NIHMS150445

Legionella pneumophila Secretes an Endoglucanase That Belongs to the Family-5 of Glycosyl Hydrolases and Is Dependent upon Type II Secretion

Abstract

Examination of cell-free culture supernatants revealed that Legionella pneumophila strains secrete an endoglucanase activity. L. pneumophila lspF mutants were deficient for this activity, indicating that the endoglucanase is secreted by the bacterium’s type II protein secretion system. Inactivation of celA, encoding a member of the family-5 of glycosyl hydrolases, abolished the endoglucanase activity in L. pneumophila culture supernatants. The cloned celA gene conferred activity upon recombinant Escherichia coli. Thus, CelA is the major secreted endoglucanase of L. pneumophila. Mutants inactivated for celA grew normally in protozoa and macrophage, indicating that CelA is not required for the intracellular phase of L. pneumophila. The CelA endoglucanase is one of at least 25 proteins secreted by the type II system of L. pneumophila and the seventeenth type of enzyme effector associated with this pathway. Only a subset of the other Legionella species tested expressed secreted endoglucanase activity, suggesting that the type II secretion output differs among the different legionellae. Overall, this study represents the first documentation of an endoglucanase (EC 3.2.1.4) being produced by a strain of Legionella.

Keywords: Legionella pneumophila, type II protein secretion, endoglucanase, cellulase, intracellular infection

INTRODUCTION

Legionella pneumophila is a gram-negative bacterium that is ubiquitous in natural and man-made water systems, surviving planktonically, in protozoa, and as a part of multi-organism biofilms (Fields, et al., 2002, Lau & Ashbolt, 2009). Human infection occurs following the inhalation of contaminated water droplets originating from various types of aerosol-generating devices. Within the lung, L. pneumophila infects and grows within macrophages, eventually resulting in a pneumonia known as Legionnaires’ disease (Fields, et al., 2002, Diederen, 2008). The bacterium also undergoes various forms of differentiation as it transitions between intracellular and extracellular niches (Dalebroux, et al., 2009, Morash, et al., 2009). Much of the ecology and pathogenesis of L. pneumophila is mediated by secreted factors (Bruggemann, et al., 2006, Chatfield & Cianciotto, 2007, De Buck, et al., 2007, Shin & Roy, 2008, Allard, et al., 2009, Cianciotto, 2009, Isberg, et al., 2009). For the secretion of enzymes and other proteins, L. pneumophila utilizes type II secretion, type IVA secretion, and type IVB secretion (Cianciotto, 2005, Vincent, et al., 2006, Bandyopadhyay, et al., 2007, Al-Khodor, et al., 2009, Rasis & Segal, 2009), and the genome database suggests the existence of type I and type V secretion (Jacobi & Heuner, 2003, Cazalet, et al., 2004).

Type II protein secretion (T2S) promotes the physiology of many environmental bacteria and the virulence of a variety of human, animal, and plant pathogens (Cianciotto, 2005). In T2S, proteins destined for secretion are first translocated across the inner membrane and into the periplasm by the Sec or Tat pathway and then a pseudopilus acts to push the proteins through an outer membrane (secretin) pore (Johnson, et al., 2006, Buddelmeijer, et al., 2009, Korotkov, et al., 2009). For L. pneumophila, T2S helps the bacterium to grow and survive at low temperature as well as within amoebae, macrophages, and the lung (Hales & Shuman, 1999, Liles, et al., 1999, Rossier, et al., 2004, Cianciotto, 2005, DebRoy, et al., 2006, Rossier, et al., 2008, Söderberg, et al., 2008, Rossier, et al., 2009). Recently, a two-dimensional polyacrylamide gel electrophoresis comparison of wild type and lsp mutant supernatants revealed that the T2S secretome of L. pneumophila includes at least twenty-five proteins (DebRoy, et al., 2006). Among the proteins that were identified was a protein that was predicted to encode endoglucanase activity. L. pneumophila is well known for elaborating many enzymes that degrade protein substrates (Rossier, et al., 2008) but several studies have indicated its ability to also utilize carbohydrates (Weiss, et al., 1980, Tesh, et al., 1983, Bruggemann, et al., 2006, Fonseca, et al., 2008), therefore we sought to investigate this new, predicted activity. Using mutational and cloning analysis, we demonstrate that L. pneumophila secretes, through its T2S system, an endoglucanase that belongs to the family-5 of glycosyl hydrolases.

MATERIALS AND METHODS

Strains and media

L. pneumophila strain 130b (ATTC strain BAA-74, also known as AA100 or Wadsworth) served as our wild-type strain (Rossier, et al., 2004). A mutant of 130b containing a kanamycin-resistance (KmR) cassette inserted into lspF (NU275) was previously described (Rossier, et al., 2004). Other strains tested were L. pneumophila Philadelphia-1 (ATCC 33512), Dallas-1E (ATCC 33216), and Concord-3 (ATCC 35096), as well as L. erythra (ATCC 35303), L. feeleii (ATCC 35072), L. longbeachae (ATCC 33462), and L. parisiensis (= ATCC 35299). Legionellae were routinely cultured in buffered yeast extract (BYE) broth or on buffered charcoal yeast extract (BCYE) agar (Rossier, et al., 2004). Growth in broth was assessed by measuring the optical density of the culture at 660 nm. Escherichia coli DH5α and DH5α λ pir (Invitrogen, Carlsbad, CA), hosts for recombinant plasmids, were grown on LB agar (Rossier, et al., 2004). Antibiotics were added to media at the following concentrations (μg per ml): ampicillin, 100; chloramphenicol, 6 for L. pneumophila and 30 for E. coli; gentamicin, 2.5; and kanamycin, 25 for L. pneumophila and 50 for E. coli. Chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Sequence analysis, gene cloning, and mutant constructions

