Bacterial tail-specific proteases (Tsps) have been attributed a wide variety of functions including in-tracellular virulence, cell wall morphology, proteo-lytic signal cascades and stress response. This study tested the hypothesis that Tsp has a key function for the transmissive form of Legionella pneumophila. A tsp mutant was generated in Legionella pneumophila 130b and the characteristics of this strain and the isogenic wild-type were examined using a range of growth and proteomic analyses. Recombinant Tsp protein was also produced and analyzed. The L. pneumophila tsp mutant showed no defect in growth on rich media or during thermo-osmotic stress con-ditions. In addition, no defects in cellular morphol-ogy were observed when the cells were examined using transmission electron microscopy. Purified recombinant Tsp was found to be an active protease with a narrow substrate range. Proteome analysis using iTRAQ (5% coverage of the proteome) found that, of those proteins detected, only 5 had different levels in the tsp mutant compared to the wild type. ACP (Acyl Carrier Protein), which has a key role for Legionella differentiation to the infectious form, was reduced in the tsp mutant; however, tsp- was able to infect and replicate inside macrophages to the same extent as the wild type. Combined, these data dem-onstrate that Tsp is a protease but is not essential for Legionella growth or cell infection. Thus, Tsp may have functional redundancy in Legionella.

Key words: intracellular; iTRAQ; Legionella; pro-tease; tail-specific protease

Introduction

　Legionella pneumophila, the causative agent of Legion-

naires’ disease, is an aerobic gram-negative bacterium with fastidious growth requirements (Horwitz and Silverstein, 1980). The organism is a unique intracellular pathogen that evades host immune defences by parasitising human macrophages (Horwitz and Silverstein, 1980). L. pneumoph-ila exhibits a biphasic lifecycle in which it alternates between a non-motile, thin walled replicative form (replicative) and a motile, thick walled infectious form (transmissive) (reviewed Edwards et al. (2009)). When nutrient levels and other conditions are favorable in host cells, L. pneumophila alters cellular functions to replicate maximally. When nutrients become limiting, however, intracellular bacteria produce factors to alter morphologically to the transmissive form (Byrne and Swanson, 1998). This is mediated by the stringent response (Potrykus and Cashel, 2008) (reviewed Newton et al. (2010)).　The Tsp protease has been implicated in the Legionella transmissive phase by two different studies. Bruggemann and co-workers examined the transcriptomic changes for the transmissive phase relative to the replicative phase of L. pneumophila Colby strain and found that tsp was up-regulat-ed 54-fold (Bruggemann et al., 2006). Secondly, the Lqs (Legionella quorum sensing) gene is a key transcriptional activator for the transmissive phase and lqs mutants were found to have lower levels of tsp mRNA and protein (Tiaden et al., 2008). Tsp (tail-specific protease) was first identified from E. coli as a periplasmic serine protease which selective-ly degrades proteins with specific hydrophobic C-terminal sequences (Silber et al., 1992; Keiler and Sauer, 1995), impacting on cell division and morphology during stress (Hara et al., 1991), and has also been implicated in potentiat-ing long-chain fatty acid transport in E. coli (Azizan and Black, 1994). Tsp is emerging as a virulence factor in other intracellular bacteria. tsp mutants in the pathogen Brucella suis were unable to survive intracellularly, and had altered cell morphology (Bandara et al., 2005). Tsp (CT441) and a Tsp-like protease (CPAF) have been characterized to be secreted proteases with host cell substrates for the intracel-

Full Paper

Characterization of the tail-specific protease (Tsp) from Legionella(Received November 13, 2013; Accepted March 26, 2014)

Amba Lawrence,1,# Simon K. Nicholls,1,# Scott H. Stansfield,1 and Wilhelmina M. Huston1,＊1 Institute of Health and Biomedical Innovation, and School of Biomedical Sciences,

Queensland University of Technology, Q Block, 60 Musk Ave, Kelvin Grove QLD 4059, Australia

＊ Corresponding author: Wilhelmina (Willa) May Huston, Q Block, 60 Musk Ave, Kelvin Grove, QLD 4073, Australia.Tel: +61 7 31386258　　Fax: +61 7 31386030　　E-mail: w.huston@qut.edu.au#These authors contributed equally to the manuscript.

