Outer membrane protein and biotype analysis of non-serotypable strains of Haemophilus influenzae.

Strains of Haemophilus influenzae (n = 161) were isolated from inpatients with symptoms of pulmonary infection. Conventional tests showed that 144 strains were non-serotypable and all belonged to one of eight biotypes. The common biotypes were 2 (41%), 3 (27.1%), 1 (13.2%) and 5 (10.4%). The outer membrane protein (OMP) profiles of 59 non-serotypable strains were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). A comparison of OMP profiles suggested a possible association between several strains belonging to biotype 2. Although no clear correlation was established between biotype or OMP profile cluster groups and the age or clinical state of the patients from whom the strains were isolated, SDS-PAGE analysis was a useful technique for the epidemiological study of non-serotypable H influenzae.     

Distinct antigenic and genetic properties of the immunoglobulin A1 protease produced by Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever in Brazil.

All examined Haemophilus influenzae biogroup aegyptius isolates of the clone associated with Brazilian purpuric fever (the BPF clone) produced type 2 immunoglobulin A1 (IgA1) proteases encoded by identical iga genes that were distinct from the iga genes of other Brazilian H. influenzae biogroup aegyptius isolates. A partial nucleotide sequence analysis revealed close similarities to the iga genes of H. influenzae serotype c and one noncapsular H. influenzae biotype III strain isolated from a case of conjunctivitis in Tunisia, suggesting an evolutionary relationship. Epitopes recognized by neutralizing antibodies differed for the IgA1 proteases of the BPF clone and of other H. influenzae strains, including Brazilian H. influenzae biogroup aegyptius isolates from patients with noninvasive conjunctivitis. The low probability of developing cross-reacting neutralizing antibodies to the IgA1 protease of the BPF clone may contribute to the pathogenic potential of this virulent phenotype in Brazil.     

Outer membrane protein and biotype analysis of pathogenic nontypable Haemophilus influenzae.

The techniques of biotype determination and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane protein preparations were applied to 35 epidemiologically unrelated isolates of pathogenic nontypable Haemophilus influenzae. Three of five isolates obtained from the blood of unrelated newborns with sepsis had concordant major outer membrane from the blood of unrelated older children or adults with bacteremia had concordant major outer membrane protein profiles, distinct from the common profile of neonatal strains, and were biotype II. The outer membrane protein profiles of the remaining 5 isolates from blood, 2 isolated from cerebrospinal fluid, and 23 isolated from middle ear aspirates of children with otitis media were unique, although each isolate had peptides with apparent molecular weights of 16,000 and 31,500. These results suggest that a subset of nontypable isolates associated with bacteremia has distinctive strain markers. Their pathogenicity may relate to a prediction for colonizing the female genital tract in the case of the common neonatal strain or an increased ability to evade host defenses.     

Haemophilus influenzae type b isolates show antigenic variation in a major outer membrane protein.

Antigenic variation of the outer membrane proteins among isolates of Haemophilus influenzae was examined by immunoblotting. Rabbit antisera were raised against six strains of H. influenzae type b and tested against outer membrane preparations of 50 isolates. The principal outer membrane band was not reactive on immunoblotting, so its antigenic heterogeneity could not be examined. Most of the other outer membrane proteins shared common determinants among all strains tested. Absorption of serum with heterologous bacteria removed antibody to nearly all proteins, confirming the extensive cross-reactivity among isolates. The greatest antigenic variation was seen in one major outer membrane band, a heat-modifiable, Zwittergent-soluble protein with a molecular weight of 49,000 to 51,000. One antiserum reacted with the 49,000-to-51,000-molecular-weight protein of the homologous isolate only; the remaining five antisera showed differing patterns of reactivity with heterologous 49,000-to-51,000-molecular-weight proteins. We were able to divide the 50 H. influenzae isolates into 13 antigenic groups based on their reaction patterns. The antigenic groupings may provide an epidemiological tool for studying the prevalence and transmission of strains of H. influenzae type b.     

Outer membrane protein profiles of Haemophilus pleuropneumoniae.

