University of Montana
ScholarWorks at University of Montana
Biological Sciences Faculty Publications
Biological Sciences
1-1987
Characterization of Protein-I from Serum-Sensitive
and Serum-Resistance Transformants of NeisseriaGonorrhoeae
Ralph C. Judd
University of Montana - Missoula, [email protected]
Milton Tam
Keith Joiner
Follow this and additional works at: http://scholarworks.umt.edu/biosci_pubs
Part of the Biology Commons
Recommended Citation
Judd, Ralph C.; Tam, Milton; and Joiner, Keith, "Characterization of Protein-I from Serum-Sensitive and Serum-Resistance
Transformants of Neisseria-Gonorrhoeae" (1987). Biological Sciences Faculty Publications. Paper 109.
http://scholarworks.umt.edu/biosci_pubs/109
This Article is brought to you for free and open access by the Biological Sciences at ScholarWorks at University of Montana. It has been accepted for
inclusion in Biological Sciences Faculty Publications by an authorized administrator of ScholarWorks at University of Montana. For more information,
please contact [email protected].
*~
INFECTION AND IMMUNITY, Jan. 1987, p. 273-276
0019-9567/87/010273-04$02.00/0
Copyright C) 1987, American Society for Microbiology
Vol. 55, No. 1
Characterization of Protein I from Serum-Sensitive and
Serum-Resistant Transformants of Neisseria gonorrhoeae
RALPH C.
JUDD,'*
MILTON TAM,2 AND KEITH JOINER3
Department of Microbiology, University of Montana, Missoula, Montana 598121; Genetic Systems Corp., Seattle,
Washington 981212; and Laboratory of Clinical Investigations, National Institute of Allergy and Infectious Diseases,
Bethesda, Maryland 208923
Received 21 July 1986/Accepted 14 October 1986
The protein lAs of serum-sensitive (FA635) and serum-resistant (FA638) transformants of Neisseria
gonorrhoeae, which have identical pedigrees, have been shown to be different by the use of a monoclonal
antibody and were also shown to be different by proteinase K cleavage and primary structural and surface
peptide mapping. The difference in structure is within the surface-exposed region of the molecule. The only
other difference observed between the two strains was a very slight difference in lipooligosaccharide silver
staining in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that protein I alone
or in combination with lipooligosaccharide may significantly contribute to serum resistance.
The protein IA (P.IA) subclass of protein I (P.1) of
Neisseria gonorrhoeae has been associated with serum
resistance (5) and disseminated gonococcal infection (4, 7,
9). Transformation studies by Hildebrandt et al. (8) demonstrated that N. gonorrhoeae becomes resistant to serum
killing when the organisms acquire a P.IA. Further studies
by Cannon et al. (5), Spratt et al. (18), and Shafer et al. (17)
suggest that three separate serum antibody complement
(sac) loci are distinct from the P.1 locus, although they are
closely linked. The gene product of the sac locus was not
identified, although recent data from several laboratories
suggest that changes in lipooligosaccharide (LOS) determinants are associated with alterations in the sac locus.
To address the effect of variations in the sac locus on
corhplement consumption and binding by N. gonorrhoeae,
one of us (K.J.) initially undertook studies with a pair of
transformants generated by J. Cannon and P. F. Sparling
(University of North Carolina, Chapel Hill) by transforming
the protein IB (P.IB)-bearing strain F62 with DNA from the
P.IA-bearing strain FA130 (Rif Spcr Strr). Selection for Spcr
resulted in the generation of an exquisitely serum-sensitive
strain, FA635 (5; J. F. Puentes, R. C. Judd, and K. Joiner,
mahuscript in preparation), and a serum-resistant strain,
FA638 (J. G. Cannon, personal communication). The P.Is of
these strains appeared to be identical in apparent molecular
weight (aMW). Therefore, P.1 was discounted as a factor in
serum resistance. However, when immunoblot analyses are
performed by using the 4G5 monoclonal antibody (MAb), a
P.IA-screening antibody used in serovar determinations (16,
20), 4G5 is not observed to bind to the P.1 of strain FA635,
whereas it does bind to the P.1 of strain FA638 (13).
Furthermore, 4G5 binds to whole FA638 organisms but not
to FA635 cells, as assessed by coagglutination and by
direct-binding studies with 125I-labeled 4G5 (Puentes et al.,
in preparation).