None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work.

J. Gen. Appl. Microbiol., 60, 95‒100 (2014)doi 10.2323/jgam.60.95©2014 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation

LAWRENCE et al.96

lular pathogen Chlamydia (Zhong et al., 2001; Lad et al., 2007). Tsp was shown to have an ‘accessory’ site-1 protease role in a proteolytic signalling cascade that releases a sigma factor in Pseudomonas aeruginosa (Reiling et al., 2005). A similar role for Tsp was discovered in Bacillus subtilis in the σw antibiotic resistance cascade (Martin et al., 1993; Prince et al., 2005; Makinoshima and Glickman, 2006; Westers et al., 2006). Given the transcriptional data implicating tsp with the transmissive phase of Legionella and the virulence roles of Tsp proteases identified for other bacteria, this project aimed to test the hypothesis that Legionella tsp encodes for a protease which has a critical role in the transmissive phase.

Materials and Methods

　Legionella strains and culture conditions.　 Bacterial strains and plasmids are indicated in Table 1. L. pneumophi-la serogroup 1 strain 130b was the wild-type strain used for this study (sourced from the ATCC). E. coli cells were grown in Luria-Bertani (LB) broth or on LB agar (Sambrook et al., 1989) (ampicillin and chloramphenicol at 100 µg/ml and 25 µg/ml, respectively). L. pneumophila strains were grown in chemically defined medium (CDM) (Pine et al., 1979) with or without salt at 37 or 42°C. Salt-free CDM was prepared by omitting salt, halving the MOPS concentration, and using 1/10th KH2PO4. Cultures were grown at 37 or 42°C at 220 rpm; OD660nm was recorded over a 30 h time period. L. pneumophila strains were grown either in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES; Sigma Aldrich) buffered yeast extract (AYE) broth at 37°C or on buffered charcoal yeast extract (BCYE) agar at 37°C (kanamycin and/or chloramphenicol at 25 µg/ml and 6 µg/ml respectively).　Tissue culture conditions and intracellular infection by Legionella.　The human acute monocytic leukemia cell line THP-1 was cultured in RPMI 1640 medium, 10% fetal calf serum (FCS) (Gibco) in 5% CO2 37°C. Cells were seeded into 24-well tissue culture trays at a density of 5×105 cells/well and pre-treated with 10-8 M phorbol 12- myristate 13-acetate (PMA) for 48 h in 5% CO2 at 37°C, to induce differentiation. Stationary-phase L. pneumophila cells were added at an MOI of 5 and incubated for 2 h. Cells were then treated with 100 µg/ml gentamycin to kill any external bacterial cells. THP-1 cells were washed three times with PBS and treated with 0.1% digitonin in PBS for lysis, releas-

