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Jan 1, 2010 - like motifs, repeats and domains or other characteristics involved in ... was assessed by the Gimenez staining.25 C. burnetii-free supernatants were diluted ... protein name .... several Web-based software, freely available from the “ExPASy .... Our task was to identify low-abundance bacterial proteins from an.
Proteomic Screening for Possible Effector Molecules Secreted by the Obligate Intracellular Pathogen Coxiella burnetii Georgios Samoilis,†,‡ Michalis Aivaliotis,§,# Iosif Vranakis,†,‡ Anastasia Papadioti,†,‡ Yiannis Tselentis,‡ Georgios Tsiotis,*,† and Anna Psaroulaki‡ Division of Biochemistry, Department of Chemistry, University of Crete, P.O. Box 2208, GR-71003 Voutes, Greece, Department of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Medical School, University of Crete, GR-71409 Heraklion, Greece, and Department of Membrane Biochemistry, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany Received July 10, 2009

Abstract: Coxiella burnetii is a Gram-negative, gammaproteobacteria with nearly worldwide distribution, and it is the pathogenic agent of Q-fever in man. It is an obligate intracellular parasite that is highly adapted to reside within the eukaryotic phagolysosome. In fact, it is the only known intracellular bacterium that manages to survive and replicate within a fully formed, acidic phagolysosome. C. burnetti possesses a functional Type 4 Secretion System (T4SS), similar to the Dot/Icm system of Legionella pneumophila. Up to date there have been no reports for the effector molecules secreted by Coxiella’s T4SS. These are speculated to have quite different roles than the effectors of other intracellular pathogens, since there is no need for phagosomal arrest or escape in the case of Coxiella. In this study, we have investigated the cytoplasm of Vero cells infected with C. burnetti strain Nine Mile Phase II. We have identified by mass spectrometry (ESI-MS/MS) several C. burnetti proteins that bear typical characteristics of effector molecules. Most of the identified proteins were also very alkaline, something which is supportive for a protective strategy that has evolved in this bizarre pathogen against acidic environments. Keywords: Coxiella burnetii • secretome • intracellular bacteria • effectors

Introduction Coxiella burnetii, the causative agent of Q fever,1-3 is an obligate intracellular pathogen belonging to the gamma subdivision of Proteobacteria. Because of its high infectivity rates (one organism can cause infection), high levels of resistance in various stressful conditions (heat, drying, disinfection), and mode of transmission (via infectious aerosols), C. burnetii has * To whom correspondence should be addressed. Georgios Tsiotis, Division of Biochemistry, Department of Chemistry, University of Crete, P.O. Box 2208, GR-71003 Voutes, Greece. Tel. +30 2810 545006. Fax +30 2810 545001. E-mail: [email protected]. † Department of Chemistry, University of Crete. ‡ Department of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Medical School, University of Crete. § Max Planck Institute of Biochemistry. # Present address: Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology, Heraklion, Crete, Greece. 10.1021/pr900605q

