Eur. J. Biochem. 72, 35-40 (1977)
Ribitol-Containing Lipopolysaccharides from Proteus mirabilis and Their Serological Relationship Jobst GMEINER, Hubert MAYER, Inge FROMME, Krystyna KOTELKO, and Krystyna Z Y C H Institut fur Mikrobiologie, Fachbereich Biologie, Technische Hochschule Darmstadt, Max-Planck-Institut fur Immunbiologie, Freiburg, and Instytut Mikrobiologii, Uniwersytet Lodzki (Received September 17, 1976)
Ribitol phosphate was recently identified as a constituent of lipopolysaccharides obtained from Proteus mirubilis strain D52 giving 1 :4-anhydroribitol during acid hydrolysis (Gmeiner, 1975). Two other Proteus mirabilis strains belonging to serogroups 0 1 6 and 0 3 3 were shown previously to contain an unknown compound X as lipopolysaccharide constituent (Kotelko et ul., 1975). In this report the identification of compound X as 1 : 4-anhydroribotol by gas-liquid chromatography, mass spectrometry and mass fragmentography is described. Serological investigations using passive hemagglutination, hemagglutination inhibition and semiquantitative precipitin reactions indicate strongly that ribitol plays a role in the serological specificity of the respective lipopolysaccharides. The recent chemical analysis of 31 different 0 serogroups from Proteus mirabilis has led to their classification into 11 distinct chemotypes [l]. Chemotypes V and XI, comprising serogroups 016 and 033, were found to contain an apparently identical unknown constituent (compound X) characterized by a low retention time in gas-liquid chromatography. Quite recently, Gmeiner [2] described the identification of ribitol phosphate as one of the main constituents of lipopolysaccharides extracted from strain D52 of Proteus mirabilis. In agreement with earlier reports [3] it was shown that ribitol phosphate, as well as lipopolysaccharides containing ribitol phosphate, liberate upon acid hydrolysis not only ribitol but also 1 : 4-anhydroribitol. The suggestion [2] that the unknown constituent found in acid hydrolysates of 016 and 0 3 3 lipopolysaccharides could indeed be 1 : 4-anhydroribitol formed from the original constituents ribitol or ribitol phosphate during hydrolysis was further investigated. We report here the unequivocal identification of the unknown constituent of 016 and 033 lipopolysaccharides as 1 : 4-anhydroribitol by mass spectrometric analysis of its peracetate. The concomitant appearance of ribitol in these two samples showed that either ribitol or ribitol phosphate is the actual lipopolysaccharide constituent of these two serotypes. Further studies were carried out to determine whether the occurrence of this unusual constituent in 016, 033 and D52 lipopolysaccharides could be confirmed
by serological cross reaction tests. The serological investigations performed include passive hemagglutination, hemagglutination inhibition and semi-quantitative precipitin reactions. MATERIALS AND METHODS Bacterial Strains
In addition to Proteus mirabilis strain D52, the following serologically classified strains 141 were used (0-serogroups in parentheses) : F485 (016), U510 (033), F16 (05), F120 (014) and F27 (07). Lipopolysaccharides
Lipopolysaccharides were extracted by the hot phenol/water procedure and purified by preparative ultracentrifugation. From strain D52 an additional lipopolysaccharide (lipopolysaccharide 11) was isolated from the supernatant after ultracentrifugation as described previously [ 5 ] . Antisera
Anti-0 sera were obtained by immunizing White Vienna rabbits weighing 2.5 kg at 4-day intervals with heat-killed bacterial suspensions (1.5 x lo1' cells/ ml, 100 "C, 2 h) in doses of 0.25, 0.5 and 1.0 ml. The animals were bled on day 9 after the last injection.
36
Gas-Liquid Chromatography Neutral sugars were examined as alditol acetates by gas-liquid chromatography. Hydrolysis was carried out with 0.1 N HCl at 100 "C for 48 h [6] and neutralization with ion-exchange resin Amberlite IRA 410 ( H C 0 3 form). Reduction was done with NaB2H4 in 2 H 2 0and acetylation according to Sawardeker et al. [7]. Chloroform solutions were injected into a Varian Aerograph fitted with a glass column (0.32 x 152 cm) filled with 3 % ECNSS-M on Gas-chrom Q (100120 mesh) at 160 "C and a nitrogen flow of 28 ml/min. The method of Morrison [8] was used for gas-chromatographic discrimination of ribose which might originate from contaminating RNA, and ribitol from lipopolysaccharide. Aldoses in the lipopolysaccharide hydrolysate were converted to acetylated aldononitriles, whereas alditols were converted to alditol acetates. The neutralized hydrolysate was first treated with 5 % hydroxylammoniumchloride (100 "C, 15min) in pyridine (0.25 ml/mg hydrolysate) and then for an additional 60 min at 100 "C with acetic anhydride (0.25 ml/mg hydrolysate). Aldoximes formed in the first step of the reaction were subsequently dehydrated and acetylated to peracetyl aldononitriles in a second step. Gas chromatography was performed on the same ECNSS-M column and at the same temperature as described above for separation of alditol acetates. Mass Spectrometry For mass spectrometry a Finnigan gas chromatography/mass spectrometry quadrupol system (model 3200E) with an all-glass interface was used in connection with a Finnigan (model 6000) data and graphic output system. The gas chromatograph was equipped with a U-shaped glass column (0.2 x 152 cm) packed with the above described ECNSS-M phase. Gas chromatography/mass spectrometry conditions were as follows. Column temperature: 160 "C, program 4 "C/min; injection port temperature: 200 "C; transfer line temperature : 240 "C ; ion source temperature : 50 "C; electron energy 70 eV; 40-400 atomic mass units range; A/V sensitivity and a helium flow of 25 ml/min. Serological Methods Hemagglutination. Passive hemagglutination and hemagglutination inhibition were carried out according to Schmidt et al. [6] using the Takatsy system for antisera dilutions. Absorption of Serum
0.8 ml of serum D52, diluted 1 : 2, was added to 0.4 ml of packed human erythrocytes ORh', coated
Ribitol-Containing Lipopolysaccharides from Proteus miruhilis
with alkali-treated lipopolysaccharide 0 1 6 or 033, respectively. The absorption was carried out for 1.5 h at room temperature and repeated several times if necessary. The absorbed serum was used for passive hemagglutination tests. Semi-Quantitative Precipitin Assay Lipopolysaccharide solutions (1 mg/ml) were heated for 2 min in a boiling water bath; then, 2- 3 pl triethylamine was added to obtain a clear solution without any pellet when centrifuged (5000 rev./min). 100 pl of serial double dilutions of the respective lipopolysaccharides were added to 100 p1 of undiluted serum for the precipitin reaction. The highest dilutions of antigen still giving a visible precipitin ring were recorded.
