508 Lenka Hernychová1 Jirˇí Stulík1 Petr Halada2 Alesˇ Macela1 Michal Krocˇa1 Torsten Johansson3 Michal Malina1 1
Institute of Radiobiology and Immunology, Purkyne Military Medical Academy, Hradec Králové 2Institute of Microbiology, Czech Academy of the Sciences of Czech Republic, Prague, Czech Republic 3 Department of Microbiology, National Defence Research Establishment, Umea, Sweden
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Construction of a Francisella tularensis two-dimensional electrophoresis protein database We have started the construction of a two-dimensional database of the proteome of Francisella tularensis, a bacterium that is responsible for the highly pathogenic disease tularemia. The genome of this intracellular pathogen is not completely sequenced yet and, currently, information about only 66 proteins is available from NCBI database. We have analyzed the F. tularensis live vaccine strain by two-dimensional gel electrophoresis with immobilized pH 3–10 gradient in the first dimension and 9–16% gradient or tricine SDS-PAGE in the second dimension. In both cases about 2000 spots were detected. Furthermore, we compared the protein pattern of the nonvirulent F. tularensis live vaccine strain with protein profiles of two wild type clinical isolates and more than 50 differentially expressed proteins were counted. The separated proteins are going to be identified by peptide mass fingerprinting. However, due to the lack of complete genome sequence data only eight proteins were unambiguously identified. Among them, acid phosphatase and the most basic isoform of a hypothetical 23 kDa protein are characteristic only for virulent strains. Keywords: Two-dimensional gel electrophoresis / Francisella tularensis / Protein database / Proteome PRO 0048
1 Introduction Francisella tularensis is a facultative intracellular bacterium causing a serious disease known as tularemia in small rodents and in humans exposed to infected animals, Arthropods or insects. In humans, tularemia can be a potentially fatal disease, especially if the causative agent is highly virulent (type-A strains). Host defense against tularemia is cell mediated and it depends on interaction of infected macrophages with Francisella-specific T cells. This interaction augments the microbicidal capacity of infected cells which is a prerequisite for the elimination of invading microbes [1]. Despite intensive research on pathogenesis of tularemia infection there are still great gaps in knowledge of molecular mechanisms regulating microbial invasion, resistance to hostile environment and intracellular multiplication of microbes. Furthermore, there is a lack of information describing the immunologically relevant constituents of bacterium associated with the induction of protective immunity. Finally, the identification of molecules enabling discrimination of virulent strains of type A from strains of type B that have limited virulence for rabbits and men is missing.
Correspondence: J. Stulík, Institute for Radiobiology and Immunology, Purkyneˇ Military Medical Academy, Trˇebešská 1575, 500 01 Hradec Králové, Czech Republic E-mail:
[email protected] Fax: +420-49-551-30-18 Abbreviations: LVS, live vaccine strain
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001
Recently the sequencing of F. tularensis strain Schu4 genome has been started. Currently, 1.83 Mb of assembled sequence data organized into 353 sequence contigs are accessible (http://microbios1.mds.qmw.ac.uk/ft/) and gene functions were putatively assigned to 1289 open reading frames [2]. The next logical step that should follow genome sequencing is the analysis of functional complement of genetic information – the proteome. Until now only a few proteins of the F. tularensis microbe have been identitied. One of them is a 17 kDa lipoprotein named TUL4 identified using a combination of several molecular methods and finally expressed by recombinant Salmonella typhimurium chi 4072 [3]. TUL4 is a T cellreactive membrane component of various strains of the genus Francisella and it seems to be Francisella-specific because no hybridization or Western blot reactivity was seen with probes designed for other Gram-positive and Gram-negative bacteria [4]. Due to its specificity and immunogenicity TUL4 was used for the construction of specific immunostimulating complexes (ISCOMs) or for a new immunochromatographic membrane-based handheld assay (HHA) for the early detection of F. tularensis [5, 6]. The exposure of the F. tularensis live vaccine strain (LVS) to stressful stimuli led to the identification of a group of stress proteins involving the 75 kDa homolog of DnaK, the 60 kDa homolog of GroEL (Cpn60), and 10 kDa homolog of GroES (Cpn10) of Escherichia coli [7]. Zuber and coworkers [8] identified DnaJ protein homologous to the respective protein of E. coli that also belongs to the 1615-9853/01/0404–508 $17.50+.50/0
