Feb 16, 1988 - GEOFFREY A. WEINBERG,"2 DWIGHT A. TOWLER,3 AND ROBERT S. ... and Rehn (3) and later found in other members of the family.
JOURNAL OF BACTERIOLOGY, Sept. 1988, p. 4161-4164 0021-9193/88/094161-04$02.00/0 Copyright © 1988, American Society for Microbiology
Vol. 170, No. 9
Lipoproteins of Haemophilus influenzae Type b GEOFFREY A. WEINBERG,"2 DWIGHT A. TOWLER,3 AND ROBERT S. MUNSON, JR.l.2.4* Edward Mallinckrodt Department of Pediatrics,1 Department of Microbiology and Immunology,4 and Department of Biological Chemistry,3 Washington University School of Medicine, and Division of Infectious Diseases, Children's Hospital,2 St. Louis, Missouri 63110 Received 16 February 1988/Accepted 31 May 1988
Haemophilus influenzae type b Minn A produced 12 lipoproteins with apparent molecular weights of between 14,000 and 67,000. The lipoproteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of delipidated extracts of cells grown in [3Hlpalmitate. When the delipidated cell extracts were subjected to acid methanolysis, tritium was quantitatively recovered as palmitate and methyl palmitate, indicating that the [3H]palmitate had not been degraded and reincorporated into nonlipid material during cell growth. One of the lipoproteins comigrated with outer membrane protein (OMP) P6. OMP P6 was purified from [3lH]palmitate-labeled cells. The purified protein preparation contained both amide- and ester-linked fatty acids. We conclude that (i) H. influenzae type b produces several lipoproteins, and (ii) one of these lipoproteins is OMP P6, a protein under consideration as a vaccine component.
Lipoproteins in the cell envelopes of several procaryotes have been described. The best characterized is the murein lipoprotein, originally described in Escherichia coli by Braun and Rehn (3) and later found in other members of the family Enterobacteriaceae (13). The NH2-terminal cysteine of murein lipoprotein is covalently modified with diacylglycerol via a thioether linkage [glycerylcysteine, S-(propane-2',3'diol)-3-thio-2-aminopropionic acid] and with an amide-linked fatty acid moiety (8). A second group of bacterial lipoproteins is the peptidoglycan-associated lipoproteins found in several gram-negative bacteria including E. coli (5, 13, 14). These proteins are closely, but noncovalently, associated with peptidoglycan and contain the same NH2-terminal modification as murein lipoprotein. Membrane lipoproteins that are not associated with peptidoglycan have also been described previously (9, 15). Finally, both gram-negative and gram-positive bacteria contain membrane-bound lipoproteins that may be secreted as exoenzymes, either with (23) or without (10, 21) the NH2-terminal lipid modification. Detailed investigation of bacterial lipoproteins has led to important insights into posttranslational modification and processing of exported proteins (reviewed in references 25 and 26). In this report we have demonstrated that Haemophilus influenzae type b produces at least 12 lipoproteins. One of these lipoproteins is the outer membrane protein (OMP) we designated P6 (16). This protein has been designated a peptidoglycan-associated lipoprotein by Green et al. (7). P6 is of potential interest as a vaccine component, since antisera directed against P6 are active both in an in vitro bactericidal assay (7, 18) and in an in vivo passive protection model (7, 16). (This work was presented in part at the 87th Annual Meeting of the American Society for Microbiology, Atlanta, Ga., 1 to 6 March 1987.) MATERIALS AND METHODS
Bacterial growth and labeling. H. influenzae type b Minn A to mid-log phase in brain heart infusion broth supplemented with 2 [xg each of hemin and NAD per ml;
was grown
*
Corresponding author.
cells were harvested and frozen as previously described (2, 17). For labeling experiments, [9,10-3H]palmitic acid (28.5 Ci/mmol [1,054 GBq/mmol]; DuPont, NEN Research Products, Boston, Mass.) was included in the growth medium as follows. [3H]palmitic acid in ethanol was added to a sterile 250-ml flask, and the solvent was evaporated under a nitrogen stream. The palmitic acid was then dissolved in sterile warm broth. An inoculum of log-phase cells was added such that the A600 of the culture was 0.1. The culture was then allowed to grow for 90 min (approximately three generations) at 37°C before the cells were harvested. The final concentration of [3H]palmitic acid was 22 to 25 ,uCi/ml (814 to 925 kBq/ml). Delipidation of [3H]palmitate-labeled cells. The frozen cell pellet yielded from the growth of 18 ml of cells labeled with [3H]palmitate was thawed and suspended in 0.2 ml of 1% sodium dodecyl sulfate (SDS) at room temperature. Lipids that were not covalently attached to proteins were removed by repetitive chloroform-methanol extraction. Chloroformmethanol (2:1 [vol/vol], 10 ml) was added, the mixture was vortexed for 60 s, and the insoluble residue was separated from the solvent by centrifugation. The pellet was solubilized in 1% SDS and treated with chloroform-methanol for eight additional times as described above. No further radioactivity was extracted by the ninth extraction, indicating that all soluble lipids had already been removed. Excess solvent was removed from the precipitate with a stream of nitrogen at room temperature, and the precipitate was suspended in 1% SDS. Analysis of protein-bound fatty acids by HPLC. Radiolabeled delipidated cells were subjected to acid methanolysis, petroleum ether extraction, and reverse-phase high-pressure liquid chromatography (HPLC) as previously described (22, 24). Suspended delipidated cells (20 ,ul) were treated with 1 ml of 83% methanol-2 M HCl under nitrogen at 95°C for 18 to 22 h. The reaction solution was then extracted five times with 1 ml of petroleum ether. Radioactivity remaining in the aqueous phase (amino acids) and in the combined organic phase (fatty acids and fatty acid methyl esters) was determined by liquid scintillation counting of portions. The identities of the fatty acids covalently bound to protein were determined by comparison of the reverse-phase HPLC absorbance elution profile of added fatty acid-fatty acid 4161
4162
WEINBERG ET AL.