RESULTS Characterization ofAnhydrorihito1 Gas chromatographic separation of alditol acetates of Proteus mirabilis 016 and 0 3 3 lipopolysaccharides has been described [l] and was confirmed by the present studies. Mass spectra from both the NaBH4- as well as the NaB'H4-reduced alditol acetate mixtures were taken. The expected mass shifts due to the deuterium label were clearly evident with hexoses and heptoses, i.e. with galactose, L-glyceroand D-glycero-D-manno-heptoses but not with "ribose" nor with compoundX. This was strongly in favour of ribitol being the real constituent in the respective lipopolysaccharides and of X being its anhydro derivative formed by acid hydrolysis. The peracetates of authentic ribitol and 1 : 4-anhydroribitol added to the corresponding alditol acetate mixtures showed identical retention times and positions of the substances (Rxylitol= 0.64 for ribitol and 0.24 for anhydroribitol and X). The mass spectra of the peracetates of X and 1 : 4-anhydroribitol were identical, thus giving the final proof of their chemical identity. The mass spectrum of the peracetate of 1 : 4-anhydroribitol as well as the expected fragmentation scheme is given in Fig. 1. As usual with alditol acetates, the acetylion (m/e = 43) served as base peak. The main primary fragment was mje = 187, from which secondary fragments at m/e = 85, 115 and 127 were formed by elimination of either acetic acid ( M , = 60), ketene ( M , = 42), formaldehyde ( M , = 30) or combinations thereof. The even fragments at m/e = 200, 140 and 98 were apparently obtained by the same rules from the non-observable molecular peak (m/e = 260). The amount of anhydroribitol formed during acid hydrolysis in relation to ribitol was different for each lipopolysaccharide as shown in Table 1, which
37
J. Gmeiner, H. Mayer, I. Fromme, K. Kotelko, and K. Zych
1oa
FH2I - -
2u
CHOAc
CHOAc
Y
L
1 I
50
150
250
200
m le
Fig. 1. Muss spectrum ofperuceiyluied 1 :4-unhydroribitol
Table 1. Sugur composition o ~ l ~ p o p o l y ~ u c c h u r ~ d e . ~ d0clA = 3-deoxy-~-munno-octu~osonic acid ; LD-Hep = L-glycero-D-munno-heptose; DD-Hep Serogroup
Strain
d0clA
x
LD-Hep
_
~
016 033
D52 I I1 F485 U510
4.1 1.1 2.4 3.3
5.6 3.8 6.7 4.7
05 014 07
F16" F120" F27"
1.7 2.0 1.6
3.1 3.9 3.3
a
DD-Hep
___ __ 0.4 tr
2.2 1.5
1.8 2.3
UA
Glc
~_ _ _
-
Gal
=
D-glycero-D-munno-heptose; tr
GlcN
GalN
-
Ribitol
-
6.9 3.2 6.9 3.3
8.0 4.2 10.8 4.2
3.5 10.2 1.9 8.3
6.1 20.9 6.4 22.1
2.5 tr 15.0 4.2
20.3 4.5 10.4
1.3 12.0 1.4
2.1 13.4 1.2
13.6 13.7 3.6
4.3 1.3 18.0
=
Anhydro- P ribitol
- ---
0.9 10.1 1.8 3.6
trace
0.2 1.9 1.4 1.2
Lys
~
1.7 3.3 2.6 3.3 1.2 2.6 0.8
-
+ + -
+ +
Data taken from reference [13].
gives the sugar composition of the lipopolysaccharides investigated. Since ribose and ribitol (after alditol acetate formation) could easily originate from contaminating RNA, the values were obtained by the technique of Morrison, which allows for the simultaneous discrimination and quantitation of corresponding sugars and sugar alcohols [8,9]. Previous studies had shown that gas-liquid chromatography on ECNSS-M readily separates anhydroribitol acetate, ribitol acetate and acetylated ribononitrile, as well as the acetylated aldononitriles of glucose, galactose and heptose. The identity of the individual peaks were assured by comparison with appropriate standards. In addition, the peaks were analyzed by mass spectrometry and
monitored by mass fragmentography making use of fragment m/e = 242 which is characteristic for acetylated aldononitriles [9] and of fragment mje = 145 which is common to both aldononitriles and alditol acetates (see Fig. 2). Acetylated 1 : 4-anhydroribitol contained neither fragment m/e = 242 nor fragment m/e = 145 in a substantial amount (see Fig. 1). Serological Investigations The recognition of compound X as originating from ribitol or its phosphate in Proteus mirahilis serogroups 016 and 0 3 3 raised the question as to whether this constitutent could be localized in the
Ribitol-Containing Lipopolysaccharides from Proteus mirabilis
38
I i
A
B CH20Ac
I
11 3
H- C -0AC
361'
H- C -0AC
I
(145
(98) 2891 ACO-
c -n
ACO-
,217
n- c L
I
n- c
-OAC
289'
C -H
213 -OAC
,2289
242
1451
145 I
n- c
-OAC
H- C -0Ac 136 1
CH20AC
I
7 31
CH20Ac
Fig. 2. Fractionation scheme of perucetyl glucononitrile ( A ) and glucitol hexaucetate ( B ) . Primary fragments in parentheses do not show up usually in the mass spectra [9]
Table 2. Cross hemagglutinations of lipopolysaccharides Lipopol ysaccharide
Reciprocal titer of - ___- ___.- - --serum anti-DS2 serum ant~-016
~
D52-I1 D52-I 033 016
5120 2560 1280 320
640 160 80 2560
05 014
< 10 20
< 10 20
0-specific lipopolysaccharide part and thus play a role in the serological specificity of these two lipopolysaccharides as well as the lipopolysaccharide fractions isolated form Proteus mirabilis strain D52. Lysine was recently described by Gromska and Mayer as a constituent of the 0-specific determinant of the lipopolysaccharide from Proteus mirabilis strain 1959 [lo]. Because of the presence of lysine in lipopolysaccharides of serogroups 0 1 6 and 0 3 3 and its absence from lipopolysaccharide of strain D52, it was necessary to compare, in suitable systems, the reactivity of some other Proteus lipopolysaccharides, lacking both ribitol and lysine or ribitol alone (see Table 1).