 2010 American Chemical Society

been classified as a category B possible agent for bioterrorism.4-7 Human Q fever, usually acquired by inhalation of infectious aerosols produced by domestic animals, exhibits highly variable clinical manifestation.8,9 The disease/infection can be either acute (ranging from a flu-like syndrome to severe pneumonia requiring intensive care), or chronic (manifesting mainly as endocarditis or hepatitis), or it can even lead to asymptomatic seroconversion. C. burnetii is highly adapted for surviving and replicating within the eukaryotic phagolysosome.10,11 In fact, it is the only intracellular bacterium, known to date, that survives and replicates within a fully formed phagolysosome, thus, within a highly acidic environment (pH∼4.7-5.2) containing hydrolytic enzymes and other denaturing factors.11-13 A wide range of cell lines such as fibroblasts, epithelial cells and macrophages are the target cells.5 The pace of discovery of virulence determinants for this pathogen has been slow, in part, because methods for creation of defined mutations have been unavailable. Moreover, genomic analysis can be even misleading in many cases, due to the highly dynamic nature of the C. burnetii genome, which seems to be still undergoing reduction, having numerous mobile elements, pseudogenes and hypothetical proteins.14 Several recent studies have addressed at the proteome and transcriptome levels the aspects of the pathogenesis and survival mechanisms that C. burnetii has evolved.15-17 One feature in particular has received a great amount of attention, and that is the possession and expression of several components of the Secretion System Type IV (T4SS).15-19 Bacterial secretion systems have generally been studied intensively and there are several reasons for this. Studying such systems has, and can provide us with vital information on: (a) the hostpathogen interactions needed to establish an intracellular lifestyle, (b) gene acquisition and genome evolution of the pathogen in relation to its close-related organisms and its host, and (c) the actual cell biology of the eukaryotic host.20,21 Effector molecules secreted by bacterial SSs commonly contain/ bear eukaryotic motifs involved in protein-protein interactions and recently has been reported that C. burnetii use a type IV secretion system to deliver into eukaryotic cells a large number of different bacterial proteins containing ankyrin repeat homology domains.22,23 This is totally expected since the parasite’s effector proteins need to establish specific interactions with components of its eukaryotic host, in order to interfere with Journal of Proteome Research 2010, 9, 1619–1626 1619 Published on Web 01/01/2010

technical notes cellular systems and promote its intracellular parasitism and hence survival and replication. However, one could expect such a finding since C. burnetii is the only bacterium, facultative intracellular or not, known to date not to block fusion of lysosomes with phagosomes. Thus, there is no need for this pathogen to secrete effector molecules to prevent phagolysosomal fusion and maturation. It has to be mentioned here that, since C. burnetii exhibits a rather unusual life cycle within the host cell compared to other intracellular parasites, it is expected that this bacterium will express and secrete effector molecules that “highjack” the cellular systems of the host in a different manner. For example, members of the Legionella and Brucella species, which are intracellular parasites that reside within the eukaryotic phagosome, express and secrete effector molecules that prevent the fusion between lysosomes and phagosomes, thus, avoiding a proteolytic denaturing attack by the host.24 It is nearly certain that these types of effector molecules will not be expressed by Coxiella, where the creation of a mature and fully formed phagolysosome, and thus the fusion between lysosomes and phagosomes, is essential for the survival and replication of the parasite. In the present study, we have investigated in vitro C. burnetii proteins identified in the cytosol of infected fibroblasts. Our results constitute a screening of proteins possibly being secreted by the reference strain Nine Mile, phase II, at the protein level. Moreover, a wide bioinformatic-based research was performed across all C. burnetti proteins identified in this study, in order to locate within protein sequences eukaryoticlike motifs, repeats and domains or other characteristics involved in pathogenesis and/or survival mechanisms. As far as we know, this is the first report concerning the effector molecules of this bizarre pathogen, at the protein level.

Experimental Procedures Cultivation of C. burnetii. C. burnetii strain Nine Mile phase II was grown in confluent Vero cell lines (African green monkey kidney fibroblasts; CCL-81; ATCC), cultivated in Minimum Essential Medium Eagle (MEM; Gibco Laboratories), supplemented with 4% fetal bovine serum (FBS; Gibco Laboratories), and 1% L-Glutamine (Gibco Laboratories), at 35 °C and 5% CO2. All laboratory procedures involving the handling of live bacteria were carried out in a Biosafety Level 3 (BSL-3) laboratory according to international SOPs. Purification of Cytoplasmic Fraction from Infected Cells. Infected cells were collected upon 90-95% infection (∼10-12 days post-infection (p.i.)). Briefly, the culture medium was discarded and the infected cell monolayer was washed twice with K36 buffer (16.5 mM KH2PO4, 33.3 mM K2HPO4, 100 mM KCl, 15.5 mM NaCl, pH 7.0). Infected Vero cells were detached using glass beads and finally obtained in 10 mL of K36. Samples were centrifuged at 2600g for 45 min at 4 °C, and the pellet was resuspended in 2 mL of K36 with Protease Inhibitor Cocktail (PIC, Sigma). Infected Vero cells were fractionated by at least 20 passages through a syringe with a fine needle attached to it. The samples were centrifuged twice at 150g for 10 min, in order to discard unfractionated cells. K36 buffer containing PIC was added to the supernatant to a final volume of 10 mL. One volume of 25% sucrose in K36 buffer was added and the samples were centrifuged at 3850g for 30 min at 4 °C. The supernatant was obtained and the presence of C. burnetii was assessed by the Gimenez staining.25 C. burnetii-free supernatants were diluted 5-fold with Tris-HCl, pH 6.8, and ultracentrifuged at 150 000g for 90 min at 4 °C, in a fixed-angle 1620

