Outer Membrane Protein Composition in Disease Isolates of Haemophilus influenza: Pathogenic and Epidemiological

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1 INFECTION AND IMMUNITY, Dec. 1980, p /80/ /09$02.00/0 Vol. 30, No. ; Outer Membrane Protein Composition in Disease Isolates of Haemophilus influenza: Pathogenic and Epidemiological Implications MARILYN R. LOEB* AND DAVID H. SMITH Department of Pediatrics, University of Rochester Medical Center, Rochester, New York The outer membrane protein composition of 50 disease isolates of Haemophilus influenzae has been determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All strains, including 28 strains of serotype b, one strain each of serotypes a, c, d, e, and f, and 17 untypable strains, had an outer membrane protein composition typical of gram-negative bacteria, i.e., these membranes contained two to three dozen proteins with four to six proteins accounting for most of their protein content. Variation in the mobility of these major outer membrane proteins from strain to strain was common but not universal; the observed patterns provided useful data and new insight into the epidemiology of type b disease. The basic findings can be summarized as follows: (i) All 50 strains possessed three proteins (one minor and two major) each having identical mobilities. The other proteins, both major and minor, varied in mobility. (ii) All type b strains possessed a fourth (major) protein of identical mobility. (iii) The 28 type b strains, on the basis of the mobility of the six major outer membrane proteins, could be divided into eight subtypes. Of all the other strains examined, both typable and untypable, only the serotype a strain belonged to one of these subtypes. (iv) The untypable strains showed considerable variation in the mobilities of their major outer membrane proteins. Of these 17 strains, 13 had an additional major outer membrane protein not present in encapsulated strains. (v) The outer membrane protein composition of a single strain remained unchanged after many passages on solid media, but varied with the growth phase. (vi) The outer membrane protein composition of isolates obtained from nine patients during an epidemic of type b meningitis varied, indicating that a single strain was not responsible for the epidemic. At least five different strains were responsible for these nine cases. (vii) Identical outer membrane protein compositions were observed in the following: in a type b strain and a mutant of this strain deficient in capsule production, indicating that the level of capsule synthesis is not obviously related to outer membrane protein composition; in type b strains isolated from different anatomic sites of patients acutely ill with meningitis, indicating that the strain associated with bacteremia is the same as that isolated from the cerebrospinal fluid; in type b strains isolated from siblings who contracted meningitis at about the same time, indicating infection with the same strain; and in type b strains isolated from the initial and repeat infection of a single patient, suggesting that reinfection was due to the same strain. Encapsulated Haemophilus influenza is a cause of serious systemic invasive disease in humans, young children being most vulnerable. Of the six capsular types (types a through f), type b is responsible for more than 95% of all invasive disease caused by this bacterium (23). Because antibodies to the type b capsule were found to be protective both in vitro as evidenced by their bactericidal and opsonic activity (3, 10, 24) and in vivo as revealed by the protective role of passive immunization in humans (1) and in infant rats (22), a vaccine of purified capsular material was developed and tested in humans. 709 Although providing more than 90% protection in recipients older than 18 months, the vaccine unfortunately was ineffective in the age group most at risk, children under 18 months (20). The failure of immunization with capsule to protect the very young has stimulated this laboratory to investigate the role of other cell surface components, i.e., outer membrane proteins and lipopolysaccharide, in the pathogenesis of H. influenzae disease. These somatic (i.e., noncapsular) antigens are also of interest because of problems resulting from disease caused by unencapsulated H. influenza, e.g., otitis media and

