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Outer membrane proteins of Brucella
BRUCELLA AND BRUCELLOSIS: AN UPDATE
87
[7~ VERSTREATE,D.R., CREASY,M.T., CAVENEY,N.T., BALDWIN,C.L., BEAn, M.W. & WINTER, A.J., Outer membrane proteins of Brucella abortus: isolation and characterization. Infect. Immun., 1982, 35, 979-989. [8] WINTER, A.J., VERSTREATE, D.R., HALL, C.E., JACOBSON, R.H., CASTLEMAN, M.P., MEREDITH,M.P. & MCLAUGHL1N,C.A., Immune response to porin in cattle immunized with whole cell, outer membrane and outer membrane protein antigens of Brucella abortus combined with ~rehalose dimycolate and muramyl dipeptide adjuvant, rnfect. Immun., 1983, 42, 1159-1167.
OUTER MEMBRANE PROTEINS OF BRUCELLA by A.J. Winter
New York State College o f Veterinary Medicine, Cornell University, Ithaca, N Y 14853 (USA) This review will be concerned with the physical and chemical properties of the major outer membrane proteins of Brucella species. The principal outer membrane proteins of Brucella spp. are porins [2, 3, 6, 7, 8], group 3 proteins [7, 81 and a lipoprotein covalently linked to peptidoglycan [4]. Brucella porins were designated group 2 proteins by Verstreate et al. [8] before their functional characterization as porins was defined. Killed cells were disrupted in a high pressure cell and extracted with N-lauroyisarcosinate. Group 2 proteins were then extracted from the insoluble residue (crude cell envelopes) by a dipolar ionic detergent (Zwittergent 3-14) but this extraction was extremely ip~fficient unless the cell envelopes were first subjected to digestion with egg white lysozyme [8]. The requirement for lysozyme digestion to release outer membrane proteins of Brucella from peptidoglycan, noted also by Dubray and his co-workers [1, 2], must be ascribed in part to alterations in the cell envelope resulting from chemical or physical agents used to kill cells prior to their extraction [$]. In like maturer, the extraction of grcup 2 proteins from killed cells by boiling SDS was found to be very inefficient [2, 8],
whereas extraction by this method of cell envelopes prel:ared directly from living organisms was much more effective [6]. Group 2 proteins appear to exist in the native state as tfimers which are reduced to monomers in boiling SDS [8]. The monomers generally give rise in SDS-FAGE to 2 or 3 closely spaced bands [2, 3, 7, 8, 9], but it is not established whether these represent different species of polypepfides. The tight association of group 2 [5, 8] as well as group 3 [8] proteins with LPS may be responsible in part for this apparent heterogeneity. The molecular weights assigned originally by Verstreate et al. [$] were 41 to 43 kilodaltons (Kd}. However, in light ::,f work in othel laboratories [1, 2, 3, 6] and our own [7, 9], a range of 35 to 40 Kd appears more accurate. Group 2 proteins of B. abortus [8] as well as B. melitens~ and B. canis [7] resembled each other closely in amino acid compoz.;tion and bore an overall resemblance to OmpF of E. coli [8]. Douglas et al. [3] demonstrated by functional assays that group 2 proteins were porins, with porin channels ranging slightly among Brucet.la species and generally comparable in size to .*.hoseof OmpF. To date, there is no published information on the occurrence of iron-
88
2 nd F O R U M i N A I I C R O B I O L O G Y
dependent porins in Brucclla. The relative proportions of porin to group 3 proteins in smooth or rough strains of B. abortus was abc ut 1/1, bvA i~ B. melitensi~; B. ovis and B. can;.~'. ~l~e~.roportion of porin was substantially ~ower [7]. In B. melitensis, disso~iatio'a from smooth to rough forms was accompanied by a dramatic decrease in the porin to group 3 ratio [7]. The porins of 49 strains of B. abortus shared a common antigenic determinant [9] which was also possessed by a majority of the 9 strains :ested of B. melitensis, B. ovis and B canis [7]. Group 3 proteins were released from the cell envelope under the same conditions employed for extraction of pori~s [1, 2, 8]. it was hypothesized that gre~;p 3 r,) :,~:eins were counterparts c._t E. coli ~.