To analyze more carefully the P.Is of these strains,
bacteria were grown on clear typing media (19). Whole-cell
(WC) lysates of strains FA635 and FA638 as well as two
*
Untreated
i
- 0
to$
PKdigest
;,
: 2'
An
co
3:
94k
68k
44 k
P.I
30k
21k
,<
~
~
~~~~
~14
FIG. 1. SDS-PAGE of whole cells of N. gonorrhoeae. An
acrylamide gel (15%) was used to separate WC lysates (A) and
whole cells digested with PK (B). The N. gonorrhoeae strains used
were JS1, JS3, FA635 (635), and FA635 (638). Digestion times were
15 min for JS1 and JS3, 30 min for FA638, and 60 min for FA635. PK
fragments of P.Is are identified with stars. The gel was stained with
Coomassie brilliant blue, and the molecular weight markers were
from the Bio-Rad low-molecular-weight marker kit. Molecular sizes
are expressed in kilodaltons (K).
Corresponding author.
273
274
_~
NOTES
INFECT. IMMUN.
p
3
M4
5
8
*
4
0
......
:X:
6_
0_,w,
'I
n
0-_6
.
3
,5
a
3
_
...
...
8
5
i.
,
:f.
u,
.f
I
uB
U
0
in
0
0
0
c:
FIG. 2. a-Chymotryptic 1251I-labeled peptide maps (primary [10] structure) and surface peptide maps of P.Is. N. gonorrhoeae JS1 (panels
1), JS3 (panels 3), FA635 (panels 5), and FA638 (panels 8) are presented. Note the differences in both primary structure and surface exposure
of the P.Is from strains FA635 and FA638. TLE, Thin-layer electrophoresis; TLC, thin-layer chromatography. The origins (0) are in the lower
right corners.
well-characterized laboratory strains, JS1, a P.IB-bearing
strain (11), and JS3, a P.IA-bearing strain (11), generously
provided by John Swanson, Rocky Mountain Laboratories,
Hamilton, Mont.), were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 11, 14)
(Fig. 1A). To confirm that both the FA635 and FA638 strains
were P.IA-bearing strains, proteinase K (PK) cleavage of
intact bacteria was performed essentially as described by
Barrera and Swanson (2). However, the time required for
PK (20 pug/ml) to cleave the P.IA varied appreciably with the
strains used; whereas the P.1 of JS1 could be completely
cleaved in 15 min, JS3 P.1 was only partially cleaved after 15
min. The FA638 P.1 was still only partially cleaved at 30 min,
and FA635 required 60 min of incubation to partially cleave
the P.I. Almost no PK cleavage of the FA635 P.I was
observed after 15 min of incubation, whereas complete P.I
cleavage could not be accomplished in strains JS3, FA635,
and FA638, since membrane integrity was destroyed before
complete P.1 cleavage. Results of PK cleavage are shown in
Fig. 1B. Whereas both the FA635 and FA638 strains bore a
P.IA, as assessed by the cleavage of a single fragment of
about 1.5 kilodaltons from P.1 with PK treatment, the P.I of
B
A
635
I4Le
C
3563
aO
635
638
I|t
"I
9 t.
¶
L
:E
e
-4
-."-
#
5 fAR
t
0
SURFACE
0
0
*TLC-
FIG. 3. High-resolution ax-chymotryptic '25I-labeled peptide maps and surface peptide maps of P.1s. from N. gonorrhoeae FA635 (635) and
FA638 (638). Two methods of protein radioiodination were used, i.e., chlorlamine-T (A) and iodogen (B). Both procedures indicate the
presence of minor peptide migrational differences (stars) and small differences in radioemission (e). Surface peptide maps (C) show a clear
difference in the surface-exposed region of the molecules (arrows). TLE, thin-layer electrophoresis; TLC, thin-layer chromatography. The
origins (0) are in the lower right corners.
NOTES
VOL. 55, 1987
strain FA635 was slightly smaller in aMW (35,500) than the
P.A of strains JS3 (35,600) or FA638 (35,000), as was the
resultant large PK fragment (Fig. 1, stars). The characteristic
P.IB cleavage pattern (2) was observed in strain JS1, in
which the P.1 was completely cleaved, resulting in two large
membrane-bound fragments (Fig. 1, stars) of aMW of about
22,500 and 14,500.
Further characterization of the P.Is involved performing
primary structural (11) and surface (12) peptide maps on the
P.Is of all four strains. The characteristic P.IB structural
map of strain JS1 and the characteristic P.IA structural maps
of strains JS3, FA635, and FA638 (12) are shown in Fig. 2.