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Figure 1. Tricine SDS-PAGE analysis of the cytoplasmic proteome of uninfected Vero cells (lane 1), C. burnetii infected Vero cells (lane 2) and whole cells of C. burnetii (lane 3). A total of 70 µg of protein was loaded in each lane.

rotor. Proteins in the supernatant were considered to be the hydrophilic fraction of the cytosol, and were acetone precipitated (1:3, sample/ice-cold acetone, at -20 °C overnight). Samples were centrifuged the next day at 10 000g for 15 min at 4 °C, and the pellet was resuspended in Tris-HCl, pH 6.8. C. burnetti which was used as a control, was isolated by renografin density centrifugation according to Samoilis et al.16 and was prepared for SDS electrophoresis by using the procedure described above. Protein concentrations were determined by the Bradford assay.26 SDS Polyacrylamide Gel Electrophoresis. The protocol applied here was carried out according to the original reference by Laemmli27 with slight modifications. Approximately 50 µg of protein from each sample was incubated in a volume of 2× sample buffer (0.125 M Trsi-HCl, pH 6.8, 20% (v/v) glycerol, 40% (v/v) of a 10% SDS solution, 10% (v/v) β-mercaptoethanol, 5% (v/v) of 1% bromophenol blue solution) at 40 °C for 1 h, and then loaded into the gel wells. A 4% polyacrylamide gel was used for the stacking of proteins, which were then separated in a 12% polyacrylamide gel. Electrophoresis was carried out at constant current (30 mA/gel) in electrophoresis buffer (10×: 250 mM Tris-HCl, 1.2 M glycine, 1% (w/v) SDS, pH 8.3), until the dye front reached the lower end of the gel. Proteins were fixed on the gels with 45% methanol, 1% glacial acetic acid for at least 4 h with gentle agitation. Home-made colloidal Coomassie brilliant blue (17% (w/v) ammonium sulfate, 34% (v/v) methanol, 0.5% (v/v) glacial acetic acid, 0.1%

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Possible Effector Molecules Secreted by C. burnetii Table 1. Proteins Identified no.