ing the bacterial cells. Serial dilutions were plated onto BCYE media and colonies counted (72 h).　Molecular methods and cloning.　 Generation of the Legionella tspΔkn (tsp-) construct for mutation was conducted in E. coli. The plasmid consisted of a 4 kb region surrounding the tsp gene on the L. pneumophila 130b genome into the pBLUEScript vector, with a deletion and insertion of the kanamycin cassette. Construction was as follows. The primers used were TspMutF1 5′-gggaattcg gcactcaccgtattggaagg-3′ and TspMutR1 5′-gggaattctggaaac cgttgcacatcttcc-3′. The pBluescript tspΔkn mutation vector was generated by deletion of an ～800 bp internal tsp region using Acc1 and blunt insertion of the kanamycin cassette. Legionella tsp mutation was generated by natural transfor-mations of Legionella (Aragon et al., 2001). The comple-mentation vector was generated by PCR amplification of the tsp gene and promoter (pMIPTspFwd 5′-GGTCTAGAATGG TTATTAAGAAACTTTTTTCCAGTAC-3′ and pMIPTspRev 5′-GGAAGCTTTTAGTGATGATGATGATGATGTCGT TAGCTAACGCCATTCC-3′). The PCR product was cloned into the pMIP plasmid using restriction enzyme digest (sites underlined).　The protein expression vector was generated by PCR amplification using primers LpTspExpFNde1 (5′-TTCATA TGTCGGCTGAAGAAACCAATTCAAATTATTC-3′) and LpTspExpRXho1 (5′-TTCTCGAGTCTGTTAGCTAACGC CATTCCTTCC-3′). The PCR product was cloned in frame with the N-terminal pelB signal sequence and C-terminal hexaHis tag on the pET22b expression vector (Novagen), using the primer-encoded restriction enzyme sites. All vectors were confirmed by restriction enzyme digest, PCR when appropriate, and sequence analysis.　Western blots.　 Polyclonal anti-serum was generated commercially through IMVS (South Australia) using the purified recombinant Tsp protein. Serum reactivity and specificity was confirmed using immunoblots against recombinant Tsp, E. coli Tsp expression lysates, and Legionella extracts.　iTRAQ protocol.　 Labelling of L. pneumophila was performed using an Applied Biosystems iTRAQ reagents 8-plex kit. For L. pneumophila total cell extracts, strains were grown in triplicate to an equal OD660nm. Pellets were pulse sonicated (Microson ultrasonic cell disruptor) for a total of 3 min in the presence of 0.2% (w/v) sodium dodecyl sulphate (SDS) and 150 mM NaCl. Samples were buffer exchanged in PBS using dialysis cassettes, reduced with

Table 1.　Strains and plasmids used in this study.

Strain/plasmid Genotype/purpose of use Source/reference

pET22b His-tagged protein expression vector NovagenpET22btsp Tsp recombinant protein expression vector This studypMIP Legionella plasmid for complementation (E. Hartland, University of Melbourne)pMIPtsp Legionella Tsp plasmid for complementation This studypBLUEScripttspΔkn Vector to generate tsp- deletion mutant This studyLegionella pneumophila 130b Wild-type strain (N. Cianciotto, Northwestern University)Legionella pneumophila 130b tspΔkn (tsp－) This studyLegionella pneumophila 130b pMIP This studyLegionella pneumophila 130b pMIPtsp This studyLegionella pneumophila 130b tspΔkn pMIP This studyLegionella pneumophila 130b tspΔkn pMIPtsp This studyE. coli BL21 DE3 Protein expression strainE. coli JM109 Cloning strain

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10 mM dithiothreitol (DTT) and heated at 95°C for 25 min; 10 mM iodoactamide was added and incubated at room temperature for 30 min before addition of trypsin for 48 h at 37°C. A concentration of 0.5‒1 mg/ml protein digest was labelled per mass tag following manufacturer’s instructions. Samples were sent to the Molecular and Cellular Proteomics Mass Spectrometry Facility (University of Queensland) where MS/MS spectra were acquired using SCX-RP HPLC MS/MS. MS/MS spectra were matched using multiple search engines (OMSSA/X!Tandem) against a combined L. pneumophila and common Repository of Adventitious Proteins (The Global Proteome Machine; version 1.0) protein database including reverse protein sequences as decoy matches. Matched spectra were analyzed using the TransProteomic Pipeline (TPP; version 4.2) and associated embedded tools. iTRAQ quantitation proceeded using the Libra quantitation routines embedded in TPP with statistical analysis performed in the R statistical environment using publicly available routines (LIMMA) to account for multiple testing correction in iTRAQ quantitation. Only proteins with a ±0.3 log fold change (FC) or greater were considered as significant.　Electron microscopy.　 L. pneumophila cultures at 30 h were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer. Pellets were washed several times in cacodylate buffer and postfixed for 1 h in 1% osmium tetroxide. After washing with distilled water, 2% uranyl acetate was added. Pellets were dehydrated in graded concentrations of alcohol and embedded in resin before thin sections were placed onto copper grids. Thin sections were examined with a JEOL 1200EX Transmission Electron Microscope (TEM) (Analyt-ical Electron Microscopy Facility, Queensland University of Technology) at varying magnifications.　Protein production and biochemical assays.　 The Tsp expression was conducted using BL21 (DE3) by growing cultures at 30°C with 0.5 mM IPTG added at an OD600nm of 0.5. The protein was purified using Talon Resin (Clontech) using standard protocols and dialyzed into PBS or Tris buffer (pH 7.0) as appropriate. Purified protein was examined for purity by SDS PAGE and BCA assays conducted to determine protein concentration. Protease assays were conducted using known concentrations of protein and substrate in Tris buffer (pH 7.0). Peptide assays were on substrates with a pNA (para-nitroanilide) group and were monitored using the xMark microplate spectrophotometer (Bio-Rad) at 405 nm for 45 min (37°C). Assays for proteoly-sis of β-casein were incubated with 2 µg of protease and 10 µg substrate for 60 min in the stipulated buffer at 37°C prior to addition of SDS PAGE loading buffer, and boiling samples for PAGE Coomassie analysis.