DNA and protein sequences were analyzed using Lasergene (DNASTAR). The Clustal method of Lasergene Megalign was used for protein alignments. Protein homologs were identified in the database using programs based on BLAST (Altschul, et al., 1990). Conserved domains were identified by searching the conserved domain database (Marchler-Bauer, et al., 2007). Signal sequences were identified using SignalP (Nielsen, et al., 1997), and further predictions about the cellular location of proteins were done using PSORTb (Gardy, et al., 2005). DNA was isolated as described previously (Rossier, et al., 2004). Primers for amplifying DNA from strain 130b were designed based on the Philadelphia-1 genome database (Chien, et al., 2004). A 2052-bp fragment containing celA as the only intact ORF was amplified using primers OR138 (5′-GGAATTCGTTAGCTGGCCAC) and OR139 (5′-CCTCTTAATGCTGCTACCTG) and ligated into pGEM-T Easy (Promega, Madison, WI) to yield pGCelA. A1825-bp fragment containing celA was amplified using primers MP22 (5′-TCACTCCATATGGTACCCACTCAA) and OR139 and cloned into pMMB2002 (Rossier, et al., 2004) to yield pMCelA. Allelic exchange was used to make celA mutants. To that end, a 1193-bp fragment containing the 5′ portion of celA was amplified using primers OR138 and MP2 (5′-ATTGACCTGATGCACTGCCT) and ligated into pGEM-T Easy, yielding pGCelA(N). The plasmid was digested with BstEII, which cuts 108-bp after the celA start, and then ligated to a KmR gene to yield pGCelA(N)::Km. Next, the SacI-SphI fragment of pGCelA(N)::Km containing the disrupted gene was cloned into pRE112 (Edwards, et al., 1998) yielding pRECelA(N)::Km. pRECelA(N)::Km was electroporated into 130b, and mutants were selected as before (Rossier, et al., 2004).

Detection of enzyme activities

To assess secreted endoglucanase activity, we grew legionellae in BYE broth to late-log phase and then assayed cell-free culture supernatants for activity against carboxymethylcellulose (CMC) using the zymogram method (Schwarz, et al., 1987). 15 μl of supernatants were electrophoresed through non-denaturing 12% polyacrylamide gel containing 0.1% CMC. Next, the gel was incubated at 37° for 18 h and then stained with 1 mg/ml Congo Red and destained with 1M NaCl. As confirmed using purified cellulase of Aspergillus niger, yellow clearings against the red background were indicative of a CMC-hydrolysis. In order to assess cloned activity, E. coli lysates, which were obtained as before (DebRoy, et al., 2006, Rossier, et al., 2009), were placed into wells in 1% agarose containing 1% CMC. After 3 d of incubation at 37°C, the plates were stained with 0.5% Congo Red and then destained with 1M NaCl. In order to quantitate the amount of endoglucanase activity in L. pneumophila BYE cultures, 20 μl of 100-fold concentrated supernatants (DebRoy, et al., 2006) were incubated with 1% CMC in water at 37° for 20 hours with shaking, and then the amount of reducing sugars generated was determined using the dinitrosalicylic acid method (Miller, 1959, Percival Zhang, et al., 2006). The amount of reducing sugars in supernatant samples was expressed as glucose equivalents based on a glucose standard curve. To gauge substrate specificity, recombinant E. coli lysates were incubated with CMC, Avicel, or birchwood xylan in distilled water for 20 hours at 37° with shaking, and the release of reducing sugars was measured by the 2, 2′-bicinchroninate method modified (Zhang & Lynd, 2005). To assess cellobioside activity, L. pneumophila supernatants or E. coli lysates were incubated with 0.05 mM 4-methylumbelliferyl-β-D-cellobioside in 0.05 M sodium acetate pH 5.0 at 37° for 20 hours, and then the amount of released 4-methylumbelliferone was determined fluorometrically using excitation and emission wavelengths of 360 nm and 455 nm, respectively (Chernoglazov, et al., 1989, Barr & Holewinski, 2002). The presence of secreted lipase and phosphatase activities in L. pneumophila supernatants was determined as before (Aragon, et al., 2000, Aragon, et al., 2001, Aragon, et al., 2002).

Infection assays

To examine L. pneumophila growth in protozoa, H. vermiformis and A. castellanii were infected as previously described (Rossier, et al., 2008). Thus, 104 CFU were added to wells containing 105 amoebae and then, at various times post-inoculation, the numbers of bacteria per co-culture were determined by plating dilutions on BCYE agar. To assess L. pneumophila growth in macrophages, human U937 cells were infected as previously described (Rossier, et al., 2004). Briefly, monolayers containing 106 macrophages were inoculated with 105 CFU, and then, at various times, the numbers of bacteria in the monolayer were determined by plating on BCYE agar. For infection of A/J mice, 6-8-week-old females (Jackson Lab) were inoculated intratracheally with a 25 μl suspension containing 106 CFU of a 1:1 ratio of wild type and mutant strains (Rossier, et al., 2004, DebRoy, et al., 2006). One and three days later, infected lungs were homogenized, and the numbers of bacteria and the ratio of wild type to mutant were determined by plating. Animal experiments were approved by the Animal Care and Use Committee of Northwestern University.

