- Email: [email protected]
Low molecular mass protein patterns in mycobacterial culture filtrates and purified protein derivatives
IMMUNOLOGY AND MEDICAL MICROBIOLOGY
ELSEVIER
FEMS Immunology and Medical Microbiology 23 (1999) 21-25
Low molecular mass protein patterns in mycobacterial filtrates and purified protein derivatives Sharon
S. Rowland
‘, Jennifer
L. Ruckert
2, Patrick
J. Cummings
culture *
Department of Medical and Research Technology, University of Maryland School of Medicine, Allied Health Building. Room 405A. 100 Penn Street, Baltimore, MD 21202. USA
Received 2 March 1998: received in revised form 2 October 1998; accepted 6 October 1998
Abstract Mycobacterial polypzptides from 2 kDa to 14 kDa may be involved in host response to infection and be useful for new diagnostics and vaccines. Tris-tricine SDS-polyacrylamide gel electrophoresis separation of proteins in tuberculin purified protein derivative (PPD) and in 6-&week culture filtrates of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and other Mycobacteriurn species demonstrated as many as 10 low molecular mass bands. Common and distinct bands were observed among different species and PPD. These low molecular mass culture filtrate proteins may represent potential diagnostic reagents and vaccines for Mycobacterium tuberculosis. 0 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords:
Mycobacteriunz
tuberculosis: Culture filtrate protein: Purified protein derivative
’ Present address: McKesson BioServices, 7501 Standish Place, Rockville, MD 20855, [ISA.
USA as a diagnostic indicator of exposure to M. tuberculosis, lacks specificity and is not useful in countries which routinely vaccinate against tuberculosis [2,3]. Other Mycobacterium spp., such as M. aviumlintracellulare, are important pathogens in AIDS patients and must be distinguished from M. tuberculosis [4]. In addition, there is a great need to improve the current BCG-based tuberculosis vaccine and to develop faster and more accurate means of identifying mycobacterial infections [1,2,5]. The extracellular proteins of M. tuberculosis, released into culture broth during in vitro growth, are believed to play a role in infection and immunity and are of particular interest [6-121. The tuberculin PPD, which produces a marked delayed hypersensi-
’ Present address: Medlmmune. 35 West Watkins Mill Road,
tivity
Gaithersburg, MD 20878, USA.
sists of a complex mixture
1. Introduction
Tuberculosis,
caus,ed by Mycobacterium
tuberculo-
sis, is the leading cause of death due to an infectious
disease in the world and has been declared a global emergency by the ‘World Health Organization. A primary goal of mycobacterial research is to identify M. tuberculosis antigens which can be used for new vaccines and diagnostics [l]. The tuberculin purified protein derivative (PPD) test, which is used in the * Corresponding author. Tel.: +l (410) 706-2627; Fax: +l (410) 706-0073; E-mail: [email protected]
reaction
in individuals
with tuberculosis,
of extracellular
con-
proteins
092%8244/99/$19.00 0 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved PII: SO928-8244(98)001 13-S
22
S.S. Rowhmd et al. I FEMS
Immunology
from A4. tuberculosis stationary phase liquid culture medium. PPD is prepared by heat sterilization of 6-week-old M. tuberculosis grown in Sauton medium, followed by filter sterilization and protein concentration using ultrafiltration or ammonium sulfate precipitation [3]. Considering the widespread use of this immunological reagent it is surprising little is known about the active components of PPD [3,13151. Much work has been done fractionating M. tuberculosis proteins and studying the immune response [ 16-201. However, protein antigens below 10 kDa have not been studied in detail, partly because they do not separate well using standard SDSPAGE, are present in low amounts in culture filtrate, are not easily visualized with stains, and have low immunogenicity. The goal of this study was to separate the low molecular mass proteins present in stationary phase culture filtrates of A4. tuberculosis and other Mycobacterium species and tuberculin PPD using Tris-tricine SDS-PAGE, to visualize the polypeptides with a silver stain/Coomassie blue stain combination and to compare the profiles.
2. Materials and methods 2.1. Bacteriul strains strains of M. tuberculosis H37Rv (25618) (25177). M. tuberculosis Erdman (35801), M. bovis BCG Tokyo (35737) and clinical isolates of M. chelonae, M kansasii, and M. gordonae were used throughout these studies. ATCC
M. tuberculosis H37Ra
2.2. Growth
ofbacteria
Bacterial cultures were maintained on LowensteinJensen slants (Becton-Dickinson Microbiology Systems, Cockeysville, MD). Growth was transferred to Sauton’s agar plates (L-asparagine, 0.4%: citric acid, 0.2%; KzHPOd, 0.5%; MgS04 7Ha0, 0.5%; ferric ammonium citrate, 0.005%; glycerol, 4.0% v/ v; pH to 7.5 with NHdOH; 1.5% agar) and incubated in air at 37°C. Five colonies were suspended in saline and 0.5 ml of loosely packed cells was inoculated into 150 ml of Sauton’s broth. Broth cultures were incubated in air at 37°C without shaking for 6-8 weeks.