Results

Legionella tsp is not required for growth during stress conditions or to maintain cellular morphology　A L. pneumophila 130b tsp deletion mutant generated for the purposes of this study was found to have no defects during growth on AYE broth (Fig. 1A). As tsp mutants in other bacteria showed a defect during growth under thermo- and osmotic stress conditions (Hara et al., 1991; Bandara et al., 2005), L. pneumophila tsp mutant growth was compared

to that of the wild type under a variety of conditions. Whilst the tsp- strain did not grow as well as the wild type on standard chemically defined media, this was not statistically significant (paired t-test, data not shown). No significant difference was observed between the growth of the wild type and tsp- under any of the osmotic or thermo-osmotic conditions tested (data not shown). Cells grown in CDM were examined by thin section transmission electron micros-copy (TEM) and no morphological difference was observed for tsp mutants compared to the wild-type cell morphology (Fig. 1B, C). The cells from both appeared healthy, exhibited pleomorphism with the presence of both long and short rods, were non-motile and contained large β-hydroxybutyrate vacuoles. No cell wall defects were observed and clear septum could be seen forming between dividing cells when several images were examined (data not shown).　The biphasic morphologies of Legionella have been reported to have differing responses to salt, with the replica-

Fig. 1.　 tsp mutants do not have any apparent growth or morphologi-cal defects.

　(A) Growth curve of Legionella strains on AYE broth. L. pneumoph-ila 130b (wild-type) is represented by filled circles, tsp- : open circles, L. pneumophila 130b pMIP: filled triangles, L. pneumophila 130b pMIPtsp: open triangles, tsp- pMIP: filled squares, tsp- pMIPtsp: open squares. Sigmoidal curves have been fitted to the data for each strain (no significant differences detected). (B/C) tsp- legionella does not have any cellular morphology defects. TEMs of (c) L. pneumophi-la 130b and (c) tsp- strains from growth on CDM media. (D) Legio-nella tsp- is not as salt sensitive as the wild-type strain at 30 h (post exponential growth). The cfu ml-1 from 10 and 30 h broth growth of the cultures serial diluted onto BCYE plates (±100 mM NaCl). The strains and times are indicated below the graph. (E) The growth curve of the Legionella 130b and tsp- strains on AYE and AYE 100 mM NaCl. (F) Tsp protein is expressed throughout the growth curve and is higher with 100 mM NaCl. Western blots for Tsp. The samples (1‒14 AYE, 15 AYE, and 16 AYE 100 mM NaCl) are 1. WT 12 h; 2. tsp- 12 h; 3. WT 16 h; 4. tsp－ 16 h; 5. WT 20 h; 6. tsp－ 20 h; 7. WT 24 h; 8. tsp－ 24 h; 9. WT 33 h; 10. tsp－ 33 h; 11. WT 48 h; 12. tsp- 28 h; 13. Marker; 14. Recombinant Tsp protein; 15. WT 0 mM NaCl 30 h; and 16. WT 100 mM NaCl 30 h. The sizes of the molecular weight marker bands are indicated to the right.