The surface peptide maps also show the very different
exposure patterns of P.IAs and P.IBs. Differences can be
seen among the P.Is of strains JS3, FA635, and FA638 in
both primary structure and surface exposure. However, the
P.Is of FA635 and FA638 are so similar that higherresolution peptide maps are needed to identify structural
differences clearly (Fig. 3). Two protein iodination procedures were used, i.e., chloramine-T iodination (6; Fig. 3A),
which produces iodination of very high specific activity but
also results in protein cleavage, especially at tryptophan (1);
and iodogen iodination (15; Fig. 3B), which results in lower
specific activities but is less damaging to the proteins. Both
procedures revealed structural differences between the P.Is
of FA635 and FA638 (Fig. 3). Clear differences are seen in
the surface-exposed regions of the molecule (Fig. 3C, arrows), confirming the differences observed by MAb-binding
(13) and PK digestion.
The P.Is of strains FA635 (serum sensitive) and FA638
(serum resistant) are indeed different and may, therefore,
have an influence on serum resistqnce. However, other
surface structures could also differ between these strains.
Immunoblot analysis (13) demonstrated that H8 antigen (3)
exhibits identical antigenic characteristics and migration in
SDS-PAGE, suggesting that H8 is the same in each strain.
Possible differences in LOS were assessed by silver staining
WC lysates of the four strains by the procedure of Tsai and
Frasch (21; Fig. 4). The only difference in silver staining
patterns between strains FA635 and FA638 was in the LOS
region, where a slight but reproducible difference in LOS
staining can be seen.
The data presented here indicate that there is a difference
in the surface-exposed regions of the P.Is in strains FA635
and FA638. This is supported by the following lines of
evidence: (i) PK digestion, in which different aMW fragments were generated and the P.I of strain FA635 was more
resistant to PK digestion than was the P.1 of FA638; (ii) MAb
binding, in which the 4G5 MAb failed to bind to the P.1 of
strain FA635; and (iii) primary structural and surface peptide
mapping. The only other difference we could discern in the
outer membrane of strains FA635 and FA638 was a slight
variation in LOS staining.
The structural differences in the P.Is of strains FA635 and
FA638 appear to occur at the exposed N terminus of the
molecule (13). This change alters antibody binding and could
alter serum sensitivity by chatnging the binding of bactericidal antibodies directed primarily against P.A (9, 10). A
combination of P.A variation and LOS alteration, which
could affect antibody binding to LOS, or LOS variation
alone might alter serum sensitivity. In every case in which a
P.1 alteration spontaneously occurs (P.1 alterations remain
anecdotal and retrospective), a LOS alteration is also observed (R.C.J., personal observation), suggesting a link
between P.I and LOS expression. This might relate to the
observation that the serum resistance sac-i locus, which
3
275
5 8
;68k
44k
...
S~~p.l
303k
21 k
i.1
k
*
] LOS
FIG. 4. SDS-PAGE of WC lysates of N. gonorrhoeae stained for
LOS, then counterstained with Coomassie brilliant blue. An
acrylamide gel (15%) was used to separate WC lysates of N.
gonorrhoeae strains JS1 (lane 1), JS3 (lane 3), FA635 (lane 5), and
FA638 (lane 8). The gel was silver stained, revealing staining
differences in LOS. Clear differences in LOS migration are seen
between strains JS1 and JS3; both are different from FA635 and
FA638. Only a slight difference in LOS staining is seen between
FA635 and FA638, with the bottom LOS band staining more heavily
in FA635.
may encode for LOS structure, is very close to but not
identical with the nmp-2 P.1 locus (5).
The results of this paper show that an extremely minor
variation in P.1 structure coupled with a minor LOS difference is sufficient to alter serum sensitivity in N. gonorrhoeae. Only through the construction of isogenic strains
varying in P.1 and serum sensitivity but identical at all other
loci will it be possible to unequivocally relate P.I phenotype
to serum resistance and serum sensitivity.
This work was supported by Public Health Service grants 5RO1AI21236-02 and 3RO1-AI21236-02S1 from the National Institute of
Allergy and Infectious Diseases, by University of Montana research
grant 1117-M, and by Stella Duncan Memorial Research Institute.
LITERATURE CITED
1. Alexander, N. M. 1973. Oxidation and oxidative cleavage of
tryptophanyl peptide bonds during iodination. Biochem. Biophys. Res. Commun. 54:614-621.