and Medical Microbiology
23 (1999) 21-25
2.3. Culture jiltrate preparation Cultures were centrifuged after 6-8 weeks and the filtrate passed through a low protein binding 0.2 pm filter. The culture filtrate was placed in 1000 molecular mass cut-off dialysis tubing (Spectra/Par cellulose ester membrane, Spectrum, Houston, TX) and placed in sucrose at 4°C until concentrated 30 times (1.2 mg ml-‘). PPD (FDA US Standard-2) was a gift from Michael Brennan, FDA, Center for Biologics Evaluation and Research, Bethesda, MD. 2.4. SDS-PAGE
and gel staining
Tris-tricine SDS-PAGE electrophoresis was performed according to Schagger and von Jagow [21]. Gels (22 X 22 cm, 1.5 mm thick) were poured in three layers with a resolving gel of 16% T, 3% C, a 3-cm spacer gel of 10% T, 3% C, and a stacking gel of 4% T, 3% C. The anode buffer consisted of 0.2 M Tris, pH 8.9 and the cathode buffer consisted of 0.1 M Tris, 0.1 M tricine, 0.1% SDS, pH 8.25. Low molecular mass markers were from Gibco BRL (Life Technologies, Rockville, MD). Wells contained 50 ~1 of 30 x concentrated culture filtrates or 200 yg of PPD. Electrophoresis gels were run at 30 V for 14-20 h in a water cooled electrophoresis chamber. After electrophoresis, gels were fixed in 50% methanol, 10% acetic acid for 1 h. Gels were then silver stained using Daiichi Silver Stain (Integrated Separation Systems, Natick, MA). Following silver staining, the gels were placed in 0.025% Coomassie blue in 10% acetic acid overnight. Gels were destained with 10% acetic acid.
3. Results and discussion Fig. 1 demonstrates low molecular mass proteins present in PPD and M. tuberculosis H37Rv culture filtrate. The lines at the side of the figure indicate polypeptide bands (below 14 kDa) in M. tuberculosis H37Rv culture filtrate and the lines with arrows indicate polypeptide bands of a similar size to those present in PPD. There are at least 10 bands present in the M. tuberculosis H37Rv filtrate in the 2-14-kDa size range. Five of these are in the region between 2 kDa and 5.5 kDa. Most of the mycobacterial anti-
23
S.S. Rowland et al. I FEMS Immunology and Medical Microbiology 23 (1999) 21-25
kDa 43.2 29.3 18.4 14.0
5.5
2.8
Fig. 1. Comparison of low molecular mass protein profiles of PPD and M. /uberculosisH37Rv (Mtb) culture filtrate. Proteins were separated by Tris-tricine SDS-PAGE and double stained with silver stain and Coomassie blue. Lane I, molecular mass markers: lane 2. PPD: lane 3, Mtb. Filtrate was concentrated 30X from a &week Sauton broth culture.
gens studied to date have been greater than 14 kDa in size [22,23]. Only a few mycobacterial protein antigens with a lower molecular mass have been reported. A 1ZkDa protein was identified in PPD and shown to have homology to a lo-kDa protein from M tuberculosis and 13CG and the GroES stress protein [24,25]. A 6-kDa secreted protein referred to as ESAT-6 has been identified in short-term culture filtrates of M. tuberculosis and shown to induce potent T-cell response in a murine model of TB infection, and in M. bouis infected cattle [21,26,27]. However, ESAT-6 is absent fr’om the vaccine strain M. bovis BCG [28]. Another report indicated that a gel filtration fraction containing I-3-kDa non-proteinaceous molecules from an M. tuberculosis H37Rv cell lysate activated human T cells [29]. These reports support the concept that small molecular mass mycobacterial molecules may be important in protective immunity and that their composition may vary among mycobacterial strains and species. The PPD protein profile in Fig. 1, lane 2, reveals three distinct bands between 14 kDa and 5 kDa which appear to be present both in PPD and in the M. tuberculosis H37Rv filtrate (marked by arrowheads). In addition, there are two distinct PPD polypeptide bands in the 45-kDa region which do not appear to be present in the M.
tuberculosis H37Rv, lane 3. Typically, SDS-PAGE analysis of PPD results in a smear with poorly defined protein bands [3,14,30]. Use of Tris-tricine SDS-PAGE combined with silver staining followed by Coomassie blue staining allows visualization of small molecular mass polypeptides. Our results show the presence of distinct bands in PPD. These bands can now be isolated and their role in immunity examined. Fig. 2 contrasts the polypeptide profiles of M. tuberculosis Erdman, M. bovis BCG, and M. tuberculosis H37Rv. The profile of M. bovis BCG is very similar to that of M. tuberculosis; however, some differences can be observed. In particular, there is a prominent band in the M. tuberculosis filtrates (lanes 1 and 4) at approx. 10 kDa which is either absent or at a greatly reduced level in the M. bovis BCG filtrate (lane 3). In addition, there appear to be fewer polypeptide bands in the 6-8-kDa region of the M. bovis BCG filtrate. Fig. 3 contains the profiles of M. tuberculosis H37Ra, M. chelonae, M. kansasii, and M. gordonae. The results show that the stationary phase culture filtrates of all Mvcobacterium spp. tested contain small molecular mass proteins. There are both diskDa 43.2 29.3 18.4 14.0
5.5 2.8
I
1
Fig. 2. Comparison of low molecular mass protein profiles of M. tuberculosis Erdman, M. bovis BCG, and M. tuberculosis H3lRv. Proteins were separated by Tris-tricine SDS-PAGE and double stained with silver stain and Coomassie blue. Lane 1. M. tuberculosis Erdman; lane 2, molecular mass markers: lane 3. A4. bovis BCG; lane 4, A4. tuberculosis H37Rv. Filtrates were concentrated 30x from 6-&week Sauton broth cultures.