Proteomics 2001, 1, 508–515 chaperon family produced by F. tularensis. Acid phosphatases (Acp) functioning as suppressors of the intracellular burst are released by intracellular pathogens to enhance their survival after engulfment by phagocytes. Reilly and coworkers [9] described the identification of a novel burst-inhibiting acid phosphatase from F. tularensis that was easily released from the bacterial cell. The other identified proteins of F. tularensis then involve MinD, an ATPase activator of MinC that functions as inhibitor of cell division [10] and FopA, a major outer membrane protein of F. tularensis [11]. Finally, Golovliov and coworkers [12] described a hypothetical 23 kDa protein whose amount increased during the growth of the F. tularensis attenuated live vaccine strain (LVS) within the macrophage-like murine cell line J774. In this study we have launched the analysis of the tularemia proteome. First, we separated cellular proteins of the F. tularensis LVS strain and started their identification using a peptide mass fingerprinting approach. The LVS strain exhibits reduced virulence in humans and is currently in use as an experimental vaccine for laboratory workers. However, it remains virulent for mice, and murine tularemia caused by LVS is commonly used to study the mechanism of anti-Francisella resistance [13]. Furthermore, we performed comparative proteome analysis of the F. tularensis LVS strain and two wild F. tularensis strains, designated as 108 and 176, that were isolated from blood specimens of patients.
2 Material and methods 2.1 Chemicals and materials Multiphor II, Multitemp II thermostatic circulator, Dry-Strip kit, Immobiline DryStrips with nonlinear pH gradient 3–10, 18 cm, Pharmalytes pH 8–10.5 and pH 3–10 were from Amersham Pharmacia Biotech (Uppsala, Sweden); a 6000 V power supply was purchased from Serva (Heidelberg, Germany). Second-dimensional gels were cast and run in a Bio-Rad (Hercules, CA, USA) Multi Cell and Western blotting was done using a Bio-Rad Trans-blot apparatus. Acrylamide, urea, CHAPS, DTT and tricine were from USB (Amersham Pharmacia Biotech). 1.4Bis(acryloyl)piperazine (PDA), SDS, TEMED and ammonium persulfate were from Bio-Rad (Richmond, CA, USA). PVDF and proteinase inhibitors were purchased from Boehringer (Mannheim, Germany). Tris, agarose, thiourea, TCA, glycine, formaldehyde, silver nitrate, methanol, acetic acid, citric acid, bicinchoninic acid (BCA) assay reagents, iodoacetamide and caprylyl sulfobetain (SB 3–10) were from Sigma (St. Louis, MO, USA). Tributyl phosphine (TBP) was from Fluka (Buchs, Switzerland).
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Deionized water prepared with a Milli-Q system (Millipore) was used for all buffers.
2.2 Bacterial cultures and sample preparation F. tularensis LVS (ATCC 29684, American Type Culture Collection, Rockville, MD, USA) was cultured on McLeod agar supplemented with bovine haemoglobin and Iso VitaleXTM (Becton-Dickinson, San Jose, CA, USA) for 20 h at 36.67C. Two wild type strains of F. tularensis, designated as 108 and 176, were isolated from the blood specimens of patients according to Provenza [14]. A suspension of microbes was prepared in cold PBS, centrifuged, and the pellets then immediately homogenised in lysis buffer (137 mM NaCl, 10% glycerol, 1% p-octyl-b-Dglucopyranoside, 50 mM NaF, 1 mM Na3VO4 and proteinase inhibitors). The membranes of the microbes were degraded by repeated cycles of an freeze-thawing in nitrogen. The undisrupted microbes were eliminated from lysates by centrifugation. Extracted proteins were precipitated overnight in 20% TCA in acetone (–187C) containing 0.2% DTT [15] and then solubilized in IEF buffer (9 M urea, 4% CHAPS, 70 mM DTT and 2% carrier ampholytes pH 9–11). Protein concentration in IEF buffer was determined by modified BCA assay [16].