2. Barrera, O., and J. Swanson. 1984. Proteins IA and IB exhibit
different surface exposures and orientations in the outer membrane of Neisseria gonorrhoeae. Infect. Immun. 44:565-568.
3. Cannon, J. G., W. J. Black, I. Nachamkin, and P. W. Stewart.
1984. Monoclonal antibody that recognizes an outer membrane
antigen common to the pathogenic Neisseria species but not to
most nonpathogenic Neisseria species. Infect. Immun. 43:
994-999.
4. Cannon, J. G., T. M. Buchanan, and P. F. Sparling. 1983.
Confirmation of association of protein I serotype of Neisseria
gonorrhoeae with ability to cause disseminated infection. Infect. Immun. 40:816-819.
5. Cannon, J. G., T. J. Lee, L. F. Guymon, and P. F. Sparling.
276
6.
7.
8.
9.
10.
11.
12.
13.
NOTES
1981. Genetics of serum resistance in Neisseria gonorrhoeae:
the sac-i genetic locus. Infect. Immun. 32:547-552.
Elder, J. H., R. A. Pickett II, J. Hampton, and R. A. Lerner.
1977. Radioidination of proteins in single polyacrylamide gel
slices. J. Biol. Chem. 252:6510-6515.
Hildebrandt, J. F., and T. M. Buchanan. 1978. Identification of
an outer membrane protein associated with gonococci capable
of causing disseminated infection, p. 138-141. In G. F. Brooks,
E. C. Gotschlich, K. K. Holmes, W. D. Sawyer, and F. E.
Young (ed.), Immunology of Neisseria gonorrhoeae. American
Society for Microbiology, Washington, D.C.
Hildebrandt, J. F., L. W. Mayer, S. P. Wang, and T. M.
Buchanan. 1977. Neisseria gonorrhoeae acquire a new outer
membrane protein when transformed to resist serum bactericidal activity. Infect. Immun. 20:267-273.
James, J. F., E. Zurlinden, C. J. Lammel, and G. F. Brooks.
1982. Relation of protein I and colony capacity to serum killing
of Neisseria gonorrhoeae. J. Infect. Dis. 145:37-43.
Joiner, K. A., K. A. Warren, J. Tam, and M. M. Frank. 1985.
Monoclonal antibodies directed against gonococcal protein I
vary in bactericidal activity. J. Immunol. 134:3411-3419.
Judd, R. C. 1982. 251I-peptide mapping of protein III isolated
from four strains of Neisseria gonorrhoeae. Infect. Immun.
37:622-631.
Judd, R. C. 1982. Surface peptide mapping of protein I and
protein III of four strains of Neisseria gonorrhoeae. Infect.
Immun. 37:632-641.
Judd, R. C. 1986. Evidence for N-terminal exposure of the
protein IA subclass of Neisseria gonorrhoeae protein I. Infect.
INFECT. IMMUN.
Immun. 54:408-414.
14. Laemmli, U. K. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature (London)
227:680-685.
15. Markwell, M. A. K., and C. F. Fox. 1978. Surface-specific
iodination of membrane proteins of viruses and eucaryotic cells
using 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Biochemistry 17:4807-4817.
16. Sandstrom, E. G., K. C. S. Chen, and T. M. Buchanan. 1982.
Serology of Neisseria gonorrhoeae: coagglutination serogroups
WI and WIT/Ill correspond to different outer membrane protein
I molecules. Infect. Immun. 38:462-470.
17. Shafer, W. M., L. F. Guymon, and P. F. Sparling. 1982.
Identification of a new genic site (sac-3+) in Neisseria gonorrhoeae that affects sensitivity to normal human serum. Infect.
Immun. 35:764-769.
18. Spratt, S. K., F. Jones, T. E. Shockley, and J. H. Jackson. 1980.
Cotransformation of a serum resistance phenotype with genes
for arginine biosynthesis in Neisseria gonorrhoeae. Infect.
Immun. 29:287-289.
19. Swanson, J. 1982. Colony opacity and protein II compositions of
gonococci. Infect. Immun. 37:359-368.
20. Tam, M. R., T. M. Buchanan, E. G. Sandstrom, K. K. Holmes,
J. S. Knapp, A. W. Siadak, and R. C. Nowinski. 1982. Serological classification of Neisseria gonorrhoeae with monoclonal
antibodies. Infect. Immun. 36:1042-1053.
21. Tsai, L. M., and C. E. Frasch. 1982. A sensitive silver stain for
detecting lipopolysaccharides in polyacrylamide gels. Anal.
Biochem. 119:115-119.