Unraveling Persistent Host Cell Infection with Coxiella burnetii by

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Unraveling Persistent Host Cell Infection with Coxiella burnetii by Quantitative Proteomics Iosif Vranakis,† Pieter-Jan De Bock,‡,§ Anastasia Papadioti,|| Georgios Samoilis,†,|| Yannis Tselentis,† Kris Gevaert,‡,§ Georgios Tsiotis,*,|| and Anna Psaroulaki† †

)

Department of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Medical School, University of Crete, GR-71110 Heraklion, Greece ‡ Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium § Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium Division of Biochemistry, Department of Chemistry, University of Crete, P.O. Box 2208, GR-71003 Voutes, Greece

bS Supporting Information ABSTRACT: The interaction between the immune system and invading bacteria is sufficient to eradicate microorganisms for the majority of bacterial infections, but suppression of the microbicidal response leads to reactivation or chronic evolution of infections and to bacterial persistence. To identify the cellular pathways affected by bacterial persistence, we applied the MS-driven combined fractional diagonal chromatography (COFRADIC) proteomics technique for a comparative study of protein expression in the C. burnetii strains Nine Mile (NM) and its respective strain (NMper) isolated from 18 months persistently infected cell cultures. In total, three different proteome comparisons were performed with the total bacterial proteome, potentially secreted bacterial proteins, and the eukaryotic infected proteome being assessed. Our results revealed that among the 547 identified bacterial proteins, 53 had significantly altered levels of expression and indicated potential metabolic differences between the two strains. Regarding differences in the secreted proteins between both strains and different modulation of the host cell, machineries reflect at least large rearrangements of both bacterial and eukaryotic proteomes during the persistent model of infection when compared to the acute one, which emphasizes that C. burnetii orchestrates a vast number of different bacterial and eukaryotic host cell processes to persist within its host. KEYWORDS: C. burnetii, persistent infection, comparative proteomics, methionine-COFRADIC

’ INTRODUCTION Intracellular organisms have evolved several stratagies to resist the microbicidal mechanisms of their infected hosts, particularly to the lethal consequences of phagosome-lysosome fusion.1 Some organisms escape from the phagosomes, whereas others prevent the fusion of these organelles with lysosomes. Coxiella burnetii, the agent of Q fever, although previously believed to resist the attack of the lysosomal environment by adapting to the acidic environment of a parasitophorous vacuole with phagolysosomal characteristics,2,3 now appears to reside in an acidic compartment with characteristics of late endosomes and not lysosomes (absence of cathepsin D).4 Finally, the revolutionary work by Omsland and co-workers altered the main characteristic of C. burnetii from obligatory intracellular to facultative intracellular bacterium.5,6 C. burnetii has the ability to induce persistent infections both in humans and animals.7 Chronically infected animals shed bacteria in feces, urine and, in pregnant females, in vast numbers in the placenta leading to abortion or low fetal birth weight.8 C. burnetii r 2011 American Chemical Society

can induce chronic infections in humans, especially in immunocompromised patients or during pregnancy.9 In vitro, C. burnetiiinfected eukaryotic cells may be maintained in persistent cultures for prolonged periods of time without the addition of fresh cells or bacteria, provided that the cell incubation medium is changed regularly.3,10 13 The slow intracellular multiplication of C. burnetii, which is similar to that of eukaryotic cells, may partly explain why the bacterium does not damage infected cells despite prolonged infection. In vitro infected cell models have provided essential information on the intracellular behavior of this pathogen.10,12,13 Roman et al. proposed a model for persistent cell infection in which when an infected cell divides, one daughter cell receives the unique C. burnetii vacuole whereas the other remains uninfected.3 The finding that uninfected cells are still present in infected cell cultures after several months of infection is compatible with such Received: May 9, 2011 Published: July 27, 2011 4241

dx.doi.org/10.1021/pr200422f | J. Proteome Res. 2011, 10, 4241–4251

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Figure 1. Flow diagram depicting the design of the study.

a model. Using the murine J774 macrophage-like cell line persistently infected with the Nine Mile isolate of C. burnetii, Akporiaye et al. defined the properties of the environment in which the bacteria survived. They reported acid phosphatase activity and an acidic pH of 5.21 and concluded that C. burnetii was located in vacuoles of phagolysosomal origin.10 Hackstadt and Williams provided an explanation for the survival of this bacterium in such a hostile environment.2 Metabolism and growth of the bacteria are activated at acidic pH, as shown by the enhanced incorporation of several metabolites under such conditions.14 16 Maurin and his co-workers confirmed the acidity of the vacuoles in which C. burnetii grows.17 They extended the results of Akporiaye et al. by demonstrating that acidic pH is maintained in the vacuolar growth chamber during early propagation of the bacteria and after maintenance of the persistent infection for as long as 153 days postinoculation with the Nine Mile isolate.10 Nevertheless, Yeaman and co-workers reported variability in antibiotic susceptibility of C. burnetii during persistent infection; antibiotics were more effective in the early stage of propagation of bacteria than after establishment of persistent infection, suggesting that early diagnosis and antibiotic therapy may be important in preventing chronic disease.18 The observation that more freshly infected cells are more susceptible to antibiotics than long-term infected cells also suggests the existence of an either genetic or metabolic differentiation between both types of bacteria, possibly explaining increased antibiotic resistance. Bacterial secretion of effector proteins that functionally mimic the activities of eukaryotic proteins is a well-established virulence mechanism used to manipulate a variety of host cell processes. Examples of such host cell processes that are manipulated toward the microorganism’s benefit include cell survival signaling and vesicular trafficking.19 During C. burnetii infection, the bacterium actively manipulates biogenesis of the phagolysosomal origin vacuole where it multiplies, as well as other host cell processes that presumably ensure a stable replication niche for the duration of the bacterium’s lengthy infectious cycle.20 Bacterial protein effectors of these processes are likely delivered to the host cytosol by the organism’s Dot/Icm type IV secretion system (T4SS).