RESULTS

Identification of an endoglucanase activity secreted by L. pneumophila

Previously, we identified a protein that is present in wild-type strain 130b but not T2S mutant supernatants and is annotated in L. pneumophila databases (http://genolist.pasteur.fr/LegioList/) as being similar to endoglucanases (DebRoy, et al., 2006). In strains Philadelphia-1, Lens, Paris, and Corby, its corresponding gene is lpg1918, lpl1882, lpp1893, and lpc1372, respectively (Cazalet, et al., 2004, Chien, et al., 2004, Glockner, et al., 2007). The gene is monocistronic, with an upstream ORF encoding a hypothetical protein, and a downstream ORF encoding 3-deoxy-manno-octulosonate cytidylyltransferase. Compatible with its secretion, the 53-kDa protein had a Sec-dependent signal sequence (DebRoy, et al., 2006). The mature protein sequence contained an Asn-Glu-Pro domain, which, in known endoglucanases, is essential for activity (Baird, et al., 1990). For reasons below, we designated the protein and its gene as CelA and celA. Because Legionella had not been previously investigated for endoglucanases, we assayed supernatants from wild-type strain 130b for activity against the cellulose derivative CMC, as has been done in order to operationally define many endoglucanases (Molhoj, et al., 2002). The supernatant material readily cleaved CMC in a zymogram (Fig. 1A), indicating that L. pneumophila indeed secretes an endoglucanase activity. An lspF mutant defective for T2S expressed less activity in its supernatants than parental 130b did (Fig. 1A). To quantitate the amount of enzymatic activity in the supernatants, we measured the numbers of reducing sugars generated by hydrolysis of CMC using the dinitrosalicylic acid method (Fig. 2A). Once again, the T2S mutant had substantially less activity than its parental wild type. The residual activity seen for the mutant in the two different assays was likely due to cell lysis in the cultures, as we have observed before when monitoring other T2S-dependent activities (Rossier, et al., 2004, DebRoy, et al., 2006, Rossier, et al., 2008, Rossier, et al., 2009). Taken together, these data document that L. pneumophila secretes an endoglucanase that is dependent upon T2S. That both CelA and endoglucanase activity were lacking in lspF mutant supernatants suggested that CelA encodes the activity.

FIG. 1
Endoglucanase activity secreted by wild-type and mutant strains of L. pneumophila. Supernatants obtained from broth cultures were electrophoresed through a non-denaturing gel containing CMC. After staining, a clear area revealed endoglucanase activity. ...
FIG. 2
Reducing sugars generated by wild-type and mutant strains of L. pneumophila. Concentrated supernatants were incubated with CMC, and then the amount of reducing sugars was quantified using the dinitrosalicylic acid method. The samples tested include (A) ...

Influence of celA on L. pneumophila endoglucanase activity

In order to determine if CelA is in fact the secreted endoglucanase, we used allelic exchange to construct a set of celA mutants from strain 130b. Three independent mutants, NU353, NU354, and NU355, were obtained. Similar to other lsp mutants (Rossier, et al., 2004), the celA mutants grew normally at 37° in BYE broth and on BCYE agar (data not shown), indicating that celA is not required for extracellular growth under standard conditions. When cultured in broth, mutant supernatants contained normal levels of acid phosphatase and lipase (data not shown), indicating the strains do not have general defects in T2S (Aragon, et al., 2001, Rossier & Cianciotto, 2001, Aragon, et al., 2002). The celA mutants did not display the altered colony morphology or reduced efficiency of plating at 25-17°C exhibited by lsp mutants (Rossier, et al., 2004, Söderberg, et al., 2004, Söderberg, et al., 2008). To examine secreted endoglucanase activity, we grew the legionellae in BYE broth to a similar stage in the late-log phase and then assayed cell-free supernatants for activity against CMC using both the zymogram and dinitrosalicylic acid methods. In contrast to wild type, the celA mutants completely lacked activity (Fig. 1B and and2B).2B). Since the two mutants tested behaved similarly and since celA is monocistronic, these data indicate that the loss of activity was due to inactivation of celA and not a second-site mutation or polar effect. That celA encodes an endoglucanase was confirmed when lysates from recombinant E. coli expressing celA, whether on a high-copy or low-copy number vector, cleaved CMC, while samples from E. coli containing only vector did not (Fig. 3). In addition, the E. coli expressing celA also cleaved microcrystalline cellulose (Avicel) but not xylan (Fig. 4) or cellobioside (data not shown). Because the celA mutants completely lacked reactivity, CelA is the only endoglucanase active against CMC that is secreted by L. pneumophila strain 130b under standard growth conditions.

FIG. 3
CelA activity expressed by recombinant E. coli. Lysates obtained from E. coli DH5α, DH5α containing vector pMMB2002, and DH5α carrying the celA gene cloned into either high-copy number pGEM-T Easy (i.e., pGCelA) or low-copy number ...
FIG. 4
Reducing sugars generated by recombinant E. coli. Lysates from E. coli expressing the pGEM-T Easy vector (gray bars) or pGCelA (black bars) were incubated with CMC, Avicel, or xylan, and then the amount of reducing sugars generated was defined as net ...