24
S.S. Rowlmd et al. IFEMS
Immunology and Medico1 Microbiology
kDa 43.2
29.3
18.4
14.0
,73 (1999) ZI-_‘5
mass mycobacterial proteins identified in this study will now be purified and examined for a potential role in the immune response to mycobacterial infections. The potential of M. tuberculosis secreted proteins as vaccines has recently been demonstrated with plasmid vaccines containing antigen 85 (30-32 kDa), 65-kDa heat shock protein, and ESAT-6 [34,35]. This suggests that mycobacterial secreted proteins may be useful components in a new vaccine against M. tuberculosis.
Acknowledgments 5.5
2.8
Fig. 3. Comparison tuberculosis H37Ra;
of low molecular
mass protein
This research was supported by the American Lung Association of Maryland and a Graduate Research Assistantship to J.L. Ruckert from the Graduate School, University of Maryland, Baltimore, MD.
profiles of M.
M. chelonae. M. kamasii and M. gordonae.
Proteins were separated by Tris-tricine SDS-PAGE and double stained with silver stain and Coomassie blue. Lane 1, M. tuberculosis H37Ra; lane 2, M. cl&onae; lane 3, M. kansasii: lane 4. M. gordonne: and lane 5, molecular mass markers. Filtrates were concentrated 30X from 66S-week Sauton broth cultures.
tinct and common bands between species. It is interesting to note that M. kansasii (lane 3). which can cause primary tuberculosis, has a low molecular mass profile with greater similarity to that of M. tuberculosis (lane 1) than does M. chelorzae (lane 2), an opportunistic mycobacterium which causes tissue infections. Interestingly, M. kansasii is also one of the few pathogenic mycobacterial species that also have the ESAT-6 gene [28]. In conclusion, these results demonstrate the presence of low molecular mass polypeptides from 2 kDa to 14 kDa in stationary phase culture filtrates of a variety of Mycobacterium spp. and in PPD. Other bacteria are known to secrete low molecular proteins, such as bacteriocins, hemolysins, and peptide antibiotics, which range from 1 kDa to 4 kDa in size [31-331. Our results indicate that mycobacteria also produce small proteins in this size range. We have found consistent polypeptide profiles from @-week stationary phase Sauton broth culture filtrates of each Mycobacterium species. These low molecular
References [l] Harth, G.. Lee. B.-Y. and Horowitz. M.A. (1997) High-level heterologous expression and secretion in rapidly growing nonpathogenic mycobacteria of four major Mycobacterium tuberculosis extracellular proteins considered to be leading vaccine candidates and drug targets. Infect. Immun. 65, 2321-2328. 121Huebner. R.E., Schein, M.F., Cauthen, G.M., Geiter, L.J., Selin, M.J., Good. R.C. and O’Brien, R.J. (1992) Evaluation of the clinical usefulness of mycobacterial skin test antigens in adults with pulmonary mycobacterioses. Am. Rev. Respir. Dis. 145, 1160-l 166. [31Klausen, J.. Magnusson, M.. Andersen, A.B. and Koch. C. (1994) Characterization of purified protein derivative of tuberculin by use of monoclonal antibodies: isolation of a delayedtype hypersensitivity reactive component from M. tuberculosis culture filtrate. Stand. J. Immunol. 40, 3455349. [41Nair. J., Rouse, D.A. and Morris, S.L. (1993) Nucleotide sequence analysis and serologic characterization of a 27-kilodalton Mycobacterium intracellulare lipoprotein. Infect. Immun. 61, 10741081. [51Marshall, B.G., Chambers, M.A., Wangoo, A., Shaw. R.J. and Young, D.B. (1997) Production of tumor necrosis factor and nitric oxide by macrophages infected with live and dead mycobacteria and suppression by an interleukin-lo-secreting recombinant. Infect. Immun. 65, 1931-1935. 161Andersen, P., Askgaard, D., Ljungqvist. L., Bennedsen. J. and Heron, I. (1991) Proteins released from Mycobacterium ruherculosis during growth. Infect. Immun. 59. 1905-1910. [71Collins. F.M., Lamb. J.R. and Young, D.B. (1988) Biological activity of protein antigens isolated from Mycobacterium tuberculosis culture filtrate. Infect. Immun. 56, 1260-1266.