Former studies indicated that C. burnetii interferes with host cell apoptosis upon infection of mammalian cells.21,22 Up to date, the only C. burnetii proteins that have been demonstrated to be delivered into host cells by the Dot/Icm system are those belonging to the Ankyrin repeat23 family of effectors containing ankyrin repeat homology domains (ARHDs).24,25 However, the role and function of these effectors during bacterial infection remain unknown. The differences in virulence between phase I (virulent) and phase II (avirulent) clonal variants of C. burnetii has long been established.26 However, several researchers, as the ones of this manuscript, take advantage of the absence of virulence of phase II bacteria and the lack of need of live animals to deduct important indications concerning the virulent form of C. burnetii. Thus, all C. burnetii strains mentioned herein are in phase II. In this study searching for bacterial determinants that may be differentiated during persistently infected cells from C. burnetii, C. burnetii-infected eukaryotic cells were maintained in culture for 18 months (NMper) and the proteome content from such isolated bacteria was compared with the respective reference strain (NM) cultured for 10 days. Such a comparison could help explain the observed increased antibiotic resistance, provide information on factors that might be altered during a persistent infection, or identify bacterial determinants that could possibly influence the clinical outcome of Q fever. In addition, a second study was undertaken in parallel to determine the modulation of the host cell systems by identifying and comparing the eukaryotic host cell proteins infected by NM (V10) or NMper (Vper). Finally, modulation of the host cell systems toward the pathogen’s benefit, a third comparative study, was carried out during which bacterial proteins were identified within the cytoplasm of infected by either NM (NMsec) or NMper (NMper/sec) host cells in search of possible secreted effector proteins. The design of the study is depicted in Figure 1. Further, a bioinformaticsbased screening was performed across C. burnetti proteins identified within the cytosol of infected fibroblasts to locate within protein sequences eukaryotic-like motifs, repeats, and domains or other characteristics involved in pathogenesis and/or survival mechanisms to aid identification of potentially secreted 4242

dx.doi.org/10.1021/pr200422f |J. Proteome Res. 2011, 10, 4241–4251

Journal of Proteome Research effector proteins. The additional quantitative comparison among possibly secreted effector proteins by NMper and NM could add to our knowledge on persistent host cell infection by C. burnetii.

’ MATERIALS AND METHODS C. burnetii Propagation

C. burnetii Nine Mile (RSA439) phase II reference strain for Q fever was propagated in confluent African Green Monkey kidney fibroblasts (Vero; ATCC no. CCL-81) in 225 cm2 angled neck flasks (Corning Inc., Corning, NY). Infected cells were cultured in minimum essential medium (MEM; Gibco Laboratories) supplemented with 4% fetal bovine serum (FBS; Gibco Laboratories, Gaithersburg, MD) and 2 mM L-glutamine (Gibco Laboratories) at 35 °C in a 5% CO2 incubator. Infection was monitored by Gimenez staining27 and IFA using antiserum (Q fever-Positive control, Panbio Inc., Columbia, MD) to C. burnetii. C. burnetiiinfected Vero cells were maintained in culture consecutively for 18 months by changing the culture medium once a week. The course of infection was monitored by the methods described above, and sterility checks were performed every time the medium was changed. The bacteria isolated from the 18-month continuous culture period were named Nine Mile persistent (NMper). C. burnetii Nine Mile (RSA439) phase II reference strain for Q fever was propagated once more under identical conditions and culture media in Vero cells until the infection rate was >90% as determined by Gimenez staining and direct immunofluorescence (∼10 12 days). The bacteria isolated following the 10-days culture period were named NM. Bacteria and Bacterial Protein Isolation

C. burnetii strains NM and NMper were isolated from their host cells using renographin (Ultravist 370; 0.769 g/mL iopromide; Schering) density gradient ultracentrifugation as described elsewhere.28,29 Renographin clean bacteria resuspended in K36 buffer (16.5 mM KH2PO4, 33.5 mM K2HPO4, 100 mM KCl, 15.5 mM NaCl) containing a protease inhibitor cocktail (SigmaAldrich) underwent five 5-min freeze thaw cycles (from 196 to 37 °C), and the protein concentration was determined by the Bradford assay. All manipulations involving viable bacteria were performed in a Biosafety Level III laboratory. Infected Host Cell Cytosol Isolation