ARh+ was made impossible by the observation that normal uncoated human erythrocytes A reacted with the anti-016 rabbit antiserum, the reciprocal titer being as high as 160. The same serum reacted to a similar titer with human erythrocytes A coated with lipopolysaccharide 0 5 which did not cross react at all with lipopolysaccharide 0 16 when coated on human erythrocytes O R h + . Therefore, in our tests human erythrocytes of group ORh' were used for coating with alkali-treated lipopolysaccharides. The results of passive cross hemagglutination tests are presented in Table 2. Two anti-0 sera, D52 and 0 1 6 exhibiting high homologous hemagglutinin titers were selected for the study. The reciprocal titer in the third serum (033) did not exceed the value 640. As can be seen, there were strong cross reactions between D52-I, D52-I1 and 0 3 3 lipopolysaccharides. The cross reactivity between 0 1 6 and D52-I and I1 lipopolysaccharides was less strong, and only a very weak cross reaction existed between lipopolysaccharides 0 1 6 and 033. It must, however, be emphasized that no cross reactions between lipopolysaccharides D52-I, D52-11,016 and 0 3 3 on the one hand and lipopolysaccharides 0 5 or 0 1 4 on the other could be observed. Hemagglutination Inhibition
Pussi ve Hemugglu tina tion It may occur that some of the Proteus lipopolysaccharides possess determinants cross reacting with sheep as well as with human erythrocytes. In our investigations the use of human erythrocytes of group
Two systems were used. The first was serum D52, diluted to 1 : 1280, versus human erythrocytes of group ORh' coated with alkali-treated lipopolysaccharide D52-11; and the second was serum 016, diluted to 1 : 640, versus human erythrocytes ORh+ coated with
39
J . Gmeiner, H. Mayer, I. Fromme, K. Kotelko, and K. Zych
alkali-treated lipopolysaccharide 016. For the inhibition of anti-0 antibodies a number of non-alkalitreated lipopolysaccharides in decreasing concentrations was employed. The results are given in Table 3. They agree principally with those shown in Table 2. Table 3. Inhibition ofhemagglutinating systems 0 5 2 and 0 1 6 n.i. = no inhibition ( > 250 pg/ml) Serum
Lipopolysaccharide antigen
Lipopolysaccharide inhibitor
D52, 1 : 1280
D52-I1
D52-I1 D52-I 033 016
Minimal inhibiting dose pg/ml 0.45 1.9 0.9 62.5
016
n.i. n.i.
016 D52-11
0.11 125.0
Semi-quantitu tive Ring Precipitin A ssuy
014 07
n.i. n.i.
To substantiate the observed cross reactions an additional test, the semi-quantitative ring assay, was carried out. The results are presented in Table 5. In this assay not only the titers of cross reactions but also their intensity under identical conditions was compared. The different reaction intensities of lipopolysaccharide 0 16 in heterologous antiserum in comparison with lipopolysaccharide 033 were clearly evident.
Table 4. Passive hemagglutination feSt in absorbed serum D52 n.h. = serum fully absorbed, no hemagglutination Serum
Lipopolysarcharide antigen used for absorption
LipopolyReciprocal saccharide titer used as indicator antigen
D52
033
033 D52 016
n.h. 20 n.h.
016 D52 033
n.h. 640 640
016
~
In order to confirm the suggestion that the same structure might be responsible for the cross reactivity of all three Proteus chemotypes examined, the absorption of D52-serum with lipopolysaccharide 0 16 or 033 was performed. The results of hemagglutination are shown in Table 4. The results presented in this Table, compared with the data shown in Tables 2 and 3, confirm the great similarity, if not identity, of immunodeterminant structures of D52 and 033 lipopolysaccharides. A certain degree of structural similarity between lipopolysaccharide 016 and D52 was evident; the absorption of anti-D52 serum with lipopolysaccharide 0 16 diminished its reciprocal titer in the homologous system from 5120 to 640. The antigenic relationship between lipopolysaccharides 0 1 6 and 0 3 3 was weakly expressed.
07
014 0 16
Absorption of Antiserum
~~
DISCUSSION Ribitol phosphate, known as a constituent of teichoic acids in a number of gram-positive bacteria, was recognized for the first time as a component of lipopolysaccharides isolated from Proteus mirabilis
~~~
Table 5. Semi-quantitative ring precipitin assay ~
Lipopolysaccharideantigen
Serum D52 (undiluted) titer at pg/ml
D52-I D52-I1 033 016 -~
-_016 D52-I D52-I1 033 014
-
_.
