Microbial Pathogenesis 1992 ; 13 : 243-249
The 17 kDa lipoprotein and encoding gene of Francisella tularensis LVS are conserved in strains of Francisella tularensis A . Sjostedt,' 2 K . Kuoppa,' T . Johansson' and G . Sandstrom' 2 'Department of Microbiology, National Defence Research Establishment, Umea and 2 Department of Infectious Diseases, University of Umea, Sweden (Received June 2, 1992 ; accepted in revised form June 30, 1992)
Sjostedt, A . (Dept of Microbiology, National Defence Research Establishment, Umea, Sweden), K . Kuoppa, T . Johansson and G . Sandstrom . The 17 kDa lipoprotein and encoding gene of Francisella tularensis LVS are conserved in strains of Francisella tularensis. Microbial Pathogenesis 1992 ; 13 : 243-249 . A T-cell-stimulating 17 kDa protein of the vaccine strain Francisella tularensis LVS has previously been cloned, sequenced and shown to be a lipoprotein . In the present study, it was investigated whether the protein, denoted TUL4, and its gene are present in various strains of the genus Francise/la . By Western blot analysis, it was demonstrated that a TUL4-specific monoclonal antibody bound to a protein present in each of the Francisella strains . The immunoreactive proteins had an M, of 17 kDa in all F. tu/arensis strains and in the strain Francisella novicida, whereas the M, in strains of Francisella philomiragia was 20 kDa . When genomic preparations were probed with a radioactive DNA fragment of F . tularensis LVS encoding TUL4, hybridization was demonstrated in all strains of Francise/la, although the F. philomiragia strains did not hybridize under conditions of high stringency . The hybridizing chromosomal DNA fragment of the F. phi/omiragia strains was larger than that of the other Francisella strains . No hybridization or Western blot reactivity was seen when various other Gram-negative and Gram-positive bacteria were probed . In summary, the 17 kDa lipoprotein of F. tularensis LVS appears to be Francisella-specific and present in the species F. tularensis and F. novicida, whereas an immunologically related protein is present in F. philomiragia . Key words : 17 kDa lipoprotein ; F . tularensis LVS ; genus ; Francisella .
Introduction Francise//a is a small, Gram-negative, coccoid rod . The genus comprises three species . The facultative intracellular bacterium Francise//a tularensis causes a zoonotic disease known as tularemia .' Francise//a novicida has predominantly been isolated in the environment, although it has been reported to cause human disease on a few occasions .' Also, Francisella philomiragia has only rarely been associated with human disease . It has been isolated from immunocompromised humans and from individuals near drowning . 3 This species was originally designated as Yersinia phi/omiragia, but it has subsequently by analysis of its fatty acid composition, as well as by serological 2 .3 examination, been referred to as Francisella . 0882-4010/92/090243+07 $08 .00/0
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Strains of the species F . to/arensis are usually divided into two types that differ from each other in virulence, geographic distribution and in animal reservoirs .4 ,5 Francise//a tularensis subspecies tularensis, also known as type A, is believed to be restricted to North America .` Most strains of the species are highly virulent for mammals ."' Francisella tularensis subspecies palaearctica, also denoted type B, is found in Europe and Asia, but also in North America ."' It has a moderate or low virulence and human fatalities are extremely rare . It was recently demonstrated, that the differentiation of these two types according to virulence and biochemical characteristics is by no means unambiguous . 6 Moreover, it was found that 16S rRNA of strains of F. tularensis isolated outside the U .S .A . hybridized with a probe specific to F. tularensis type A . 6 To clarify these ambiguities and obtain a more precise classification, more data on the genetic and molecular relationships of strains belonging to the species F . tularensis and the genus Francise/la would be valuable . In the 1930s, work was initiated in the U .S .S .R . for the purpose of developing attenuated live vaccine strains of F. tularensis .' One of the vaccine strains was subsequently transferred to the U .S .A .' It has been demonstrated that SDS-PAGEseparated protein profiles of the vaccine strain, known as F. tularensis LVS, and of virulent strains of the species are similar .' Virtually all available data regarding the protein composition of Francise//a strains originate from studies on T-cell stimulating membrane proteins of F . tularensis LVS .' -" In particular, a major 17 kDa membrane protein of the vaccine strain has been studied . The encoding gene has been cloned in Escherichia co/i and its amino acid sequence deduced ." , " The protein is lipid-modified and recognized by T-cells from a majority of F. tularensis-primed individuals ." In the present study, the presence of the 17 kDa lipoprotein and the encoding gene among strains of Francise/la and other bacterial genera was investigated .
