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A novel fimbrial haemagglutinin produced by a strain of Salmonella of serotype Salinatis

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A novel fimbrial haemagglutinin produced by a strain of Salmonella of serotype Salinatis
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  FEMS Microbiology Letters 57 (1989) 29-34 29 Published by Elsevier FEM 03401 novel fimbrial haemagglutinin produced by a strain of almonella of serotype Salinatis D.E. Yakubu, B.W. Senior and D.C. Old Department of Medical Microbiology University of Dundee Medical School Ninewells Hospital Dundee DD1 9S Y U.K. Received 1 August 1988 Accepted 26 August 1988 Key words: Salmonella; Serotype, Salinatis; Fimbriae; Mannose-resistant and eluting; Haemagglutinin 1. SUMMARY A strain of Salmonella of serotype Satinatis, that produced a mannose-resistant and eluting haemagglutinin (MREHA) when cultured at 37 ° C but not at 18°C, was examined by electron mi- croscopy after negative staining. Production of this MREHA, previously described as being non- fimbrial, was correlated with the presence of thin fimbriae which had an external diameter of 3.6 nm. The purified Salinatis thin fimbriae had an estimated Mr of 19 kDa. This fimbrial MREHA was not produced by strains of the antigenically related serotypes Duisburg and Sandiego. 2. INTRODUCTION Two kinds of fimbriae have been described in SalmOnellae. The majority (approx. 80%) of strains produce type-1 fimbriae which are 7-8 nm in diameter, have an axial hole and are associated with a mannose-sensitive haemagglutinin (MSHA) Correspondence to: D.C. Old, Department of Medical Microbi- ology, Ninewells Hospital, Dundee DD1 9SY, U.K. [1,2]. A few strains produce type-3, or type 3-like, fimbriae [2] which are 3-4 nm in diameter, un- channelled and are associated with different kinds of mannose-resistant haemagglutinins (MRHAs) [3-51. In this paper we describe and characterize a novel kind of fimbrial haemagglutinin produced by a strain of Salmonella of subspecies 1 of the rare serotype Salinatis [6]. 3. MATERIALS AND METHODS 3.1. Bacterial strains The strains examined belonged to different serotypes of S. enterica subsp, enterica [7]. Strain 263 of serotype Salinatis from the collection of Professor J.P. Duguid had been received originally as strain 151 from Dr. P.R. Edwards in 1948 and is also strain NCTC6247 from the National Col- lection of Type Cultures, London, U.K. Its anti- genic formula is now considered to be 4,12:d:e,h: e,n,z15 [8]. We also examined 12 strains of serotype Duisburg (1,4,12,27:d:e,n,z15) and four strains of serotype Sandiego (4,12:e,h:e,n,z15) received from Professor L. Le Minor, Service des Ent&obact6ries, Institut Pasteur, Paris, France. 0378-1097/89/ 03.50 © 1989 Federation of European Microbiological Societies  30 3.2. Culture conditions The media and methods of culture used were essentially those described before [9]. Thus, in tests for production of MSHA, bacteria were grown serially at 37°C for 48-h periods in Oxoid nutrient broth no. 2. For production of MRHA, bacteria were grown for 48 h in phosphate-buffered broth (PBB), pH 7.0, or for 24 h on phosphate- buffered agar (PBA), pH 7.0, at 18°C or at 37 o C. 3.3. Haemagglutination tests Bacteria were harvested and tested for their haemagglutination titres. MRHA titres were ex- pressed as the haemagglutinating power (HP) [10] estimated in tests with the most-sensitive species of red cell agglutinated; MSHA titres were ex- pressed similarly in tests with guinea-pig red cells [101. 3.4. Electron microscopy Portions of bacterial cultures, taken before HA testing, were prepared and negatively stained with 2% (w/v) ammonium molybdate (pH 7.0), 0.3% phosphotungstic acid (pH 6.8) or 1% (w/v) uranyl acetate (pH 4.0) by previously described methods [11] and examined with a Jeol 100CX transmission electron microscope. The detailed procedure for immune electron microscopy of type-1 fimbriae with type-1 fimbrial antiserum prepared against strain 625F of serotype Typhimurium has been described [11]. 3.5. Isolation and purification of fimbriae Bacteria from twelve 24-h PBA cultures grown at 37°C were harvested in 10 ml nutrient broth and the resultant suspension (c 1012 bacteria/ml) heated at 60 °C for 20 min to detach fimbriae [12]. After sedimentation of bacteria by centrifugation (3000 × g for 20 min), fimbriae in the supernatant were applied to an anion-exchange Mono Q col- umn and chromatographed by a fast protein liquid chromatography (FPLC) system (Pharmacia). Fimbriae were eluted with a linear gradient of 0-0.5 M NaC1 in 50 mM Tris-HC1 buffer (pH 8.0). 