LAWRENCE et al.98

tive phase (exponential) being salt resistant and the post-exponential (transmissive phase) being salt sensitive (Byrne and Swanson, 1998; Bachman and Swanson, 2004). Cultures grown on AYE broth were subcultured onto high salt (100 mM NaCl, BCYE plates) and standard media (BCYE plates) at the exponential (10 h) and post-exponen-tial phase/transmissive phase (30 h). The wild-type strain was significantly salt sensitive (p＜0.001) at 30 h but not the tsp mutant (Fig. 1D). The tsp mutant also appeared slightly less salt sensitive at 10 h compared to the wild-type strain (not significant). The impact of 100 mM salt in rich media (AYE) on the growth of the strains was monitored through-out the growth curve. Interestingly, the wild-type and tsp- strains had very similar growth reductions when cultured continuously in the presence of 100 mM NaCl (Fig. 1E).　The protein levels of Tsp were monitored throughout the growth curve by Western blots. A pair of bands correspond-ing to the predicted mass of the Tsp protein was detected from 16‒33 h in the Legionella 130b wild-type cultures and not in the tsp mutant (Fig. 1F). The Tsp protein levels from L. pneumophila 130b wild-type cultures grown in broth in the presence and absence of supplemented 100 mM NaCl were also examined using Western blot at 30 h. There appeared to be slightly higher levels of the Tsp protein during wild-type growth with 100 mM NaCl at 30 h (or transmissive phase) (Fig. 1F, lane 16) compared to standard media, even though the optical density of the culture was lower (OD660 nm : no salt: 0.31 ± 0.0035, 100 mM salt: 0.249 ± 0.0064).

L. pneumophila tsp− is not impaired in macrophage infection or intracellular growth　The ability of the Legionella strains to infect and replicate in THP-1 macrophages was tested. There was no difference in infection or intracellular replication of tsp- compared to the wild type in this model (Fig. 2). The ability of L. pneumophila 130b and tsp- to infect and grow in Acantham-oeba castellanii was also examined and there was no signifi-cant difference between the viable bacterial yield of the wild type and tsp- at 2, 24, 48 or 72 h (data not shown).

Tsp is an active protease which is not involved in universal proteome changes for Legionella　Tsp is bioinformatically predicted to have a signal sequence, and to be anchored to the cytoplasmic membrane, suggesting a similar periplasmic/cytoplasmic membrane localization to that of other bacterial Tsps. Purified recombi-nant Tsp generated for this study cleaved β-casein, resulting in one predominant band approximately ～7 kDa smaller and the activity was not substantially improved by addition of CaCl2 and MgCl2 (Fig. 3). The activity of Tsp was then screened in 100 mM Tris pH 7.0, 5 mM CaCl2 against a series of peptides which were already available in house. The protease showed a very narrow range of activity and was only able to cleave one of these peptides (Table 2). Other substrates with the same immediate C-terminal or P1 residue (Val) were not cleaved by Tsp.　iTRAQ, a multiplexed protein quantitation strategy developed by Ross et al. (2004), was used to simultaneously determine relative protein levels in the two strains under investigation in this study. Triplicate independent samples of both L. pneumophila 130b wild type and tsp- were cultured and labelled using the iTRAQ multiplex reagent multiplex kit. 6,063 spectra were obtained by MS/MS analysis of the samples. The spectra were used for protein identification and quantification from which 102 proteins, representing approx-imately 5% of the proteome, were identified with a false discovery rate of ＜0.05. The majority of proteins identified in this spectrum was not significantly different between the wild type and tsp mutant (Table 3 for examples, not all data shown). These constituted housekeeping genes including those involved in DNA replication, transcription, and translation (elongation factor G, ribosomal protein L1 and transcription termination), and proteins involved in cell wall

Fig. 2.　 L. pneumophila tsp- is not impaired in macrophage infection or intracellular growth.

　The viable Legionella from intracellular infections (0) and replica-tion within THP-1 macrophages (24 and 48 h). The strains shown on the figure are L. pneumophila130b (indicated by filled circles) and L. pneumophila tsp- (open circles).

Table 2.　Peptides tested for Legionella Tsp proteolysis activity.