Results Occurrence of the 17 kDa lipoprotein and of the encoding gene in strains of Francisella The presence of the gene of the 17 kDa lipoprotein in various strains of Francisella was studied by Southern blot analysis, using a 732-bp, TUL4-specific DNA fragment as a probe . Chromosomal DNA was prepared from an environmental isolate of F. novicida and from 12 F. tularensis strains listed in Table 1 . DNA was digested with EcoRl (data not shown), or Clal and Pvull [Fig . 1 (a) and (b)] . Irrespective of enzyme combination used, all bacterial strains showed identical hybridization patterns at 42°C, indicating that the genes are at least 85% homologous in these strains ." Two F. philomiragia strains were also included, one patient isolate and an animal isolate . Hybridization was detected to DNA of these strains only at 21 °C [Fig . 1 (c)], but not at 42°C [Fig . 1 (b)] . Hybridization at 21 °C is detectable only if there are less than 30% mismatches . 15 The hybridization was detected to a chromosomal DNA fragment of an approximate size of 2 .0 kb, thus, considerably larger than the 0 .7 kb chromosomal fragment of the other Francise//a strains that hybridized to the probe . Irrespective of stringency, there was no hybridization to chromosomal DNA of the following bacterial species : E. coli, Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus, Yersinia enterocolitica, Listeria monocytogenes and Legionella pneumophila (data not shown) . Lysates of the bacterial strains were analysed by Western blotting to demonstrate the presence of immunoreactive proteins . A monoclonal antibody (MAb) directed against TUL4 invariably recognized a 17 kDa protein in the strains belonging to the species F. tularensis and F . novicida (Fig . 2) . In F . philomiragia, the antibody recognized
17 kDa lipoprotein in strains of F .
to/arensis
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Representation of the 17 kDa lipoprotein and encoding gene of F. to/arensis Table 1 LVS among strains of Francisella and other bacteria
Number 1 . 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 .
Strain, origin F. tularensis LVS, vaccine strain F. tularensis B38, from human F. tularensis Vavenby, from hare F. tularensis Schu A, from human F. tularensis SBL R45, from human F. tularensis FOA1, from human F. tularensis 543, from hare F. tularensis Jap4, from human F. tularensis S-2, from human F. tularensis Oniwa, from human F . tularensis Ebina, from human F. novicida, environmental isolate F . philomiragia, from muskrat F . philomiragia, from human Y. enterocolitica 8081 -C E. coil DH1 S. typhimurium LT2 S. aureus B . subtilis L . monocytogenes L . pneumophila
Type
Reference
Hybridizatio'
B A A A B B A A A A A A A A
ATCC 29684 ATCC 6223 6 8 6 CCUG 17299 6 6 6 6 2 ATCC 15482 CCUG 19700 CCUG 12603 TF Wetzler 24 ATCC 19585 ATCC 6538 ATCC 6633 ATCC 15527 ATCC 33152
++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + -
Western blot + + + + + +
+ + + + + +
-
'The typing is based on RNA hybridization to 1 6S rRNA genes of strains of Francisella ( M . Forsman, personal communication) .' The distinction between the two species are based on the presence of North American strains of a nucleotide G (type A) and in F. to/arensis LVS a nucleotide A (type B) in position 1153 of the genes . °(++) Hybridization to the TUL4-specific probe was obtained at 42°C; (+) hybridization was obtained at 21'C, but not at 42'C .
a 20 kDa protein [Fig . 2(b)] . No proteins were recognized by the monoclonal in strains of the other bacterial species included in this study (data not shown) .
Discussion All strains of Francise//a studied in this report express immunologically related proteins with M,s of 17 or 20 kDa, irrespective of their virulence and geographical origin . The strains represent the three species of Francisella . The TUL4-specific MAb showed no reactivity with other bacterial species . Similarly, in earlier studies using polyclonal serum specific to the 17 kDa lipoprotein, no reactivity to E. co/i and S . typhimurium has been noted ." , " Irrespective of the stringency, the 17 kDa TUL4-specific probe hybridized with all strains of F. tularensis and F. novicida, but only at lower stringency to strains of F. phi/omiragia . The probe was strictly genus-specific, as no signal was seen when chromosomal DNA from bacteria of other species was probed . It has previously been demonstrated that the amino acid sequence of the 17 kDa lipoprotein shows some similarity to the sequence of the PAL lipoprotein of E. co/i, although when aligned, only 15% of the amino acids are identical ." Due to this low similarity, no hybridization to E. co/i could be expected under the conditions used . In similarity to PAL, it is possible that TUL4 is an essential constituent of the bacterial cell wall, which may explain the conservation of the protein in F. to/arensis . The presence of either the 17 kDa protein or the immunologically related 20 kDa protein in all strains of Francise//a, but not in bacteria of other species, renders it a potential target for detection of Francise//a in immunological assays . The demonstration of an immunologically related protein in F. phi/omiragia and also some DNA homology
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Fig . 1 . Southern blot hybridization of chromosomal DNA of strains of Francise!/a . Chromosomal DNA of various strains of Francise//a were separated in an agarose gel and transferred to nitrocellulose . A Clal-Pvull 0 .7 kb DNA fragment of F. to/arensis LVS encoding TUL4 was used as a probe . (a) and (b) are the results of hybridizations at 42°C . The strains in (c) are the same as in (b), but hybridization was carried out at 21°C . Lanes : (a) 1, F. tularensis LVS ; 2, F. tularensis R45 ; 3, F. tularensis B38 ; 4, F. tularensis Vavenby ; 5, F. novicida . (b) 1, F. tularensis Schu A ; 2, F. tularensis CCUG 17299 ; 3, F. tularensis P 13863 ; 4, F. tularensis Jap4 ; 5, F. tularensis S-2 ; 6, F. tularensis Oniwa ; 7, F. tularensis Ebina ; 8, F. tularensis 543 ; 9, F. tularensis CCUG 19700 ; 10, F. to/arensis CCUG 12603 . Numbers on the left indicate molecular masses in kb .