Outer membrane protein profiles of Haemophilus pleuropneumoniae were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cells were disrupted by sonication, and outer membrane-enriched fractions were prepared by differential centrifugation and selective solubilization of the inner membrane with sodium N-lauroyl sarcosinate. Colony type, growth medium, time of harvest, and in vitro or in vivo passage had no appreciable effect on the protein profiles of the strains examined. Seven patterns were distinguished among the reference strains of the nine capsular serotypes. These patterns were based on the mobility of the major outer membrane proteins migrating in the 39,000- to 44,000-molecular-weight region of the gel, a 16K to 16.5K protein, and a heat-modifiable 29K protein. Strains of serotypes 1 and 9 had identical outer membrane protein profiles, as did strains of serotypes 2 and 6. The reference strains of the remaining five serotypes each had a distinct pattern. The outer membrane protein profiles of 95 field isolates belonging to serotypes 1, 5, 7, and 9 from swine in the midwestern United States were determined and compared with the reference patterns. The results indicate that the population of H. pleuropneumoniae is clonal, with three predominant clones distinguished by both serotype and outer membrane protein profile responsible for the majority of H. pleuropneumoniae disease occurring in swine in the United States.     

Stable, conserved outer membrane epitope of strains of Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever.

Brazilian purpuric fever is a rapidly fatal childhood disease associated with a clonal strain of Haemophilus influenzae biogroup aegyptius. We describe a conserved, surface-exposed epitope present on 95% of H. influenzae biogroup aegyptius isolates that are associated with Brazilian purpuric fever. This epitope, defined by reaction with the monoclonal antibody 8G3, is on or associated with the 48-kDa heat-modifiable P1 protein. The epitope is absent on strains of H. influenzae biogroup aegyptius that are not associated with Brazilian purpuric fever but is present on one strain of H. influenzae biotype II. None of 81 other Haemophilus strains tested reacted with 8G3. The sensitivity and specificity of the 8G3 monoclonal antibody in detecting Brazilian case-clone strains of H. influenzae biogroup aegyptius associated with Brazilian purpuric fever are 95 and 99%, respectively. Immunoelectron microscopy revealed that the epitope is surface exposed, and N-terminal amino acid sequencing of an 8G3-reactive P1 protein from a strain of H. influenzae biogroup aegyptius showed 100% correlation with the published N-terminal amino acid sequence of a P1 protein of H. influenzae type b. The virulence of the organism in an infant rat model of bacteremia was not dependent on the expression of this epitope.     

Expression of an immunoreactive 72 kDa protein in strains of Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever

Brazilian purpuric fever (BPF) is a newly described pediatric syndrome that results in significant morbidity and mortality. BPF is caused by specific phenotypic strains of Haemophilus influenzae biogroup aegyptius that are capable of intravascular survival. Immunoblotting of outer membrane proteins of H. influenzae biogroup aegyptius with normal human serum showed that most virulent strains of H. influenzae biogroup aegyptius associated with BPF expressed an immunologically prominent protein at 72 kDa. A corresponding protein in avirulent isolates migrated at 79 kDa. Although a minor component on SDS-PAGE analysis of the outer membrane, specific antibody against this protein is present in high concentrations in normal human serum.     

Purification and characterization of a pilin specific for Brazilian purpuric fever-associated Haemophilus influenzae biogroup aegyptius (H. aegyptius) strains.

Brazilian purpuric fever (BPF) is a recently described fatal pediatric disease caused by systemic infection with Haemophilus influenzae biogroup aegyptius. Previous studies have shown that all H. influenzae biogroup aegyptius strains isolated from BPF cases and case contacts share several unique phenotypic and genotypic characteristics that differentiate them from other H. influenzae biogroup aegyptius strains isolated from conjunctivitis cases in Brazil. One key characteristic of this BPF clone is reactivity in a BPF-specific monoclonal antibody enzyme-linked immunosorbent assay. We have purified and partially characterized a pilin, referred to as the 25-kilodalton (kDa) protein. Aggregates of this protein contain a heat-labile epitope which is recognized by a monoclonal antibody used in the BPF-specific enzyme-linked immunosorbent assay. The protein has a molecular weight of approximately 25,000, is insoluble in most detergents, and fractionates with outer membrane vesicles after LiCl extraction. Biochemical analysis of the 25-kDa protein shows it to have an amino acid composition similar but not identical to that of the H. influenzae type b pilin. The sequence of 20 N-terminal amino acids of the 25-kDa protein shows almost complete homology with the N terminus of the H. influenzae type b pilin and the types 1 and P pilins of Escherichia coli. Transmission electron microscopic analysis of the purified protein shows the presence of filamentous structures similar in morphology to those of H. influenzae pili. Reactivity between the 25-kDa protein and the BPF-specific monoclonal antibody is demonstrated by Western blotting (immunoblotting) and colloidal gold-enhanced immunoelectron microscopy. Hemadsorption analysis shows that expression of this protein is associated with increases in piliated cells and enhanced binding of these cells to human erythrocytes. These studies indicate that expression of the 25-kDa protein is a characteristic unique to the BPF clone and suggest that this protein plays a role in the pathogenesis of BPF.     