3.6. Polyacrylamide gel electrophoresis Column fractions and protein markers of known molecular weight were analysed by discontinuous SDS-PAGE by a modification of the method of Laemmli [13] as described elsewhere [14]. 4. RESULTS 4.1. Haemagglutinins When Salinatis strain 263 was grown on PBA or in PBB at 37 o C, it produced an MRHA which agglutinated the red cells of species of fowl, guinea pig and ox (i.e. pattern FGO) but not those of horse, man, pig or sheep. The bacteria 'eluted' from the red cells as the temperature of the ag- glutinated mixture was increased to 60 ° C. That unusual property of 'elution', together with the observation that MRHA was not produced by cultures of Salinatis grown in PBB or on PBA at 18°C, indicated that the MRHA of Salinatis was of the 'mannose-resistant and eluting' (MRE) type [15,16]. None of 12 strains of Duisburg or 4 strains of Sandiego produced MREHA under these or other cultural conditions tested (data not shown). After three or four 48-h serial subcultures in broth at 37°C, Salinatis produced MSHA the activity of which was inhibited by a-methyl-D- mannoside. This MSHA was not formed when Salinatis was grown in PBB or on PBA under conditions favouring production of MREHA. All strains of Duisburg and Sandiego also produced MSHA when grown serially in broth. 4.2. Fimbriae When MSHA-MREHA + cultures of Salinatis grown on PBA or in PBB at 37 °C were examined by electron microscopy, approx. 60% of the bacteria were found to carry numerous thin fimbriae which had an external diameter of 3.6 nm and were non-channelled, i.e. did not have an axial hole (Fig. la). Bacteria from PBA or PBB cultures grown at 18°C, however, did not form these thin fimbriae. When MSHA + MREHA- cul- tures of Salinatis, i.e. those obtained after the third or fourth serial 48-h subculture in broth, were examined, most of the bacteria bore thick fimbriae which were of external diameter 7-8 nm and had an axial hole. In immune electron mi- croscopy (antibody labelling) tests with type-1  31 Fig. 1. Salinatis strain 263 showing: (a) thin fimbriae of external diameter 3.6 nm, and stained with uranyl acetate; (b) thick, type-1 fimbriae labelled with Typhimurium type-1 fimbrial antiserum, and stained with ammonium molybdate. Bar marker, 100 nm. A E3 fimbrial antiserum prepared against Typhimurium kDa strain 625F, the thick fimbriae of Salinatis were strongly coated with antibody suggesting that they were type-1 fimbriae (Fig. lb). The thick fimbriae 97 4 of strains of Duisburg and Sandiego were also coated, albeit less strongly, with Typhimurium 66.2 625F antiserum. 42 7 31 0 21 5 14.4 4.3. Purification of thin fimbriae Isolation and purification of thin fimbriae was attempted only on MSHA-MREHA + cultures of strain 263 grown at 37°C on PBA or PBB, i.e. conditions favouring the production of thin fimbriae. The O.D. 28o profile of the material eluted with NaC1 from the Mono Q column, loaded with the supernatant obtained after the Salinatis sus- pension had been heated and centrifuged, revealed only one peak eluting at 0.3 M NaC1. Electron microscopy of this material showed that it con- tained thin fimbriae only and haemagglutination tests showed that the purified fimbriae ag- glutinated in MRE fashion the red cells of fowl, guinea pig and ox (FGO). SDS-PAGE of that same material showed the thin fimbriae to be of protein and of size 19 kDa (Fig. 2). Fig. 2. Coomassie blue-stained SDS-polyacrylamide gel of thin fimbriae of Salinatis strain 263 (lane B). The M r values (kDa) of protein standards (i.e., from top, phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, soya-bean trypsin inhibitor and lysozyme) are indicated on the left (lane A). 5. DISCUSSION Production of MREHA is a rare phenomenon among salmonellae [1]. It is of some interest,  32 therefore, that of approx. 