Ferrochelatase activity and protoporphyrin IX utilization in Haemophilus influenzae.

Previous research showed that the heme-requiring human pathogen Haemophilus influenzae lacks the first six of the seven enzymes required for heme synthesis, starting with the precursor, 5-amino levulinic acid. In this study, I demonstrated either directly or by reasonable inference that all 57 strains of H. influenzae examined, including 2 unable to grow on protoporphyrin IX, possess ferrochelatase, which catalyzes heme formation by insertion of Fe2+ into the protoporphyrin IX nucleus and which is the last enzyme in the heme synthetic pathway. Further, I showed that this enzyme can also function in the reverse direction, releasing Fe2+ from heme.

Isolation of the outer membrane of Branhamella catarrhalis.

The emergence of Branhamella catarrhalis as an important human pathogen has stimulated interest in investigations of the outer membrane (OM) of the bacterium. In this study, the OM of B. catarrhalis was isolated and partially characterized. Radiolabelled cells were lysed and fractionated by isopycnic centrifugation in a continuous sucrose gradient. Five fractions were identified. Fraction A consisted of OM fragments of varying density. Fractions B and C were OM of a discrete density containing some cytoplasmic membrane. Fraction D was cytoplasmic membrane and Fraction E contained smaller less dense fragments of cytoplasmic membrane. The protein composition of the Branhamella OM is typical for that of Gram-negative bacteria in that approximately 10 to 20 proteins were present with six to eight of these proteins predominating. Having isolated and partially characterized the OM by sucrose density centrifugation, five simpler techniques for isolating OM were employed and the preparations compared to OM isolated on the gradient. Techniques that are based on differential detergent solubility of OM and cytoplasmic membrane were ineffective in isolating OM of B. catarrhalis. By contrast, techniques that involved collection of OM vesicles were successful in isolating OM of B. catarrhalis. Collection of vesicles from broth culture supernatants and EDTA-heat-induced vesicles were identified as convenient and reliable methods for isolating OM. Isolating and partially characterizing the OM of B. catarrhalis represents an initial step in a systematic study of outer membrane antigens of the bacterium.

A comparison of the adherence of fimbriated and nonfimbriated Haemophilus influenzae type b to human adenoids in organ culture.

Adherence of fimbriated and nonfimbriated variants of a single strain of Haemophilus influenzae type b to organ cultures of human adenoidal tissue was measured by three assays, two of which were quantitative. In one assay, the adherence of radioactively labeled bacteria was measured; the numbers of CFU of bacteria per gram of adenoidal tissue were 16.0 +/- 6.7 for fimbriated bacteria and 10.2 +/- 4.0 for nonfimbriated bacteria (P less than 0.05). In the second assay, adherent CFU were determined directly; the results were 23.4 +/- 17.2 CFU/g of tissue for fimbriated bacteria and 5.1 +/- 2.2 CFU/g for the nonfimbriated variant (P less than 0.02). By combining data from the two assays it appears that fimbriated and nonfimbriated bacteria do not compete for the same site on the tissue, and that the adherent bacteria do not change their state of fimbriation under the assay conditions used. In contrast, the third assay, scanning electron microscopy, showed very poor adherence of nonfimbriated bacteria. Fimbriated bacteria, on the other hand, adhered in clusters to nonciliated epithelial cells. Overall, the data indicate that fimbriae enhance adherence of H. influenzae type b to a type of tissue that is a normal site of human colonization and that nonfimbriated bacteria adhere by a distinctly different mechanism.

Unexpected effects of absorbed normal rabbit serum and bovine serum albumin on survival of Haemophilus influenzae type b in the infant rat.

In the course of using the infant rat model to determine the ability of various rabbit antisera to protect against challenge by Haemophilus influenzae type b we made two unexpected observations. In these experiments 4-day-old rats were inoculated s.c. on the dorsum with either rabbit serum or physiological buffers (sham serum) and then were challenged the next day with H. influenzae type b injected i.p. Bacteremia, as a marker for disease, was measured 24 h later on day 6. We observed the following. (i) Pre-immune, i.e., normal rabbit serum, containing minimal levels of antibodies to outer membrane proteins and depleted of antibodies to capsule and lipopolysaccharide, nevertheless significantly (P less than 0.01) protected the rats from challenge with H. influenzae type b when compared to a sham inoculation of buffer; (ii) In the absence of a serum inoculation on day 4 (a buffer was used as a sham serum inoculation), the levels of bacteremia obtained after inoculation with bacteria on day 5 depended upon the composition of the buffer in which the H. influenzae inoculum was suspended. Use of phosphate buffered saline (PBS) resulted in higher levels of bacteremia than PBS containing 0.5% bovine serum albumin (PBS-BSA) (P less than 0.001), i.e. the BSA apparently acted to protect the rats from H. influenzae infection. In fact the use of PBS-BSA as an inoculum buffer masked the protective effect noted above of the absorbed normal rabbit serum.