The purification of the cytoplasmic fraction from infected Vero cells (either by NM or NMper) was performed as described by Samoilis et al.30 In brief, C. burnetii infected fibroblasts were centrifuged at 2600 g for 10 min at 4 °C after which the pellet was resuspended in 2 mL K36 buffer containing a protease inhibitor cocktail (Sigma) and ruptured by several passages through a fine needle syringe. Samples were centrifuged twice at 150 g for 10 min at 4 °C. The pellet containing unfractionated infected cells and cellular debris was discarded and K36 buffer containing protease inhibitor cocktail was added to the supernatant to a final volume of 10 mL. Twenty-five percent sucrose in K36 buffer was added to the samples in a 1:1 ratio after which the samples were centrifuged for 30 min at 3850 g at 4 °C. The supernatant was collected and assessed using the Gimenez stain for the presence of bacteria. Supernatants free of bacteria were diluted 5-fold with Tris-HCl pH 6.8 and centrifuged at 150 000 g for 90 min at 4 °C. Cytosolic proteins present in the supernatant were precipitated using three volumes of ice-cold acetone per one volume of supernatant at 20 °C overnight and centrifuged the next day at 10 000 g for 15 min at 4 °C. Cytoplasmic protein

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pellets were resuspended in phosphate buffer pH 7.0, and the protein concentration was determined by the Bradford assay. Isolation, Identification, and Quantification of Differentially Labeled Methionine-Containing Peptides by COFRADIC

The methionine-COFRADIC procedure was performed on both NM and NMper whole lysates as described elsewhere. The method is a peptide-based protein identification technique and was applied to select and identify methionine peptides. The methionine peptides are selected by two repeated reverse phase HPLC runs with an oxidation step in between.31 Proteomes were digested with endoproteinase Lys-C, and the peptides were labeled by N-propionylation as described previously.32 The NM proteome digest was labeled with 12C3-propionyl whereas the NMper proteome digest was labeled with 13C3-propionyl. Each peptide thus had a label on its N-terminal alpha-amino group and on its C-terminal lysine epsilon-amino group, evoking a difference of 6 Da between light and heavy peptides. Samples were mixed in a 1:1 ratio and analyzed by LC MS/MS on an Ultimate 3000 HPLC system (Dionex, Amsterdam, The Netherlands) in-line connected to a LTQ Orbitrap XL mass spectrometer (Thermo Electron, Bremen, Germany). Instrument settings for LC MS/MS analysis and generation of MS/MS peak lists were as described elsewhere.33 MS/MS peak lists were searched with Mascot using the Mascot Daemon interface (version 2.2.0, Matrix Science). The Mascot search parameters were as follows. Searches were performed in a C. burnetii database downloaded from Uniprot on June 17th 2009 (containing 1815 protein entries). Lys-C/P was set as the used protease with one missed cleavage allowed, and the mass tolerance on the precursor ion was set to (10 ppm and on fragment ions to (0.5 Da. S-Carbamidomethylation of cysteine and oxidation of methionine (to its sulfoxide) were set as fixed modifications. In addition, Mascot’s C13 setting was set to 1. The light and heavy labels were defined in Mascot’s quantitation method. Peptide quantifications were carried out using the Mascot Distiller Quantitation Toolbox (version 2.2.1). The quantification method details were as follows: constrain search, yes; protein ratio type, average; report detail, yes; minimum peptides, 1; protocol, precursor; allow mass time match, yes; allow elution shift, no; all charge states, yes. Ratios for identified proteins were calculated by comparing the XIC peak areas of all matched light peptides with those of the heavy peptides, and the results were verified by inspection of MS spectra using the in-house developed Rover tool.34 This methionine-COFRADIC procedure was also used for analyzing the cytosolic proteomes of Vero cells infected by either NM or by NMper. Now, the NM-infected Vero proteome digest was labeled with 12C3-propionyl, while NMper-infected Vero proteome digest with 13C3-propionyl, and LC MS/MS analysis was performed as described above. First, the MS/MS spectra were searched in the C. burnetii database to identify possibly secreted proteins of the bacterium into the cytoplasm of the host cell. Second, the spectra were searched in the Swiss-Prot database, with taxonomy set to “Other primates” to identify Vero cell proteins with identical parameters. The quantitation method described above was used for comparing eukaryotic proteins from infected Vero cells and possibly secreted bacterial proteins from both bacterial strains. Protein Analysis by Bioinformatics

Analysis of the amino acid sequences of all identified proteins apart from the eukaryotic proteins that were not up- or down regulated was carried out using several Web-based software tools, 4243



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with the proteins from the respective reference strain cultured for 10 days (here referred as NM). Throughout the 18-month time period of the culture, asymmetric cell division initially described by Baca and co-workers11 was observed through the cytopathic effect of the persistently infected Vero cells. In particular, although 8 10 days post infection ∼90% of the cells were enlarged (infected) (Figure 2c), the following 20 days a “reduction” of the infection percentage was noted. In fact, one could argue that there were periods (2 4 days) that there was no infected Vero cytopathic effect (Figure 2). Bacterial Proteome Comparison