1 2 3

4

5 6 7 8 1 2 3 4 5 6 7 8 9

10

11 12

1

2 3 4 5

6 7

8 9 10

protein name

signal peptide

motifs/domains/repeats/ profiles

Proteins Identified in All Three Samples NADH dehydrogenase subunit I + - no relevant profile/ motif/ repeat 5-formyltetrahydrofolate cyclo-ligase family - no relevant profile/ motif/ repeat protein ribonuclease R - bipartite nuclear localization signal profile - S1 domain profiles - UPF0103 protein (unknown function with human homologues) - typical RNA-DNA interaction domains 30S ribosomal protein S2 - coiled coil domain - uncertain lysine-rich region profile lipid binding properties alcohol dehydrogenase (NADP+) - located in a region of the genome coding for T4SS proteins Orf 145, hypothetical protein QpH1_p21 - coiled coil domain - plasmid encoded DNA repair protein RecN - coiled coils domains - leucine rich repeats hypothetical protein CburD_01001397 + - uncertain kinase sites Proteins Identified in Two of the Samples drug resistance transporter, EmrB/QacA family - many multidrug resistance profiles - Major Facilitator Superfamily (MFS) profile putative uncharacterized protein A35_A0191 - no relevant profile profile/ motif/ repeat putative uncharacterized protein - possible nucleic acid binding properties stress induced protein, putative - protein secE/sec61-gamma signature phosphoribosylamine--glycine ligase - no relevant profile profile/ motif/ repeat chaperonin, 60 kDa - coiled coil domain phosphoglyceromutase - no relevant profile profile/ motif/ repeat serine hydroxymethyltransferase - no relevant profile profile/ motif/ repeat sensor protein - many regulatory (protein interactions) and signal transduction (two-component systems) profiles 27 kDa outer membrane protein + - potential Tat signal - DSBA-like thioredoxin domain - uncertain eukaryotic motifs acyl-CoA dehydrogenase family protein, - uncertain basic region leucin zipper degenerate - uncertain HAT (Half-A-TPR) repeats Putative uncharacterized protein CBU_1209 + - uncertain prenyl group binding site (CAAX box) - uncertain Pumilio RNA-binding repeat and homology domain profiles Proteins Identified in One of the Samples - possible Tat signal - helix-hairpin-helix motif (gene regulatory proteins) - uncertain eukaryotic motifs ATPase, AAA family domain protein - possible regulatory domains for protein (Uncharacterized protein CBU_1189) denaturation ferrous iron transport protein - ferrous-binding and transport protein amino acid permease family protein + - binding and transport protein (Amino acids, peptides and amines) hypothetical protein A35_A0301 - uncertain Big-1 (bacterial Ig-like domain 1) domain profile - uncertain WD-40 repeat putative uncharacterized protein CBU_1508 - leucine repeats putative uncharacterized protein CBU_1210 - serine-rich region with unknown substrate - uncertain staphylococus-like repeat of an extracellular protein putative uncharacterized protein CBU_1823 - coiled coil domain cell division protein FtsL + - involved in cell division ribosomal protein L7/L12 + - possible multiple subcellular localization sites (cytoplasmic membrane > periplasmic membrane > outer membrane) - involved in protein synthesis and modification A/G-specific adenine glycosylase

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Table 1. Continued no.

protein name

signal peptide

motifs/domains/repeats/ profiles

- cellular role: detoxification - possible Tat signal - GST_C Glutathione S-transferase, C-terminal domain - no relevant profile/ motif/ repeat - possible multiple subcellular localization sites (cytoplasmic membrane > periplasmic > extracellular > outer membrane) - involved in protein synthesis and modification - coiled coil domains - TPR repeat detected - signal transduction: Two-component systems - leucine repeat - no relevant profile/ motif/ repeat - cellular role: toxin production and resistance - multidrug pump - translocation of unknown substrates across the membrane - uncertain Nebulin repeat profile which binds actin - mobile and extrachromosomal element functions: Transposon functions - leucine repeat detected - coiled coil domains - no relevant profile/ motif/ repeat - leucine repeat - uncertain marR-type HTH domain - DNA binding domain of the LexA SOS regulon repressor - coiled coil domain - protein involved in a SOS response - no relevant profile/ motif/ repeat - no relevant profile/ motif/ repeat - possible Tat signal - transport and binding protein: unknown substrates - uncertain leucine-rich repeat variant - involved in protein synthesis and modification - central intermediary metabolism: One-carbon metabolism

11

glutathione S-transferase family protein

-

12 13

alcohol dehydrogenase, zinc-containing 50S ribosomal protein L22

+

14

sensor protein (sensory box histidine kinase/ response regulator)

-

15 16 17

putative uncharacterized protein CBU_0328 putative uncharacterized protein CBU_0018 transporter, AcrB/AcrD/AcrF family

-

18

ABC transporter, permease protein

-

19

transposase for insertion sequence element IS1111A

-

20 21 22 23

putative uncharacterized protein CBU_1314 pyridine nucleotide-disulfide oxidoreductase linear gramicidin synthetase subunit C putative uncharacterized protein CBU_0343

-

24

UvrABC system protein B (Protein uvrB)

-

25 26 27 28

putative uncharacterized protein CBU_1455 putative uncharacterized protein putative uncharacterized protein CBU_0012 ABC transporter, permease protein