2 710 LOEB AND SMITH INFECTr. IMMUN. bronchitis. Evidence from our laboratory and others demonstrates that antibodies to somatic antigens are widely prevalent among healthy adults (3, 17), are induced by infection (5), have bactericidal and opsonic activity in vitro (4, 8, 10), and protect infant rats from sepsis and meningitis (4, 18) in a model that mimics human disease (16). Recently, this laboratory developed an enzyme-linked immunosorbent assay technique for distinguishing anti-protein antibody from antilipopolysaccharide antibody and obtained data suggesting that some outer membrane proteins of H. influenzae are immunogenic in humans. We found an age-related titer of anti-protein antibodies in healthy subjects and a uniform appearance of such antibodies in convalescent children. The titers in convalescent children equal those of healthy adults and are independent of anticapsular antibody responses (R. A. Insel et al., submitted for publication). We have also initiated an ultrastructural and biochemical analysis of the cell envelope of H. influenzae, as recently reported (M. R. Loeb, et al., submitted for publication). Using type b strain Eag as our reference strain, we have demonstrated that the cell envelope ofh. influenzae growing logarithmically in aerated liquid medium is typical of gram-negative bacteria (6) in that there is an inner (cytoplasmic) and an outer membrane. However, there are some distinctive differences. The H. influenzae outer membrane is quite rugose, and the inner membrane is so fragile that gentle techniques had to be developed to separate outer and inner membrane. In addition, the inner membrane has an unusual ultrastructure. The outer membrane, our main focus of interest, is quite typical of most gramnegative bacteria. It contains phospholipid, lipopolysaccharide, and protein. The protein composition as shown by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS- PAGE) is also typical (9, 12) in that there are about two to three dozen polypeptides, with seven polypeptides accounting for most of the protein. To further our understanding of the role of H. influenza outer membrane proteins, we have determined the polypeptide composition of outer membranes from 50 additional disease isolates, including 28 type b, 17 untypable, and one each of types a, c, d, e, and f. These strains came from various geographic and anatomic sites and were selected to enable us to evaluate the use of outer membrane protein composition for subtyping H. influenzae and for understanding the epidemiology of the disease. In addition, knowledge of the extent of differences in outer membrane protein composition among strains is required before considering the use of one or more of these proteins in a vaccine. MATERIALS AND METHODS Cell culture. The 50 isolates examined were all kept as frozen skimmed milk stock cultures in this laboratory. Methods for maintaining stocks and growing cultures have been described (Loeb et al., submitted for publication). Briefly, cells were grown with aeration at 370C in brain heart infusion medium (Difco Laboratories) containing 2 jig of nicotinamide adenine dinucleotide and 10 fig of hemin per ml. Isolation of outer membranes. As described in detail previously (Loeb et al., submitted for publication), cells were grown to late log phase, collected, washed, and sonicated. Cell envelopes were obtained by differential centrifugation and extracted with 2% Triton X-100 (21; Loeb et al., J. Bacteriol., in press) to yield a residue rich in outer membrane proteins and free of inner membrane. SDS-PAGE. The polypeptide composition of membrane fractions was determined by SDS-PAGE (13) on gels containing 10% acrylamide as described previously (Loeb et al., submitted for publication). RESULTS Polypeptide composition of outer membranes from encapsulated H. influenza. Initially, the outer membrane protein composition of 17 isolates of type b obtained from patients in Huntsville, Ala., New York, N.Y., and Boston, Mass., over the last 12 years, plus one of Hattie Alexander's strains isolated in New York before 1945, were examined by SDS-PAGE. Typical results for 13 of these strains plus a mutant of strain Eag deficient in production of capsule appear in Fig. 1, lanes 1 to 14. Note the overall similarity of pattern. Each strain contained a few major outer membrane proteins with the same or similar molecular weights plus several minor proteins. This was also true for single isolates of H. influenzae of capsular types a, c, d, e, and f (lanes 15 to 19, respectively). For purposes of reference the major polypeptides of strain Eag are labeled a through f and have the following approximate molecular weights: a, 46,000; b, 38,000; c, 37,000; d, 34,000; e, 28,000; and f, 26,000. (Note that the major band consists of two polypeptides, b and c; these are separated when less material is applied to the gel [Loeb et al., submitted for publication].) In addition, strain Eag had a smaller polypeptide of 15,000 molecular weight, protein g, that appeared on 15% gels (data not shown). This polypeptide, polypeptide e, and a minor polypeptide appearing above protein e, protein h were present in all serotypes. Protein d, on the other hand, had identical mobility only in the serotype b