between the two genes, is a further demonstration that F. phi/omiragia is related to the other species of Francise//a . Originally, F. philomiragia was believed to belong to the genus Yersinia . In studies on Legione//a, similar findings have been made, i .e . the presence of immunologically related proteins has been demonstrated in various species of the genus, despite that the DNA homologies between the encoding genes are variable ." DNA probes specific to the TUL4-encoding gene may be useful for detection of strains of the species F. tularensis, although no differentiation of the two types will be possible . Indeed, primers complementary to the 17 kDa lipoprotein gene have been used in the polymerase chain reaction (PCR) to demonstrate the presence of F. tularensis LVS in infected mice ." The present findings are also of interest with regard to previous taxonomic studies on F. tularensis . It has been demonstrated that probes specific to 16S rRNA of F. to/arensis can be utilized to specifically detect strains of
17 kDa lipoprotein in strains of F. to/arensis ( a)
I
2
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4
24 7
5 10
69 .046-0-
30 .0-
21 .521 .514.314 .3-
Fig . 2 . Western blot analysis of whole cell extracts . Cell extracts were separated by SDS-PAGE and after transfer to nitrocellulose, a MAb raised against TUL4 of F . tularensis LVS was used as a probe . Lanes : (a) 1, F. tularensis LVS ; 2, F . tularensis R45 ; 3, F . tularensis B38 ; 4, F . tularensis Vavenby ; 5, F . novicida. (b) 1, F. tularensis Schu A ; 2, F . tularensis CCUG 17299 ; 3, F . tularensis P 13863 ; 4, F . tularensis Jap4 ; 5, F . tularensis S-2 ; 6, F . tularensis Oniwa ; 7, F. tularensis Ebina ; 8, F. tularensis 543 ; 9, F. tularensis CCUG 19700 ; 10, F. to/arensis CCUG 12603 . Numbers on the left indicate molecular masses in kDa . the species, as well as to differentiate between the two different types of F . tu/arensis .s,19 Either identification based on 16S rRNA or assays based on detection of the TUL4gene seems feasible for identification and detection of strains belonging to F . tularensis . The conservation of the 17 kDa lipoprotein in strains of F. to/arensis is also interesting with regard to previous studies on the role of the protein as a T-cell immunogen . The 17 kDa lipoprotein elicits a T-cell response in a majority of primed individuals and the proliferating T-cells secrete IFN-y, the presence of which is crucial for killing of F . tularensis in vivo .14 .20,21 As the lipoprotein is conserved in all virulent F . tularensis strains examined, the protein may contribute to the induction of a protective cellmediated immunity to tularemia . Materials and methods Bacterial strains and plasmids . Bacterial strains used in this study are listed in Table 1 . Plasmid pTUL4-7, which is a derivative of pACYC1 84, has been described elsewhere ." Media and enzymes . Francisella strains and L . monocytogenes were cultivated on modified Thayer-Martin agar22 for 1-3 days at 37'C . Legionella pneumophila was grown on Legionella BYCE medium23 for 2-3 days at 37'C . All other bacterial strains were grown overnight on Luria agar .24 Restriction endonucleases were purchased from Pharmacia AB, Uppsala, Sweden . DNA techniques. Manipulations of DNA were performed by use of standard techniques ." Chromosomal DNA was prepared as described by Marmur .26 To isolate the DNA fragment used as a probe, pTUL4-7 was digested with C/al and Pvull, and DNA was separated by
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electrophoresis in a 1% agarose gel (Seakem GTG) . The resulting 732 by DNA fragment was recovered by electroelution . The electroeluted DNA fragment was labelled with 7-[ 32 P]dATP by Multi-prime labelling (Amersham International) according to instructions given by the manufacturer . Probe . The 732-bp C/al-Pvull DNA fragment used as a probe encompassed the complete 450 by gene encoding TUL4 in positions 43-493, No other open reading frames are contained within the DNA fragment .