Role of celA in L. pneumophila infection

On many occasions, we have observed that T2S mutants of strain 130b are impaired for infection of H. vermiformis and A. castellanii amoebae and human U937 cell macrophages (Liles, et al., 1999, Rossier & Cianciotto, 2001, Rossier, et al., 2004, Rossier & Cianciotto, 2005, DebRoy, et al., 2006, Rossier, et al., 2008, Söderberg, et al., 2008, Rossier, et al., 2009). Thus, to assess the role of type II-dependent endoglucanase activity in intracellular infection, we compared strain 130b and the celA mutants for their ability to infect these three, disparate hosts. Over the course of 72 hours, wild type and NU353 showed comparable growth in H. vermiformis (Fig. 5A) and A. castellanii (data not shown). The celA mutant also grew normally in the macrophage line (Fig. 5B). When the other celA mutants were tested, they grew like wild type did in amoebae and macrophages (data not shown). These data indicate that CelA is not required for intracellular infection. To ascertain the in vivo significance of the T2S endoglucanase, we analyzed NU353 for its relative ability to grow and survive in the lungs of A/J mice, using a competition assay that had detected the in vivo growth defects of other T2S mutants (Rossier, et al., 2004, DebRoy, et al., 2006). Because L. pneumophila chitinase mutants were unexpectedly defective in this assay (DebRoy, et al., 2006), we thought it possible that a cellulase, though traditionally viewed, like chitinase, as only being relevant in environmental niches, might be beneficial to L. pneumophila in the lung. However, the celA mutant persisted in the lungs as well as wild type did (data not shown), indicating that celA is not required for lung infection.

FIG. 5
Intracellular infection by a celA mutant of L. pneumophila. (A) H. vermiformis amoebae and (B) U937 cell macrophages were infected with wild-type strain 130b (•) and celA mutant NU353 (Δ) and then, at various times post-inoculation, the ...

Secreted endoglucanase activity in other legionellae

The detection of endoglucanase activity in the supernatants of several other wild-type strains of L. pneumophila (Fig. 6A) indicates that secreted endoglucanases are conserved in the L. pneumophila species. Compatible with this, a recent proteomic analysis of culture supernatants from the Philadelphia-1 strain detected a spot corresponding to CelA (Galka, et al., 2008). The Legionella genus includes 51 species besides L. pneumophila (Diederen, 2008). Therefore, we examined a sampling of other legionellae for endoglucanase activity (Fig. 6B). Whereas strains of L. erythra, L. feeleii, and L. parisiensis expressed activity, a strain of L. longbeachae lacked it, indicating that some but not all Legionella species secrete endoglucanase.

FIG. 6
Endoglucanase activity secreted by other strains of L. pneumophila and other species of Legionella. Culture supernatants were electrophoresed through a non-denaturing gel containing CMC. After staining, a clear area revealed activity. (A) L. pneumophila ...

DISCUSSION

We have defined CelA as a type II-secreted endoglucanase of L. pneumophila, based upon its strong activity against CMC and more modest activity against Avicel (Percival Zhang, et al., 2006). BLASTP results indicate that CelA is a member of glycosyl hydrolase (GH) family-5 (Beguin, 1990, Wilson & Irwin, 1999). GH enzymes cleave the glycosidic bond in carbohydrates and are organized into 113 families based on amino acid similarity (Henrissat, et al., 2008). Family-5 is the largest group of GH and is comprised of mainly endoglucanases (EC 3.2.1.4) that cleave β-glucosidic bonds at any point along a cellulose molecule (Beguin, 1990, Wilson & Irwin, 1999). Although many environmental bacteria and fungi produce these endoglucanases (Lynd, et al., 2002), this is the first documentation of such an enzyme being made by a strain of Legionella. CelA showed sequence similarity, with E values ranging from 1 × 10−7 to 7 × 10−4, to endoglucanases of various bacteria, including CelC of Pseudomonas fluorescens, Xft818 of Xylella fastidiosa, and Cel5A of Acidothermus cellulolyticus (Ferreira, et al., 1991, Geelen, et al., 1995, Baker, et al., 2005, Wulff, et al., 2006). The Legionella enzyme displayed comparable levels of similarity to putative endoglucanases predicted from the genomes of Cellvibio japonicus, Clavibacter michiganensis, Reinekea blandensis, Ruminococcus albus, Saccharophagus degradans, and Xanthomonas axonopodis and oryzae (da Silva, et al., 2002, Xu, et al., 2004, Pinhassi, et al., 2007, DeBoy, et al., 2008, Holtsmark, et al., 2008, Salzberg, et al., 2008, Weiner, et al., 2008). Given the relatively modest level of sequence similarity between CelA and other known endoglucanases, it is possible that CelA is a novel Legionella enzyme that may have additional activities. CelA is the sixth example of an endoglucanase being secreted through a T2S system, with the other examples being from Erwinia carotovora, Dickeya dadantii (formerly E. chrysanthemi), Ralstonia solanacearum, X. campestris, and X. oryzae (Hu, et al., 1992, Chapon, et al., 2001, Corbett, et al., 2005, Liu, et al., 2005, Jha, et al., 2007).