S.S. Rowland et al. IFEMS Immunology and Medical Microbiology 23 (1999) 21-25 [8] Harboe, M. and Wiker, H.G. (1992) The 38-kDa protein of Mycobacteriunr tubercur’osis: A review. J. Infect. Dis. 166, 874884. [9] Nagai. S., Wiker, H.G., Harboe, M. and Kinomoto. M. (1991) Isolation and partial characterization of major protein antigens in the culture fluid of Mvcobacterium tuberculosis. Infect. Immun. 59. 3722382. [lo] Wiker, H.G. and Harboe. M. (1992) The antigen 85 complex: a major secretion product of Mycobacterium tuberculosis. Microbiol. Rev. 56, 6488661. [ll] Manta, C., Lyashchenko, K., Wiker, H.G., Usai, D., Colangeli, R. and Gennaro, M.L. (1997) Molecular cloning. purification, and serological characterization of MPT63. a novel antigen secreted by M.vcobncteriutn tuberculosis. Infect. Immun. 65. 16-23. [12] Bassey. E.O.E., Catty. D., Kumararatne, D.S. and Raykundalia, C. (1996) Candidate antigens for improved serodiagnosis of tuberculosis. Tubercle Lung Dis. 77. 136145. [13] Haagsma, J. and Angus. R.D. (1995) Tuberculin production in Mycobacterium hovis infection in animals and humans. In: Thoen, C.O. and Steele, J.H. (Ed%). pp. 73391. Iowa State University Press. Ames. IA. [14] Harboe, M., Wiker. HG. and Lachmann, P.J. (1990) Carrier effect of concanavalin A reactive and non-reactive material in tuberculin PPD. Stand. J. Immunol. 32, 263-271. [15] Huebner. R.E., Schein. M.F. and Bass, J.B. Jr. (1993) The tuberculin skin test. Clin. Infect. Dis. 17, 7688775. [16] Abou-Zeid. C., Smith, I., Grange, J., Steele, J. and Rook, G. (1986) Subdivision of daughter strains of Bacille CalmetteGuerin (BCG) according to secreted protein patterns. J. Gen. Microbial. 132, 3047-3053. [17] Andersen. A.B.. Anderren, P. and Ljungqvist, L. (1992) Structure and function of a 40.000-molecular-weight protein antigen of A(vcobacterium tuberculosis. Infect. Immun. 60. 23172323. [18] Jackson, M., Portnoi, I)., Catheline, D.. Dumail, L.. Rauzier, J., Legrand, P. and Gicquel, B. (1997) Mycobacterium tuberculosis Des protein: an immunodominant target for the humoral response of tuberculous patients. Infect. Immun. 65. 288332889. 1191 Peake, P., Gooley. A. and Britton, W.J. (1993) Mechanism of interaction of the 85B secreted protein of Mvcobacterium bovis with fibronectin. Infect Immun. 61. 48284834. [20] Sorensen, A.L., Nagai. S., Houen, G.. Andersen, P. and Andersen. A.B. (1995) Purification and characterization of a lowmolecular-mass T cell antigen secreted by Mycobacterium tuberculosis. Infect, Immun. 63, 1710-1717. 1211 Schagger. H. and von Jagow, F. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electtophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166. 3688379. [22] Young, D.B. (1988) Structure of mycobacterial antigens. Br. Med. Bull. 44, 5622583.
25
[23] Young. D.B., Kaufmann, S.H.E., Hermans, P.W.M. and Thole, J.E.R. (1992) Mycobacterial protein antigens: a compilation Mol. Microbial. 6, 133-145. [24] Ben-Nun, A., Mendel. I., Sappler, G. and Kerlero de Rosbo. N. (1995) A 12-kDa protein of Mvcobacterium tuberculosis protects mice against experimental autoimmune encephalomyelitis. J. Immunol. 154, 2939-2948. [25] Shinnick. T.M., Plikaytis. B.B., Hyche. A.D., Van Landingham, R.M. and Walker. L.L. (1989) The Mycobacterium tuberculosis BCG-a protein has homology with the Escherichio coli GroES protein. Nucleic Acids Res. 17, 1254. [26] Andersen, P., Andersen. A.B., Sorensen, A.L. and Nagai. S. (1995) Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice. J. Immunol. 154, 3359-3372. [27] Pollack, J.M. and Andersen, P. (1997) Predominant recognition of the ESAT-6 protein in the first phase of infection with Mq‘cobacterium bovis in cattle. Infect. Immun. 65, 25872592. [28] Harboe, M., Oettinger, T., Wiker, H.G., Rosenkrands, I. and Andersen. P. (1996) Evidence for occurrence of the ESAT-6 protein in Mycobarteriutn tuberculosis and virulent Mvcobacterium bovis and for its absence in Mycobacteriurn hovis BCG. Infect. Immun. 64. 16. [29] Pfeffer, K., Schoel, B., Plesnila, N., Lipford. G.B., Kromer. S., Deusch, K. and Wagner, H. (1992) A lectin-binding, proteaseresistant mycobacterial ligand specifically activates V9+ human T cells. J. Immunol. 146. 5755583. [30] Affronti, L.F., Porrello, V. and Gupta. S. (1990) Trace elements incorporated into the culture medium of Mycobacterium tuberculosis promote the presence of tuberculoprotein C in the preparation of purified protein derivatives. Microbios 63, 101-107. [31] Aymerich, R., Holo, H., Havarstein, L.S., Hugas, M., Garriga, M. and Nes, I.F. (1996) Biochemical and genetic characterization of enterocin A from Enterococctrs ftrecizrm, a new antilisterial bacteriocin in the pediocin family of bacteriocins. Appl. Environ. Microbial. 62. 1676-1682. [32] Chatterjee. S.. Chatterjee. S., Lad. S.J.. Phansalkar, MS.. Rupp. R.H. and Ganguli. B.N. (1991) Mersacidin, a new antibiotic from Bacillus: fermentation. isolation. purification and chemical characterization. J. Antibiot. 45, 832-837. [33] Donvito. B.. Etienne. J., Denoroy. L.. Greenland, T., Benito, Y. and Vandenesch, F. (1997) Synergistic hemolytic activity of Stapllylococcus lugdunensisis mediated by three peptides encoded by a non-agr genetic locus. Infect. Immun. 65, 955 100. [34] Lowrie, D.B., Silva. C.L., Colston, M.J.. Ragno, S. and Tascon, R.E. (1997) Protection against tuberculosis by a plasmid DNA vaccine. Vaccine 15, 8344838. [35] Ulmer, J.B., Liu, M.A., Montgomery, D.L.. Yawman, A.M., Deck, R.R.. Dewitt, C.M., Content, J. and Huygen, K. (1997) Expression and immunogenicity of Mvcobacterium tuberculosis antigen 85 by DNA vaccination. Vaccine 15, 7922794.