Cross-reactivity of surface-exposed epitopes of outer membrane antigens of Haemophilus influenzae type b.

The cross-reactivity of exposed surface epitopes of outer membrane proteins from a spectrum of Haemophilus influenzae type b isolates that varied in their evolutionary distance from each other and in their outer membrane protein composition was analyzed by using an immunoblot assay. The results for outer membrane proteins a, n, and b/c were as follows. (i) A total of 13 of 14 strains possessing a protein a with similar mobilities on gels (i.e., the same apparent molecular weight) as protein a of strain Eag absorbed antibodies to protein a of strain Eag. These strains represented a broad spectrum on a scale of evolutionary distance. (ii) In contrast, only one of seven strains possessing a protein a with different mobilities absorbed these antibodies. (iii) Of five isolates close to strain Eag on the evolutionary scale, the four with a protein n with the same mobility as protein n of strain Eag absorbed antibodies to protein n of strain Eag. (iv) In contrast, of five isolates distant from strain Eag on the evolutionary scale, none absorbed antibodies to protein n, including one strain that had a protein n of the same mobility as that of strain Eag. (v) All strains that absorbed antibodies to protein b/c also absorbed antibodies to lipopolysaccharide, and the reverse of this was also true. Evolutionary distance and mobility of protein b/c on gels were not factors. Control experiments indicated that this result was an artifact due to the strong association of lipopolysaccharide with protein b/c on the gel and subsequent blot. The important conclusions from these experiments, especially pertinent for consideration of these proteins in either whole or peptide vaccines, are that proteins with apparently identical molecular weights can possess different surface-exposed epitopes, that proteins with different molecular weights can possess cross-reactive surface-exposed epitopes, and that some surface-exposed epitopes have been conserved even though the bacterium has undergone evolutionary divergence. In addition, experiments were also performed to determine whether H. influenzae type b strains maintained their integrity during the absorption step, i.e., incubation in antiserum. Strain Eag, which was used as a prototype type b strain, released a small proportion of its membrane (0.13%), but this did not result in exposure of epitopes that were usually buried. In contrast, strain S2, an unencapsulated mutant of strain Eag, was quite unstable, releasing three times as much membrane and a large proportion of its periplasmic proteins.

Protection of infant rats from Haemophilus influenzae type b infection by antiserum to purified outer membrane protein a.

Protein a (46,000 molecular weight [46K]) was purified from outer membranes of Haemophilus influenzae type b by a relatively simple procedure. Spontaneously shed outer membranes from a 24-h, 12-liter culture of an unencapsulated variant of strain Eag were combined with outer membranes released from the cells by Tris buffer and extracted with the nonionic detergent octylpolyoxyethylene. The extract was then subjected to open column chromatography on Sephacryl S-200 and Trisacryl-carboxymethyl to yield 7.5 mg of protein a from 180 mg of outer membrane protein. Approximately 99% of the protein in this preparation was protein a; in addition, the preparation contained 1.25% (wt/wt) lipopolysaccharide and had a residual detergent/protein ratio of 1.6:1 (wt/wt). Antibodies to the preparation were induced in rabbits by using alum as an adjuvant. As determined by immunoblotting, the great preponderance of antibodies induced were specific for protein a. However, very low levels of antibodies to several other outer membrane components, which were not apparent on gels of the pure preparation of protein a, were also induced. Preimmune and postimmune sera, after depletion of antibodies to capsular polysaccharide and lipopolysaccharide, were tested for biological activity against H. influenzae type b. Compared with preimmune serum, postimmune serum was bactericidal in vitro against strain Eag (the only strain tested) and offered significant protection (P less than 0.01) to infant rats against infection by all four strains tested, two of which had a protein a that was larger (47K) than the 46K protein a in the preparation. These results indicate that protein a should be considered as a vaccine to prevent H. influenzae type b disease.

Limited genetic diversity of Haemophilus influenzae (type b).

170 strains of Haemophilus influenzae (serotype b), isolated largely from patients with invasive disease from differing temporal and geographic origins were characterized using the combined approaches of DNA hybridization and outer membrane protein classification. Hybridization of a DNA probe to a region of the chromosome involved in the expression of type b capsular polysaccharide revealed that 163 (96%) isolates had one of three distinct, but closely related, chromosomal restriction fragment length polymorphisms (RFLPs). Each polymorphism was associated with its own distinctive set of outer membrane protein subtypes, indicating that the majority of H. influenzae (type b) isolates have evolved from common ancestors, giving rise to globally distributed organisms that have clonal characteristics.