Southern blot analysis . Ten micrograms of chromosomal DNA of the strains listed in Table 1 were digested with C/al and Pvull or EcoRl and separated by agarose gel electrophoresis . Blotting was performed essentially as described by Southern ." Prehybridization was performed at 42°C, using a previously described technique ." High stringency hybridization was performed in 50% formamide at 42°C in 5xSSC . For low stringency conditions, the same hybridization solution was used at 21'C . Monoclonal antibody. A MAb raised against the 149 amino acid, 17 kDa lipoprotein TUL4 of F. tularensis LVS was kindly provided by B . Lilliehook, National Defence Research Establishment, Umea, Sweden . The epitope recognized by the antibody has been mapped to the region of amino acids 86-115 . 14
SDS-PAGE and Western blot analysis . Proteins of bacterial lysates were separated by SDSPAGE on gels containing a linear gradient of acrylamide (10-17 .5%) . 29 Western blot analysis was performed as described by Swanson et al." After transfer to nitrocellulose filter, the TUL4specific MAb was added at a dilution of 1 :25 as primary antibody . After incubation, secondary alkaline phosphatase-conjugated antibodies were added .
We thank G . Bostrom for help with the preparation of the illustrations and M . Forsman for a critical reading of the manuscript .
References 1 . Hopla CE . The ecology of tularemia . Adv Vet Sci Comp Med 1974 ; 18 : 25-53 . 2 . Ohara S, Sato T, Homma M . Serological studies on Francisella tularensis, Francisella novicida, Yersinia philomiragia, and Brucella abortus . Int J Syst Bacteriol 1974 ; 24 : 191-6 . 3 . Hollis DG, Weaver RE, Steigerwalt AG, Wenger JD, Moss CW, Brenner DJ . Francisella philomiragia comb . nov . (formerly Yersinia philomiragia) and Francisella tularensis biogroup novicida (formerly Francisella novicida) associated with human disease . J Clin Microbiol 1989 ; 27 : 1601-8 . 4 . Olsufiev NG, Emelyanova OS, Dunayeva TN . Comparative study of strains of Bacterium tularense . II . Evaluation of criteria of virulence of Bacterium tularense in the old and the new world and their taxonomy . J Hyg Epidemiol Microbiol Immunobiol 1959 ; 3 : 138-49 . 5 . Eigelsbach HT, McGann VG . Gram-negative aerobic cocci . In : Krieg NR, Holt JG, eds . Bergey's manual of systemic bacteriology . Baltimore : Williams & Wilkins Co . 1984 ; 394-9 . 6 . Sandstrom G, Sjostedt A, Forsman M, Pavlovich NV, Mishankin BN . Characterization and classification of strains of Francisella tularensis isolated in the Central Asian focus of the Soviet Union and in Japan . J Clin Microbiol 1992 ; 30 . 172-5 . 7 . Gaiskii NA, Admareva N D, Litstik TG . Rapidity of the appearance and length of persistance of immunity after vaccination with tularemia vaccine . J Microbiol Epidemiol Immunol 1947, 7 : 46--51 . 8 . Eigelsbach HT, Downs CM . Prophylactic effectiveness of live and killed tularemia vaccines . I . Production of vaccine and evaluation in the white mouse and guinea pig . J Immunol 1961, 87 : 415-25 . 9 . Sjostedt A, Sandstrom G, Tarnvik A . Several membrane polypeptides of the live vaccine strain Francise//a tularensis LVS stimulate T cells from naturally infected individuals . J Clin Microbiol 1990 ; 28 : 43-8 . 10 . Surcel H-M, Sarvas M, Helander IM, Herva E . Membrane proteins of Francisella tularensis LVS differ in ability to induce proliferation of lymphocytes from tularemia-vaccinated individuals . Microb Pathogen 1989 ;7 :411-7 . 11 . Sandstrom G, Tarnvik A, Wolf-Watz H . Immunospecific T-lymphocyte stimulation by membrane proteins from Francisella tularensis. J Clin Microbiol 1987 ; 25 : 641-4 . 12 . Sjostedt A, Sandstrom G, Tarnvik A, Jaurin B . Molecular cloning and expression of a T-cell stimulating membrane protein of Francisella tularensis . Microb Pathogen 1989 ; 6 : 403-14 . 13 . Sjostedt A, Sandstrom G, Tarnvik A, Jaurin B . Nucleotide sequence and T-cell epitopes of a membrane protein of Francisella tularensis . J Immunol 1990 ; 145 : 311-7 . 14 . Sjostedt A, Tarnvik A, Sandstrom G . The T-cell stimulating 1 7-kilodalton protein of Francisella to/arensis LVS is a lipoprotein . Infect Immun 1991 ; 59 : 3163-8 .