Peptide sequence Activity (ΔAbs405(nm) min-1 µg protein-1)

GGVVPV-pNA 0MPVVPV-pNA 0GGGEHTV-pNA 0GGGSFGR-pNA 0GGGAAPL-pNA 0MFKLI-pNA 0PMFKLI-pNA 0DMPFKLL-pNA 0DMPFKLV-pNA 0.46DMPFKLP-pNA 0

Fig. 3.　Tsp is an active protease able to cleave β-casein.　Coomassie stained PAGE gel of β-casein digests by purified recom-binant Tsp. The lanes are 1. Tsp protein with β-casein (100 mM Tris pH 7.0); 2. Tsp protein with β-casein (100 mM Tris, 5 mM MgCl2 pH 7.0); 3. Tsp protein with β-casein (100 mM Tris, 5 mM CaCl2 pH 7.0);, 4. Tsp protein (100 mM Tris pH 7.0); and 5.β-casein (100 mM Tris pH 7.0). The approximate molecular weights of the bands are in-dicated to the right of the figure.

Legionella tail-specific protease 99

integrity and division (rod-shaped determining protein and septum site determining protein), metabolism (succinyl CoA synthetase) and virulence (macrophage infectivity potentia-tor (MIP)) and major outer membrane protein (MOMP)). Five proteins were significantly different between the tsp mutant and wild type (greater than ±0.3 log fold change) (Table 3). Two of these proteins were fatty acid pathway constituents. One of these proteins, Lpl1429, has very little functionality described as yet. The other protein, ACP, has been characterized in L. pneumophila to be a critical component of fatty acid and lipid biosynthesis and was detected to be reduced by approximately 4-fold in the tsp mutants.

Discussion

　A diverse range of functions has been attributed to bacteri-al tail-specific proteases (Tsps), including thermo-osmotic growth, cell division, cell wall maintenance, and proteolytic signalling cascades (Keiler et al., 1995; Beebe et al., 2000; Lad et al., 2007). Previous evidence that Legionella Tsp is regulated by Lqs and highly expressed in the transmissive phase (Bruggemann et al., 2006; Tiaden et al., 2008) led to the hypothesis tested in this study that Tsp may have a critical function for Legionella transmission.　This hypothesis was tested by generating and analyzing a tsp mutant in L. pneumophila 130b. There was no impact of the tsp- mutation on the ability of the Legionella to grow on rich media, or under thermo-osmotic stress conditions, and no impact on cell morphology or cell division was apparent when the appearance of the cells was examined by transmis-sion electron microscopy. The tsp- mutation affected the salt sensitive property of the transmissive form of Legionella, and the Tsp protein level was increased in the presence of salt at this growth phase. This lack of salt sensitivity was only evident when the strains were cultured to this phase and tested for salt sensitivity and not when the entire growth curve was conducted in the presence of salt. This may suggest that the Tsp salt sensitivity role is very growth phase-specific or can be compensated for by conducting the entire growth curve in the presence of salt. Furthermore, in our

hands L. pneumophila 130b wild-type did not show the expected corresponding salt resistance during the replicative form. Potentially this phenotypic difference is not as marked in the 130b strain or more stringent salt conditions than those used here are required to accurately assess the phenotype. Certainly these data do not support a global stress response, cell division, or osmolarity-protection function for Legionel-la Tsp. Therefore, the function of Legionella Tsp appears to be distinct to that described for E. coli and Brucella.　Tsp was confirmed to be a protease during this study and the proteome of the tsp mutant was examined in order to identify native Tsp substrates or the impact of Tsp on the Legionella proteome. Using iTRAQ, triplicate independent samples of both L. pneumophila wild-type and tsp mutant were examined. From this analysis, 102 proteins were identi-fied, representing approximately 5% of the proteome, of which only five proteins were found to be different between the wild type and tsp mutant, two of which are predicted fatty acid pathway constituents. ACP (4-fold less in tsp mutants) has been characterized in L. pneumophila to be a critical component of fatty acid and lipid biosynthesis. ACP contributes to the regulatory events which control the differ-entiation between the two phases of Legionella (Dalebroux et al., 2009). The enzymes RelA and SpoT initiate the stringent response by generation of the alarmone ppGpp (guanosine tetraphosphate) which co-ordinates differentia-tion at the level of transcription (Dalebroux et al., 2009). SpoT activates either when fatty acids are excessive or when their biosynthesis is perturbed, via a direct interaction with ACP (Edwards et al., 2009). When ACP can no longer bind SpoT, L. pneumophila fails to differentiate to its infectious form (Dalebroux et al., 2009). However, given that the Legionella tsp mutants were not impaired in intracellular infections and the ACP levels were perturbed only by 0.57 (±0.21) log fold, any Tsp function in fatty acid maintenance is not essential for initiation of the stringent response. Given that Tsp is predicted to be periplasmic and ACP is known to be cytoplasmic, it seems unlikely that ACP is a direct substrate of Tsp. This may indicate that similar to Prc in P. aeruginosa, Legionella tsp may be an accessory protease which is not essential but may contribute to or amplify a