Figure 2. Vero cells at various stages during their infection by C. burnetii as seen by IFA and at 60 magnification. (a) Uninfected Vero cells. (b) C. burnetii infection 1 day post infection. Most of the Vero cells are still uninfected with the vast majority of the bacterium remaining extracellular. (c) C. burnetii infection 10 days postinfection. Most of the Vero cells are infected by the intracellular bacterium, which multiplies occupying a large part of the infected cell. (d) C. burnetii infection 18 months postinfection. Although Vero cells are in continuous culture for 18 months, the infection rate has been reduced compared to the 10 days postinfection figure. Note the number of uninfected Vero cells (red fluorescence) among the infected ones (green fluorescence). One can also observe the enlargement of the infected Vero cell to accommodate the vast numbers of the intracellular bacteria.

freely accessible from the “ExPASy Proteomics Server” of the Swiss Institute of Bioinformatics (SIB) (http://au.expasy.org/). The purpose of this analysis was to calculate the molecular weight (MW) and isoelectric point (pI) of each protein using the software ProtParam Tool (http://au.expasy.org/tools/protparam.html) and Compute pI/Mw Tool (http://au.expasy.org/ tools/pi_tool.html). Additionally, C. burnetii proteins identified from the cytosol of infected Vero cells were screened using the protein subcellular localization prediction software Psortb v.3.0 at http://www.psort.org/psortb/index.html. Proteins with extracellular, multiple location sites, unknown and cytoplasmic with low extracellular scores, were further analyzed with two additional protein subcellular localization prediction software tools, namely CELLO (http://cello.life.nctu.edu.tw/) and PSLpred (http://www.imtech.res.in/raghava/pslpred/), and with the SignalP software (http://www.cbs.dtu.dk/services/SignalP/) to predict signal peptide cleavage sites. Concurrently, these “possibly secreted” proteins were examined by REP (http://www.embl. de/∼andrade/papers/rep/search.html) and SMART (http:// smart.embl-heidelberg.de/) for the presence of specific repeats (i.e., ankyrin repeats) and known and/or unknown motifs, domains and other characteristics, respectively.

’ RESULTS AND DISCUSSION C. burnetii proteins isolated from persistently infected Vero cells (namely NMper) were identified and quantitatively compared

Based on the selection of methionine-containing peptides, 19,452 MS/MS spectra have been identified and following stringent filtering,35 547 proteins, corresponding to more than 30% of all ORFs, common to both strains (NM and NMper) were identified (Supplementary Table 1, Supporting Information) (Table 1). The 547 proteins were assigned to 20 functional categories (Figure 3) based on the criteria reported in Samoilis et al.28 This protein set covered a wide range of cellular functions, suggesting an unbiased sampling of bacterial proteins. Concerning the quantitative comparison among NM and NMper, following statistical analysis, all proteins or peptides with a ratio less than 0.718 or higher than 5.088 were considered as significantly up- or down-regulated (p < 0.05), respectively, with higher ratios indicating proteins that are more abundant in the NM strain, whereas proteins with lower ratios are more abundant in the NMper strain. In total, 53 proteins were found to be significantly regulated in either of the two strains, with 44 overexpressed in NM (Supplementary Table 2, Supporting Information) and just 8 in NMper (Supplementary Table 3, Supporting Information). The observed broad distribution of peptide ratios that can be also depicted in Figure 4 can be interpreted as though we are dealing with two completely different proteomes. Since this cannot be the case here, it can only be indicative of the large proteome rearrangements that occur during the persistent model of infection. Adaptation to Atypical Condition. The ability of C. burnetii adapting to unusual and/or stressful conditions, such as those prevailing in the microenvironment of the phagosome is of the utmost importance for its survival. During this study we identified six proteins with known role in this process one of which, the starvation sensing protein (Q83C56) was overexpressed in NM. According to the literature this protein is overexpressed under nutrient starvation and is a general indication for the start of the adaptation of bacteria to stressful conditions.36 The stationary phase of bacterial growth is a period of quiescent, nongrowth during which the bacterium dramatically alters patterns of gene expression to allow extended cell survival in the absence of nutrients. Thus, starvation is the major signal regulating entry into stationary phase. In C. burnetii, Large Cell Variants (LCVs) are transformed to Small Cell Variants (SCVs) during the stationary phase of the bacterium. The transition into stationary phase is directed by key transcription factors, the activity or production of which is stimulated by starvation.37 In consequence, the NMper under-expressed starvation sensing protein (Q83C56) could be an indication of the bacterium’s strategy to avoid its stationary phase leading to persistent infection. Cell Envelope. Six of the identified under-expressed proteins in NMper are cell envelope proteins. DTDP-glucose-4,6-dehydratase 4244


C3-propionyl

points above the threshold C3-propionyl 13

12

C3-propionyl

C3-propionyl 13

12

11062

6763

5,064 (75%)

8376 (75%)