Variable adherence of fimbriated Haemophilus influenzae type b to human cells.

The attachment of isogenic fimbriated and nonfimbriated Haemophilus influenzae type b variants to human cells was studied by using a radioactive assay and an indirect immunofluorescent assay. As described previously, fimbriated H. influenzae variants adhered to a greater extent than nonfimbriated variants to human buccal epithelial cells (2.1 and 0.29 bacteria per cell, respectively, as determined by the radioactive assay [P less than 0.05]; 7.6 and 1.6 bacteria per cell, respectively, as determined by the immunofluorescent assay [P less than 0.01]). As the concentration of fimbriated bacteria was increased, so were the numbers of adherent bacteria; in contrast, increasing the bacterial concentration had a much smaller effect on adherence of nonfimbriated H. influenzae type b. The distribution of bacteria on the buccal cells also differed. Whereas 37% of the buccal cells failed to bind nonfimbriated H. influenzae type b, failure to bind was observed for only 4% of the buccal cells exposed to fimbriated H. influenzae. In contrast, adherence to human foreskin fibroblasts was low regardless of the presence of fimbriae. On the other hand, fimbriated H. influenzae type b adhered less well than nonfimbriated variants to HEp-2 cells (1.6 and 3.8 bacteria per cell, respectively, as determined by the radioactive assay [P less than 0.05]; 1.3 and 4.8 bacteria per cell, respectively, as determined by the immunofluorescent assay [P less than 0.02]). Whereas adherence to HEp-2 cells increased considerably as the concentration of nonfimbriated bacteria was increased, there was only a small enhancement of adherence with an increase in the concentration of fimbriated H. influenzae type b. Furthermore, only 16% of the HEp-2 cells failed to bind nonfimbriated H. influenzae type b, whereas 50% failed to bind fimbriated H. influenzae type b. These data indicate that H. influenzae type b may contain two adhesins. One is associated with fimbriae and enables adherence to buccal cells, whereas the other is nonfimbrial and is associated with adherence to HEp-2 cells. It is not known whether either of these adhesins plays a role in pathogenesis.

Immunoblot method for identifying surface components, determining their cross-reactivity, and investigating cell topology: results with Haemophilus influenzae type b.

Methods currently used for identifying exposed membrane components of gram-negative bacteria can give false positive results, and also do not provide information on nonprotein components such as lipopolysaccharide and polysaccharide capsules. A method, described within, has been developed to overcome these limitations. Briefly an outer membrane preparation derived from encapsulated (type b) Haemophilus influenzae was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated components were then transferred electrophoretically to nitrocellulose. The nitrocellulose was cut into vertical strips, which were then each incubated with rabbit antiserum to the whole bacterium or with the same antiserum after absorption with any of the following: the same strain of H. influenzae, a capsule-deficient mutant of that strain, other strains of H. influenzae, or other bacteria. The strips were then incubated with 125I-protein A, and the bound antibodies were detected by autoradiography. The autoradiograph of the strip exposed to unabsorbed antisera revealed the identity of those individual outer membrane components that bound antibodies. A comparison of the intensity of the various bands on this strip with those on the strips exposed to absorbed antisera was then used to identify (1) surface-exposed components, (2) those components occluded by capsule, and (3) cross-reactivity of exposed components. This method should be applicable to other cells and subcellular particles. Its major disadvantage is that it can provide false negative results.

A hemadsorption method for detection of colonies of Haemophilus influenzae type b expressing fimbriae.

Although fimbriated variants of Haemophilus influenzae type b have recently been described, cultures of most clinical isolates contain only a small proportion of fimbriated forms. Because colonies of fimbriated and nonfimbriated cells are visually indistinguishable, a rapid, simple method was developed for the identification and quantitation of colonies of fimbriated H influenzae. This procedure, also applicable to other bacteria (for example, Escherichia coli), involves transferring the colonies from agar to nitrocellulose disks and incubating the disks in a suspension of red blood cells. Colonies that contain predominantly fimbriated bacteria bind the red blood cells and appear as red dots on the nitrocellulose. This nitrocellulose hemadsorption method is described, as well as its applicability for determining the proportion of fimbriated cells in a culture, the kinetics of enrichment of fimbriated forms during enrichment procedures, and the transition rate from the nonfimbriated to the fimbriated state.

Lactoperoxidase and Iodo-Gen-catalyzed iodination labels inner and outer membrane proteins of Haemophilus influenzae.