-
-
-
-
1000
500
250
125
62 5
31 2
15 6
78
+++ +++ +++ +
+++ +++ ++ +
+++ +++ ++ +
+++ +++ ++ +
++ +++ ++
+ ++ + +
+ + + +
+ + +-
+
Serum 0 1 6 (undiluted) titer at pg/ml ___________~___~________ 1000 500 250 125 62.5 31 2 15 6 78 ~-
+++ + + + -
~
_____ 19 -
+-
-
-
-
-
- ~ ~ _ _ _ _ _ _ _ _ _ _ _ - _
-~
~
39
~
+++ + + +
+++ + + +
++ + + +
++ + + +
++ + + +
-
-
-
-
-
+ +
+ +
-
-
-
-
-
-~ 39 19 _ _ _ ~ -
-
-
-
-
-
-
-
-
-
40
J. Gmeiner, H. Mayer, I. Fromme, K . Kotelko, and K Zvch : Ribitol-Containing Lipopolysaccharides from Proteus mirahitis
strain D52 [2]. The mode of isolation seemed to indicate that ribitol was linked by a phosphodiester bond to the sugar polymer as a side branch in contrast to ribitol phosphate found within the polysaccharide main chain of the specific polysaccharide from Pneumococcus, type 6 1101. The formation of 1 : 4-anhydroribitol during acid hydrolysis of the lipopolysaccharide or of isolated ribitol or ribitol phosphate was used for identification. By the same criteria and by mass spectrometric analysis and mass fragmentography, ribitol has now been identified as a constituent of two other lipopolysaccharides derived from Proteus mirabilis, strains F485 and U510 belonging to serogroups 0 1 6 and 033, respectively. It was therefore of interest to see whether ribitol contributes to the serological specificity of lipopolysaccharides containing it. An intense cross reactivity between D52-I, D52-I1 and 0 3 3 lipopolysaccharides was clearly evident. A much lower cross reactivity existed between D52 and 016 lipopolysaccharides and only very weak cross reactions were detected between 0 3 3 and 0 1 6 lipopolysaccharides. No cross reaction at all was found in the case of three other Proteus mirabilis lipopolysaccharides which, with the exception of ribitol, possess similar qualitative compositions as D52, 0 1 6 and 0 3 3 lipopolysaccharides. The results presented strongly suggest that ribitol plays a role in the serological specificity of the respective lipopolysaccharides. Nothing is yet known about the mode of linkage of ribitol in serogroups 0 1 6 and 033. Acid hydrolysis of 0 1 6 lipopolysaccharide yielded much more anhyribitol in relation to ribitol compared with 0 3 3 or D52 (see Table 1). Substitution of ribitol, i.e. by phosphate, greatly enhances anhydro formation during acid hydrolysis [2,3]. On the other hand, the mode of linkages involved and their susceptibility towards acid hydrolysis would strongly influence anhydro formation. Therefore, no prediction about the ribitol linkage in serogroups 0 1 6 and 0 3 3 can be made from the analytical data. However, the low cross reactivity of serogroup 0 1 6 lipopolysaccharide might be an indication of a different substitution of ribitol in
this strain, partly masking its immunodeterminant group. In this context it is of interest to note that lipopolysaccharide I1 of strain D52, which is not sedimented by ultracentrifugation and which has a much higher content of strain specific constituents, i.e. ribitol, showed the same serological specificity and even exhibited similar hemagglutination titers as lipopolysaccharide I. Whether similar fractions could be obtained from serogroups 0 1 6 or 0 3 3 has not been investigated. It should also be mentioned that the lipopolysaccharides of serogroups 014 and 0 7 which contain lysine like 0 16 and 0 3 3 lipopolysaccharides, failed to give cross reactions, indicating that lysine detected in a number of Proteus lipopolysaccharides [ 1,121 and even in the antigen determinant of some of them [I 11 (Gromska, Kaca and Kotelko, unpublished results) does not always play the same role in all the products containing it. The investigations by J. G . were supported by the Deurschr Forschungsgemeinscha~z.The serological investigations were partly supported by a Grant of the Polish Academy of Sciences.
REFERENCES 1 . Kotelko, K., Fomme, I. & Sidorczyk, Z. (1975) Bull. Acad. Pol. Sci. CI. V I 23, 249- 256. 2. Gmeiner, J. (1975) Eur. J . Biochem. 58, 627-629. 3. Baddiley, J., Buchanan, J . G. & Carss, B. (1957) J . Chem. Soc. Lond. 4138-4139. 4. Sidorczyk, Z . & Kotelko, K. (1973) Arch. Immunol. Ther. ESP. 21,829-838. 5. Gmeiner, J. (1975) Eur. J . Biochem. 58, 621 -626. 6. Schmidt, G., Fromme, I. & Mayer, H. (1970) Eur. J . Biochem. 14,357 - 366. 7. Sawardeker, J. S., Sloneker, J . H . & Jeanes, A. (1967) Anal. Chem. 37,1602- 1604. 8. Morrison, I. M. (1975) J . Chromatogr. 108, 361 -364. 9. Dmitriev, B. A., Backinowsky, L. V., Chizhov, 0. S., Zolotarev, B. M . & Kochetkov, N. K. (1971) Curhohydr. Res. 19, 432 -435. 10. Rebers, P. A. & Heidelberger, M. (1961) J . Am. Chem. Sue. 83. 3056- 3059. 11. Gromska, W . & Mayer, H. (1976) Eur. J . Biochem. 62, 391399. 12. Sidorczyk, Z., Kaca, W. & Kotelko, K . (1975) Bull. Acad. Pol. Sci. CI. I I 33, 603 - 609. 13. Sidorczyk, Z. (1971) Thesis, University of Lodz.