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15 . Davis RW, Botstein D, Roth JR . A manual for genetic engineering : advanced bacterial genetics . Coldspring Harbor, NY : Cold Spring Harbor Press 1980 ; 220-1 . 16 . Sjostedt A, Sandstrom G, Tarnvik A . Humoral and cell-mediated immunity in mice to a 1 7-kilodalton lipoprotein of Francisella tularensis expressed by Salmonella typhimurium . Infect Immun 1992 ; 60 : 2855-62 . 17 . Ott M, Bender L, Chirinos E, Ehret W, Hacker J . Phenotype versus genotype of the 1 9-kD peptidoglycan associated protein of Legionella (PpIA), among Legionellae and other Gram-negative bacteria . Microb Pathogen 1991 ; 11 : 357-65 . 18 . Long GW, Narayanan RB, Fortier AH, Nacy CA, Oprandy JJ . Polymerase chain reaction assay for detection of Francisella tularensis. Am J Trop Med Hyg 1991, 45 : 110 . 19 . Forsman M, Sandstrom G, Jaurin B . Identification of Francisella species and discrimination of type A and type B strains of F . to/arensis by 16S rRNA analysis . Appl Env Microbiol 1990 ; 56 : 949-55 . 20 . Sjostedt A, Eriksson M, Sandstrom G, Tarnvik A . Various membrane proteins of Francisella tularensis induce interferon- ;• production in both CD4 and CD8 T cells of primed humans . Immunology 1992 ; 76 : 584-92 . 21 Leiby DA, Fortier AH, Crawford RM, Schreiber RD, Nacy CA . In vivo modulation of the immune response to Francisella tularensis LVS by administration of anticytokine antibodies . Infect Immun 1992 ; 60 : 84-9 . 22 . Sandstrom G, Tarnvik A, Wolf-Watz H, Lofgren S . Antigen from Francisella tularensis : non-identity between determinants participating in cell-mediated and humoral reactions . Infect Immun 1984; 45 : 101-6 . 23 . Edelstein PH . Improved semiselective medium for isolation of Legione//a pneumophila from contaminated clinical and environmental specimens . J Clin Microbiol 1981 ; 14 : 298-303 . 24 Bertani G . Studies on lysogenesis . I . The mode of phage liberation by lysogenic Escherichia co/i. J Bacteriol 1951 ; 62 : 293- 300 . 25 . Maniatis T, Frisch EF, Sambrook J . Molecular cloning : a laboratory manual . Cold Spring Harbor, NY : Cold Spring Harbor Laboratory Press 1982 . 26 . Marmur J . A procedure for the isolation of deoxyribonucleic acid from microorganisms . J Mol Biol 1961 ; 3 : 208--18 . 27 Southern EM . Detection of specific sequences among DNA fragments separated by gel electrophoresis . J Mol Biol 1975 ; 98 : 503-17 . 28 . Jaurin B, Cohen SN . Streptomyces lividans RNA polymerase recognizes and uses Escherichia coli transcriptional signals . Gene 1984 ; 28 : 83-91 . 29 . Laemmli UK . Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 1970 ; 227 : 680--5 . 30 . Swanson J, Mayer LW, Tam MR . Antigenicity of Neisseria gonorrhoeae outer membrane protein(s) III detected by immunoprecipitation and Western blot transfer with a monoclonal antibody . Infect Immun 1982 ; 38 : 668--72 .
The 17 kDa lipoprotein and encoding gene of Francisella tularensis LVS are conserved in strains of Francisella tularensis A . Sjostedt,' 2 K . Kuoppa,' T . Johansson' and G . Sandstrom' 2 'Department of Microbiology, National Defence Research Establishment, Umea and 2 Department of Infectious Diseases, University of Umea, Sweden (Received June 2, 1992 ; accepted in revised form June 30, 1992)
Sjostedt, A . (Dept of Microbiology, National Defence Research Establishment, Umea, Sweden), K . Kuoppa, T . Johansson and G . Sandstrom . The 17 kDa lipoprotein and encoding gene of Francisella tularensis LVS are conserved in strains of Francisella tularensis. Microbial Pathogenesis 1992 ; 13 : 243-249 . A T-cell-stimulating 17 kDa protein of the vaccine strain Francisella tularensis LVS has previously been cloned, sequenced and shown to be a lipoprotein . In the present study, it was investigated whether the protein, denoted TUL4, and its gene are present in various strains of the genus Francise/la . By Western blot analysis, it was demonstrated that a TUL4-specific monoclonal antibody bound to a protein present in each of the Francisella strains . The immunoreactive proteins had an M, of 17 kDa in all F. tu/arensis strains and in the strain Francisella novicida, whereas the M, in strains of Francisella philomiragia was 20 kDa . When genomic preparations were probed with a radioactive DNA fragment of F . tularensis LVS encoding TUL4, hybridization was demonstrated in all strains of Francise/la, although the F. philomiragia strains did not hybridize under conditions of high stringency . The hybridizing chromosomal DNA fragment of the F. phi/omiragia strains was larger than that of the other Francisella strains . No hybridization or Western blot reactivity was seen when various other Gram-negative and Gram-positive bacteria were probed . In summary, the 17 kDa lipoprotein of F. tularensis LVS appears to be Francisella-specific and present in the species F. tularensis and F. novicida, whereas an immunologically related protein is present in F. philomiragia . Key words : 17 kDa lipoprotein ; F . tularensis LVS ; genus ; Francisella .