2.3 2-DE electrophoresis Commercial strips with nonlinear immobilized pH 3–10 gradient were used for isoelectric focusing. These strips were swollen in rehydration buffer containing 2 M thiourea, 5 M urea, 2% CHAPS, 2% SB 3–10, 2 mM TBP, 40 mM Tris base and 0.5% Pharmalytes pH 3–10 overnight. For analytical 2-DE 100 mg of protein was loaded in the first dimension. In the second dimension either gradient 9–16% SDS-PAGE or 16.5%T, 6%C tricine-SDS-PAGE was used. After electrophoresis, proteins were visualized by silver staining [17] and then the gels were scanned using a laser densitometer (4000 6 5000 pixels, 12 bits/ pixel; Molecular Dynamics, Palo Alto, USA) linked to a SunSparc s-s20 workstation (Sun Microsystems, Mountain View, USA). The 2-DE image computer analysis was carried out using a Melanie II package, Version 2.2 (BioRad). Proteins separated by 2-DE were quantitated in terms of their relative spot volumes. Correspondence analysis and Student t-test that are implemented in Melanie 2.2 were used for classification of samples and for determination of significant differences at the levels of p 5 0.05. The quantitative data from two independently prepared samples for each tularemic strain were submitted to statistical analysis. The isoelectric points and molecular weights of individual proteins were approximated using polypeptide SDS-PAGE standards (BioRad).
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Table 1. The list of proteins identified by MALDI-MS Protein no.
37 a) 191 1068 770 2017 1999 2011 1255
Mr pI (kDa)
73.6 56.7 14.0 56.1 24.6 24.6 24.8 40
4.8 4.9 4.7 5.9 5.6 5.8 6 4.7
Seq. Protein description coverage
24% 42% 16% 23% 40% 45% 30% 26%
Protein level in
DNAK, Heat Shock Protein 70 GroEL Protein Macrophage growth locus B Acid phosphatase Hypothetical 23 kDa protein Hypothetical 23 kDa protein Hypothetical 23 kDa protein Cell division protein FtsZ
LVS
Wild type strains
+ + + – + ++ – +
+ + + + ++ + + +
a) Spot numbers derived from reference gel
2.4 In-gel digestion For the micropreparative 2-DE gels up to 1 mg of protein was loaded on IPG strips. Selected spots stained by Coomassie Blue were then excised and covered with 100 mL of 100 mM N-ethylmorpholine, 100 pmL acetonitrile and 10 mL of 2 M DTT for 10 min at 457C. The wash was repeated. Then the gel pieces were convered with 100 mL of acetonitrile for 5 min (sonicating) until they shrank and blanched. After that they were vacuum dried in a Speed Vac (Eppendorf, Germany). 20 mL of 20 mg/mL protein sequencing grade Promega porcine trypsin (Promega, Madison, WI, USA) in 25 mM ammonium bicarbonate and 1% acetonitrile was added to each gel piece. After incubation at 377C for 12 h under shaking the supernatant was removed and the gel pieces were washed with 20 mL 0.5% aqueous TFA/acetonitrile (2:1). The combined supernatants were evaporated in the Speed Vac and dissolved in 4 mL 0.5% aqueous TFA/acetonitrile (2:1) for mass spectrometrical analysis.
base searching was performed using ProFound – Peptide Mapping (Short Form). The search parameters were NCBInr database, all species, 10 to 80 kDa, 4 to 9 pI, trypsin (2 missed cleavages) and mass tolerance of 0.5 Da.