J. Gmeiner, Institut fur Mikrobiologie, Fachbereich Biologie der Technische Hochschule Darmstadt, SchnittspahnstraRe 9, D-6100 Darmstadt, Federal Republic of Germany H. Mayer and I. Fromme, Max-Planck-Institut fur Immunobiologie, Stiibeweg 51, D-7800 Freiburg-Zahringen, Federal Republic of Germany
K . Kotelko and K.Zych, Instytut Mikrobioiogii, Uniwersytet todzki, PL-90-237 Lodz, Banacha 12, Poland
Ribitol-Containing Lipopolysaccharides from Proteus mirabilis and Their Serological Relationship Jobst GMEINER, Hubert MAYER, Inge FROMME, Krystyna KOTELKO, and Krystyna Z Y C H Institut fur Mikrobiologie, Fachbereich Biologie, Technische Hochschule Darmstadt, Max-Planck-Institut fur Immunbiologie, Freiburg, and Instytut Mikrobiologii, Uniwersytet Lodzki (Received September 17, 1976)
Ribitol phosphate was recently identified as a constituent of lipopolysaccharides obtained from Proteus mirubilis strain D52 giving 1 :4-anhydroribitol during acid hydrolysis (Gmeiner, 1975). Two other Proteus mirabilis strains belonging to serogroups 0 1 6 and 0 3 3 were shown previously to contain an unknown compound X as lipopolysaccharide constituent (Kotelko et ul., 1975). In this report the identification of compound X as 1 : 4-anhydroribotol by gas-liquid chromatography, mass spectrometry and mass fragmentography is described. Serological investigations using passive hemagglutination, hemagglutination inhibition and semiquantitative precipitin reactions indicate strongly that ribitol plays a role in the serological specificity of the respective lipopolysaccharides. The recent chemical analysis of 31 different 0 serogroups from Proteus mirabilis has led to their classification into 11 distinct chemotypes [l]. Chemotypes V and XI, comprising serogroups 016 and 033, were found to contain an apparently identical unknown constituent (compound X) characterized by a low retention time in gas-liquid chromatography. Quite recently, Gmeiner [2] described the identification of ribitol phosphate as one of the main constituents of lipopolysaccharides extracted from strain D52 of Proteus mirabilis. In agreement with earlier reports [3] it was shown that ribitol phosphate, as well as lipopolysaccharides containing ribitol phosphate, liberate upon acid hydrolysis not only ribitol but also 1 : 4-anhydroribitol. The suggestion [2] that the unknown constituent found in acid hydrolysates of 016 and 0 3 3 lipopolysaccharides could indeed be 1 : 4-anhydroribitol formed from the original constituents ribitol or ribitol phosphate during hydrolysis was further investigated. We report here the unequivocal identification of the unknown constituent of 016 and 033 lipopolysaccharides as 1 : 4-anhydroribitol by mass spectrometric analysis of its peracetate. The concomitant appearance of ribitol in these two samples showed that either ribitol or ribitol phosphate is the actual lipopolysaccharide constituent of these two serotypes. Further studies were carried out to determine whether the occurrence of this unusual constituent in 016, 033 and D52 lipopolysaccharides could be confirmed
by serological cross reaction tests. The serological investigations performed include passive hemagglutination, hemagglutination inhibition and semi-quantitative precipitin reactions. MATERIALS AND METHODS Bacterial Strains
In addition to Proteus mirabilis strain D52, the following serologically classified strains 141 were used (0-serogroups in parentheses) : F485 (016), U510 (033), F16 (05), F120 (014) and F27 (07). Lipopolysaccharides
Lipopolysaccharides were extracted by the hot phenol/water procedure and purified by preparative ultracentrifugation. From strain D52 an additional lipopolysaccharide (lipopolysaccharide 11) was isolated from the supernatant after ultracentrifugation as described previously [ 5 ] . Antisera
Anti-0 sera were obtained by immunizing White Vienna rabbits weighing 2.5 kg at 4-day intervals with heat-killed bacterial suspensions (1.5 x lo1' cells/ ml, 100 "C, 2 h) in doses of 0.25, 0.5 and 1.0 ml. The animals were bled on day 9 after the last injection.
36
Gas-Liquid Chromatography Neutral sugars were examined as alditol acetates by gas-liquid chromatography. Hydrolysis was carried out with 0.1 N HCl at 100 "C for 48 h [6] and neutralization with ion-exchange resin Amberlite IRA 410 ( H C 0 3 form). Reduction was done with NaB2H4 in 2 H 2 0and acetylation according to Sawardeker et al. [7]. Chloroform solutions were injected into a Varian Aerograph fitted with a glass column (0.32 x 152 cm) filled with 3 % ECNSS-M on Gas-chrom Q (100120 mesh) at 160 "C and a nitrogen flow of 28 ml/min. The method of Morrison [8] was used for gas-chromatographic discrimination of ribose which might originate from contaminating RNA, and ribitol from lipopolysaccharide. Aldoses in the lipopolysaccharide hydrolysate were converted to acetylated aldononitriles, whereas alditols were converted to alditol acetates. The neutralized hydrolysate was first treated with 5 % hydroxylammoniumchloride (100 "C, 15min) in pyridine (0.25 ml/mg hydrolysate) and then for an additional 60 min at 100 "C with acetic anhydride (0.25 ml/mg hydrolysate). Aldoximes formed in the first step of the reaction were subsequently dehydrated and acetylated to peracetyl aldononitriles in a second step. Gas chromatography was performed on the same ECNSS-M column and at the same temperature as described above for separation of alditol acetates. Mass Spectrometry For mass spectrometry a Finnigan gas chromatography/mass spectrometry quadrupol system (model 3200E) with an all-glass interface was used in connection with a Finnigan (model 6000) data and graphic output system. The gas chromatograph was equipped with a U-shaped glass column (0.2 x 152 cm) packed with the above described ECNSS-M phase. Gas chromatography/mass spectrometry conditions were as follows. Column temperature: 160 "C, program 4 "C/min; injection port temperature: 200 "C; transfer line temperature : 240 "C ; ion source temperature : 50 "C; electron energy 70 eV; 40-400 atomic mass units range; A/V sensitivity and a helium flow of 25 ml/min. Serological Methods Hemagglutination. Passive hemagglutination and hemagglutination inhibition were carried out according to Schmidt et al. [6] using the Takatsy system for antisera dilutions. Absorption of Serum
0.8 ml of serum D52, diluted 1 : 2, was added to 0.4 ml of packed human erythrocytes ORh', coated
Ribitol-Containing Lipopolysaccharides from Proteus miruhilis
with alkali-treated lipopolysaccharide 0 1 6 or 033, respectively. The absorption was carried out for 1.5 h at room temperature and repeated several times if necessary. The absorbed serum was used for passive hemagglutination tests. Semi-Quantitative Precipitin Assay Lipopolysaccharide solutions (1 mg/ml) were heated for 2 min in a boiling water bath; then, 2- 3 pl triethylamine was added to obtain a clear solution without any pellet when centrifuged (5000 rev./min). 100 pl of serial double dilutions of the respective lipopolysaccharides were added to 100 p1 of undiluted serum for the precipitin reaction. The highest dilutions of antigen still giving a visible precipitin ring were recorded.