2200 known Salmonella serotypes [17], one as exotic as Salinatis should possess this ability. The correlation between pro- duction of MREHA and the presence of thin fimbriae was established by culture of Salinatis on phosphate-buffered media at different temper- atures and by our observation that detached fimbriae agglutinated in MRE fashion the same red cell species as MSHA-MREHA + bacteria. We also confirmed earlier observations [1] that MSHA-MREHA + Salinatis bacteria adhered in a mannose-independent manner to a wide range of epithelial cells including human buccal cells and mouse duodenal and colonocyte cells (data not shown); and we showed that MSHA+MREHA - Salinatis bacteria, like other salmonellae [18], ad- hered in large numbers to cultured epithelial cells such as HeLa and HEp2 in a mannose-sensitive manner associated with type-1 fimbriae (data not shown). The srcinal description of Salinatis MREHA as being non-fimbrial [1] was based on gold-pal- ladium shadowing techniques which, though satisfactory for the detection of type-3 (4.5 nm) fimbriae of Klebsiella species [19], did not allow demonstration of the thinner (3.6 nm) fimbriae of Salinatis. Some MREHAs of Escherichia coli are destroyed by formaldehyde [15,16], but for- maldehyde-sensitivity seems an unlikely explana- tion for the failure to demonstrate the thin fimbriae of Salinatis because their associated MREHA is remarkably stable to formaldehyde (37°C for 18 h) [1]. Since its first detailed description [6], Salinatis, though known to be antigenically complex, has generally been considered to be a diphasic salmonella (4,12:d,e,h:d,e,n,z15). More recently, however, other workers have suggested that Salinatis, a rare serotype represented by only one strain (Professor L. Le Minor, personal communi- cation), might be a triphasic flagellar (4,12:d:e,h: e,n,z~5 ) variant strain of the commonly isolated serotype Duisburg (1,4,12,27:d:e,n,z15) [8]; thus, Salinatis has been deleted from the most recent Kauffmann-White schema [17]. However, Salinatis might equally well be a triphasic variant strain derived from Sandiego (4,12:e,h:e,n,zas); indeed, Edwards and Brunet recovered a strain biochem- ically and serologically indistinguishable from Sandiego after in vitro cultivation of Salinatis in the presence of H-d flagellar antiserum [6]. With our failure to demonstrate MREHA production by strains of Duisburg or Sandiego, the srcin of Salinatis remains obscure. Although many strains of salmonellae produce non-fimbrial MRHAs, some of them diffusible products [18,20], it is clear that the MREHA of Salinatis is not one of them. Again, its MREHA pattern is unlike that of other MREHAs de- scribed, for example, in Escherichia coli or Haemophilus species [15,16,21], and so we are unable to suggest a likely srcin of this unusual fimbrial haemagglutinin. REFERENCES [1] Duguid, J.P., Anderson, E.S. and Campbell, I. (1966) J. Pathol. Bacteriol. 92, 107-138. [2] Duguid, J.P. and Old, D.C. (1980) in Bacterial adherence (Beachey, E.H., ed.), pp. 187-217, Chapman and Hall, London. [3] Rohde, R., Aleksid, S., Miiller, G., Plavsic, S. and Aleksid, V. (1975) Zbl. Bakteriol. Hyg., 1 Abt., Orig. A 230, 38-50. [4] Aleksid, S, Rohde, R., Aleksid, V. and Mtiller, G. (1978) Zbl. Bakteriol. Hyg., I Abt., Orig A 241,427-437. [5] Adegbola, R.A., Old, D.C. and Aleksid, S. (1983) FEMS Microbiol. Lett. 19, 233-238. [6] Edwards, P.R. and Bruner, D.W. (1942) J. Bacteriol. 44, 289- 300. [7] Le Minor, L. and Popoff, M.Y. (1987) Int. J. Syst. Bacteriol. 37. 465-468. [8] Le Minor, L. and Bockemiihl, J. (1987) Ann. Inst. Pasteur/Microbiol. 138, 343-348. [9] Old, D.C. and Crichton, P.B. (1986) J. Med. Microbiol. 21,337-342. [10] Old, D.C. (1985) in The virulence of Escherichia coli (Sussman, M., ed.), pp. 287-313, Academic Press, London. [ll] Adegbola, R.A. and Old, D.C. (1982) Infect. Immun. 38, 306-315. [12] Orskov, 1., Orskov, F. and Birch-Andersen, A. (1980) Infect. Immun. 27, 657-666. [13] Laemmli, U.K. (1970) Nature 227, 680-685. [14] Old, D.C., Tavendale, A. and Senior, B.W. (1985) J. Med. Microbiol. 20, 203-214. [15] Duguid, J.P., Smith, I.W., Dempster, G. and Edmunds, P.N. (1955) J. Pathol. Bacteriol. 70, 335-348. [16] Duguid, J.P., Clegg, S. and Wilson, M.I. (1979) J. Med. Microbiol. 12, 213 227.  [17] Le Minor, L. and Popoff, M.Y. 1988) in Antigenic for- mulas of the Salmonella serovars, 5th rev.), pp. 1-146, WHO Collaborating Centre for Reference and Research on Salmonella Institut Pasteur, Paris, France. [18] Tavendale, A., Jardine, C.K.H., Old, D.C. and Duguid, J.P. 1983) J. Med. Microbiol. 16, 371-380. 33 [19] Duguid, J.P. 1959) J. Gen. Microbiol. 21,271-286. [20] Halula, M.C. and Stocker, B.A.D. 1987) Microbial Patho- genesis 3, 455-459. [21] Scott, S.S. and Old, D.C. 1981) FEMS Microbiol. Lett. 10, 235-240.

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