As a group, endoglucanases perform a variety of functions, but, because it is secreted, CelA is most likely involved in the catabolism of exogenous polysaccharides. In degrading complex polysaccharides like cellulose, CelA would most probably act in concert with other enzymes, including other secreted glucosidases that are predicted from in silico analysis of the L. pneumophila genome database (DebRoy, et al., 2006, Percival Zhang, et al., 2006). One of the end-products of this sort of catabolism is glucose (Percival Zhang, et al., 2006), and early studies had indicated that L. pneumophila can utilize glucose (Weiss, et al., 1980, Tesh, et al., 1983). Therefore, we investigated the impact of CelA on L. pneumophila growth, but found that the celA mutants were not impaired for replication in broth and agar media, U937 cells, amoebae, or mice. Although these data suggest that CelA is not critical for L. pneumophila growth, it is possible that the protein promotes the growth of L. pneumophila in natural aquatic niches that contain higher levels and/or different types of polysaccharides, including cellulose produced by plants, amoebae, or other bacteria (Barrett & Alexander, 1977, Aksozek, et al., 2002, Linder, et al., 2002, Recouvreux, et al., 2008). Overall, the fact that L. pneumophila secretes an endoglucanase, in addition to a chitinase (DebRoy, et al., 2006), suggests that environmental legionellae survive part of the time as free-living saprophytes vs. being strict intracellular parasites of protozoa. Beyond its role in L. pneumophila ecology, CelA may have some utility in the industrial arena, as is the case for other endoglucanases and cellulases (Wilson & Irwin, 1999, Lynd, et al., 2002).

We can now add endoglucanase activity and CelA to the expanding list of functions that are linked to L. pneumophila T2S; that is, the secretion of more than 25 exoproteins and at least 17 different types of enzymatic activities, with the previously defined activities representing a metalloprotease, chitinase, ribonuclease, lysophospholipase A, cholesterol acyltransferase, phospholipase A, two aminopeptidases, two acid phosphatases, two phospholipases C, three lipases, and a surfactant (Hales & Shuman, 1999, Liles, et al., 1999, Aragon, et al., 2000, Aragon, et al., 2001, Flieger, et al., 2001, Rossier & Cianciotto, 2001, Aragon, et al., 2002, Flieger, et al., 2002, Rossier, et al., 2004, Banerji, et al., 2005, DebRoy, et al., 2006, DebRoy, et al., 2006, Rossier, et al., 2008, Stewart, et al., 2009). Thus, the L. pneumophila system is arguably providing us with the broadest appreciation for the impact of T2S on bacterial physiology, ecology, and pathogenesis (Cianciotto, 2009).

Although the connection between type II secretion, endoglucanase activity, and CelA is currently limited to L. pneumophila, we suspect that it holds for a number of other legionellae based upon the presence of endoglucanase activity in the culture supernatants of most of the other species tested as well as the conservation of lsp genes within the Legionella genus (Rossier, et al., 2004). However, the absence of activity in cultures of L. longbeachae indicates that some species lack expression of CelA or a related endoglucanase. Interestingly, previous examinations found that another Legionella species, L. micdadei, lacked other T2S-dependent activities (Stewart, et al., 2009). Thus, the picture is emerging in which the different species of Legionella, though all possessing the T2S machinery, are not all equivalent in terms of their secretion output. The further elucidation of these differences may over time help to explain differences in the ecology and pathogenicity of the different legionellae.

Acknowledgments

We thank past and present members of the Cianciotto lab, especially Jenny Dao and Marilyn Wells, for their technical assistance and helpful comments. This work was supported by NIH grant AI43987 awarded to N. P. C.