RESULTS Characterization ofAnhydrorihito1 Gas chromatographic separation of alditol acetates of Proteus mirabilis 016 and 0 3 3 lipopolysaccharides has been described [l] and was confirmed by the present studies. Mass spectra from both the NaBH4- as well as the NaB'H4-reduced alditol acetate mixtures were taken. The expected mass shifts due to the deuterium label were clearly evident with hexoses and heptoses, i.e. with galactose, L-glyceroand D-glycero-D-manno-heptoses but not with "ribose" nor with compoundX. This was strongly in favour of ribitol being the real constituent in the respective lipopolysaccharides and of X being its anhydro derivative formed by acid hydrolysis. The peracetates of authentic ribitol and 1 : 4-anhydroribitol added to the corresponding alditol acetate mixtures showed identical retention times and positions of the substances (Rxylitol= 0.64 for ribitol and 0.24 for anhydroribitol and X). The mass spectra of the peracetates of X and 1 : 4-anhydroribitol were identical, thus giving the final proof of their chemical identity. The mass spectrum of the peracetate of 1 : 4-anhydroribitol as well as the expected fragmentation scheme is given in Fig. 1. As usual with alditol acetates, the acetylion (m/e = 43) served as base peak. The main primary fragment was mje = 187, from which secondary fragments at m/e = 85, 115 and 127 were formed by elimination of either acetic acid ( M , = 60), ketene ( M , = 42), formaldehyde ( M , = 30) or combinations thereof. The even fragments at m/e = 200, 140 and 98 were apparently obtained by the same rules from the non-observable molecular peak (m/e = 260). The amount of anhydroribitol formed during acid hydrolysis in relation to ribitol was different for each lipopolysaccharide as shown in Table 1, which
37
J. Gmeiner, H. Mayer, I. Fromme, K. Kotelko, and K. Zych
1oa
FH2I - -
2u
CHOAc
CHOAc
Y
L
1 I
50
150
250
200
m le
Fig. 1. Muss spectrum ofperuceiyluied 1 :4-unhydroribitol
Table 1. Sugur composition o ~ l ~ p o p o l y ~ u c c h u r ~ d e . ~ d0clA = 3-deoxy-~-munno-octu~osonic acid ; LD-Hep = L-glycero-D-munno-heptose; DD-Hep Serogroup
Strain
d0clA
x
LD-Hep
_
~
016 033
D52 I I1 F485 U510
4.1 1.1 2.4 3.3
5.6 3.8 6.7 4.7
05 014 07
F16" F120" F27"
1.7 2.0 1.6
3.1 3.9 3.3
a
DD-Hep
___ __ 0.4 tr
2.2 1.5
1.8 2.3
UA
Glc
~_ _ _
-
Gal
=
D-glycero-D-munno-heptose; tr
GlcN
GalN
-
Ribitol
-
6.9 3.2 6.9 3.3
8.0 4.2 10.8 4.2
3.5 10.2 1.9 8.3
6.1 20.9 6.4 22.1
2.5 tr 15.0 4.2
20.3 4.5 10.4
1.3 12.0 1.4
2.1 13.4 1.2
13.6 13.7 3.6
4.3 1.3 18.0
=
Anhydro- P ribitol
- ---
0.9 10.1 1.8 3.6
trace
0.2 1.9 1.4 1.2
Lys
~
1.7 3.3 2.6 3.3 1.2 2.6 0.8
-
+ + -
+ +
Data taken from reference [13].
gives the sugar composition of the lipopolysaccharides investigated. Since ribose and ribitol (after alditol acetate formation) could easily originate from contaminating RNA, the values were obtained by the technique of Morrison, which allows for the simultaneous discrimination and quantitation of corresponding sugars and sugar alcohols [8,9]. Previous studies had shown that gas-liquid chromatography on ECNSS-M readily separates anhydroribitol acetate, ribitol acetate and acetylated ribononitrile, as well as the acetylated aldononitriles of glucose, galactose and heptose. The identity of the individual peaks were assured by comparison with appropriate standards. In addition, the peaks were analyzed by mass spectrometry and
monitored by mass fragmentography making use of fragment m/e = 242 which is characteristic for acetylated aldononitriles [9] and of fragment mje = 145 which is common to both aldononitriles and alditol acetates (see Fig. 2). Acetylated 1 : 4-anhydroribitol contained neither fragment m/e = 242 nor fragment m/e = 145 in a substantial amount (see Fig. 1). Serological Investigations The recognition of compound X as originating from ribitol or its phosphate in Proteus mirahilis serogroups 016 and 0 3 3 raised the question as to whether this constitutent could be localized in the
Ribitol-Containing Lipopolysaccharides from Proteus mirabilis
38
I i
A
B CH20Ac
I
11 3
H- C -0AC
361'
H- C -0AC
I
(145
(98) 2891 ACO-
c -n
ACO-
,217
n- c L
I
n- c
-OAC
289'
C -H
213 -OAC
,2289
242
1451
145 I
n- c
-OAC
H- C -0Ac 136 1
CH20AC
I
7 31
CH20Ac
Fig. 2. Fractionation scheme of perucetyl glucononitrile ( A ) and glucitol hexaucetate ( B ) . Primary fragments in parentheses do not show up usually in the mass spectra [9]
Table 2. Cross hemagglutinations of lipopolysaccharides Lipopol ysaccharide
Reciprocal titer of - ___- ___.- - --serum anti-DS2 serum ant~-016
~
D52-I1 D52-I 033 016
5120 2560 1280 320
640 160 80 2560
05 014
< 10 20
< 10 20
0-specific lipopolysaccharide part and thus play a role in the serological specificity of these two lipopolysaccharides as well as the lipopolysaccharide fractions isolated form Proteus mirabilis strain D52. Lysine was recently described by Gromska and Mayer as a constituent of the 0-specific determinant of the lipopolysaccharide from Proteus mirabilis strain 1959 [lo]. Because of the presence of lysine in lipopolysaccharides of serogroups 0 1 6 and 0 3 3 and its absence from lipopolysaccharide of strain D52, it was necessary to compare, in suitable systems, the reactivity of some other Proteus lipopolysaccharides, lacking both ribitol and lysine or ribitol alone (see Table 1).