Difference in structure between type b and nontypable Haemophilus influenzae populations

The extent of chromosomal genetic variability and the genetic structure of Haemophilus influenzae populations was analyzed. A total of 119 isolates from humans in G?teborg, Sweden, and Birmingham, Ala., and 16 strains from a type culture collection were characterized for capsular type, biotype, outer membrane protein profile, and enzyme electrophoretic type (ET). The results of this study indicate that the bacteria identified as H. influenzae are a genetically extremely variable array of organisms. For the six enzymes studied, the estimated mean genetic diversity was 0.57 (approximately 20% higher than the corresponding estimate for Escherichia coli). Two lines of evidence indicate that despite its ability to recombine by transformation, H. influenzae maintains a largely clonal population structure. Although there is considerable potential for generating different genotypes, there were only 88 distinct ETs among the 135 strains, and isolates of the same ET and biotype were recovered at frequencies greater than would be anticipated at random. This evidence for a clonal population structure holds for uncapsulated as well as capsulated strains. However, these data also suggest that the stability of H. influenzae clones (clone persistence time) may be less than that of the nontransforming species E. coli. The ET data indicate that there is somewhat less variability among H. influenzae strains that express the same capsular antigens, biotype, and outer membrane proteins than among randomly chosen isolates. Nevertheless, there is substantial genetic variation among isolates within each of these classes and combinations thereof. There is also variation in these typing characteristics among strains of the same ET. These observations and those on genetic variability and population structures have implications for the characterization of H. influenzae isolates in clinical and epidemiological studies.     

Fur and iron transport proteins in the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius.

The Brazilian purpuric fever (BPF) clone of Haemophilus influenzae biogroup aegyptius causes a fatal septicaemic disease, resembling fulminant meningococcal sepsis, in children. When isolate F3031 was grown under iron-limiting conditions, the presence of several iron-regulated proteins of 38-110 kDa was revealed by electrophoretic analysis and a Fur homologue was shown by immunoblotting. Dot-blot assays and immunoblotting indicated that BPF cells bound human transferrin and contained transferrin-binding proteins in the outer membrane. However, the binding activity and the biosynthesis of these proteins were detected even under iron-rich conditions. Immunoblot analysis demonstrated the presence of a periplasmic protein related to the ferric iron-binding protein A (FbpA), the major iron-binding protein described in Neisseria spp. However, the FbpA homologue in strain F3031 was constitutively expressed and was smaller than the periplasmic protein detected in H. influenzae type b strain Eagan. The periplasm of strain F3031 also contained a protein related to the Streptococcus parasanguis FimA protein which recently has been shown to be involved in iron acquisition in Yersinia pestis. Although the Eagan and F3031 FimA homologues had a similar mol. wt, of 31 kDa, the expression of the BPF fimA-like gene was not regulated by the iron concentration of the culture medium.     

Frequency and properties of naturally occurring adherent piliated strains of Haemophilus influenzae type b.

We found that 41 of 75 (55%) children with Haemophilus influenzae type b disease (70 cases of meningitis, 2 of cellulitis, 2 of septic arthritis, and 1 of epiglottitis) and 2 of 120 (1.7%) children with upper respiratory infection were colonized with H. influenzae type b in the nasopharynx (NP). Of these 43 NP strains from children with systemic H. influenzae type b disease, 7 (16%) adhered to human buccal epithelial cells. The strains isolated from the systemic site of all children, including children from whose NP adherent bacteria were isolated, did not adhere to buccal epithelial cells in vitro. Each adherent NP strain had biotype (I), serotype (b), and antibiotic susceptibility (sensitive) similar to that of the corresponding nonadherent systemic isolate. With one exception, all NP-systemic pairs had similar major outer membrane proteins. Six of the seven NP strains had a protein band in the whole cell lysate preparation with a molecular weight between 22,000 and 23,000, which could not be seen in the nonadherent cerebrospinal fluid strains. Electron micrographs of all adherent strains showed that more than 95% of the organisms examined were highly piliated, whereas the nonadherent strains were not piliated. All piliated strains agglutinated human erythrocytes. Adherence to buccal epithelial cells and agglutination of erythrocytes could not be blocked by mannose or alpha-methyl-D-mannoside. We speculate that piliation is not important for NP colonization by H. influenzae type b and that the loss of pili may be required for host invasion.     