~:~A on the basis of their general resemb;ance to OmpA in amino acid composi,ion and apparem shong binding to peptidoglycan, even though there was no evidence in group 3 proteins of heat modifiability [8]. In support of this idea, group 3 proteins did not function as porins [31 and have recently been found to display heat modifiability in the absence of zwitterionic detergents (I. Moriyon, personal communication, 1986). Group 3 proteins of B. melitensis, B. ovis and B. canis resembled those of B. abortus in amino acid composition and apparent molecular weights and, as already mentioned, constituted the predominant outer membrane protein of those species [7]. In B. melitensis, moreover, a portion of group 3 p~otein appeared to occur as a relatively stable dimer which required boiling in buffers containing high ratios of SDS to protein to achieve
its dissociation [7~ The group 3 proteins of 49 strains ot ~_L abortus were antigenically relate~ to each other [9] and t6 representative st.:ains of B. melitensis, B. ovis and B. canis [7]. Gomez.-Miguel and Moriyon [4] recently demonstrated the presence in B. abortus and B. ovis of a lipoprotein covalently linked to peptidoglycan. The trypsin-digested fragments, with an apparent molecular weight of 8,000, bore an overall resemblance to the corresponding lipoprotein of E. coli in size, isoelectric point, amino acid composition and the presence of both ester- and amide-linked fatty acids [4]. Other minor outer membrane lZ~roteins of Brucella spp. have been reported. Group ~ proteins rmlged h~ molecular weight from about 88 to 04 Kd, were heat-modifiable, copurified with a portion of f~roup 3 [8] and occurred in all strains of B. abortus [8, 9], as well as B. melitensis, B. o vi.¢ and B. canis [7] which were tested. Moriyon and Berman [5] detected a number of proteins exposed on the cell surface of B. abortus which were not extracted with zwitterionic detergents. Dubray and Charriaut [2] subjected crude cell envelopes to boiling SDS and demonstrated a prominent band at 43 Kd in SDS-PAGE gels. Its relationship to the porin proteins which required lysozyme digestion for their extraction was not established. In conclusion, Brucella species possess porins and a liporotein covalently bound to peptidoglycan which are very similar to those of E. coll. A tifird major outer membrane protein appears to correspond to OmpA of E. coil
References.
[!] DVBRAY,G. & i~EZAI~D,G., Isolation of three Brucella abortus cell-wall antigens protective in murine experimental brec,Aiosis. Ann. Rech. Pet., 1980, i l , 367-373. [2] DUBRAY,G. & CHARR)AUT,C., Evidence of three major polypeptide species 6nd two major polysaccharide species in the Brucelle outer membrane. Ann. Rech. Vet., 1983, 14, 3!1-318. [3] DOUGLAS,J.T., ROSENBERG,E.Y., NIKAI90, H., VERSTREATE,D.R. & WINTEr~, A.J., Porins of Brucella species. Infect. Immun., 1984, 44, 16-21.
BRUCELLA AND BRUCELLOSIS: AN UPDATE
89
[4] GOMEZ-MIOUEL,M.J. & MORIYON,1., Demonstration of a peptidoglycan-linked [5] [6] [7] [8]
[9]
lipoprotein and characterization of its trypsin fragment in the outer membrane of Brucetla spp. Infect. Immun., 1986, 53, 678-684. MORIYON, [. ~1; BERMAN, D.T., Effects of non-ionic, ionic and dipolar ionic detergents and EDTA on the Brucella cell envelope. J. Bact., 1982, 152, 822-828. MORIYON,I. & BERMAN,D.T., Isolation, purification and partial characterization ot Brucella abortus matrix protein. Infect. Immun., 1983, 39, 394-402. SANTOS,J.M., VERSTREATE,D.R., PERERA, V.Y. & WINTEn, A.J., Outer membrane proteins from rough strains of four Brucella species. Infect. Immun., 1984, 46, 188-194. VERSTREATE,D.R., CREASY,M.T., CAV::'.NEY,N.T., BALDWIN,C.L., BLAB, M.W. t~ WINTER,A.J., Outer membrane proteins of Brucella abortus: isolation and characterization. Infect. Immun., 1982, 35, 979-989, VERSTREATE,D.R. 8£ WINTER, A.J., Comparison of sodium dodec~t sulfatepolyacrylamide gel electrophoresis profiles and antigenic relatedness among outer membrane proteins of 49 Brucella aborlus strains. !nfect. Immun., 1984, 46, 182-187.