-

29

ribosomal protein S5

-

30

adenosylhomocysteinase

-

(w/v) Coomassie G250) was used to stain the gels overnight with gentle agitation.28 Destaining of the gels was done with 3-4 × 10 min washes with ddH2O, and gentle agitation. Mass Spectrometry Analysis and Protein Identification. Mass spectrometry analysis involved a liquid chromatography system on-line connected (CapLC, Waters, Milford, MA) inline with a Q-TOF instrument (Q-TOF Ultima, Waters, Milford, MA). The methodology was performed according to Gevaert et al.29 with modifications from Klein et al.30 Salt removal was done through a micro-RP column.31 Multiply charged ions were selected for fractionation. For the measurements, the masses scanned were in the range of 300-1500 m/z. After nano LCESI-MS/MS, the CID-spectra obtained were converted to the Mascot 2.2.0 (Matrix Science, London, U.K.)28 acceptable pkl format using ProteinLynx software 2.0. These peak lists were used in protein identification performing two individual searches using Mascot. The first search was performed using protein database NCBI (release 20070212), over all entries (4 565 699 sequences, 1 571 958 806 residues), and the second search was 1622

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performed using protein database NCBI (release 20070212) for the taxonomy of Proteobacteria (purple bacteria) (1 335 474 sequences). Search parameters were: 2 missed cleavage sites, 1.60 Da peptide tolerance, 0.6 Da MS/MS tolerance, carbamidomethylation of cysteines as a fixed modification, and oxidation of methionine and protein N-terminal acetylation as variable modifications. Results are based on a “probabilitybased MOWSE score algorithm”. The MS/MS ion score threshold for peptide identification was 30 with a minimum of 4 identified fragments. In addition, MS/MS spectra were manually checked and annotated. Protein Sequence Analysis. Analysis of the amino acid sequences of all identified proteins was carried out using several Web-based software, freely available from the “ExPASy Proteomics Server” of the Swiss Institute of Bioinformatics (SIB) (http://au.expasy.org/). The purpose of this analysis was to reveal the physicochemical properties and other characteristics of each identified protein. In particular, the molecular weight (MW), isoelectric point (pI) and the hydrophobicity (GRAVY

Possible Effector Molecules Secreted by C. burnetii

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score) were calculated (theoretical values) 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). Known and/or unknown motifs, domains and other characteristics were revealed using the software SMART (http:// smart.embl-heidelberg.de/) and Motif Scan (http://myhits.isbsib.ch/cgi-bin/motif_scan). Subcellular localization of all identified proteins was predicted by PSORTb v.2.0 (http:// www.psort.org/), transmembrane helices by HMMTOP (http:// www.enzim.hu/hmmtop/), signal peptides by SignalP 3.0 (http://www.cbs.dtu.dk/ services/SignalP/) and TatP 1.0 (http:// www.cbs.dtu.dk/services/TatP/), motifs such as Leucine Zippers, Leucine repeats and Coiled-coil domains by 2ZIP (http:// 2zip.molgen.mpg.de/index.html), COILS (http://www.ch.embnet. org/software/COILS_form.html) and Parcoil2 (http://groups. csail.mit.edu/cb/paircoil2/), and specific repeats (TPR, ankyrin repeats, etc.) by REP (http://www.embl-heidelberg.de/ ~andrade/papers/rep/search.html).