1297

2016

612

949

peptides with a score less than 10

peptides shorter than 8 amino acids

710

313

547

NMpersistent

by NM vs Proteins from Vero infected by

secreted proteins

Proteins from Vero infected

NM persistent

whole lysate

NM secreted proteins vs

99% confidence interval

peptides with a FALSE ratio

812 2094 14733 (75%) 19452 C3-propionyl 13

C3-propionyl Endoproteinase Methionine-

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COFRADIC Lys-C

proteins

12

NM whole lysate vs

NM persistent

filtration

criteria proteins

no. of unique no. of unique

peptides containing spectra

no. of methionine no. of obtained

labeling proteome

digestion technique study

Table 1. Synopsis of the Protein Identification Results from Methionine-COFRADIC Following Filtration of the Spectra

spectra

no. of identified


(Q93N67) and GDP-mannose-4,6-dehydratase (Q93N54) are the starting points of two de novo biosynthetic pathways that lead to the synthesis of L-rhamnose and L-fucose, respectively. Both monosaccharides are found in polysaccharides of the bacterial cell wall and specifically in the lipopolysaccharide (LPS).38 Another under-expressed protein in NMper involved in the cell envelope was UDP-N-acetylglucosamine 2-epimerase (Q83D96), which catalyzes the interconversion of UDP-Nacetylglucosamine (GlcNAc) and UDP-N-acetylmannosamine (ManNAc). It provides bacteria with a source of activated ManNAc residues that are used for the biosynthesis of a variety of cell surface polysaccharides.39 Yet another under-expressed protein in NMper with a predicted cellular role related to the cell envelope is undecaprenyl pyrophosphate synthetase (Q83BV5). This protein is associated with the transport of lipids for the synthesis of peptidoglycan. Researchers have suggested this specific protein as a potential target of antibiotics in Helicobacter pylori40 with encouraging results, which underlines the importance of this protein in the life cycle of bacteria. The remaining two proteins ADP-heptose-LPS heptosyltransferase II (Q83B54) and polyprenyl-phosphate beta-D-mannosyltransferase (B5U8Q5) that were under-expressed in NMper are involved in the lipopolysaccharide biosynthetic process of the bacterium. DNA Metabolism (Replication, Recombination, Repair). Seven protein down-regulated in NMper are involved in DNA metabolism. The DNA polymerase III beta chain (Q83FD7), DNA polymerase III alpha subunit (Q83C00) and the DNA primase (Q83BB7) participate in a complex network of interacting proteins and enzymes required for DNA replication. Two proteins which participate in DNA repair were found downregulated, namely the mismatch repair protein mutL (Q83CM9) and the recA protein (Q83CQ4). While the mutL is involved in the repair of DNA mismatches, the biological role of recA is in the DNA damage, recombination, repair and SOS response. The remaining DNA-binding protein fis (Q83EI3) and the exodeoxyribonuclease 7 large subunit (Q83C85) that were under-expressed in NMper are involved in regulation of transcription and in DNA catabolism, respectively. Transcription. Three proteins that are associated with regulation of transcription are under-expressed in NMper. The membrane sensor protein dTDP-4-dehydro-6-deoxy-D-glucose 4-aminotransferase (Q93N46) and nucleotide-sugar aminotransferase (B5U8Q1) with transaminase activity belong to the degT/dnrJ/eryC1 family. The third under-expressed protein is the two-component response regulator (Q83AA0) which is part of one of the predominant signal transduction mechanisms employed by microbes to modulate their gene expression repertoire in order to adapt to changing environments. This signal transduction mechanism is a phosphotransfer pathway commonly referred to as ‘two-component’ signal transduction system (TCSTS). It typically consists of a sensor histidine kinase41 and a response regulator (RR), and has been found across all three domains of life; Bacteria, Archaea and Eukarya.42 Although most bacterial TCSTS use a simple phosphotransfer pathway from one HK to one RR, C. burnetii employs a multiplestep phosphorelay involving a phosphotransfer scheme of His Asp His Asp which is predominant in eukaryotes and less common in prokaryotes.43 Energy Metabolism. Two proteins which are involved in carbohydrate metabolism are under-represented in the NMper strain. The pyruvate dehydrogenase E1 component alpha subunit (B5U8Q8) is involved in glycolysis with an acetyl-transferring 4245



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Figure 3. Identified C. burnetii proteins grouped according to their predicted cellular function.

Figure 4. Statistical analysis for the determination of overexpression limits showing also the broad distribution of peptide ratios.

activity, and the second one, transaldolase (Q83DM4), participates in carbohydrate metabolic processes and particularly in the nonoxidative phase of the pentose phosphate pathway. Transaldolase44 is a key enzyme of the reversible nonoxidative

branch of the pentose phosphate pathway (PPP) that is responsible for the generation of NADPH to maintain glutathione at a reduced state namely GSH and, thus, to protect cellular integrity from reactive oxygen intermediates (ROIs).23 Additionally, 4246



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Secretome Comparison

Figure 5. IFA photo from the isolated intracellular bacteria following separation from the host cell components.