Typing of urogenital, maternal, and neonatal isolates of Haemophilus influenzae and Haemophilus parainfluenzae in correlation with clinical source of isolation and evidence for a genital specificity of H. influenzae biotype IV.

Over a period of 6 years, 114 strains of Haemophilus influenzae and Haemophilus parainfluenzae were isolated from genital, mother-infant, or neonatal infections. Their serotypes, biotypes, antibiotic resistance phenotypes, and outer membrane protein (OMP) electrophoretic patterns were characterized and correlated with the various clinical outcomes. Genital H. influenzae and H. parainfluenzae appeared to behave mostly as opportunistic pathogens; for instance, 62% of the cases of endometritis or pelvic inflammatory disease were related to the presence of an intrauterine device. However, as seen clearly in one case, the strains may be sexually transmitted. The analysis of OMP patterns proved to be a very convenient method to seek evidence for the sexual origin of the infection. H. influenzae was more often involved in complicated genital infections than was H. parainfluenzae. Nontypeable and biotype II H. influenzae strains were the more frequent isolates, except in pelvic inflammatory diseases, in which biotype I prevailed, and in mother-infant infections, in which one-fourth of the cases were due to biotype IV. Characterization of H. influenzae isolates did not support a general concept of specific genital strains. However, strains of biotype IV clearly stood out with two characteristics: (i) a peritrichous fimbriation and (ii) a very peculiar homogeneous OMP pattern comprising an OMP of molecular weight approximately 18,000 unique to this biotype. These characteristics were also found in H. influenzae biotype IV strains isolated from genital infections in the United States and used as controls. H. influenzae biotype IV strains may thus correspond to a group somewhat adapted to the genital tract.     

Conservation of immune responses to proteins isolated by preparative polyacrylamide gel electrophoresis from the outer membrane of nontypeable Haemophilus influenzae.

Outer membrane proteins P2, P4, and P6 and two with molecular masses of 26 and 28 kDa have been purified from a strain of nontypeable Haemophilus influenzae by a preparative form of polyacrylamide gel electrophoresis (PAGE). Outer membrane protein P6, with a molecular mass of 16 kDa (determined by sodium dodecyl sulfate [SDS]-PAGE) was purified by both native PAGE and SDS-PAGE from three strains of nontypeable H. influenzae and one strain of type b H. influenzae. The same conditions were required for purification from each strain. The suitability of proteins isolated by these methods was assessed by studying the immune response of rats immunized with P6 in incomplete Freund's adjuvant into the Peyer's patches. P6 purified by either native PAGE or SDS-PAGE did not differ significantly from P6 purified by gel filtration and anion-exchange chromatography in the ability to enhance pulmonary clearance of live bacteria. This study also investigated the effects of SDS on P2 immunological responses in vivo and the effects of the reagents Zwittergent and sodium lauryl sarcosinate on outer membrane protein lymphocyte-proliferative responses in vitro. It was found that the presence of SDS in the immunization emulsion enhanced the antigen-specific cell-mediated response but suppressed the antigen-specific antibody responses. The presence of residual traces of Zwittergent in an outer membrane protein preparation inhibited antigen-specific cell-mediated proliferation, whereas extraction of outer membrane proteins with sodium lauryl sarcosinate did not inhibit antigen-specific proliferation. These results demonstrate that preparative PAGE is a suitable method for the purification of proteins from the outer membrane of H. influenzae required for investigation of their immunological significance as vaccine candidates and that traces of reagents used during protein purification may play an important role in determining the success of in vivo and in vitro studies.     

Major subtypes of invasive Haemophilus influenzae from 1983 to 1985 in Atlanta, Ga.