Results and Discussion Our experimental design was based on three simple facts about C. burnetii and the current available methodology. The first one is that C. burnetii does possess a functional Type IV Secretion System, as it was shown in earlier studies by other laboratories,18,19 and our team.16 Therefore, the bacterium is expected to express and secrete to the cytoplasm of the host cell effector molecules that exhibit the ability to reorganize and direct the various cellular systems of the host toward the parasite’s benefit. Effector molecules bear specific motifs that allow them to interact with the proteins and/or protein complexes of a eukaryotic cell, thus, the host cell. The second fact is that many of these characteristic motifs, domains, and amino,acid repeats can be detected by various Web-based software that has been developed. However, the identification of possible effector molecules also as substrates of a secretion system requires highly specialized approaches. However, and this is the third fact, these molecules can still be detected and identified in the cytoplasm of the host cell by another simple, fast, and reliable approach: mass spectrometry. On the basis of the three facts mentioned above, the main concept of this study was to generate a list of possible C. burnetii effector molecules by identifying C. burnetii proteins in the cytoplasm of infected cells and then analyzing their amino acid sequences in silico in order to reveal any motifs, domains, and/or repeats possibly present. In silico analysis of each individual identified C. burnetii protein could point toward specific mechanisms of pathogenesis and/or survival that this parasite has evolved. It has to be mentioned here that, since C. burnetii exhibits a rather unusual life cycle within the host cell compared to other intracellular parasites, it is expected that this bacterium will express and secrete effector molecules that “highjack” the cellular systems of the host in a different manner. For example, members of the Legionella and Brucella species, which are intracellular parasites that reside within the eukaryotic phagosome, express and secrete effector molecules that prevent the fusion between lysosomes and phagosomes, thus, avoiding a proteolytic denaturing attack by the host.32-34 It is nearly certain that these types of effector molecules will not be expressed by Coxiella, where the creation of a mature and fully formed phagolysosome, and thus the fusion between lysosomes and phagosomes, is essential for the survival and replication of the parasite.

Figure 2. SDS-PAGE analysis of three different biological samples (A, B, and C) from the cytoplasm of Vero cells infected with C. burnetii strain Nine Mile, phase II. Every sample was divided into 19 bands which were excised from the gel and digested with trypsin (in-gel tryptic digestion).

In the experimental approach designed here, the following goals were set: (a) to separate the intracellular bacteria from the components of the host cell and then isolate only the hydrophilic proteins that are present in the host’s cytoplasm in the mildest possible way so that the bacteria remain intact, (b) to analyze the isolated proteins in the less possible restrictive way (in physicochemical terms), and (c) to use a powerful identification method in terms of reliability. Concerning the first part, after a series of experiments, a quite reliable and most importantly repeatable protocol was reached for the isolation of hydrophilic proteins from the cytoplasm of infected cells. The methodology developed was mild enough so that the intracellular bacteria remained intact. Tricine-SDS-PAGE analysis of the cytoplasm proteome of the uninfected Vero cell, C. burnetii infected Vero cells and C. burnetii was performed in order to investigate the potentials of the developed method. All samples were prepared using the same extraction protocol (see Experimental Procedures). As shown in Figure 1, differJournal of Proteome Research • Vol. 9, No. 3, 2010 1623

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Figure 3. Percentage of C. burnetii identified proteins that possibly bear a eukaryotic motif and/or contain domains/profiles/repeats that are involved in survival and/or pathogenesis. In all three categories, this percentage exceeds 50% which is indicative for their possible role as effector molecules.

Figure 4. pI distribution among C. burnetii identified proteins in the three different protein categories. In all three of them, the percentage of alkaline proteins reached nearly or over 60%. This is in line with the predicted percentage of basic proteins in the theoretical proteome of C. burnetii.

ences between the cytoplasm of the Vero cells and the C. burnetti are clear indicating the absence of the most highabundance proteins of the C. burnetii in the isolated proteome. On the other hand, no significant difference are observed in the C. burnetti infected Vero cells and uninfected Vero cells indicating the most of the proteins are host proteins. Our task was to identify low-abundance bacterial proteins from an eukaryotic cytosolic preparation where the ratio of eukaryotic versus bacterial proteins is high. This was achieved using a higher degree of accuracy, especially MS/MS, coupled with a peptide separation system (nLC), in order to overcome the difficulties arising from the complexity of the sample. This was clearly demonstrated in our results, since no structural proteins of the bacterium were identified (Table 1). The only exception included some outer membrane proteins for which it is expected to find their fragments outside from the bacterium. The isolated proteins were resolved by SDS-PAGE in which a highly repeatable protein pattern was achieved (Figure 2). Although this method’s resolution is limited (the proteins are 1624