a methionine import ATP-binding protein metN (Q83F44) was under-expressed in NMper. It belongs to the ABC transporter family containing the family’s signature domain (LSGGQKQRVAIARAL)45 and part of the ABC transporter complex MetNIQ involved in methionine import, and thus responsible for energy coupling to the transport system. One more protein associated with transport and binding of sugars, the phosphoenolpyruvate-protein phosphotransferase (Q83BF9), was identified only in NM. Although in many bacteria the phosphotransferase system (PTS) is associated with transport and phosphorylation of sugars, in the phylum of Proteobacteria, PTS proteins have regulatory properties in terms of gene expression, metabolism and chemotaxis. These properties are the result of different phosphorylation states of proteins according to the PTS-sugar substrate to be transferred. Participation of different forms of PTS proteins in distinct interactions with numerous other proteins results in a large number of regulatory functions.46 Therefore, although it is likely that the function of the C. burnetii phosphoenolpyruvate-protein phosphotransferase is associated with the transport of sugars, its exact role and the reason for its observed expression only in NM remains unknown. Intermediary Metabolism. The NMper under-expressed proteins, 3-oxoacyl-[acyl-carrier protein] reductase (Q93N41) and rubredoxin-NAD(+) reductase (Q83EN9) are oxidoreductases. The first one participates in fatty acid biosynthesis and the second in fatty acid metabolism. The following sulfate adenylyltransferase (B5U8Q7) and methyltransferase (Q83DM1) are transferases, the first one participating in three metabolic pathways, purine metabolism, selenoamino acid metabolism and sulfur metabolism, while the second one by transferring a methyl group from a donor to an acceptor (methylation), participates in numerous biological pathways, including signal transduction, biosynthesis, protein repair and gene silencing.47 Finally, 15 proteins under-expressed and nine overexpressed in NMper have yet unknown cellular functions, indicative of the additional work that needs to be done to that direction.

The approach for the identification of secreted C. burnetii proteins was adapted from Samoilis et al.48 aimed at separating intracellular bacteria from the host cell components and then isolate the hydrophilic proteins that are present in the host’s cytoplasm in the mildest possible way so that the bacteria remain intact. The applied methodology was indeed mild enough so that the intracellular bacteria remained intact as was determined by IFA (Figure 5). Concerning the acquired spectra from cytosols of Vero cells infected by NM (NMsec) or NMper (NMper/sec), these were searched in the Swiss-Prot database, with taxonomy set to C. burnetii to identify possibly secreted proteins of the bacterium into the cytoplasm of its host cell. This search resulted to 6763 spectra getting identified, 5064 of which contained methionine (75%), finally cumulating to 1297 unique peptides and 612 unique proteins. After quality filtering described above, 3405 identifications remained, covering 582 unique peptides and 313 unique proteins (Table 1). As in NM and NMper whole lysates analysis, the 313 identified proteins were assigned to functional categories28 (Supplementary Table 4, Supporting Information). Out of the 313 identified proteins, 250 were overexpressed during NMper infection and 63 proteins were identified in equal expression levels among the two strains (Supplementary Table 4, Supporting Information). Figure 6 indicates the log2 peptide ratios of C. burnetii (red bars) in comparison with the log2 peptide ratios of identified eukaryotic proteins (blue bars). As it can be observed there are a lot more C. burnetii proteins identified in the NMper infected Vero sample. Following analysis using Psortb v.3.0 software out of the 313 bacterial proteins that were identified in the cytosol of the infected host cells 108 proteins indicated extracellular, multiple location sites, unknown or cytoplasmic location with low extracellular scores highlighting the possibility of their secreted nature. These 108 proteins were further analyzed using the software mentioned previously. Out of these 108 proteins, 79 were overpresent in the NMper strain. Out of these 108 proteins, 10 proteins were identified as extracellular by PSLpred software and just 4 by CELLO software, while 14 proteins had either a strong or a weak signal peptide as indicated by SignalP software (Supplementary Table 5, Supporting Information), increasing the possibilities that these proteins are secreted. Although the method for isolation of C. burnetii secreted proteins developed by Samoilis and co-workers (2010) could not be characterized as highly efficient (as termed by the large number of identified nonsecreted proteins) following analysis of the proteins that were identified using bioinformatics approaches, several identified proteins became of interest. Initially all hypothetical exported proteins were subjected to a similarity search using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the database of C. burnetii’s closest relatives namely L. pneumophilla and F. tularensis. Out of the results of this search, which are summarized in Table 2, the highest score was attained by CBU_1984 with a similarity percentage of 73% with the L-PSP (mRNA) endoribonuclease of L. pneumophilla. Endoribonucleases are known for their role in inhibition of protein synthesis.49 Is it possible that this C. burnetii’s protein has a similar function within the cytoplasm of the infected host cell? Another identified bacterial protein within the cytoplasm of infected Vero cells that is of interest is the enhanced entry protein enhC (Q83CH9). This particular protein possesses 18 TPR repeats and a leucine-rich region as indicated by the REP 4247



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Figure 6. Log2 peptide ratios of C. burnetii (red bars) in comparison with the log2 peptide ratios of identified eukaryotic proteins (blue bars).