References

  • Aksozek A, McClellan K, Howard K, Niederkorn JY, Alizadeh H. Resistance of Acanthamoeba castellanii cysts to physical, chemical, and radiological conditions. J Parasitol. 2002;88:621–623. [PubMed]
  • Al-Khodor S, Kalachikov S, Morozova I, Price CT, Abu Kwaik Y. The PmrA/PmrB two-component system of Legionella pneumophila is a global regulator required for intracellular replication within macrophages and protozoa. Infect Immun. 2009;77:374–386. [PMC free article] [PubMed]
  • Allard KA, Dao J, Sanjeevaiah P, et al. Purification of legiobactin and the importance of this siderophore in lung infection by Legionella pneumophila. Infect Immun. 2009;77:2887–2895. [PMC free article] [PubMed]
  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. [PubMed]
  • Aragon V, Kurtz S, Cianciotto NP. Legionella pneumophila major acid phosphatase and its role in intracellular infection. Infect Immun. 2001;69:177–185. [PMC free article] [PubMed]
  • Aragon V, Rossier O, Cianciotto NP. Legionella pneumophila genes that encode lipase and phospholipase C activities. Microbiol. 2002;148:2223–2231. [PubMed]
  • Aragon V, Kurtz S, Flieger A, Neumeister B, Cianciotto NP. Secreted enzymatic activities of wild-type and pilD-deficient Legionella pneumophila. Infect Immun. 2000;68:1855–1863. [PMC free article] [PubMed]
  • Baird SD, Hefford MA, Johnson DA, Sung WL, Yaguchi M, Seligy VL. The Glu residue in the conserved Asn-Glu-Pro sequence of two highly divergent endo-beta-1,4-glucanases is essential for enzymatic activity. Biochem Biophys Res Commun. 1990;169:1035–1039. [PubMed]
  • Baker JO, McCarley JR, Lovett R, et al. Catalytically enhanced endocellulase Cel5A from Acidothermus cellulolyticus. Appl Biochem Biotechnol. 2005;121–124:129–148. [PubMed]
  • Bandyopadhyay P, Liu S, Gabbai CB, Venitelli Z, Steinman HM. Environmental mimics and the Lvh type IVA secretion system contribute to virulence-related phenotypes of Legionella pneumophila. Infect Immun. 2007;75:723–735. [PMC free article] [PubMed]
  • Banerji S, Bewersdorff M, Hermes B, Cianciotto NP, Flieger A. Characterization of the major secreted zinc metalloprotease- dependent glycerophospholipid:cholesterol acyltransferase, PlaC, of Legionella pneumophila. Infect Immun. 2005;73:2899–2909. [PMC free article] [PubMed]
  • Barr BK, Holewinski RJ. 4-Methyl-7-thioumbelliferyl-beta-D-cellobioside: a fluorescent, nonhydrolyzable substrate analogue for cellulases. Biochem. 2002;41:4447–4452. [PubMed]
  • Barrett RA, Alexander M. Resistance of cysts of amoebae to microbial decomposition. Appl Environ Microbiol. 1977;33:670–674. [PMC free article] [PubMed]
  • Beguin P. Molecular biology of cellulose degradation. Annu Rev Microbiol. 1990;44:219–248. [PubMed]
  • Bruggemann H, Cazalet C, Buchrieser C. Adaptation of Legionella pneumophila to the host environment: role of protein secretion, effectors and eukaryotic-like proteins. Curr Opin Microbiol. 2006;9:86–94. [PubMed]
  • Bruggemann H, Hagman A, Jules M, et al. Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila. Cell Microbiol. 2006;8:1228–1240. [PubMed]
  • Buddelmeijer N, Krehenbrink M, Pecorari F, Pugsley AP. Type II secretion system secretin PulD localizes in clusters in the Escherichia coli outer membrane. J Bacteriol. 2009;191:161–168. [PMC free article] [PubMed]
  • Cazalet C, Rusniok C, Bruggemann H, et al. Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity. Nat Genet. 2004;36:1165–1173. [PubMed]
  • Chapon V, Czjzek M, El Hassouni M, Py B, Juy M, Barras F. Type II protein secretion in gram-negative pathogenic bacteria: the study of the structure/secretion relationships of the cellulase Cel5 (formerly EGZ) from Erwinia chrysanthemi. J Mol Biol. 2001;310:1055–1066. [PubMed]
  • Chatfield CH, Cianciotto NP. The secreted pyomelanin pigment of Legionella pneumophila confers ferric reductase activity. Infect Immun. 2007;75:4062–4070. [PMC free article] [PubMed]
  • Chernoglazov VM, Jafarova AN, Klyosov AA. Continuous photometric determination of endo-1, 4-beta-D-glucanase (cellulase) activity using 4-methylumbelliferyl-beta-D-cellobioside as a substrate. Anal Biochem. 1989;179:186–189. [PubMed]
  • Chien M, Morozova I, Shi S, et al. The genomic sequence of the accidental pathogen Legionella pneumophila. Science. 2004;305:1966–1968. [PubMed]
  • Cianciotto NP. Type II secretion: a protein secretion system for all seasons. Trends Microbiol. 2005;13:581–588. [PubMed]
  • Cianciotto NP. Many substrates and functions of type II protein secretion: Lessons learned from Legionella pneumophila. Future Microbiol. 2009 In press. [PMC free article] [PubMed]
  • Corbett M, Virtue S, Bell K, et al. Identification of a new quorum-sensing-controlled virulence factor in Erwinia carotovora subsp. atroseptica secreted via the type II targeting pathway. Mol Plant Microbe Interact. 2005;18:334–342. [PubMed]
  • da Silva AC, Ferro JA, Reinach FC, et al. Comparison of the genomes of two Xanthomonas pathogens with differing host specificities. Nature. 2002;417:459–463. [PubMed]
  • Dalebroux ZD, Edwards RL, Swanson MS. SpoT governs Legionella pneumophila differentiation in host macrophages. Mol Microbiol. 2009;71:640–658. [PubMed]
  • De Buck E, Anne J, Lammertyn E. The role of protein secretion systems in the virulence of the intracellular pathogen Legionella pneumophila. Microbiol. 2007;153:3948–3953. [PubMed]