3 Results and discussion
2.5 Mass spectrometry
Whole cell preparations of F. tularensis LVS were separated on nonlinear pH 3–10 IPG strips. In the second dimension, we applied either 9–16% gradient SDS-PAGE (Fig. 1A) or a gel system with tricine as trailing ion (Fig. 1B). Approximately 2000 spots were counted with the Melanie software in both gels. We repeated the 2-DE separation of soluble tularemic proteins several times and in all cases we obtained nearly identical protein patterns. Comparing the acidic part of gel with the basic one, it is evident that tularemic proteins accumulate preferentially in the acidic part of gel. A similar distribution of bacterial proteins was described by Jungblut et al. [18] for 2-DE protein patterns of mycobacterial species.
The spectra were recorded by using a time-of-flight delayed extraction MALDI mass spectrometer (VoyagerDE STR, Perseptive Biosystems, Framingham, MA, USA). The samples were mixed in an Eppendorf tube with the same volume of the matrix solution a-cyano-4hydroxycinnamic acid (in 50% acetonitrile in 0.5% TFA). Two mL of the mixtures were applied to a plated sample holder and introduced into the mass spectrometer after drying. The spectra were obtained in reflector mode by counting 100 laser shots with the 20 kV acceleration voltage, 75% grid voltage, 0.02 guide wire voltage, 100 ns delay time and low mass gate at 500 m/z. Data-
Selected proteins from both gradient as well as tricine SDS-PAGE were submitted to peptide mass fingerprinting using MALDI-MS. Up to now, we have analyzed 55 spots covering either the most distinct proteins or proteins exhibiting differential expression comparing vaccine and wild type strains. The positions of all analyzed proteins are depicted on Figs. 1A, 1B and Fig. 3. Although we obtained mass spectra of very good quality (Fig. 2) in all but eight spots the identification was hampered by the incompleteness of the genome sequence database of F. tularensis strain Schu4. Therefore, we were able to annotate only eight proteins (Table 1).
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Figure 1. The representative silver-stained 2-DE maps of F. tularensis LVS as (A) gradient 9–16% SDS-PAGE and (B) 16.5%T, 6%C Tricine-SDS-PAGE. All identified proteins are indicated. Arrows show the positions of the spots whose MALDI-TOF analyses provided mass spectra of good quality; asterisks then denote the spots whose MALDI-TOF analyses were unsuccessful. Four pattern sectors comprising characteristic protein alterations between the wild type and the vaccine F. tularensis strains are boxed.
Figure 2. Peptide mass fingerprint of DnaK protein obtained by MALDI-MS.
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Figure 3. The representative silver stained 2-DE maps of wild type Francisella tularensis strain, designated as 176, that was isolated from a blood sample of patient with tularemia. All identified proteins are indicated. Arrows show the positions of the spots whose MALDI-TOF analysis provided mass spectra of good quality; asterisks then denote the spots whose MALDI-TOF analyses were unsuccessful.
Four identified proteins are expressed nearly at the same level in vaccine and wild type strains. Two of them, DnaK and GroEL, are members of the molecular chaperone system of F. tularensis. The DnaK protein belongs to the heat shock protein 70 family and its nucleotide and the deduced amino acid sequences share significant homology with its E. coli counterpart [8]. The mass spectrum of a third protein matched with the products of a macrophage growth locus. This locus consists of an operon of two genes designated mgIAB whose products regulate the expression of genes affecting the survival and growth within macrophages [19]. The last protein cell division protein FtsZ, is essential for initiation of cell division [20]. The molecular basis for the difference in virulence between F. tularensis type A highly virulent and type B weakly virulent strains in man is still unknown. Karlsson et al. [2] described the absence of genes located on plasmids pOM1 and pNFL 10 isolated from low virulence strains of F. tularensis in the Schu4 (type-A) genome database. We have compared the protein profiles of two wild
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Figure 4. Correspondence analysis data of LVS, 108 and 176 gels. Histograms (% vol.) of the most important spots representative of a particular class of gels are shown. The spots are characterized by their group identification number (ID) and by the values of absolute contribution (in percent) to separation of gels of wild type from nonvirulent vaccine tularemic strains along the first factorial axis.