ELSEVIER
FEMS Immunology and Medical Microbiology 23 (1999) 21-25
Low molecular mass protein patterns in mycobacterial filtrates and purified protein derivatives Sharon
S. Rowland
‘, Jennifer
L. Ruckert
2, Patrick
J. Cummings
culture *
Department of Medical and Research Technology, University of Maryland School of Medicine, Allied Health Building. Room 405A. 100 Penn Street, Baltimore, MD 21202. USA
Received 2 March 1998: received in revised form 2 October 1998; accepted 6 October 1998
Abstract Mycobacterial polypzptides from 2 kDa to 14 kDa may be involved in host response to infection and be useful for new diagnostics and vaccines. Tris-tricine SDS-polyacrylamide gel electrophoresis separation of proteins in tuberculin purified protein derivative (PPD) and in 6-&week culture filtrates of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and other Mycobacteriurn species demonstrated as many as 10 low molecular mass bands. Common and distinct bands were observed among different species and PPD. These low molecular mass culture filtrate proteins may represent potential diagnostic reagents and vaccines for Mycobacterium tuberculosis. 0 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords:
Mycobacteriunz
tuberculosis: Culture filtrate protein: Purified protein derivative
’ Present address: McKesson BioServices, 7501 Standish Place, Rockville, MD 20855, [ISA.
USA as a diagnostic indicator of exposure to M. tuberculosis, lacks specificity and is not useful in countries which routinely vaccinate against tuberculosis [2,3]. Other Mycobacterium spp., such as M. aviumlintracellulare, are important pathogens in AIDS patients and must be distinguished from M. tuberculosis [4]. In addition, there is a great need to improve the current BCG-based tuberculosis vaccine and to develop faster and more accurate means of identifying mycobacterial infections [1,2,5]. The extracellular proteins of M. tuberculosis, released into culture broth during in vitro growth, are believed to play a role in infection and immunity and are of particular interest [6-121. The tuberculin PPD, which produces a marked delayed hypersensi-
’ Present address: Medlmmune. 35 West Watkins Mill Road,
tivity
Gaithersburg, MD 20878, USA.
sists of a complex mixture
1. Introduction
Tuberculosis,
caus,ed by Mycobacterium
tuberculo-
sis, is the leading cause of death due to an infectious
disease in the world and has been declared a global emergency by the ‘World Health Organization. A primary goal of mycobacterial research is to identify M. tuberculosis antigens which can be used for new vaccines and diagnostics [l]. The tuberculin purified protein derivative (PPD) test, which is used in the * Corresponding author. Tel.: +l (410) 706-2627; Fax: +l (410) 706-0073; E-mail: [email protected]
reaction
in individuals
with tuberculosis,
of extracellular
con-
proteins
092%8244/99/$19.00 0 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved PII: SO928-8244(98)001 13-S
22
S.S. Rowhmd et al. I FEMS
Immunology
from A4. tuberculosis stationary phase liquid culture medium. PPD is prepared by heat sterilization of 6-week-old M. tuberculosis grown in Sauton medium, followed by filter sterilization and protein concentration using ultrafiltration or ammonium sulfate precipitation [3]. Considering the widespread use of this immunological reagent it is surprising little is known about the active components of PPD [3,13151. Much work has been done fractionating M. tuberculosis proteins and studying the immune response [ 16-201. However, protein antigens below 10 kDa have not been studied in detail, partly because they do not separate well using standard SDSPAGE, are present in low amounts in culture filtrate, are not easily visualized with stains, and have low immunogenicity. The goal of this study was to separate the low molecular mass proteins present in stationary phase culture filtrates of A4. tuberculosis and other Mycobacterium species and tuberculin PPD using Tris-tricine SDS-PAGE, to visualize the polypeptides with a silver stain/Coomassie blue stain combination and to compare the profiles.
2. Materials and methods 2.1. Bacteriul strains strains of M. tuberculosis H37Rv (25618) (25177). M. tuberculosis Erdman (35801), M. bovis BCG Tokyo (35737) and clinical isolates of M. chelonae, M kansasii, and M. gordonae were used throughout these studies. ATCC
M. tuberculosis H37Ra
2.2. Growth
ofbacteria
Bacterial cultures were maintained on LowensteinJensen slants (Becton-Dickinson Microbiology Systems, Cockeysville, MD). Growth was transferred to Sauton’s agar plates (L-asparagine, 0.4%: citric acid, 0.2%; KzHPOd, 0.5%; MgS04 7Ha0, 0.5%; ferric ammonium citrate, 0.005%; glycerol, 4.0% v/ v; pH to 7.5 with NHdOH; 1.5% agar) and incubated in air at 37°C. Five colonies were suspended in saline and 0.5 ml of loosely packed cells was inoculated into 150 ml of Sauton’s broth. Broth cultures were incubated in air at 37°C without shaking for 6-8 weeks.