3 VoL. 30, 1980 OUTER MEMBRANE PROTEINS OF H. INFLUENZAE K a-- - a bi% C- d e..e 0-45K. -~~~~~~~~5 a Alt.~~~~ A FIG. 1. Outer membrane protein composition of encapsulated H. influenzae. Protein (25 jg) was applied to each well. The major outer membrane proteins are designated as a through f, and a minor protein is designated as h. Lanes and strains: 1, Eag; 2, S2 mutant of Eag; 3, H 292; 4, H 113; 5, C 58; 6, 65-10; 7, H 234; 8, Rab; 9, H 305; 10, Mad; 11, C 47; 12, C 54; 13, C 11; 14, C 43; 15, serotype a; 16, serotype c; 17, serotype d; 18, serotype e; 19, serotype f. Strains in wells 1 through 14 are type b. Isolates were obtained from cerebrospinal fluid or blood, except for C 54, which came from the nasopharynx, and S2, which is a mutant of Eag deficient in production of capsule. strains. The other major proteins, a, b, c, and f, varied among strains. The outer membranes in Fig. 1, lanes 1 and 2, representing strain Eag and a mutant, S2, that is deficient in capsule production, were identical, indicating that changes in levels of capsule formation are not obviously reflected in outer membrane protein composition. The isolates in lanes 13 and 14 were also identical; these are considered below. For purposes of a more general comparison, the type b strains can be divided into subtypes on the basis of the mobilities of the six major proteins, a through /. These subtypes, shown diagrammatically in Fig. 2, appear in the following lanes of Fig. 1: type 1, lanes 1, 2, 4, 6, and 7; type 2, in which protein f had a faster mobility, lane 10; type 3, in which proteins b and c have faster mobilities, lanes 3, 5, and 11-14; type 4, in which protein f is missing, lane 8; type 5, in which protein a has a slower mobility, proteins b and c have a faster mobility, and protein f is absent, lane 9. Although these subtypes overlook differences between minor proteins, they are useful for comparison. For example, only seroa- b/c _N -_ e- f FIG. 2. SDS-PAGE types of type b strains. This is a diagrammatic presentation ofthe eight SDS-PAGE types found in 28 type b strains. type a of the five non-b serotypes (lanes 15 to 19) fits into any of these subtypes (subtype 5), thus underlining differences between serotypes.

4 712 LOEB AND SMITH Stability of outer membrane protein composition. The possibility that the variations observed in outer membrane protein composition were due to changes of the strains upon storage or passage was examined. Three strains were obtained from frozen storage, and each was passaged on solid medium every day for 33 days. After 5, 12, 19, and 33 passages, the strains were grown to late log phase in liquid medium; the outer membranes were isolated, and their polypeptide composition was determined. In addition, cells were cultured a second time from the frozen stocks and outer membranes obtained after five passages. The results show that each strain maintained its outer membrane protein composition throughout the 33 passages and upon a repeated culturing from the frozen stock (Fig. 3). Strains C 153 and C 154, which have an identical pattern, are discussed below. These two strains belong to a sixth SDS-PAGE subtype (designated subtype 6) on the basis of the faster mobilities of proteins b, c, and f. The third strain in Fig. 3, H 113, belongs in subtype 1. INFECT. IMMUN. The outer membrane protein composition of an individual strain can vary, however, as shown in Fig. 4, where strain Eag outer membranes obtained from log-phase (lane 1), mid-stationary-phase (lane 2), and late-stationary-phase (lane 4) cells are compared. Although the same major outer membrane proteins were present in these three preparations, there were many more minor proteins in the material from stationaryphase cells. Also, the composition differed for mid- and late-stationary phases. It is also of interest to compare the polypeptide composition of outer membrane shed into the culture medium during stationary phase with that obtained from the cells themselves. The released outer membrane from both mid- and late-stationaryphase cells (lanes 3 and 5, respectively) lacked proteins that were present in the outer membrane of the respective cells, was enriched in some other proteins, and also contained proteins not found in the cellular outer membrane. Note, in particular, that protein h, a protein found in outer membranes isolated from all cells of H. Downloaded from 'ka~ VM _*VW OM mq 4I0 _0 O ~ *_ on November 25, 2018 by guest.4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ FIG. 3. Effect of subculturing on outer membrane protein composition. The SDS-PAGE patterns of type b strains C 153, C 154, and H 113 obtained after 5, 12, 19, and 33 passages are indicated in the first, third, fourth, and fifth lanes for each strain. The second lane shows the pattern obtained after five passages of a repeat isolation from the same frozen stock.