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resolved in one dimension according to their molecular weight only), it is not excluding for proteins with uncommon physicochemical properties, as the two-dimensional electrophoresis is for highly basic, acidic and hydrophobic proteins. In the case of Coxiella, this is particularly important, since it is expected to express a large portion of proteins with such characteristics. Moreover, in order to increase the reliability of our results, three different biological samples were analyzed twice. Following this approach, we came up with three different categories of identified proteins: the ones that were identified in all three different biological samples, those that were identified in two of the samples, and finally those that were identified only in one. This categorization of our results constituted also one of the main criteria for their evaluation. Thus, proteins that were identified in all three different biological samples were considered to be the most reliable results. Additionally, in this study, an ESI-MS/MS instrument was used for the acquisition of mass spectra which, although it is not high-throughput, it can provide much more accurate results than MALDI-TOF MS

technical notes

Possible Effector Molecules Secreted by C. burnetii with PMF. In order to strengthen the validity of our results, an extended BLAST search was performed for each identified peptide. This approach ensured not only that the identified peptides belong to C. burnetii proteins, but most importantly that they are not of eukaryotic/mammalian origin. A possible explanation for the low identification scores recorded in this study could be the low concentration of these molecules in the cytoplasm of the host cell, something which is expected. Another possible explanation would be the low protein coverage scored, due to the low number of peptides identified for each protein. Finally, the fact that in such a complex sample, like the one here, a relatively low number of bacterial proteins were identified (50 in total), is also encouraging since the number of effector molecules is expected to be low. The in silico analysis of all the C. burnetii identified proteins revealed two very important and interesting findings. The first one is that in all three categories of identified proteins, the percentage of proteins bearing a characteristic motif, domain or profile of effector molecules or being involved in some way to pathogenesis and survival mechanisms, is over 50% (Figure 3). In particular, in all three protein categories nearly one-third of the identified proteins possessed coiled-coil domains, leucine repeats, and signal peptides type Tat (Table 1). This finding strengthens substantially, and in fact confirms our initial hypothesis that proteins being expressed and secreted by C. burnetii can be identified in the cytoplasm of the infected host cell. In contrast to the eukaryotic-like motifs/domains containing proteins which are Type IV effectors, protein containing signal sequence may be secreted via a Sec dependent pathway. The second very interesting finding was that, in all three protein categories produced here, the percentage of basic (alkaline) proteins was over 55% (Figure 4). Actually, in two of these categories, this percentage was over 60%. Moreover, 58% of all 50 C. burnetii proteins identified in our study had a theoretical isoelectric point (pI) over 8. In the publication of the complete genome sequence of C. burnetii,14 the researchers suggest that the highly basic theoretical proteome of the bacterium is possibly a way that this parasite has evolved to counterbalance the highly acidic niche in which it resides, that is, the eukaryotic phagolysosome. However, in one of our previous works16 where we analyzed the proteome of C. burnetii strain Nine Mile, phase II, only one-fifth of the 185 identified proteins had basic pI. This was also reflected in a similar study for phase I of the same strain.17 This observation comes into contrast with the theoretical proteome of this organism, and also with our findings in the present study. A possible explanation for this rather contradictive finding could be that proteins being secreted to the cytoplasm of the hot cell come into direct contact with the highly acidic milieu of the phagolysosome, and thus, there is a much more evident need for these proteins to counterbalance this factor (acidity). On the other hand, proteins being located within the bacterium, either as structural or functional units, do not face the challenge of an acidic environment since the organism possesses detoxification mechanisms in order to keep a physiological pH within its cytoplasm. As far as we know, this is the first study in which the alkaline nature of Coxiella’s proteome is also reflected with such accuracy in practice. In the present study, a list of possible novel effector molecules of C. burnetii has been generated. According to our knowledge, this is the first report concerning novel effector molecules of this pathogen at the protein level. Further and more sophisticated approaches, such as heterologous systems

or gene knockouts, should be taken in the future in order to reveal the actual role and the relevant importance of each candidate effector molecule for the pathogenesis mechanisms of this bizarre pathogen.