Both inner and outer membrane proteins of Haemophilus influenzae type b were labeled by iodination procedures believed to be specific for exposed surface proteins only. It is suggested that this is due to specific properties of the outer membrane of H. influenzae and that use of these procedures with other gram-negative bacteria be evaluated carefully.

Human antibody response to individual outer membrane proteins of Haemophilus influenzae type b.

To evaluate the potential of outer membrane proteins of Haemophilus influenzae as a vaccine, sera from 11 healthy persons and from 23 patients convalescing from disease caused by Haemophilus influenzae type b were assayed for antibodies to individual outer membrane proteins of a single type b isolate, strain Eag, by a gel radioimmunoassay. All 23 patients, ranging in age from 2 months to 62 years, with 17 patients being 24 months or less, had antibodies to some of these proteins in their sera (range, antibodies to 4 to 17 proteins per patient). Although the intensity and spectrum of the response varied, all patients had antibodies to one particular outer membrane protein and 19 patients had antibodies to another, with those patients 5 years and older having antibodies to more proteins than did infants (<==24 months). In the two cases examined, convalescent sera had greater amounts and broader spectra of antibodies than did acute sera. In addition, 10 of 11 healthy subjects not known to have had systemic H. influenzae disease also had antibodies to individual outer membrane proteins, with older children having greater amounts than did their younger siblings and with children showing a different spectrum of response than that for adults. Thus, antibodies to outer membrane proteins are commonly found in humans. Also, these results and those demonstrating that hyperimmune rabbit antisera to strain Eag reacted with each of five type b substrains possessing some different outer membrane proteins indicate considerable cross-reactivity among these proteins. These results encourage continued consideration of outer membrane proteins in a vaccine.

A polysaccharide-protein complex from Haemophilus influenzae type b. II. Human antibodies to its somatic components.

A polysaccharide-protein complex prepared from Haemophilus influenzae type b strain Eagan was used as test antigen in an enzyme-linked immunosorbent assay for human serum antibodies. With washing buffer that did not contain detergent, the assay detected antibody to lipopolysaccharide (LPS) and to non-LPS somatic antigens as well as to polyribosylribitolphosphate (PRP. With buffer that did contain detergent, antibodies to LPS were not detected, whereas detection of antibodies to the non-LPS somatic components and to PRP wa unimpeded. Similarly, purified LPS could be used as test antigen with the former buffer only. IgG, IgA, and IgM antibodies to non-LPS somatic antigens were prevalent in healthy adults and children, and levels increased in 14 of 15 patients recovering from meningitis due to H. influenzae type b; IgG was the predominant class. Antibodies to LPS were prevalent but at lower concentrations, and IgM was the predominant class; levels increased in 12 of the 15 patients.

Isolation and partial characterization of outer and inner membranes from encapsulated Haemophilus influenzae type b.

A method has been developed to separate the cell envelope of encapsulated (type b) Haemophilus influenzae into its outer and inner membrane components with procedures that avoided two problems encountered in fractionation of this envelope: (i) the tendency of the outer and inner membranes to hybridize and (ii) the tendency of the apparently fragile inner membrane to fragment into difficulty sedimentable units. Log phage cells, whose lipids were radioactively labeled, were lysed by passage through a French press. The lysate was applied to a discontinuous sucrose gradient, and envelope-rich material was collected by centrifugation onto a cushion of dense sucrose under carefully controlled conditions. This material was then further fractionated by isopycnic centrifugation in a sucrose gradient to yield four membrane fractions which were partially characterized. On the basis of their radioactivity, buoyant density, ultrastructure, polypeptide composition, and content of phospholipid, protein, lipopolysaccharide, and succinic dehydrogenase, these fractions were identified as follows: fraction 1, outer membrane vesicles with very little inner membrane contamination (less than 4%); fraction 2, outer membrane vesicles containing entrapped inner membrane; fraction 3, a protein-rich fraction of inner membrane; fraction 4, a protein-poor fraction of inner membrane. Fractions 3 and 4 contained about 25% outer membrane contamination.

Outer membrane protein composition in disease isolates of Haemophilus influenzae: pathogenic and epidemiological implications.

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.

Effect of growth medium on the relative polypeptide composition of cellular outer membrane and released outer membrane material in Escherichia coli.

When ratios of the major polypeptides of the outer membrane isolated from cells of Escherichia coli B grown in minimal medium containing either a single amino acid or several amino acids were compared, no difference was observed. However, the ratio of these polypeptides in outer membrane material released into the medium during logarithmic phase growth on these two media was markedly different.