Introduction Francise//a is a small, Gram-negative, coccoid rod . The genus comprises three species . The facultative intracellular bacterium Francise//a tularensis causes a zoonotic disease known as tularemia .' Francise//a novicida has predominantly been isolated in the environment, although it has been reported to cause human disease on a few occasions .' Also, Francisella philomiragia has only rarely been associated with human disease . It has been isolated from immunocompromised humans and from individuals near drowning . 3 This species was originally designated as Yersinia phi/omiragia, but it has subsequently by analysis of its fatty acid composition, as well as by serological 2 .3 examination, been referred to as Francisella . 0882-4010/92/090243+07 $08 .00/0
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Strains of the species F . to/arensis are usually divided into two types that differ from each other in virulence, geographic distribution and in animal reservoirs .4 ,5 Francise//a tularensis subspecies tularensis, also known as type A, is believed to be restricted to North America .` Most strains of the species are highly virulent for mammals ."' Francisella tularensis subspecies palaearctica, also denoted type B, is found in Europe and Asia, but also in North America ."' It has a moderate or low virulence and human fatalities are extremely rare . It was recently demonstrated, that the differentiation of these two types according to virulence and biochemical characteristics is by no means unambiguous . 6 Moreover, it was found that 16S rRNA of strains of F. tularensis isolated outside the U .S .A . hybridized with a probe specific to F. tularensis type A . 6 To clarify these ambiguities and obtain a more precise classification, more data on the genetic and molecular relationships of strains belonging to the species F . tularensis and the genus Francise/la would be valuable . In the 1930s, work was initiated in the U .S .S .R . for the purpose of developing attenuated live vaccine strains of F. tularensis .' One of the vaccine strains was subsequently transferred to the U .S .A .' It has been demonstrated that SDS-PAGEseparated protein profiles of the vaccine strain, known as F. tularensis LVS, and of virulent strains of the species are similar .' Virtually all available data regarding the protein composition of Francise//a strains originate from studies on T-cell stimulating membrane proteins of F . tularensis LVS .' -" In particular, a major 17 kDa membrane protein of the vaccine strain has been studied . The encoding gene has been cloned in Escherichia co/i and its amino acid sequence deduced ." , " The protein is lipid-modified and recognized by T-cells from a majority of F. tularensis-primed individuals ." In the present study, the presence of the 17 kDa lipoprotein and the encoding gene among strains of Francise/la and other bacterial genera was investigated .
Results Occurrence of the 17 kDa lipoprotein and of the encoding gene in strains of Francisella The presence of the gene of the 17 kDa lipoprotein in various strains of Francisella was studied by Southern blot analysis, using a 732-bp, TUL4-specific DNA fragment as a probe . Chromosomal DNA was prepared from an environmental isolate of F. novicida and from 12 F. tularensis strains listed in Table 1 . DNA was digested with EcoRl (data not shown), or Clal and Pvull [Fig . 1 (a) and (b)] . Irrespective of enzyme combination used, all bacterial strains showed identical hybridization patterns at 42°C, indicating that the genes are at least 85% homologous in these strains ." Two F. philomiragia strains were also included, one patient isolate and an animal isolate . Hybridization was detected to DNA of these strains only at 21 °C [Fig . 1 (c)], but not at 42°C [Fig . 1 (b)] . Hybridization at 21 °C is detectable only if there are less than 30% mismatches . 15 The hybridization was detected to a chromosomal DNA fragment of an approximate size of 2 .0 kb, thus, considerably larger than the 0 .7 kb chromosomal fragment of the other Francise//a strains that hybridized to the probe . Irrespective of stringency, there was no hybridization to chromosomal DNA of the following bacterial species : E. coli, Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus, Yersinia enterocolitica, Listeria monocytogenes and Legionella pneumophila (data not shown) . Lysates of the bacterial strains were analysed by Western blotting to demonstrate the presence of immunoreactive proteins . A monoclonal antibody (MAb) directed against TUL4 invariably recognized a 17 kDa protein in the strains belonging to the species F. tularensis and F . novicida (Fig . 2) . In F . philomiragia, the antibody recognized
17 kDa lipoprotein in strains of F .
to/arensis
245
Representation of the 17 kDa lipoprotein and encoding gene of F. to/arensis Table 1 LVS among strains of Francisella and other bacteria
Number 1 . 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 .