ARh+ was made impossible by the observation that normal uncoated human erythrocytes A reacted with the anti-016 rabbit antiserum, the reciprocal titer being as high as 160. The same serum reacted to a similar titer with human erythrocytes A coated with lipopolysaccharide 0 5 which did not cross react at all with lipopolysaccharide 0 16 when coated on human erythrocytes O R h + . Therefore, in our tests human erythrocytes of group ORh' were used for coating with alkali-treated lipopolysaccharides. The results of passive cross hemagglutination tests are presented in Table 2. Two anti-0 sera, D52 and 0 1 6 exhibiting high homologous hemagglutinin titers were selected for the study. The reciprocal titer in the third serum (033) did not exceed the value 640. As can be seen, there were strong cross reactions between D52-I, D52-I1 and 0 3 3 lipopolysaccharides. The cross reactivity between 0 1 6 and D52-I and I1 lipopolysaccharides was less strong, and only a very weak cross reaction existed between lipopolysaccharides 0 1 6 and 033. It must, however, be emphasized that no cross reactions between lipopolysaccharides D52-I, D52-11,016 and 0 3 3 on the one hand and lipopolysaccharides 0 5 or 0 1 4 on the other could be observed. Hemagglutination Inhibition
Pussi ve Hemugglu tina tion It may occur that some of the Proteus lipopolysaccharides possess determinants cross reacting with sheep as well as with human erythrocytes. In our investigations the use of human erythrocytes of group
Two systems were used. The first was serum D52, diluted to 1 : 1280, versus human erythrocytes of group ORh' coated with alkali-treated lipopolysaccharide D52-11; and the second was serum 016, diluted to 1 : 640, versus human erythrocytes ORh+ coated with
39
J . Gmeiner, H. Mayer, I. Fromme, K. Kotelko, and K. Zych
alkali-treated lipopolysaccharide 016. For the inhibition of anti-0 antibodies a number of non-alkalitreated lipopolysaccharides in decreasing concentrations was employed. The results are given in Table 3. They agree principally with those shown in Table 2. Table 3. Inhibition ofhemagglutinating systems 0 5 2 and 0 1 6 n.i. = no inhibition ( > 250 pg/ml) Serum
Lipopolysaccharide antigen
Lipopolysaccharide inhibitor
D52, 1 : 1280
D52-I1
D52-I1 D52-I 033 016
Minimal inhibiting dose pg/ml 0.45 1.9 0.9 62.5
016
n.i. n.i.
016 D52-11
0.11 125.0
Semi-quantitu tive Ring Precipitin A ssuy
014 07
n.i. n.i.
To substantiate the observed cross reactions an additional test, the semi-quantitative ring assay, was carried out. The results are presented in Table 5. In this assay not only the titers of cross reactions but also their intensity under identical conditions was compared. The different reaction intensities of lipopolysaccharide 0 16 in heterologous antiserum in comparison with lipopolysaccharide 033 were clearly evident.
Table 4. Passive hemagglutination feSt in absorbed serum D52 n.h. = serum fully absorbed, no hemagglutination Serum
Lipopolysarcharide antigen used for absorption
LipopolyReciprocal saccharide titer used as indicator antigen
D52
033
033 D52 016
n.h. 20 n.h.
016 D52 033
n.h. 640 640
016
~
In order to confirm the suggestion that the same structure might be responsible for the cross reactivity of all three Proteus chemotypes examined, the absorption of D52-serum with lipopolysaccharide 0 16 or 033 was performed. The results of hemagglutination are shown in Table 4. The results presented in this Table, compared with the data shown in Tables 2 and 3, confirm the great similarity, if not identity, of immunodeterminant structures of D52 and 033 lipopolysaccharides. A certain degree of structural similarity between lipopolysaccharide 016 and D52 was evident; the absorption of anti-D52 serum with lipopolysaccharide 0 16 diminished its reciprocal titer in the homologous system from 5120 to 640. The antigenic relationship between lipopolysaccharides 0 1 6 and 0 3 3 was weakly expressed.
07
014 0 16
Absorption of Antiserum
~~
DISCUSSION Ribitol phosphate, known as a constituent of teichoic acids in a number of gram-positive bacteria, was recognized for the first time as a component of lipopolysaccharides isolated from Proteus mirabilis
~~~
Table 5. Semi-quantitative ring precipitin assay ~
Lipopolysaccharideantigen
Serum D52 (undiluted) titer at pg/ml
D52-I D52-I1 033 016 -~
-_016 D52-I D52-I1 033 014
-
_.