Table 2. Results from the Protein Blast of NCBI Using the Identified Hypothetical Exported Proteins of C. burnetii and the Protein Database of Legionella and Francisella C. burnetii hypothetical exported protein

Francisella (query coverage %)

CBU_2072

glycyl-tRNA synthetase, beta subunit (21%)

CBU_1735

hypothetical protein (31%)

hypothetical protein lpl1009

CBU_1095 CBU_1869

No significant similarity hypothetical protein (42%)

chemiosmotic efflux system protein A-like protein (51%) putative M23/M37 family peptidase (22%)

CBU_1366

hypothetical protein (54%)

conserved hypothetical protein (72%)

CBU_1984

No significant similarity

L-PSP (mRNA) endoribonuclease (73%)

software. The bacterial homologue of this protein is present in L. pneumophila where it acts in the early stages of pathogen uptake.50 Although it is believed that C. burnetii’s enhC is secreted through a Dot/Icm-independent manner, it might have similar functions to eukaryotic TPR proteins as adaptor proteins in assembling signaling complexes.51 Finally, 4 proteins of the Dot/Icm type IV secretion system (T4SS) were identified; IcmE, IcmS, IcmL and IcmX. All of these were found overexpressed in NMper/sec apart from IcmE which was equally expressed between the two infection models. The functional similarities between the Dot/Icm pathogenesis systems of C. burnetii and L. pneumophila have been studied elsewhere indicating a high sequence homology of the proteins identified in this study apart from IcmX which indicated low sequence homology to its homologous L. pneumophilla protein.52 Host Cell Cytosolic Proteome Comparison

It is already established that biogenesis and maintenance of the parasitophorous vacuole in which C. burnetii multiplies require de novo C. burnetii protein synthesis. C. burnetii proteins can also actively modulate eukaryotic signaling pathways, mediating infection events. It is thus of importance to study the host cell mechanisms, in this case on the level of proteome, and the way these might be modulated by the bacterium’s effector proteins during persistent infection. In this context, cytoplasmic proteins of host cell infected by one of both strains were isolated and quantitatively compared.

Legionella (query coverage %) GTP-binding protein Era (61%)

Spectra from the analyzed cytosolic proteomes of Vero cells infected by NM or NMper were searched in the SwissProt database, with taxonomy set to “Other primates”. The result was 11,062 identifications, of which 8,376 contained methionine (75%), cumulating in 2,016 unique peptides and 949 unique proteins, the number of which was reduced to 710 following quality filtering (Table 1). Unlike for the bacterial proteins, here only the eukaryotic proteins that were assigned as being over- or under-expressed were assigned to functional categories and had their molecular weight and pI calculated (Supplementary Table 6, Supporting Information). The ratios of the peptides identified in the “Other primates” database were calculated. The statistical analysis performed on the ratios of the peptides identified in the “Other primates” database identified the limits for up and down-regulation. All proteins or peptides with a ratio less than 0.479 or higher than 5.31 were considered significantly up or down-regulated (p < 0.05). According to these limits, 44 host cell proteins were overexpressed during infection by NMper, whereas 107 eukaryotic proteins were overexpressed during the 10 day infection period by the reference strain (Supplementary Table 6, Supporting Information). Following the classification of these proteins according to their cellular function several of them are of interest and are further discussed below. Palmitoyl-protein thioesterase 1 (PPT1) (Q8HXW6) was identified as overexpressed in Vero cells that were persistently infected (Vper). It is involved in deacylation of palmitoylated 4248


Journal of Proteome Research proteins and associated with negative regulation of cell growth. Overexpressed PPT1 in human neuroblastoma (LA-N-5) cells has been shown to protect against apoptosis through increased phosphorylation of the antiapoptotic protein Akt,53 a pathway which has been previously shown to be exploited by C. burnetii for successful intracellular parasitism and maintenance of host cell viability.51 In the same context, apoptosis inhibition, a protein that may be involved in modulation of cell growth, glypican-3 (GPC3) (A5A6P7), was found overexpressed in Vper. GPC3 is a heparan sulfate proteoglycan and can regulate the activity of a wide variety of growth and survival factors. Studies with increased GPC3 expression in cell carcinoma cell lines suggest that the protein may play a protective role against apoptosis.54 On the other hand, merlin (P59750), a multifunctional protein which is involved in integrating and regulating the extracellular cues and intracellular signaling pathways that control cell fate, shape, proliferation, survival, and motility was also overexpressed in Vper. Studies have shown that increased expression of merlin not only inhibits cell proliferation but also promotes apoptosis.55 Similarly, SPARC (Q5R767), a multifunctional glycoprotein that belongs to the matricellular group of proteins was also overexpressed in Vper. It inhibits cellular proliferation by an arrest of cells in the G1 phase of the cell cycle and also regulates the activity of several growth factors, one of which is the fibroblast growth factor (FGF)-2.56 Two more proteins that were overexpressed in Vper were legumain (Q4R4T8) and cytochrome p450 3A8 (P33268). Legumain is a cysteine protease that hydrolyzes asparaginyl bonds. Mammalian legumain is known to exist in a number of mammalian tissues such as kidney (Vero cell line). With respect to physiological functions, legumain degrades several biologically active peptides and proteins such as annexin II which is abundant in the receptor-recycling compartments of endosomes/lysosomes41 taking part in many cellular functions, including apoptosis.57 Cytochrome p450 3A8 that was also identified in overexpression in Vper belongs to the superfamily of cytochrome p450 proteins (CYPs) famous for their role in the phase I metabolism of foreign compounds, including pharmaceuticals.58 The observed overexpression of cytochrome p450 3A8 in Vper could be linked to the observed increased antibiotic tolerance of C. burnetii strains from persistently infected Vero cells. Conversely, proteins of interest that were identified in overexpression in V10 (or under-expressed in Vper) are annexin A1 (A5A6M2) that promotes membrane fusion and is involved in exocytosis through actin reorganization59 and cdc42-interacting protein 4 (Q5RCJ1), a protein crucial for endocytosis.60 In conclusion, this is the first proteome study attempting to unravel the enigma behind the persistent infection of C. burnetii. Taking into account three different aspects of the infection models, that is, the total bacterial proteome, the proteome of infected cells and the collection of possibly bacterial protein secreted in the cytosol of infected cells, our study can be considered as quite comprehensive. The results reflect at least the large rearrangements of the proteome during the persistent model of infection when compared to the acute one and emphasize that C. burnetii orchestrates a vast number of different bacterial and eukaryotic host cell processes to persist within its host.