and Medical Microbiology
23 (1999) 21-25
2.3. Culture jiltrate preparation Cultures were centrifuged after 6-8 weeks and the filtrate passed through a low protein binding 0.2 pm filter. The culture filtrate was placed in 1000 molecular mass cut-off dialysis tubing (Spectra/Par cellulose ester membrane, Spectrum, Houston, TX) and placed in sucrose at 4°C until concentrated 30 times (1.2 mg ml-‘). PPD (FDA US Standard-2) was a gift from Michael Brennan, FDA, Center for Biologics Evaluation and Research, Bethesda, MD. 2.4. SDS-PAGE
and gel staining
Tris-tricine SDS-PAGE electrophoresis was performed according to Schagger and von Jagow [21]. Gels (22 X 22 cm, 1.5 mm thick) were poured in three layers with a resolving gel of 16% T, 3% C, a 3-cm spacer gel of 10% T, 3% C, and a stacking gel of 4% T, 3% C. The anode buffer consisted of 0.2 M Tris, pH 8.9 and the cathode buffer consisted of 0.1 M Tris, 0.1 M tricine, 0.1% SDS, pH 8.25. Low molecular mass markers were from Gibco BRL (Life Technologies, Rockville, MD). Wells contained 50 ~1 of 30 x concentrated culture filtrates or 200 yg of PPD. Electrophoresis gels were run at 30 V for 14-20 h in a water cooled electrophoresis chamber. After electrophoresis, gels were fixed in 50% methanol, 10% acetic acid for 1 h. Gels were then silver stained using Daiichi Silver Stain (Integrated Separation Systems, Natick, MA). Following silver staining, the gels were placed in 0.025% Coomassie blue in 10% acetic acid overnight. Gels were destained with 10% acetic acid.
3. Results and discussion Fig. 1 demonstrates low molecular mass proteins present in PPD and M. tuberculosis H37Rv culture filtrate. The lines at the side of the figure indicate polypeptide bands (below 14 kDa) in M. tuberculosis H37Rv culture filtrate and the lines with arrows indicate polypeptide bands of a similar size to those present in PPD. There are at least 10 bands present in the M. tuberculosis H37Rv filtrate in the 2-14-kDa size range. Five of these are in the region between 2 kDa and 5.5 kDa. Most of the mycobacterial anti-
23
S.S. Rowland et al. I FEMS Immunology and Medical Microbiology 23 (1999) 21-25
kDa 43.2 29.3 18.4 14.0
5.5
2.8
Fig. 1. Comparison of low molecular mass protein profiles of PPD and M. /uberculosisH37Rv (Mtb) culture filtrate. Proteins were separated by Tris-tricine SDS-PAGE and double stained with silver stain and Coomassie blue. Lane I, molecular mass markers: lane 2. PPD: lane 3, Mtb. Filtrate was concentrated 30X from a &week Sauton broth culture.
gens studied to date have been greater than 14 kDa in size [22,23]. Only a few mycobacterial protein antigens with a lower molecular mass have been reported. A 1ZkDa protein was identified in PPD and shown to have homology to a lo-kDa protein from M tuberculosis and 13CG and the GroES stress protein [24,25]. A 6-kDa secreted protein referred to as ESAT-6 has been identified in short-term culture filtrates of M. tuberculosis and shown to induce potent T-cell response in a murine model of TB infection, and in M. bouis infected cattle [21,26,27]. However, ESAT-6 is absent fr’om the vaccine strain M. bovis BCG [28]. Another report indicated that a gel filtration fraction containing I-3-kDa non-proteinaceous molecules from an M. tuberculosis H37Rv cell lysate activated human T cells [29]. These reports support the concept that small molecular mass mycobacterial molecules may be important in protective immunity and that their composition may vary among mycobacterial strains and species. The PPD protein profile in Fig. 1, lane 2, reveals three distinct bands between 14 kDa and 5 kDa which appear to be present both in PPD and in the M. tuberculosis H37Rv filtrate (marked by arrowheads). In addition, there are two distinct PPD polypeptide bands in the 45-kDa region which do not appear to be present in the M.
tuberculosis H37Rv, lane 3. Typically, SDS-PAGE analysis of PPD results in a smear with poorly defined protein bands [3,14,30]. Use of Tris-tricine SDS-PAGE combined with silver staining followed by Coomassie blue staining allows visualization of small molecular mass polypeptides. Our results show the presence of distinct bands in PPD. These bands can now be isolated and their role in immunity examined. Fig. 2 contrasts the polypeptide profiles of M. tuberculosis Erdman, M. bovis BCG, and M. tuberculosis H37Rv. The profile of M. bovis BCG is very similar to that of M. tuberculosis; however, some differences can be observed. In particular, there is a prominent band in the M. tuberculosis filtrates (lanes 1 and 4) at approx. 10 kDa which is either absent or at a greatly reduced level in the M. bovis BCG filtrate (lane 3). In addition, there appear to be fewer polypeptide bands in the 6-8-kDa region of the M. bovis BCG filtrate. Fig. 3 contains the profiles of M. tuberculosis H37Ra, M. chelonae, M. kansasii, and M. gordonae. The results show that the stationary phase culture filtrates of all Mvcobacterium spp. tested contain small molecular mass proteins. There are both diskDa 43.2 29.3 18.4 14.0
5.5 2.8
I
1
Fig. 2. Comparison of low molecular mass protein profiles of M. tuberculosis Erdman, M. bovis BCG, and M. tuberculosis H3lRv. Proteins were separated by Tris-tricine SDS-PAGE and double stained with silver stain and Coomassie blue. Lane 1. M. tuberculosis Erdman; lane 2, molecular mass markers: lane 3. A4. bovis BCG; lane 4, A4. tuberculosis H37Rv. Filtrates were concentrated 30x from 6-&week Sauton broth cultures.