5 VOL. 30, K- 45K- _ w K-~~~~~~o --- OUTER MEMBRANE PROTEINS OF H. INFLUENZAE 713 a- b c' h - -- e- _ f- " FIG. 4. Outer membrane protein composition of cells and of released outer membrane of strain Eag as a function of growth phase. SDS-PAGE of outer membranes are shown for: 1, log-phase cells; 2, midstationary-phase cells; 3, released outer membrane of mid-stationary-phase (12 h) cells; 4, late-stationaryphase (18 h) cells; 5, released outer membrane from late-stationary-phase cells. Released outer membrane was obtained as follows. Cells were removed from the culture medium by centrifugation twice at 12,000 x g for 15 min. The supernatant fluid was centrifuged at 255,000 x g for 60 min. The pellet was suspended in 0.05 M tris(hydroxymethyl)aminomethane (Tris), ph 7.8, and applied to a linear gradient of 0.77 to 2.02 M sucrose in Tris buffer. After centrifugation in an SW27 rotor at 27,000 rpm for 16 h, light-scattering material banding at a buoyant density of 1.20 to 1.22 was collected and washed once with Tris buffer. influenza examined by us, was absent in released outer membrane. Epidemiological studies of disease-producing type b isolates. The variability of outer membrane protein composition among type b strains suggested the possible use of these proteins as a tool for studying the epidemiology of H. influenzae disease. This hypothesis was supported by some of the above results. Strains C 153 and C 154 (Fig. 3), which have identical outer membrane protein composition, were obtained from twins who contracted meningitis within 3 days of each other. This observation thus supports the epidemiological data that suggest a common etiology for both cases. Similarly, the two pairs of outer membranes in lanes 11 and 12 and lanes 13 and 14 (Fig. 1) were each collected from two patients experiencing distinct repeated infections 2 and 6 months apart, respectively. (The isolates from the first infection appear in lanes 11 and 13, and those from the second infection appear in lanes 12 and 14.) Whereas the first set of isolates (lanes 11 and 12) is similar but not identical, the second set (lanes 13 and 14) is identical. These results suggest that an individual can be reinfected by the same strain. Recently, 13 cases of meningitis were diagnosed in a 6-week period in the Rochester metropolitan area, the total for the year being only approximately 30 cases. Sixteen isolates were obtained from various anatomic sites from nine of the patients. A study of the outer membrane protein composition of their isolates (Fig. 5) revealed the following. (i) Isolates obtained from the same patient but from different anatomical sites or at different times were identical: compare lanes 2, 3, and 5; 6 and 7; 8 and 9; 10 and 11; 12 and 13; 14 and 15. (ii) As with strains C 153 and C 154 (Fig. 3), isolates from two siblings were identical; compare patients A and B. (iii) The epidemic was not caused by a single strain, as indicated by the variation in outer membrane protein composition of the various isolates. However, some unrelated patients had identical strains: compare patients C and E; D, G, and I. Thus in these nine cases, patients A and B, siblings, appeared to be infected with the same strain; patients C and E appeared to be infected with another identical strain; patients D, G, and I appeared to be infected with still another identical strain; patients F and H each appeared to be infected with a unique strain. Altogether, at least five strains caused the nine cases. Also note that proteins d, e, and h each have identical mobility in all the strains; this is also true for protein g, as observed on 15% gels (data not shown). These five different strains belonged to four different SDS-PAGE subtypes. Strains in lanes 16, 17, and 18 were type 1; strains in lanes 2, 3, 5-7, 14, and 15 were type 2; strains in lanes 8, 9, 12, and 13 were an additional type, type 7, in which the mobility of protein f is slightly greater than that in type 3; and strains in lanes 10 and 11 were another additional type, type 8, in which proteins b and c have slower mobility than those in type 1. Thus, the subtyping system emphasizes the variability in the strains isolated during this epidemic.