type strains of F. tularensis, designated 108 and 176 (Fig. 3), with 2-DE protein pattern of LVS strain. First, we used correspondence analysis for computerized classification of the six gels encompassing two independently prepared samples of each tularemic strain. Using this approach the gels were divided into two classes, one class was composed of four gels of wild type strains and the second class was composed of two gels of LVS strain. The histograms of the four spots that were selected by computer as the most important ones for gel classification are shown on Fig. 4. Three spots exhibit characteristic overexpression in the nonvirulent LVS strain, the spot number 1559 predominates in wild type strains. Figs. 5A, 5B then depict differential expression of all four spots in individual samples. In the second step, the Student t-test
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Figure 5A, B. Enlargements of gel regions showing the spots that were detected by correspondence analysis as expressed differentially in the two classes of gels.
was performed on the two given classes of gels and, additionally, subtractive analysis for evaluation of qualitative alterations comparing the individual gels to the master image encompassing all detected spots was also done. Globally we found more than 50 differentially expressed spots in the two classes of gels. Up to now, we have identified two proteins exhibiting protein abundance alterations in vaccine and wild type strains. The first protein which was identified as acid phosphatase is detectable in both wild type strains but it is nearly missing in the vaccine strain (Figs. 1A, 3). This finding corresponds with the common idea that acid phosphatases belong to virulence factors of intracellular pathogens [9]. The second identified protein, a hypothetical 23 kDa protein, consists of three major isoforms (Fig. 6) exhibiting charge heterogeneity. The middle variant, according to its pI 5.78, Mr 23 kDa values, probably corresponds to the protein identified by Golovliov [12]. It was assumed that production of this protein is associated with pathogenic potency of F. tularensis and its function is associated with the adaptation of the organism to hostile environment within phagocytic cells. The authors [12] used for their in vitro study F. tularensis LVS as an infection model. We also identified the 23 kDa protein in the LVS
strain, but the level of this variant was much higher in both wild type strains. Furthermore, in comparison to the results of Golovliov’s group we identified the additional two variants of 23 kDa protein, both of them exhibiting the differential expression between vaccine and wild type strains. In comparison to the vaccine strain, the most acidic variant is split in wild type strains into two isoforms and the most basic variant occurs only in wild type strains. All these findings support the idea that the 23 kDa protein is the new virulence factor. The other examples of characteristic qualitative or quantitative alterations between cell proteins of wild type and vaccine tularemic strains are demonstrated in Fig. 7.
4 Concluding remarks In conclusion, the presented results represent basic information about the protein composition of the F. tularensis LVS strain. Work is currently underway to characterize membrane and secreted tularemic proteins. By the end of this year the F. tularensis Schu4 genome sequencing will be completed; that should speed up the process
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Figure 6. Enlargement of the gel region with isoforms of the hypothetical 23 kDa protein.
Figure 7. Enlargement of the low molecular weight region depicting additional typical alterations in protein levels between cellular proteins of nonvirulent vaccine and wild-type strains.
of protein identification. Comparison of protein spectra of the nonvirulent vaccine strain with wild type counterparts revealed unexpectedly large differences in the level of protein expression. The finding of strain-specific proteins gives a good perspective for identification of new, more accurate molecular markers suitable for early detection, identification, typing, and diagnosis of tularemia.
The authors thank Jana Michalicˇková, and Alena Firychová for excellent technical assistance. This work was supported by Grant MO 66021299105 from the Ministry of Defence and Grant No. 4700-3 Ministry of Health of the Czech Republic. Received October 30, 2000
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5 References
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