methyl ester internal standards with the HPLC elution profile of radioactivity released from [3H]palmitate-labeled protein as monitored by liquid scintillation counting (22, 24). Chromatography was performed on a ,uBondapak C18 column (Waters Chromatography Div., Millipore Corp., Milford, Mass.) eluted with 80% acetonitrile-20% Milli-Q processed water (Millipore) at 1 ml/min for the first 25 min and at 2 ml/min for 25 to 50 min. The elution of standards was monitored by A214. Analysis of 3H-fatty acid linkage to OMP P6. OMP P6 was purified to homogeneity from [3H]palmitate-labeled cells as previously described (16). The protein was then sequentially subjected to alkaline methanolysis and acid methanolysis to release ester-linked and amide-linked fatty acids, respectively (1). Portions (5 ,ul; 5.5 jig of protein; approximately 15,000 dpm of 3H) of purified P6 protein were each treated with 0.2 ml of 0.1 M KOH in methanol for 4 h at room temperature, acidified with 0.1 ml of 2 M HCl, and then extracted three times with 0.8 ml of petroleum ether. The amount of radioactivity released into the petroleum ether phase by alkaline methanolysis, representing the fatty acids bound to OMP P6 in ester linkage, was determined by liquid scintillation counting. Solvent was removed from the remaining alkaline methanol phase by evaporation. The residue was acid methanolyzed with 1.5 ml of 83% methanol-2 M HCI as described above and then extracted four times with 1.5 ml of petroleum ether. The radioactivity extracted by petroleum ether from this second, acid methanolysis represented fatty acid bound to OMP P6 by amide linkage. Other analytical methods. Protein concentration was determined by the bicinchoninic acid method according to the directions of the manufacturer (BCA Protein Assay kit, Pierce Chemical Co., Rockford, Ill.). SDS-polyacrylamide gel electrophoresis (PAGE) was performed on 11% modified Laemmli gels by the method of Lugtenberg et al. (12) as previously described (2, 17). After being stained and destained, gels were treated with En3Hance (DuPont, NEN) prior to fluorography, according to the directions of the manufacturer. The treated gels were exposed to SB film (Eastman Kodak Co., Rochester, N.Y.) at -70°C for 3 to 5 days in cassettes equipped with intensifying screens. RESULTS Demonstration of lipoproteins in H. influenzae type b. H. influenzae type b cells grown in the presence of [3H]palmitic acid were delipidated with chloroform-methanol and subjected to SDS-PAGE. The gel was then stained with Coomassie blue and processed for fluorography to detect radiolabeled proteins. Twelve labeled proteins, one of which comigrated with OMP P6, were visualized; they were designated LP1 through LP11 and P6, respectively (Fig. 1). It is conceivable that the labeled material represented nonlipid radioactivity as a result of catabolism of [3H]palmitate and the subsequent reincorporation of label into protein as 3H-amino acids (22). Thus, the fraction of the label recoverable as fatty acid was ascertained by analysis of acidmethanolyzed delipidated cells. In two separate experiments, .99% of the radioactivity present in labeled delipidated cells was recovered in the petroleum ether organic phase. Hence, the labeled proteins visualized by fluorography contained radiolabeled lipids. Because the lipids remained associated with the proteins after repeated chloroform-methanol treatment and SDS-PAGE, the lipids were assumed to be covalently bound to the proteins. Thus, the 12 labeled proteins are lipoproteins. The petroleum ether
J. BACTERIOL.
94
K-
67
K-
....
_ 43
I- P2
0IOi-
K-
*LP4 -LP5 'L P6
30 K-
w~~~P_ LP8
201 K
L
,IOp-
p ._Di
i ~~
~
~
~
~
44K.4 K-*
FIG. 1. Lipoproteins of H. influenzae type b. Lane 1, Coomassie blue-stained 11% SDS-PAGE gel of delipidated cellular protein from [3H]palmitate-labeled H. influenzae type b. Molecular weight standards are as indicated, i.e., 94K = 94,000. Lane 2, Fluorograph of lane 1, showing 3H-labeled proteins. The 12 labeled proteins visualized were designated as shown.
extract was subjected to reverse-phase HPLC; .99% of the radioactivity was recovered exclusively as palmitate methyl ester and palmitate (Table 1). Demonstration that OMP P6 is a lipoprotein. To demonstrate that the lipoprotein which comigrated with OMP P6 by SDS-PAGE was P6 (Fig. 1), P6 was purified to homogeneity from H. influenzae type b cells labeled with [3H]palmitic acid. Purified OMP P6 contained tritium (Fig. 2). The radioactivity was demonstrated to be present as 3H-fatty acid by the same methods used with delipidated cells (see above). Again, -99% of the radioactivity was recovered as fatty acid. The predominant fatty acid recovered from the [3H]palmitate-labeled P6 was palmitate, with a small fraction of myristate (Table 1). Analysis of 3H-fatty acid linkage to OMP P6. Purified radiolabeled OMP P6 was subjected to sequential alkaline TABLE 1. Recovery of radioactivity from delipidated cellular
protein and purified OMP P6 of H. influenzae type b Radioactivitya (dpm)
Protein and Expt. no.
Injected
Recovered
into
as
fatty acids
HPLC column
Total
Palmitate
Myristate
63,238 102,765
69,800 92,660
69,428 92,325