PROPERTIES OF THE OUTER MEMBRANE OF BRUCELLA by I. Moriy6n, C. Gamazo and R. Diaz
Dpto. de Microbiologga, Fac:dtad de Medicirta, Universidad de Nuvarra, Al tdo. 273, 31080 Pamplona (E9pa~a) The members of the genus Brucella are Gram-negative organisms a~c, as such, they are endowed with a cell envelope (CE) c o n s t i t u t e d by a cytoplasmic membrane and ah outer m e m b r a n e (OM) separated by a periplasmic <>. Although morphologically similar to that of other Gram-negative bacteria, this CE presents properties of its own which become apparent in extracting and purifying its major comlponents. 1 hose properties are summarized in this paper. The CE of most Gram-negative bacteria are sensitive to Tris buffers and EDTA. ~3oth agents act together to alter ~he OM barrier to the point that it leaks pe_,r~lasmic components and becomes ,p¢,meable>> te lysozyme Thus, the cc,m~ ~w,d acfio~ of Tris, EDTA and ly~o ~., ,.: in an isoto:fic medium brings a b e t , ~h<,formation of spheroplasts. In
contrast, B. abortus and B. melitensis do not form such spheroplasts under standard conditions [6]. Since EDTA acts by removing the divalent cations stabilizing the lipopolysaccharide (LPS) of the OM, bridging of negatively charged groups does not seem necessary to stabilize the LPS in Brucella. This interpretation is consistent with the fact that, while EDTA does not enhance the action of Triton-Xl00 or Zwittergents on micelles of B. abortus LPS, cation chelation is necessary for the complete dispersion of E. coli LPS by the same detergents. A!so, EDTA by itself does not remove LPS from CE of B. melitensis or B. abortus [6]. The anchorage of the OM is d e s t r o y e d in spheroplasts, and therefore, they are the material of choice to prepare ~ c OM of Gramnegat?.,e bacteria. Sirze the lack of effect
87
[7~ VERSTREATE,D.R., CREASY,M.T., CAVENEY,N.T., BALDWIN,C.L., BEAn, M.W. & WINTER, A.J., Outer membrane proteins of Brucella abortus: isolation and characterization. Infect. Immun., 1982, 35, 979-989. [8] WINTER, A.J., VERSTREATE, D.R., HALL, C.E., JACOBSON, R.H., CASTLEMAN, M.P., MEREDITH,M.P. & MCLAUGHL1N,C.A., Immune response to porin in cattle immunized with whole cell, outer membrane and outer membrane protein antigens of Brucella abortus combined with ~rehalose dimycolate and muramyl dipeptide adjuvant, rnfect. Immun., 1983, 42, 1159-1167.