  • DeBoy RT, Mongodin EF, Fouts DE, et al. Insights into plant cell wall degradation from the genome sequence of the soil bacterium Cellvibrio japonicus. J Bacteriol. 2008;190:5455–5463. [PMC free article] [PubMed]
  • DebRoy S, Aragon V, Kurtz S, Cianciotto NP. Legionella pneumophila Mip, a surface-exposed peptidylproline cis-trans-isomerase, promotes the presence of phospholipase C-like activity in culture supernatants. Infect Immun. 2006;74:5152–5160. [PMC free article] [PubMed]
  • DebRoy S, Dao J, Soderberg M, Rossier O, Cianciotto NP. Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung. Proc Natl Acad Sci USA. 2006;103:19146–19151. [PubMed]
  • Diederen BM. Legionella spp. and Legionnaires’ disease. J Infect. 2008;56:1–12. [PubMed]
  • Edwards RA, Keller LH, Schifferli DM. Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene. 1998;207:149–157. [PubMed]
  • Ferreira LM, Hazlewood GP, Barker PJ, Gilbert HJ. The cellodextrinase from Pseudomonas fluorescens subsp. cellulosa consists of multiple functional domains. Biochem J. 1991;279:793–799. [PubMed]
  • Fields BS, Benson RF, Besser RE. Legionella and Legionnaires’ disease: 25 years of investigation. Clin Microbiol Rev. 2002;15:506–526. [PMC free article] [PubMed]
  • Flieger A, Neumeister B, Cianciotto NP. Characterization of the gene encoding the major secreted lysophospholipase A of Legionella pneumophila and its role in detoxification of lysophosphatidylcholine. Infect Immun. 2002;70:6094–6106. [PMC free article] [PubMed]
  • Flieger A, Gong S, Faigle M, Stevanovic S, Cianciotto NP, Neumeister B. Novel lysophospholipase A secreted by Legionella pneumophila. J Bacteriol. 2001;183:2121–2124. [PMC free article] [PubMed]
  • Fonseca MV, Sauer JD, Swanson MS. Nutrient acquisition and assimilation strategies of Legionella pneumophila. In: Heuner K, Swanson MS, editors. Legionella: molecular microbiology. Caister Academic Press; Norfolk, UK: 2008. pp. 213–226.
  • Galka F, Wai SN, Kusch H, et al. Proteomic characterization of the whole secretome of Legionella pneumophila and functional analysis of outer membrane vesicles. Infect Immun. 2008;76:1825–1836. [PMC free article] [PubMed]
  • Gardy JL, Laird MR, Chen F, Rey S, Walsh CJ, Ester M, Brinkman FS. PSORTb v.2.0: expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis. Bioinformatics. 2005;21:617–623. [PubMed]
  • Geelen D, van Montagu M, Holsters M. Cloning of an Azorhizobium caulinodans endoglucanase gene and analysis of its role in symbiosis. Appl Environ Microbiol. 1995;61:3304–3310. [PMC free article] [PubMed]
  • Glockner G, Albert-Weissenberger C, Weinmann E, et al. Identification and characterization of a new conjugation/type IVA secretion system (trb/tra) of Legionella pneumophila Corby localized on two mobile genomic islands. Int J Med Microbiol. 2007;298:411–428. [PubMed]
  • Hales LM, Shuman HA. Legionella pneumophila contains a type II general secretion pathway required for growth in amoebae as well as for secretion of the Msp protease. Infect Immun. 1999;67:3662–3666. [PMC free article] [PubMed]
  • Henrissat B, Sulzenbacher G, Bourne Y. Glycosyltransferases, glycoside hydrolases: surprise, surprise! Curr Opin Struct Biol. 2008;18:527–533. [PubMed]
  • Holtsmark I, Takle GW, Brurberg MB. Expression of putative virulence factors in the potato pathogen Clavibacter michiganensis subsp. sepedonicus during infection. Arch Microbiol. 2008;189:131–139. [PubMed]
  • Hu NT, Hung MN, Chiou SJ, Tang F, Chiang DC, Huang HY, Wu CY. Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris. J Bacteriol. 1992;174:2679–2687. [PMC free article] [PubMed]
  • Isberg RR, O’Connor TJ, Heidtman M. The Legionella pneumophila replication vacuole: making a cosy niche inside host cells. Nat Rev Microbiol. 2009;7:13–24. [PMC free article] [PubMed]
  • Jacobi S, Heuner K. Description of a putative type I secretion system in Legionella pneumophila. Int J Med Microbiol. 2003;293:349–358. [PubMed]
  • Jha G, Rajeshwari R, Sonti RV. Functional interplay between two Xanthomonas oryzae pv, oryzae secretion systems in modulating virulence on rice. Mol Plant Microbe Interact. 2007;20:31–40. [PubMed]
  • Johnson TL, Abendroth J, Hol WG, Sandkvist M. Type II secretion: from structure to function. FEMS Microbiol Lett. 2006;255:175–186. [PubMed]
  • Korotkov KV, Pardon E, Steyaert J, Hol WG. Crystal structure of the N-terminal domain of the secretin GspD from ETEC determined with the assistance of a nanobody. Structure. 2009;17:255–265. [PMC free article] [PubMed]
  • Lau HY, Ashbolt NJ. The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water. J Appl Microbiol. 2009;107:368–378. [PubMed]
  • Liles MR, Edelstein PH, Cianciotto NP. The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila. Mol Microbiol. 1999;31:959–970. [PubMed]
  • Linder M, Winiecka-Krusnell J, Linder E. Use of recombinant cellulose-binding domains of Trichoderma reesei cellulase as a selective immunocytochemical marker for cellulose in protozoa. Appl Environ Microbiol. 2002;68:2503–2508. [PMC free article] [PubMed]
  • Liu H, Zhang S, Schell MA, Denny TP. Pyramiding unmarked deletions in Ralstonia solanacearum shows that secreted proteins in addition to plant cell-wall-degrading enzymes contribute to virulence. Mol Plant Microbe Interact. 2005;18:1296–1305. [PubMed]
  • Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev. 2002;66:506–577. [PMC free article] [PubMed]