-
-
-
-
1000
500
250
125
62 5
31 2
15 6
78
+++ +++ +++ +
+++ +++ ++ +
+++ +++ ++ +
+++ +++ ++ +
++ +++ ++
+ ++ + +
+ + + +
+ + +-
+
Serum 0 1 6 (undiluted) titer at pg/ml ___________~___~________ 1000 500 250 125 62.5 31 2 15 6 78 ~-
+++ + + + -
~
_____ 19 -
+-
-
-
-
-
- ~ ~ _ _ _ _ _ _ _ _ _ _ _ - _
-~
~
39
~
+++ + + +
+++ + + +
++ + + +
++ + + +
++ + + +
-
-
-
-
-
+ +
+ +
-
-
-
-
-
-~ 39 19 _ _ _ ~ -
-
-
-
-
-
-
-
-
-
40
J. Gmeiner, H. Mayer, I. Fromme, K . Kotelko, and K Zvch : Ribitol-Containing Lipopolysaccharides from Proteus mirahitis
strain D52 [2]. The mode of isolation seemed to indicate that ribitol was linked by a phosphodiester bond to the sugar polymer as a side branch in contrast to ribitol phosphate found within the polysaccharide main chain of the specific polysaccharide from Pneumococcus, type 6 1101. The formation of 1 : 4-anhydroribitol during acid hydrolysis of the lipopolysaccharide or of isolated ribitol or ribitol phosphate was used for identification. By the same criteria and by mass spectrometric analysis and mass fragmentography, ribitol has now been identified as a constituent of two other lipopolysaccharides derived from Proteus mirabilis, strains F485 and U510 belonging to serogroups 0 1 6 and 033, respectively. It was therefore of interest to see whether ribitol contributes to the serological specificity of lipopolysaccharides containing it. An intense cross reactivity between D52-I, D52-I1 and 0 3 3 lipopolysaccharides was clearly evident. A much lower cross reactivity existed between D52 and 016 lipopolysaccharides and only very weak cross reactions were detected between 0 3 3 and 0 1 6 lipopolysaccharides. No cross reaction at all was found in the case of three other Proteus mirabilis lipopolysaccharides which, with the exception of ribitol, possess similar qualitative compositions as D52, 0 1 6 and 0 3 3 lipopolysaccharides. The results presented strongly suggest that ribitol plays a role in the serological specificity of the respective lipopolysaccharides. Nothing is yet known about the mode of linkage of ribitol in serogroups 0 1 6 and 033. Acid hydrolysis of 0 1 6 lipopolysaccharide yielded much more anhyribitol in relation to ribitol compared with 0 3 3 or D52 (see Table 1). Substitution of ribitol, i.e. by phosphate, greatly enhances anhydro formation during acid hydrolysis [2,3]. On the other hand, the mode of linkages involved and their susceptibility towards acid hydrolysis would strongly influence anhydro formation. Therefore, no prediction about the ribitol linkage in serogroups 0 1 6 and 0 3 3 can be made from the analytical data. However, the low cross reactivity of serogroup 0 1 6 lipopolysaccharide might be an indication of a different substitution of ribitol in
this strain, partly masking its immunodeterminant group. In this context it is of interest to note that lipopolysaccharide I1 of strain D52, which is not sedimented by ultracentrifugation and which has a much higher content of strain specific constituents, i.e. ribitol, showed the same serological specificity and even exhibited similar hemagglutination titers as lipopolysaccharide I. Whether similar fractions could be obtained from serogroups 0 1 6 or 0 3 3 has not been investigated. It should also be mentioned that the lipopolysaccharides of serogroups 014 and 0 7 which contain lysine like 0 16 and 0 3 3 lipopolysaccharides, failed to give cross reactions, indicating that lysine detected in a number of Proteus lipopolysaccharides [ 1,121 and even in the antigen determinant of some of them [I 11 (Gromska, Kaca and Kotelko, unpublished results) does not always play the same role in all the products containing it. The investigations by J. G . were supported by the Deurschr Forschungsgemeinscha~z.The serological investigations were partly supported by a Grant of the Polish Academy of Sciences.
REFERENCES 1 . Kotelko, K., Fomme, I. & Sidorczyk, Z. (1975) Bull. Acad. Pol. Sci. CI. V I 23, 249- 256. 2. Gmeiner, J. (1975) Eur. J . Biochem. 58, 627-629. 3. Baddiley, J., Buchanan, J . G. & Carss, B. (1957) J . Chem. Soc. Lond. 4138-4139. 4. Sidorczyk, Z . & Kotelko, K. (1973) Arch. Immunol. Ther. ESP. 21,829-838. 5. Gmeiner, J. (1975) Eur. J . Biochem. 58, 621 -626. 6. Schmidt, G., Fromme, I. & Mayer, H. (1970) Eur. J . Biochem. 14,357 - 366. 7. Sawardeker, J. S., Sloneker, J . H . & Jeanes, A. (1967) Anal. Chem. 37,1602- 1604. 8. Morrison, I. M. (1975) J . Chromatogr. 108, 361 -364. 9. Dmitriev, B. A., Backinowsky, L. V., Chizhov, 0. S., Zolotarev, B. M . & Kochetkov, N. K. (1971) Curhohydr. Res. 19, 432 -435. 10. Rebers, P. A. & Heidelberger, M. (1961) J . Am. Chem. Sue. 83. 3056- 3059. 11. Gromska, W . & Mayer, H. (1976) Eur. J . Biochem. 62, 391399. 12. Sidorczyk, Z., Kaca, W. & Kotelko, K . (1975) Bull. Acad. Pol. Sci. CI. I I 33, 603 - 609. 13. Sidorczyk, Z. (1971) Thesis, University of Lodz.
J. Gmeiner, Institut fur Mikrobiologie, Fachbereich Biologie der Technische Hochschule Darmstadt, SchnittspahnstraRe 9, D-6100 Darmstadt, Federal Republic of Germany H. Mayer and I. Fromme, Max-Planck-Institut fur Immunobiologie, Stiibeweg 51, D-7800 Freiburg-Zahringen, Federal Republic of Germany
K . Kotelko and K.Zych, Instytut Mikrobioiogii, Uniwersytet todzki, PL-90-237 Lodz, Banacha 12, Poland