OUTER MEMBRANE PROTEINS OF BRUCELLA by A.J. Winter
New York State College o f Veterinary Medicine, Cornell University, Ithaca, N Y 14853 (USA) This review will be concerned with the physical and chemical properties of the major outer membrane proteins of Brucella species. The principal outer membrane proteins of Brucella spp. are porins [2, 3, 6, 7, 8], group 3 proteins [7, 81 and a lipoprotein covalently linked to peptidoglycan [4]. Brucella porins were designated group 2 proteins by Verstreate et al. [8] before their functional characterization as porins was defined. Killed cells were disrupted in a high pressure cell and extracted with N-lauroyisarcosinate. Group 2 proteins were then extracted from the insoluble residue (crude cell envelopes) by a dipolar ionic detergent (Zwittergent 3-14) but this extraction was extremely ip~fficient unless the cell envelopes were first subjected to digestion with egg white lysozyme [8]. The requirement for lysozyme digestion to release outer membrane proteins of Brucella from peptidoglycan, noted also by Dubray and his co-workers [1, 2], must be ascribed in part to alterations in the cell envelope resulting from chemical or physical agents used to kill cells prior to their extraction [$]. In like maturer, the extraction of grcup 2 proteins from killed cells by boiling SDS was found to be very inefficient [2, 8],
whereas extraction by this method of cell envelopes prel:ared directly from living organisms was much more effective [6]. Group 2 proteins appear to exist in the native state as tfimers which are reduced to monomers in boiling SDS [8]. The monomers generally give rise in SDS-FAGE to 2 or 3 closely spaced bands [2, 3, 7, 8, 9], but it is not established whether these represent different species of polypepfides. The tight association of group 2 [5, 8] as well as group 3 [8] proteins with LPS may be responsible in part for this apparent heterogeneity. The molecular weights assigned originally by Verstreate et al. [$] were 41 to 43 kilodaltons (Kd}. However, in light ::,f work in othel laboratories [1, 2, 3, 6] and our own [7, 9], a range of 35 to 40 Kd appears more accurate. Group 2 proteins of B. abortus [8] as well as B. melitens~ and B. canis [7] resembled each other closely in amino acid compoz.;tion and bore an overall resemblance to OmpF of E. coli [8]. Douglas et al. [3] demonstrated by functional assays that group 2 proteins were porins, with porin channels ranging slightly among Brucet.la species and generally comparable in size to .*.hoseof OmpF. To date, there is no published information on the occurrence of iron-
88
2 nd F O R U M i N A I I C R O B I O L O G Y
dependent porins in Brucclla. The relative proportions of porin to group 3 proteins in smooth or rough strains of B. abortus was abc ut 1/1, bvA i~ B. melitensi~; B. ovis and B. can;.~'. ~l~e~.roportion of porin was substantially ~ower [7]. In B. melitensis, disso~iatio'a from smooth to rough forms was accompanied by a dramatic decrease in the porin to group 3 ratio [7]. The porins of 49 strains of B. abortus shared a common antigenic determinant [9] which was also possessed by a majority of the 9 strains :ested of B. melitensis, B. ovis and B canis [7]. Group 3 proteins were released from the cell envelope under the same conditions employed for extraction of pori~s [1, 2, 8]. it was hypothesized that gre~;p 3 r,) :,~:eins were counterparts c._t E. coli ~.~:~A on the basis of their general resemb;ance to OmpA in amino acid composi,ion and apparem shong binding to peptidoglycan, even though there was no evidence in group 3 proteins of heat modifiability [8]. In support of this idea, group 3 proteins did not function as porins [31 and have recently been found to display heat modifiability in the absence of zwitterionic detergents (I. Moriyon, personal communication, 1986). Group 3 proteins of B. melitensis, B. ovis and B. canis resembled those of B. abortus in amino acid composition and apparent molecular weights and, as already mentioned, constituted the predominant outer membrane protein of those species [7]. In B. melitensis, moreover, a portion of group 3 p~otein appeared to occur as a relatively stable dimer which required boiling in buffers containing high ratios of SDS to protein to achieve
its dissociation [7~ The group 3 proteins of 49 strains ot ~_L abortus were antigenically relate~ to each other [9] and t6 representative st.:ains of B. melitensis, B. ovis and B. canis [7]. Gomez.-Miguel and Moriyon [4] recently demonstrated the presence in B. abortus and B. ovis of a lipoprotein covalently linked to peptidoglycan. The trypsin-digested fragments, with an apparent molecular weight of 8,000, bore an overall resemblance to the corresponding lipoprotein of E. coli in size, isoelectric point, amino acid composition and the presence of both ester- and amide-linked fatty acids [4]. Other minor outer membrane lZ~roteins of Brucella spp. have been reported. Group ~ proteins rmlged h~ molecular weight from about 88 to 04 Kd, were heat-modifiable, copurified with a portion of f~roup 3 [8] and occurred in all strains of B. abortus [8, 9], as well as B. melitensis, B. o vi.¢ and B. canis [7] which were tested. Moriyon and Berman [5] detected a number of proteins exposed on the cell surface of B. abortus which were not extracted with zwitterionic detergents. Dubray and Charriaut [2] subjected crude cell envelopes to boiling SDS and demonstrated a prominent band at 43 Kd in SDS-PAGE gels. Its relationship to the porin proteins which required lysozyme digestion for their extraction was not established. In conclusion, Brucella species possess porins and a liporotein covalently bound to peptidoglycan which are very similar to those of E. coll. A tifird major outer membrane protein appears to correspond to OmpA of E. coil
References.