6 _ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...,.Ys 714 LOEB AND SMITH -~~ '-~e *0 q~~p ava p 0Q a- One ab-a flfl_ 00b e_ n O 0'1g C- d- Aft: CW Vo: wo a::zmom... ag :-: :}wu,: h- Af....:.:ol- _ -A " e- _.o Afdmft I"T f - --iha mm,a -a-_ w w - w 1 1' j I ; AA nfaaa 14mmw Imommow IRROW, "Impw 4 0 / u Li a: A7 Cd D At A i i ;i o FIG. 5. Outer membrane protein composition of type b strains isolated during a meningitis epidemic. The SDS-PAGE patterns of epidemic strains are in lanes 2, 3, and 5 through 18. The capital letters designate individual patients. Patients A and B were siblings. The sources of the isolates were as follows: lanes 2, 3, 7, 9, 10, 11, 13, 15, and 17, blood; lanes 6, 8, 12, 14, 16, and 18, cerebrospinal fluid; lane 5, nasal. Lane 1 contains strain Eag; lane 4 contains an untypable strain, C 379. Polypeptide composition of outer membranes from untypable H. influenza The outer membrane protein composition was determined for 17 untypable strains isolated from patients seen in Boston between 1971 and Results with 10 of these appear in Fig. 6, lanes 2 through 11. These strains showed a greater variability in protein composition than the type b strains as manifested by mobilities of some of the minor proteins of high molecular weight as well as the mobilities of major proteins, a, b, c, d, and f Unlike in the type b strains, the mobility of protein d varied. However, as with all encapsulated strains examined (serotypes a to f), proteins e, g, and h had identical mobilities. Note also the presence in most strains of an additional major protein, protein i; this was found in 13 of the 17 untypable strains. DISCUSSION INFECT. IMMUN K la This examination of 50 disease isolates of H. influenzae has revealed some characteristic properties of the outer membranes of this pathogen and has yielded useful epidemiological information. The current data extend our initial findings on the outer membranes of a single strain of H. influenzae type b, strain Eag (Loeb, et al, submitted for publication). In those experiments outer and inner membranes were separated by isopycnic centrifugation of a French press lysate. The polypeptide composition of outer membranes isolated by this relatively laborious method was shown to be very similar to outer membrane material isolated by the simpler Schnaitman procedure (21) involving extraction of envelopes with 2% Triton X-100, thus demonstrating the validity of using this procedure with H. influenza. (It should be recalled that the Schnaitman procedure does not yield outer membrane, but rather a subtraction of outer membrane enriched in protein and partially depleted of lipopolysaccharide and phospholipid.) All 50 strains had a similar overall pattern of outer membrane proteins typical of many gramnegative bacteria. The predominant protein of the H. influenza strains, which appeared as a doublet in strain Eag, had a molecular weight that varied from 35,000 to 40,000 depending on the strain and was similar in size to the proteins

7 VOL. 30, 1980 i - inmal --- %E w-m a- - ā w - b C-* A w _ *- m4m4 - - OUTER MEMBRANE PROTEINS OF H. INFLUENZAE 715 _0 :-I: _- 25K FIG. 6. Outer membrane protein composition of untypable strains of H. influenza. Strain Eag, type b, appears in lane 1. The strain designations and anatomic sites of isolation for the SDS-PAGE patterns from untypable strains are as follows: 2, strain C 379, nose; 3, C 304, trachea; 4, C 353, sputum; 5, C 333, nose; 6, C 294, pericardium; 7, C 273, eye; 8, C 272, eye; 9, C 270, trachea; 10, C 83, sputum; 11, C 37, sputum ofpatient with chronic lung disease. of Escherichia coli and Salmonella typhimurium (19). Whether these proteins fulfill the same function in H. influenzae remains to be shown. However, the results of Medeiros and O'Brien (15) indicating greater permeability of H. influenzae to ampicillin indicate that such pores would be larger than those present in E. coli and S. typhimurium. All H. influenza strains also have a protein, analogous to protein d of strain Eag, of about 32,000 to 36,000 molecular weight, and comparable in size to protein II* in E. coli. Like protein II*, the protein in strain Eag is heat modifiable(data not shown). The presence of a molecule comparable to free lipoprotein is uncertain. All strains showed a very faint diffuse band migrating between cytochrome c and insulin on 15% gels, but further work is needed to determine whether this molecule is similar to lipoprotein and whether the faintness is due to its presence in low levels or to interference of staining by lipopolysaccharide, as observed with E. coli strain B (14). The division of the strains into subtypes based on the mobility of six major outer membrane proteins provides a convenient index of the degree of relatedness among strains. Ignoring for the time being the possibility that proteins of similar mobility may be