24
S.S. Rowlmd et al. IFEMS
Immunology and Medico1 Microbiology
kDa 43.2
29.3
18.4
14.0
,73 (1999) ZI-_‘5
mass mycobacterial proteins identified in this study will now be purified and examined for a potential role in the immune response to mycobacterial infections. The potential of M. tuberculosis secreted proteins as vaccines has recently been demonstrated with plasmid vaccines containing antigen 85 (30-32 kDa), 65-kDa heat shock protein, and ESAT-6 [34,35]. This suggests that mycobacterial secreted proteins may be useful components in a new vaccine against M. tuberculosis.
Acknowledgments 5.5
2.8
Fig. 3. Comparison tuberculosis H37Ra;
of low molecular
mass protein
This research was supported by the American Lung Association of Maryland and a Graduate Research Assistantship to J.L. Ruckert from the Graduate School, University of Maryland, Baltimore, MD.
profiles of M.
M. chelonae. M. kamasii and M. gordonae.
Proteins were separated by Tris-tricine SDS-PAGE and double stained with silver stain and Coomassie blue. Lane 1, M. tuberculosis H37Ra; lane 2, M. cl&onae; lane 3, M. kansasii: lane 4. M. gordonne: and lane 5, molecular mass markers. Filtrates were concentrated 30X from 66S-week Sauton broth cultures.
tinct and common bands between species. It is interesting to note that M. kansasii (lane 3). which can cause primary tuberculosis, has a low molecular mass profile with greater similarity to that of M. tuberculosis (lane 1) than does M. chelorzae (lane 2), an opportunistic mycobacterium which causes tissue infections. Interestingly, M. kansasii is also one of the few pathogenic mycobacterial species that also have the ESAT-6 gene [28]. In conclusion, these results demonstrate the presence of low molecular mass polypeptides from 2 kDa to 14 kDa in stationary phase culture filtrates of a variety of Mycobacterium spp. and in PPD. Other bacteria are known to secrete low molecular proteins, such as bacteriocins, hemolysins, and peptide antibiotics, which range from 1 kDa to 4 kDa in size [31-331. Our results indicate that mycobacteria also produce small proteins in this size range. We have found consistent polypeptide profiles from @-week stationary phase Sauton broth culture filtrates of each Mycobacterium species. These low molecular
References [l] Harth, G.. Lee. B.-Y. and Horowitz. M.A. (1997) High-level heterologous expression and secretion in rapidly growing nonpathogenic mycobacteria of four major Mycobacterium tuberculosis extracellular proteins considered to be leading vaccine candidates and drug targets. Infect. Immun. 65, 2321-2328. 121Huebner. R.E., Schein, M.F., Cauthen, G.M., Geiter, L.J., Selin, M.J., Good. R.C. and O’Brien, R.J. (1992) Evaluation of the clinical usefulness of mycobacterial skin test antigens in adults with pulmonary mycobacterioses. Am. Rev. Respir. Dis. 145, 1160-l 166. [31Klausen, J.. Magnusson, M.. Andersen, A.B. and Koch. C. (1994) Characterization of purified protein derivative of tuberculin by use of monoclonal antibodies: isolation of a delayedtype hypersensitivity reactive component from M. tuberculosis culture filtrate. Stand. J. Immunol. 40, 3455349. [41Nair. J., Rouse, D.A. and Morris, S.L. (1993) Nucleotide sequence analysis and serologic characterization of a 27-kilodalton Mycobacterium intracellulare lipoprotein. Infect. Immun. 61, 10741081. [51Marshall, B.G., Chambers, M.A., Wangoo, A., Shaw. R.J. and Young, D.B. (1997) Production of tumor necrosis factor and nitric oxide by macrophages infected with live and dead mycobacteria and suppression by an interleukin-lo-secreting recombinant. Infect. Immun. 65, 1931-1935. 161Andersen, P., Askgaard, D., Ljungqvist. L., Bennedsen. J. and Heron, I. (1991) Proteins released from Mycobacterium ruherculosis during growth. Infect. Immun. 59. 1905-1910. [71Collins. F.M., Lamb. J.R. and Young, D.B. (1988) Biological activity of protein antigens isolated from Mycobacterium tuberculosis culture filtrate. Infect. Immun. 56, 1260-1266.