[!] DVBRAY,G. & i~EZAI~D,G., Isolation of three Brucella abortus cell-wall antigens protective in murine experimental brec,Aiosis. Ann. Rech. Pet., 1980, i l , 367-373. [2] DUBRAY,G. & CHARR)AUT,C., Evidence of three major polypeptide species 6nd two major polysaccharide species in the Brucelle outer membrane. Ann. Rech. Vet., 1983, 14, 3!1-318. [3] DOUGLAS,J.T., ROSENBERG,E.Y., NIKAI90, H., VERSTREATE,D.R. & WINTEr~, A.J., Porins of Brucella species. Infect. Immun., 1984, 44, 16-21.
BRUCELLA AND BRUCELLOSIS: AN UPDATE
89
[4] GOMEZ-MIOUEL,M.J. & MORIYON,1., Demonstration of a peptidoglycan-linked [5] [6] [7] [8]
[9]
lipoprotein and characterization of its trypsin fragment in the outer membrane of Brucetla spp. Infect. Immun., 1986, 53, 678-684. MORIYON, [. ~1; BERMAN, D.T., Effects of non-ionic, ionic and dipolar ionic detergents and EDTA on the Brucella cell envelope. J. Bact., 1982, 152, 822-828. MORIYON,I. & BERMAN,D.T., Isolation, purification and partial characterization ot Brucella abortus matrix protein. Infect. Immun., 1983, 39, 394-402. SANTOS,J.M., VERSTREATE,D.R., PERERA, V.Y. & WINTEn, A.J., Outer membrane proteins from rough strains of four Brucella species. Infect. Immun., 1984, 46, 188-194. VERSTREATE,D.R., CREASY,M.T., CAV::'.NEY,N.T., BALDWIN,C.L., BLAB, M.W. t~ WINTER,A.J., Outer membrane proteins of Brucella abortus: isolation and characterization. Infect. Immun., 1982, 35, 979-989, VERSTREATE,D.R. 8£ WINTER, A.J., Comparison of sodium dodec~t sulfatepolyacrylamide gel electrophoresis profiles and antigenic relatedness among outer membrane proteins of 49 Brucella aborlus strains. !nfect. Immun., 1984, 46, 182-187.
PROPERTIES OF THE OUTER MEMBRANE OF BRUCELLA by I. Moriy6n, C. Gamazo and R. Diaz
Dpto. de Microbiologga, Fac:dtad de Medicirta, Universidad de Nuvarra, Al tdo. 273, 31080 Pamplona (E9pa~a) The members of the genus Brucella are Gram-negative organisms a~c, as such, they are endowed with a cell envelope (CE) c o n s t i t u t e d by a cytoplasmic membrane and ah outer m e m b r a n e (OM) separated by a periplasmic <
contrast, B. abortus and B. melitensis do not form such spheroplasts under standard conditions [6]. Since EDTA acts by removing the divalent cations stabilizing the lipopolysaccharide (LPS) of the OM, bridging of negatively charged groups does not seem necessary to stabilize the LPS in Brucella. This interpretation is consistent with the fact that, while EDTA does not enhance the action of Triton-Xl00 or Zwittergents on micelles of B. abortus LPS, cation chelation is necessary for the complete dispersion of E. coli LPS by the same detergents. A!so, EDTA by itself does not remove LPS from CE of B. melitensis or B. abortus [6]. The anchorage of the OM is d e s t r o y e d in spheroplasts, and therefore, they are the material of choice to prepare ~ c OM of Gramnegat?.,e bacteria. Sirze the lack of effect