different, that a single band may contain more than one protein, and that the b plus c doublet found in strain Eag may not be a doublet in other strains, the 28 type b strains studied could be categorized by eight SDS-PAGE subtypes. The distinction between type b and other strains is then apparent since the isolates of four of the five other serotypes and of the untypable strains do not belong to any of these subtypes. In addition, the untypable strains show considerable variation in outer membrane protein composition among each other. It is possible that if a large number of strains of any one of the other serotypes or if a group of untypable strains obtained from the same anatomic site were examined, related strains might be observed within each of these groups. In terms of the differences observed it is important to note that the variability in outer membrane proteins is not due to epigenetic or genetic instability or to problems with the methodology, since the same outer membrane protein composition was obtained after many passages and upon repeated subculturing from the frozen stock culture (Fig. 3). The data presented raise questions about the mechanism of pathogenesis of H. influenzae. The virulence of type b strains, which account for at least 95% of systemic disease, is usually ascribed to their capsules. However, the finding that type b strains possess outer membrane proteins which can be classified into groups unique to type b and that one of these proteins, d, appears to be identical only in type b strains indicates the need for evaluating the role of other cell surface molecules) in virulence. Indeed, different surface molecules may each be responsible for the various functions associated with virulence, such as attachment, invasiveness, and resistance to phagocytosis. Further, the finding of a protein uniquely associated with most untypable strains, protein i, suggests a correlation of this protein with lack of capsule. In this regard it is interesting that colonies of the four untypable strains lacking protein i can be distinguished morphologically by their slight mucoid and/or iridescent quality (P. Anderson, personal communication). The data presented also provide some information on the evolution of H. influenza strains. The presence of six capsular types itself suggests evolutionary divergence. This is further supported by the lack of relatedness of outer membrane proteins of type b to those of four other serotypes. In addition, the considerable variation among the untypable strains leaves open the

8 716 LOEB AND SMITH question of whether they evolved before encapsulated forms and subsequently gave rise to the various serotypes and/or whether they evolved from any of the serotypes. It is also of interest that all 50 strains examined possess three proteins, e, g, and h, having identical mobilities. The extent of identity of these proteins is currently being examined, but even these preliminary data indicate that these proteins have varied far less than others in the course of evolution and suggest that their functions are rigidly related to their structures. That useful epidemiological data has been obtained from these studies is evident. The outer membrane protein composition of the initial isolate from one individual infected with a type b strain was identical to that of the isolate obtained upon reinfection (Fig. 1, lanes 13 and 14), suggesting that the same strain was responsible for both infections. In a second individual, the second isolate differed slightly from the first (Fig. 1, lanes 12 and 11). However, it should be noted that the second isolate was obtained from the nasopharynx, the blood isolate having been lost. Also, isolates from siblings who became ill at the same time had identical outer membrane protein compositions (Fig. 3, strains C 153 and C 154; Fig. 5, patients A and B), suggesting direct contagion or exposure to the same source. On the other hand, only some isolates obtained during an epidemic were identical (Fig. 5), indicating that a single highly virulent strain was not responsible for the epidemic. Lastly, the finding of identical strains in the blood and cerebrospinal fluid of patients ill with meningitis (Fig. 5) demonstrates for the first time in humans that the systemic disease is associated with a single strain of type b, rather than a mixture of strains. This is the first report of epidemiological studies of disease caused by type b H. influenzae using outer membrane protein composition as the tool. Previous efforts to distinguish disease isolates on the basis of biotype were unsuccessful because most disease is caused by the same biotype (11). Efforts in our laboratory to classify type b strains by bactericidal serotyping have recently been described (2). However, this assay is cumbersome and difficult to reproduce. Also, unlike in Neisseria meningitidis, where