’ ASSOCIATED CONTENT

bS

Supporting Information Supplementary Tables 1 6. Furthermore, all parameters of the experiment were uploaded to the pride database (http://

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www.ebi.ac.uk/pride/) under experiment accession 17115. This material is available free of charge via the Internet at http://pubs. acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Tsiotis Georgios, Division of Biochemistry, Department of Chemistry, University of Crete, P.O. Box 2208, GR-71003 Voutes, Greece.

’ ACKNOWLEDGMENT This research program was supported by the University of Crete, the Greek Ministry of Education and the General Secretariat for Research and Technology PENED-3ED863. The Ghent lab acknowledges support from research grants from the Fund for Scientific Research-Flanders (Belgium) (project number G.0042.07), the Concerted Research Actions (project BOF07/GOA/012) from Ghent University and the Interuniversity Attraction Poles (IUAP06). ’ REFERENCES (1) Moulder, J. W. Comparative biology of intracellular parasitism. Microbiol. Rev. 1985, 49 (3), 298–337. (2) Hackstadt, T.; Williams, J. C. Biochemical stratagem for obligate parasitism of eukaryotic cells by Coxiella burnetii. Proc. Natl. Acad. Sci. U.S.A. 1981, 78 (5), 3240–4. (3) Roman, M. J.; Coriz, P. D.; Baca, O. G. A proposed model to explain persistent infection of host cells with Coxiella burnetii. J. Gen. Microbiol. 1986, 132 (5), 1415–22. (4) Barry, A. O.; Mege, J. L.; Ghigo, E. Hijacked phagosomes and leukocyte activation: an intimate relationship. J. Leukoc. Biol. 2011, 89 (3), 373–82. (5) Omsland, A.; Beare, P. A.; Hill, J.; Cockrell, D. C.; Howe, D.; Hansen, B.; Samuel, J. E.; Heinzen, R. A. Isolation from Animal Tissue and Genetic Transformation of Coxiella burnetii Are Facilitated by an Improved Axenic Growth Medium. Appl. Environ. Microbiol. 2011, 77 (11), 3720–5. (6) Omsland, A.; Cockrell, D. C.; Howe, D.; Fischer, E. R.; Virtaneva, K.; Sturdevant, D. E.; Porcella, S. F.; Heinzen, R. A. Host cell-free growth of the Q fever bacterium Coxiella burnetii. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (11), 4430–4. (7) Baca, O. G.; Paretsky, D. Q fever and Coxiella burnetii: a model for host-parasite interactions. Microbiol. Rev. 1983, 47 (2), 127–49. (8) Maurin, M.; Raoult, D. Q fever. Clin. Microbiol. Rev. 1999, 12 (4), 518–53. (9) Stein, A.; Raoult, D. Q fever during pregnancy: a public health problem in southern France. Clin. Infect. Dis. 1998, 27 (3), 592–6. (10) Akporiaye, E. T.; Rowatt, J. D.; Aragon, A. A.; Baca, O. G. Lysosomal response of a murine macrophage-like cell line persistently infected with Coxiella burnetii. Infect. Immun. 1983, 40 (3), 1155–62. (11) Baca, O. G.; Akporiaye, E. T.; Aragon, A. S.; Martinez, I. L.; Robles, M. V.; Warner, N. L. Fate of phase I and phase II Coxiella burnetii in several macrophage-like tumor cell lines. Infect. Immun. 1981, 33 (1), 258–66. (12) Baca, O. G.; Scott, T. O.; Akporiaye, E. T.; DeBlassie, R.; Crissman, H. A. Cell cycle distribution patterns and generation times of L929 fibroblast cells persistently infected with Coxiella burnetii. Infect. Immun. 1985, 47 (2), 366–9. (13) Burton, P. R.; Stueckemann, J.; Welsh, R. M.; Paretsky, D. Some ultrastructural effects of persistent infections by the rickettsia Coxiella burnetii in mouse L cells and green monkey kidney (Vero) cells. Infect. Immun. 1978, 21 (2), 556–66. 4249


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