Acknowledgment. This research program was supported by the University of Crete and the Greek Ministry of Education. The project is cofunded by the European Social Fund & National Resources - EPEAEKII-HRAKLEITOS. M.A. would like to thank Prof. Oesterhelt for kindly providing the infrastructure of his department for the mass spectrometry experiments. Supporting Information Available: Spectra_Coxiella_Samoilis data. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Maurin, M.; Raoult, D. Q fever. Clin. Microbiol. Rev. 1999, 12 (4), 518–553. (2) Parker, N. R.; Barralet, J. H.; Bell, A. M. Q fever. The Lancet 2006, 367 (9511), 679–688. (3) Raoult, D.; Marrie, T. J.; Mege, J. L. Natural history and pathophysiology of Q fever. Lancet Infect. Dis. 2005, 5 (4), 219–226. (4) NIAIDCategoryA.,B&CPriorityPathogenshttp://www3.niaid.nih.gov/ biodefense/bandc_priority.htm. (5) Heinzen, R. A.; Hackstadt, T.; Samuel, J. E. Developmental biology of Coxiella burnettii. Trends Microbiol. 1999, 7 (4), 149–154. (6) Madariaga, M. G.; Rezai, K.; Trenholme, G. M.; Weinstein, R. A. Q fever: a biological weapon in your backyard. Lancet Infect. Dis. 2003, 3 (11), 709–721. (7) Rotz, L. D.; Khan, A. S.; Lillibridge, S. R.; Ostroff, S. M.; Hughes, J. M. Public health assessment of potential biological terrorism agents. Emerging Infect. Dis. 2002, 8 (2), 225–230. (8) Marrie, T. J. Coxiella burnetii pneumonia. Eur. Respir. J. 2003, 21 (4), 713–719. (9) Raoult, D.; Tissot-Dupont, H.; Foucault, C.; Gouvernet, J.; Fournier, P. E.; Bernit, E.; Stein, A.; Nesri, M.; Harle, J. R.; Weiller, P. J. Q fever 1985-1998. Clinical and epidemiologic features of 1,383 infections. Medicine (Baltimore, MD, U.S.) 2000, 79 (2), 109–123. (10) Baca, O. G.; Li, Y. P.; Kumar, H. Survival of the Q fever agent Coxiella burnetii in the phagolysosome. Trends Microbiol. 1994, 2 (12), 476–480. (11) Maurin, M.; Benoliel, A. M.; Bongrand, P.; Raoult, D. Phagolysosomes of Coxiella burnetii-infected cell lines maintain an acidic pH during persistent infection. Infect. Immun. 1992, 60 (12), 5013– 5016. (12) 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– 1162. (13) 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, 3240–3244. (14) Seshadri, R.; Paulsen, I. T.; Eisen, J. A.; Read, T. D.; Nelson, K. E.; Nelson, W. C.; Ward, N. L.; Tettelin, H.; Davidsen, T. M.; Beanan, M. J.; Deboy, R. T.; Daugherty, S. C.; Brinkac, L. M.; Madupu, R.; Dodson, R. J.; Khouri, H. M.; Lee, K. H.; Carty, H. A.; Scanlan, D.; Heinzen, R. A.; Thompson, H. A.; Samuel, J. E.; Fraser, C. M.; Heidelberg, J. F. Complete genome sequence of the Q-fever pathogen Coxiella burnetii. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (9), 5455–5460. (15) Coleman, S. A.; Fischer, E. R.; Cockrell, D. C.; Voth, D. E.; Howe, D.; Mead, D. J.; Samuel, J. E.; Heinzen, R. A. Proteome and antigen profiling of Coxiella burnetii developmental forms. Infect. Immun. 2007, 75 (1), 290–298. (16) Samoilis, G.; Psaroulaki, A.; Vougas, K.; Tselentis, Y.; Tsiotis, G. Analysis of whole cell lysate from the intercellular bacterium Coxiella burnetii using two gel-based protein separation techniques. J. Proteome Res. 2007, 6 (8), 3032–3041. (17) Skultety, L.; Hernychova, L.; Toman, R.; Hubalek, M.; Slaba, K.; Zechovska, J.; Stofanikova, V.; Lenco, J.; Stulik, J.; Macela, A. Coxiella burnetii whole cell lysate protein identification by mass spectrometry and tandem mass spectrometry. Ann. N.Y. Acad. Sci. 2005, 1063, 115–122.

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