Strain, origin F. tularensis LVS, vaccine strain F. tularensis B38, from human F. tularensis Vavenby, from hare F. tularensis Schu A, from human F. tularensis SBL R45, from human F. tularensis FOA1, from human F. tularensis 543, from hare F. tularensis Jap4, from human F. tularensis S-2, from human F. tularensis Oniwa, from human F . tularensis Ebina, from human F. novicida, environmental isolate F . philomiragia, from muskrat F . philomiragia, from human Y. enterocolitica 8081 -C E. coil DH1 S. typhimurium LT2 S. aureus B . subtilis L . monocytogenes L . pneumophila
Type
Reference
Hybridizatio'
B A A A B B A A A A A A A A
ATCC 29684 ATCC 6223 6 8 6 CCUG 17299 6 6 6 6 2 ATCC 15482 CCUG 19700 CCUG 12603 TF Wetzler 24 ATCC 19585 ATCC 6538 ATCC 6633 ATCC 15527 ATCC 33152
++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + -
Western blot + + + + + +
+ + + + + +
-
'The typing is based on RNA hybridization to 1 6S rRNA genes of strains of Francisella ( M . Forsman, personal communication) .' The distinction between the two species are based on the presence of North American strains of a nucleotide G (type A) and in F. to/arensis LVS a nucleotide A (type B) in position 1153 of the genes . °(++) Hybridization to the TUL4-specific probe was obtained at 42°C; (+) hybridization was obtained at 21'C, but not at 42'C .
a 20 kDa protein [Fig . 2(b)] . No proteins were recognized by the monoclonal in strains of the other bacterial species included in this study (data not shown) .
Discussion All strains of Francise//a studied in this report express immunologically related proteins with M,s of 17 or 20 kDa, irrespective of their virulence and geographical origin . The strains represent the three species of Francisella . The TUL4-specific MAb showed no reactivity with other bacterial species . Similarly, in earlier studies using polyclonal serum specific to the 17 kDa lipoprotein, no reactivity to E. co/i and S . typhimurium has been noted ." , " Irrespective of the stringency, the 17 kDa TUL4-specific probe hybridized with all strains of F. tularensis and F. novicida, but only at lower stringency to strains of F. phi/omiragia . The probe was strictly genus-specific, as no signal was seen when chromosomal DNA from bacteria of other species was probed . It has previously been demonstrated that the amino acid sequence of the 17 kDa lipoprotein shows some similarity to the sequence of the PAL lipoprotein of E. co/i, although when aligned, only 15% of the amino acids are identical ." Due to this low similarity, no hybridization to E. co/i could be expected under the conditions used . In similarity to PAL, it is possible that TUL4 is an essential constituent of the bacterial cell wall, which may explain the conservation of the protein in F. to/arensis . The presence of either the 17 kDa protein or the immunologically related 20 kDa protein in all strains of Francise//a, but not in bacteria of other species, renders it a potential target for detection of Francise//a in immunological assays . The demonstration of an immunologically related protein in F. phi/omiragia and also some DNA homology
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Fig . 1 . Southern blot hybridization of chromosomal DNA of strains of Francise!/a . Chromosomal DNA of various strains of Francise//a were separated in an agarose gel and transferred to nitrocellulose . A Clal-Pvull 0 .7 kb DNA fragment of F. to/arensis LVS encoding TUL4 was used as a probe . (a) and (b) are the results of hybridizations at 42°C . The strains in (c) are the same as in (b), but hybridization was carried out at 21°C . Lanes : (a) 1, F. tularensis LVS ; 2, F. tularensis R45 ; 3, F. tularensis B38 ; 4, F. tularensis Vavenby ; 5, F. novicida . (b) 1, F. tularensis Schu A ; 2, F. tularensis CCUG 17299 ; 3, F. tularensis P 13863 ; 4, F. tularensis Jap4 ; 5, F. tularensis S-2 ; 6, F. tularensis Oniwa ; 7, F. tularensis Ebina ; 8, F. tularensis 543 ; 9, F. tularensis CCUG 19700 ; 10, F. to/arensis CCUG 12603 . Numbers on the left indicate molecular masses in kb .