Table 3.　iTRAQ proteomic analysis of L. pneumophila wild-type and tsp-.

Protein Putative function pa % Coverageb # Peptidesc Log FCd

ChangedeGlyceraldehyde 3-phosphate dehydrogenase Involved in TCA cycle 1 5.2 2 ( 1) (-)0.73 (±0.31)Acyl carrier protein (ACP) Type II fatty acid synthase carrier protein 1 42.7 6 ( 4) (-)0.57 (±0.21)Lpl1806 Hypothetical protein (unknown function) 1 31.4 14 ( 6) (-)0.30 (±0.00)Lpl1429 Fatty acid oxidation 1 5.7 9 ( 3) (+)0.43 (±0.21)Lpl822 Hypothetical protein (unknown function) 1 51.1 108 ( 5) (+)0.83 (±0.29)No change fF0F1 ATP synthase subunit alpha Nucleotide binding protein 1 30.2 62 (11) 0.00Chaperonin GroEL Heat-shock protein 1 60.6 446 (24) 0.01 (±0.01)Elongation factor G Protein translation 1 20.9 65 (12) 0.02 (±0.03)Major outer membrane protein (MOMP) Virulence and adherence 1 22.2 72 ( 4) 0.10 (±0.06)

a Probability value for the correct identification of the protein based on spectral analysis and OMSSA/X!Tandem searches.b Indicates the percentage of total protein sequence identified.c Number of peptides identified (and number of unique peptides).d Indicates an average log fold change between triplicate independent samples (SD) where (-) indicates lower in the mutant and (+) indicates higher in the mutant. A cut-off analysis was used whereby only samples with a log FC of (±) 0.3 were considered significantly different.

e All significantly altered proteins detected by iTRAQ between wild-type and tsp mutant with a probability = 1.00 and high % coverage.f Examples of proteins with a probability = 1.00 and highest % coverage with no detected change in levels between tsp mutant and wild type.

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proteolytic signalling cascade upstream of ACP or which impacts indirectly on ACP. This speculated mechanism of activity may account for the several lines of evidence implicating Tsp in the Legionella transmissive phase, which are contraindicated by the lack of any observed impact of tsp- mutation on the infectious capabilities and only minor phenotypic differences of the transmissive phase (lack of salt sensitivity) detected in this study. Alternatively, there may be functional redundancy for the Tsp activity within the Legionella genome, although no other Tsp homologs could be detected by BLAST searches. This study has certainly added to the somewhat enigmatic reputation of Tsp as a bacterial protease with diverse functions.

Acknowledgments

　The authors acknowledge Dr. D. Stenzel for her TEM expertise and Alun Jones (Molecular and Cellular Proteomics Mass Spectrometry Facility (University of Queensland)) for mass spectrometry analysis of iTRAQ samples. This work was supported by a Clive and Vera Ramaciotti Foundation Establishment Grant and Perpetual Foundation Medical Research Grant. The authors wish to acknowledge Professor N. Cianciotto (Northwestern University, Chicago) for providing method advice and Professor E. Hartland (University of Melbourne, Australia) for kindly supplying constructs and strains.

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