  • Marchler-Bauer A, Anderson JB, Derbyshire MK, et al. CDD: a conserved domain database for interactive domain family analysis. Nucleic Acids Res. 2007;35:D237–240. [PubMed]
  • Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem. 1959;31:427–428.
  • Molhoj M, Pagant S, Hofte H. Towards understanding the role of membrane-bound endo-beta-1,4-glucanases in cellulose biosynthesis. Plant Cell Physiol. 2002;43:1399–1406. [PubMed]
  • Morash MG, Brassinga AK, Warthan M, Gourabathini P, Garduno RA, Goodman SD, Hoffman PS. Reciprocal expression of integration host factor and HU in the developmental cycle and infectivity of Legionella pneumophila. Appl Environ Microbiol. 2009;75:1826–1837. [PMC free article] [PubMed]
  • Nielsen H, Engelbrecht J, Brunak S, von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 1997;10:1–6. [PubMed]
  • Percival Zhang YH, Himmel ME, Mielenz JR. Outlook for cellulase improvement: screening and selection strategies. Biotechnol Adv. 2006;24:452–481. [PubMed]
  • Pinhassi J, Pujalte MJ, Macian MC, Lekunberri I, Gonzalez JM, Pedros-Alio C, Arahal DR. Reinekea blandensis sp. nov., a marine, genome-sequenced gammaproteobacterium. Int J Syst Evol Microbiol. 2007;57:2370–2375. [PubMed]
  • Rasis M, Segal G. The LetA-RsmYZ-CsrA regulatory cascade, together with RpoS and PmrA, post-transcriptionally regulates stationary phase activation of Legionella pneumophila Icm/Dot effectors. Mol Microbiol. 2009;72:995–1010. [PubMed]
  • Recouvreux DO, Carminatti CA, Pitlovanciv AK, Rambo CR, Porto LM, Antonio RV. Cellulose biosynthesis by the beta-proteobacterium, Chromobacterium violaceum. Curr Microbiol. 2008;57:469–476. [PubMed]
  • Rossier O, Cianciotto NP. Type II protein secretion is a subset of the PilD-dependent processes that facilitate intracellular infection by Legionella pneumophila. Infect Immun. 2001;69:2092–2098. [PMC free article] [PubMed]
  • Rossier O, Cianciotto NP. The Legionella pneumophila tatB gene facilitates secretion of phospholipase C, growth under iron-limiting conditions, and intracellular infection. Infect Immun. 2005;73:2020–2032. [PMC free article] [PubMed]
  • Rossier O, Starkenburg S, Cianciotto NP. Legionella pneumophila type II protein secretion promotes virulence in the A/J mouse model of Legionnaires’ disease pneumonia. Infect Immun. 2004;72:310–321. [PMC free article] [PubMed]
  • Rossier O, Dao J, Cianciotto NP. The type II secretion system of Legionella pneumophila elaborates two aminopeptidases as well as a metalloprotease that contributes to differential infection among protozoan hosts. Appl Environ Microbiol. 2008;74:753–761. [PMC free article] [PubMed]
  • Rossier O, Dao J, Cianciotto NP. A type II-secreted ribonuclease of Legionella pneumophila facilitates optimal intracellular infection of Hartmannella vermiformis. Microbiol. 2009;155:882–890. [PMC free article] [PubMed]
  • Salzberg SL, Sommer DD, Schatz MC, et al. Genome sequence and rapid evolution of the rice pathogen Xanthomonas oryzae pv. oryzae PXO99A. BMC Genomics. 2008;9:204. [PMC free article] [PubMed]
  • Schwarz WH, Bronnenmeier K, Grabnitz F, Staudenbauer WL. Activity staining of cellu lases in polyacrylamide gels containing mixed linkage beta-glucans. Anal Biochem. 1987;164:72–77. [PubMed]
  • Shin S, Roy CR. Host cell processes that influence the intracellular survival of Legionella pneumophila. Cell Microbiol. 2008;10:1209–1220. [PubMed]
  • Söderberg MA, Rossier O, Cianciotto NP. The type II protein secretion system of Legionella pneumophila promotes growth at low temperatures. J Bacteriol. 2004;186:3712–3720. [PMC free article] [PubMed]
  • Söderberg MA, Dao J, Starkenburg S, Cianciotto NP. Importance of type II secretion for Legionella pneumophila survival in tap water and amoebae at low temperature. Appl Environ Microbiol. 2008;74:5583–5588. [PMC free article] [PubMed]
  • Stewart CR, Rossier O, Cianciotto NP. Surface translocation by Legionella pneumophila: A form of sliding motility that is dependent upon type II protein secretion. J Bacteriol. 2009;191:1537–1546. [PMC free article] [PubMed]
  • Tesh MJ, Morse SA, Miller RD. Intermediary metabolism in Legionella pneumophila: utilization of amino acids and other compounds as energy sources. J Bacteriol. 1983;154:1104–1109. [PMC free article] [PubMed]
  • Vincent CD, Friedman JR, Jeong KC, Buford EC, Miller JL, Vogel JP. Identification of the core transmembrane complex of the Legionella Dot/Icm type IV secretion system. Mol Microbiol. 2006;62:1278–1291. [PubMed]
  • Weiner RM, Taylor LE, 2nd, Henrissat B, et al. Complete genome sequence of the complex carbohydrate-degrading marine bacterium, Saccharophagus degradans strain 2–40 T. PloS Genet. 2008;4:e1000087. [PMC free article] [PubMed]
  • Weiss E, Peacock MG, Williams JC. Glucose and Glutamate Metabolism of Legionella pneumophila. Curr Microbiol. 1980;4:1–6.
  • Wilson DB, Irwin DC. Genetics and properties of cellulases. Adv Biochem Eng Biotech. 1999;65:1–20.
  • Wulff NA, Carrer H, Pascholati SF. Expression and purification of cellulase Xf818 from Xylella fastidiosa in Escherichia coli. Curr Microbiol. 2006;53:198–203. [PubMed]
  • Xu Q, Morrison M, Nelson KE, Bayer EA, Atamna N, Lamed R. A novel family of carbohydrate-binding modules identified with Ruminococcus albus proteins. FEBS Lett. 2004;566:11–16. [PubMed]
  • Zhang YH, Lynd LR. Determination of the number-average degree of polymerization of cellodextrins and cellulose with application to enzymatic hydrolysis. Biomacromolecules. 2005;6:1510–1515. [PubMed]