between the two genes, is a further demonstration that F. phi/omiragia is related to the other species of Francise//a . Originally, F. philomiragia was believed to belong to the genus Yersinia . In studies on Legione//a, similar findings have been made, i .e . the presence of immunologically related proteins has been demonstrated in various species of the genus, despite that the DNA homologies between the encoding genes are variable ." DNA probes specific to the TUL4-encoding gene may be useful for detection of strains of the species F. tularensis, although no differentiation of the two types will be possible . Indeed, primers complementary to the 17 kDa lipoprotein gene have been used in the polymerase chain reaction (PCR) to demonstrate the presence of F. tularensis LVS in infected mice ." The present findings are also of interest with regard to previous taxonomic studies on F. tularensis . It has been demonstrated that probes specific to 16S rRNA of F. to/arensis can be utilized to specifically detect strains of
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Fig . 2 . Western blot analysis of whole cell extracts . Cell extracts were separated by SDS-PAGE and after transfer to nitrocellulose, a MAb raised against TUL4 of F . tularensis LVS was used as a probe . Lanes : (a) 1, F. tularensis LVS ; 2, F . tularensis R45 ; 3, F . tularensis B38 ; 4, F . tularensis Vavenby ; 5, F . novicida. (b) 1, F. tularensis Schu A ; 2, F . tularensis CCUG 17299 ; 3, F . tularensis P 13863 ; 4, F . tularensis Jap4 ; 5, F . tularensis S-2 ; 6, F . tularensis Oniwa ; 7, F. tularensis Ebina ; 8, F. tularensis 543 ; 9, F. tularensis CCUG 19700 ; 10, F. to/arensis CCUG 12603 . Numbers on the left indicate molecular masses in kDa . the species, as well as to differentiate between the two different types of F . tu/arensis .s,19 Either identification based on 16S rRNA or assays based on detection of the TUL4gene seems feasible for identification and detection of strains belonging to F . tularensis . The conservation of the 17 kDa lipoprotein in strains of F. to/arensis is also interesting with regard to previous studies on the role of the protein as a T-cell immunogen . The 17 kDa lipoprotein elicits a T-cell response in a majority of primed individuals and the proliferating T-cells secrete IFN-y, the presence of which is crucial for killing of F . tularensis in vivo .14 .20,21 As the lipoprotein is conserved in all virulent F . tularensis strains examined, the protein may contribute to the induction of a protective cellmediated immunity to tularemia . Materials and methods Bacterial strains and plasmids . Bacterial strains used in this study are listed in Table 1 . Plasmid pTUL4-7, which is a derivative of pACYC1 84, has been described elsewhere ." Media and enzymes . Francisella strains and L . monocytogenes were cultivated on modified Thayer-Martin agar22 for 1-3 days at 37'C . Legionella pneumophila was grown on Legionella BYCE medium23 for 2-3 days at 37'C . All other bacterial strains were grown overnight on Luria agar .24 Restriction endonucleases were purchased from Pharmacia AB, Uppsala, Sweden . DNA techniques. Manipulations of DNA were performed by use of standard techniques ." Chromosomal DNA was prepared as described by Marmur .26 To isolate the DNA fragment used as a probe, pTUL4-7 was digested with C/al and Pvull, and DNA was separated by
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electrophoresis in a 1% agarose gel (Seakem GTG) . The resulting 732 by DNA fragment was recovered by electroelution . The electroeluted DNA fragment was labelled with 7-[ 32 P]dATP by Multi-prime labelling (Amersham International) according to instructions given by the manufacturer . Probe . The 732-bp C/al-Pvull DNA fragment used as a probe encompassed the complete 450 by gene encoding TUL4 in positions 43-493, No other open reading frames are contained within the DNA fragment .
Southern blot analysis . Ten micrograms of chromosomal DNA of the strains listed in Table 1 were digested with C/al and Pvull or EcoRl and separated by agarose gel electrophoresis . Blotting was performed essentially as described by Southern ." Prehybridization was performed at 42°C, using a previously described technique ." High stringency hybridization was performed in 50% formamide at 42°C in 5xSSC . For low stringency conditions, the same hybridization solution was used at 21'C . Monoclonal antibody. A MAb raised against the 149 amino acid, 17 kDa lipoprotein TUL4 of F. tularensis LVS was kindly provided by B . Lilliehook, National Defence Research Establishment, Umea, Sweden . The epitope recognized by the antibody has been mapped to the region of amino acids 86-115 . 14
SDS-PAGE and Western blot analysis . Proteins of bacterial lysates were separated by SDSPAGE on gels containing a linear gradient of acrylamide (10-17 .5%) . 29 Western blot analysis was performed as described by Swanson et al." After transfer to nitrocellulose filter, the TUL4specific MAb was added at a dilution of 1 :25 as primary antibody . After incubation, secondary alkaline phosphatase-conjugated antibodies were added .
We thank G . Bostrom for help with the preparation of the illustrations and M . Forsman for a critical reading of the manuscript .
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