S.S. Rowland et al. IFEMS Immunology and Medical Microbiology 23 (1999) 21-25 [8] Harboe, M. and Wiker, H.G. (1992) The 38-kDa protein of Mycobacteriunr tubercur’osis: A review. J. Infect. Dis. 166, 874884. [9] Nagai. S., Wiker, H.G., Harboe, M. and Kinomoto. M. (1991) Isolation and partial characterization of major protein antigens in the culture fluid of Mvcobacterium tuberculosis. Infect. Immun. 59. 3722382. [lo] Wiker, H.G. and Harboe. M. (1992) The antigen 85 complex: a major secretion product of Mycobacterium tuberculosis. Microbiol. Rev. 56, 6488661. [ll] Manta, C., Lyashchenko, K., Wiker, H.G., Usai, D., Colangeli, R. and Gennaro, M.L. (1997) Molecular cloning. purification, and serological characterization of MPT63. a novel antigen secreted by M.vcobncteriutn tuberculosis. Infect. Immun. 65. 16-23. [12] Bassey. E.O.E., Catty. D., Kumararatne, D.S. and Raykundalia, C. (1996) Candidate antigens for improved serodiagnosis of tuberculosis. Tubercle Lung Dis. 77. 136145. [13] Haagsma, J. and Angus. R.D. (1995) Tuberculin production in Mycobacterium hovis infection in animals and humans. In: Thoen, C.O. and Steele, J.H. (Ed%). pp. 73391. Iowa State University Press. Ames. IA. [14] Harboe, M., Wiker. HG. and Lachmann, P.J. (1990) Carrier effect of concanavalin A reactive and non-reactive material in tuberculin PPD. Stand. J. Immunol. 32, 263-271. [15] Huebner. R.E., Schein. M.F. and Bass, J.B. Jr. (1993) The tuberculin skin test. Clin. Infect. Dis. 17, 7688775. [16] Abou-Zeid. C., Smith, I., Grange, J., Steele, J. and Rook, G. (1986) Subdivision of daughter strains of Bacille CalmetteGuerin (BCG) according to secreted protein patterns. J. Gen. Microbial. 132, 3047-3053. [17] Andersen. A.B.. Anderren, P. and Ljungqvist, L. (1992) Structure and function of a 40.000-molecular-weight protein antigen of A(vcobacterium tuberculosis. Infect. Immun. 60. 23172323. [18] Jackson, M., Portnoi, I)., Catheline, D.. Dumail, L.. Rauzier, J., Legrand, P. and Gicquel, B. (1997) Mycobacterium tuberculosis Des protein: an immunodominant target for the humoral response of tuberculous patients. Infect. Immun. 65. 288332889. 1191 Peake, P., Gooley. A. and Britton, W.J. (1993) Mechanism of interaction of the 85B secreted protein of Mvcobacterium bovis with fibronectin. Infect Immun. 61. 48284834. [20] Sorensen, A.L., Nagai. S., Houen, G.. Andersen, P. and Andersen. A.B. (1995) Purification and characterization of a lowmolecular-mass T cell antigen secreted by Mycobacterium tuberculosis. Infect, Immun. 63, 1710-1717. 1211 Schagger. H. and von Jagow, F. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electtophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166. 3688379. [22] Young, D.B. (1988) Structure of mycobacterial antigens. Br. Med. Bull. 44, 5622583.
25
[23] Young. D.B., Kaufmann, S.H.E., Hermans, P.W.M. and Thole, J.E.R. (1992) Mycobacterial protein antigens: a compilation Mol. Microbial. 6, 133-145. [24] Ben-Nun, A., Mendel. I., Sappler, G. and Kerlero de Rosbo. N. (1995) A 12-kDa protein of Mvcobacterium tuberculosis protects mice against experimental autoimmune encephalomyelitis. J. Immunol. 154, 2939-2948. [25] Shinnick. T.M., Plikaytis. B.B., Hyche. A.D., Van Landingham, R.M. and Walker. L.L. (1989) The Mycobacterium tuberculosis BCG-a protein has homology with the Escherichio coli GroES protein. Nucleic Acids Res. 17, 1254. [26] Andersen, P., Andersen. A.B., Sorensen, A.L. and Nagai. S. (1995) Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice. J. Immunol. 154, 3359-3372. [27] Pollack, J.M. and Andersen, P. (1997) Predominant recognition of the ESAT-6 protein in the first phase of infection with Mq‘cobacterium bovis in cattle. Infect. Immun. 65, 25872592. [28] Harboe, M., Oettinger, T., Wiker, H.G., Rosenkrands, I. and Andersen. P. (1996) Evidence for occurrence of the ESAT-6 protein in Mycobarteriutn tuberculosis and virulent Mvcobacterium bovis and for its absence in Mycobacteriurn hovis BCG. Infect. Immun. 64. 16. [29] Pfeffer, K., Schoel, B., Plesnila, N., Lipford. G.B., Kromer. S., Deusch, K. and Wagner, H. (1992) A lectin-binding, proteaseresistant mycobacterial ligand specifically activates V9+ human T cells. J. Immunol. 146. 5755583. [30] Affronti, L.F., Porrello, V. and Gupta. S. (1990) Trace elements incorporated into the culture medium of Mycobacterium tuberculosis promote the presence of tuberculoprotein C in the preparation of purified protein derivatives. Microbios 63, 101-107. [31] Aymerich, R., Holo, H., Havarstein, L.S., Hugas, M., Garriga, M. and Nes, I.F. (1996) Biochemical and genetic characterization of enterocin A from Enterococctrs ftrecizrm, a new antilisterial bacteriocin in the pediocin family of bacteriocins. Appl. Environ. Microbial. 62. 1676-1682. [32] Chatterjee. S.. Chatterjee. S., Lad. S.J.. Phansalkar, MS.. Rupp. R.H. and Ganguli. B.N. (1991) Mersacidin, a new antibiotic from Bacillus: fermentation. isolation. purification and chemical characterization. J. Antibiot. 45, 832-837. [33] Donvito. B.. Etienne. J., Denoroy. L.. Greenland, T., Benito, Y. and Vandenesch, F. (1997) Synergistic hemolytic activity of Stapllylococcus lugdunensisis mediated by three peptides encoded by a non-agr genetic locus. Infect. Immun. 65, 955 100. [34] Lowrie, D.B., Silva. C.L., Colston, M.J.. Ragno, S. and Tascon, R.E. (1997) Protection against tuberculosis by a plasmid DNA vaccine. Vaccine 15, 8344838. [35] Ulmer, J.B., Liu, M.A., Montgomery, D.L.. Yawman, A.M., Deck, R.R.. Dewitt, C.M., Content, J. and Huygen, K. (1997) Expression and immunogenicity of Mvcobacterium tuberculosis antigen 85 by DNA vaccination. Vaccine 15, 7922794.