serotypes determined by a bactericidal assay could be related to outer membrane protein composition (7), no such relationship is found with H. influenzae. The results also suggest strategies for devising preventive immunotherapy based on outer membrane proteins. The three universal proteins hold the greatest interest at this time since a vaccine containing such a protein would hold INFECT. IMMUN. the most promise of protecting against disease caused by both encapsulated and unencapsulated H. influenzae. ACKNOWLEDGMENTS We appreciate the excellent technical assistance of Thomas J. M. McLaughlin and Suzanne Connett and the typing assistance of Mary Henning. We thank our colleagues Porter Anderson and Richard Insel for helpful discussions and for reviewing the manuscript. This work was supported by Public Health Service grant AI from the National Institute of Allergy and Infectious Diseases to D.H.S. LITERATURE CITED 1. Alexander, H. E Treatment of type b Hemophilus influenza meningitis. J. Pediatr. 25: Anderson, P., A. Flesher, S. Shaw, A. L Harding, and D. H. Smith Phenotypic and genetic variation in the susceptibility of Haemophilus influenza type b to antibodies to somatic antigens. J. Clin. Invest. 65: Anderson, P., R. B. Johnston, Jr., and D. H. Smith Human serum activities against Haemophilus influenza type b. J. Clin. Invest. 51: Anderson, P., G. Peter, R. B. Johnston, Jr., L. H. Wetterlow, and D. H. Smith Immunization of humans with polyribophosphate, the capsular antigen of Haemophilus influenza type b. J. Clin. Invest. 51: Branefors-Helander, P., 0. Nylen, and P. H. Jeppson Acute otitis media. Assay of complement-fixing antibody against Haemophilus influenza as a diagnostic tool in acute otitis media. Acta Pathol. Microbiol. Scand. Sect. B 81: Costerton, J. W., J. M. Ingram, and K.-J. Cheng Structure and function of the cell envelope of gram-negative bacteria. Bacteriol. Rev. 38: Frasch, C. E., R. M. McNelis, and E. C. Gotschlich Strain-specific variation in the protein and lipopolysaccharide composition of the group B meningococcal outer membrane. J. Bacteriol. 127: Granoff, D. M., and R. Rockwell Experimental Haemophilus influenza type b meningitis: immunological investigation of the infant rat model. Infect. Immun. 20: Johnston, K. H., K. K. Holmes, and E. C. Gotschlich The serological classification of Neisseria gonorrhoeae. I. Isolation of the outer membrane complex responsible for serotype specificity. J. Exp. Med. 143: Johnston, R. B., Jr., P. Anderson, F. J. Rosen, and D. H. Smith Characterization of human antibody to polyribophosphate, the capsular antigen of Hemophilus influenzae, type b. Clin. Immun. Immunopathol. 1: Kilian, M A taxonomic study of the genus Haemophilus, with the proposal of a new species. J. Gen. Microbiol. 93: Koplow, J., and H. Goldfine Alterations in the outer membrane of the cell envelope of heptose-deficient mutants of Escherichia coli. J. Bacteriol. -117: Laemmli, U. K Cleavage of the structural proteins during assembly of the head of bacteriophage T4. Nature (London) 227: Loeb, M. R., and J. Kilner Lipopolysaccharide interferes with the staining of lipoprotein on polyacrylamide gels. J. Bacteriol. 137: Medeiros, A. A., and T. F. O'Brien Ampicillinresistant Haemophilus influenzae type b possessing a

9 VOL. 30, 1980 TEM-type fl-lactamase but little permeability barrier to ampicillin. Lancet, i: Moxon, E. R., A. L Smith, D. R. Averill, and D. H. Smith Haemophilus influenza meningitis in infant rats after intranasal inoculation. J. Infect. Dis. 129: Mpairiwe, Y Immunity to Haemophilus influenza type b: the role of capsular antibody. J. Med. Microbiol. 4: Myerowitz, R. L, C. W. Norden, and T. A. Demchak Significance of noncapsular antigens in protection against experimental Haemophilus influenzae type b disease: cross-reactivity. Infect. Immun. 21: Nakae, T Outer membrane ofsalmoneua. Isolation of a protein complex that produces transmembrane channels. J. Biol. Chem. 251: Peltola, H., H. Kayhty, A. Sivonen, and P. H. Makela. OUTER MEMBRANE PROTEINS OF H. INFLUENZAE Haemophilus influenza type b capsular polysaccharide vaccine in children: a double blind field study of 100,000 vaccinees 3 months to 5 years of age in Finland. Pediatrics 60: Schnaitman, C. A Effect of ethlenediaminetetraacetic acid, Triton X-100, and lysozyme on the morphology and chemical composition of isolated cell walls of Escherichia coli. J. Bacteriol. 106: Smith, A. L, D. H. Smith, D. R. Averill, Jr., J. Marino, and E. R. Moxon Production of Haemophilus influenzae b meningitis in infant rats by intraperitoneal inoculation. Infect. Immun. 8: Turk, D. C., and J. R. May Haemophilus influenzae. English University Press, London. 24. Wright, J., and H. K. Ward Studies on influenzal meningitis. II. The problem of virulence and resistance. J. Exp. Med. 55: Downloaded from on November 25, 2018 by guest