We compared outer membrane protein (OMP) patterns of Haemophilus influenzae isolated in metropolitan Atlanta, Ga., from July 1983 to June 1985. Of 74 randomly selected H. influenzae serotype b, biotype I, isolates (24% of the total number of H. influenzae, and 32% of the total number of H. influenzae serotype b, biotype I, isolates), 66 (89.2%) had the same OMP pattern. Of the remaining eight, five (6.7%) had an identical OMP pattern. The other three isolates had separate and distinct patterns. A greater diversity of OMP patterns was found with H. influenzae serotype b, biotype II, and nonserotypeable H. influenzae. Of the 18 H. influenzae serotype b, biotype II, isolates (5.8% of the total number of H. influenzae isolates), 1 had an OMP pattern similar to that of the predominate biotype I OMP type, 6 (33% of the biotype II) had the same pattern, and 11 had heterogeneous patterns. Of the 19 recoverable, nonserotypeable biotype II isolates (6.8% of the total number of H. influenzae), 18 had different OMP patterns, and no pattern was similar to those observed with serotype b. These findings indicate that most H. influenzae strains isolated during this 2-year period were indistinguishable by serotype, biotype, or OMP patterns.     

Antibody response of infants to cell surface-exposed outer membrane proteins of Haemophilus influenzae type b after systemic Haemophilus disease.

The immune response of nine infants with Haemophilus influenzae type b meningitis was examined by using a radioimmunoprecipitation procedure designed to detect antibodies directed against cell surface-exposed outer membrane proteins of this pathogen. Using intrinsically or extrinsically radiolabeled intact H. influenzae type b cells with acute- and convalescent-phase human sera in this radioimmunoprecipitation system, we found that all of the infants produced an antibody response directed against several different H. influenzae type b outer membrane proteins. Anti-H. influenzae type b outer membrane protein antibodies present in convalescent sera, but not found in acute sera, were directed against cell surface-exposed H. influenzae type b outer membrane proteins. In contrast, both acute and convalescent sera contained antibody activity directed against numerous H. influenzae type b outer membrane proteins whose antigenic determinants were apparently inaccessible to antibody on intact H. influenzae type b cells. The ability of infants to develop an antibody response to cell surface-exposed, antibody-accessible H. influenzae type b outer membrane proteins indicates that these proteins may have vaccinogenic potential.     

The penicillin binding proteins of the genus Haemophilus

We questioned whether the penicillin binding protein (PBP) profiles of representative strains from the 19 species varied within the genus Haemophilus and whether these profiles would be of taxonomic value. Seventeen of the 19 representative strains studied had distinct PBP profiles; only those of H. avium and H. paragallinarum were identical. The data support the inclusion of H. aegyptius in the genus as a species related to but separate from H. influenzae and could not exclude H. somnus, H. agni, and H. equigenitalis from the genus. Comparative PBP analysis within the genus Haemophilus may therefore be useful taxonomically.     

A set of two monoclonal antibodies specific for the cell surface-exposed 39K major outer membrane protein of Haemophilus influenzae type b defines all strains of this pathogen.

Six murine plasma cell hybridomas producing monoclonal antibodies (mabs) directed against the 39,000-molecular-weight (39K) major outer membrane protein of Haemophilus influenzae type b were employed in the antigenic analysis of the 39K protein. The initial characterization of the mabs by radioimmunoprecipitation analysis showed that four of these mabs reacted with antigenic determinants of the 39K protein that are exposed on the bacterial cell surface and accessible to antibody. The other two mabs reacted with antigenic determinants of the 39K protein that are either not exposed on the H. influenzae type b cell surface or not accessible to antibody (internal determinants). A total of 126 clinical isolates of H. influenzae type b obtained from pediatric research centers throughout the United States were examined for reactivity with the six mabs by using a solid-phase radioimmunoassay in which bacterial colony growth from agar plates was placed on filter paper and used as antigen. The reactivities of these strains with two of the mabs recognizing cell surface-exposed antigenic determinants of the 39K protein were used to divide the 126 strains into four different groups. Group 1 strains reacted with mab 12D9, group 2 strains reacted with mab 4C4, group 3 strains reacted with both mabs 12D9 and 4C4, and group 4 strains (only one was found) did not react with either mab. The reactivities of two other mabs recognizing cell surface-exposed antigenic determinants of the 39K protein were used to further divide the four groups into eight subgroups. A single mab recognizing an internal antigenic determinant of the 39K protein reacted with every H. influenzae type b strain examined in this study. These data indicate that only limited antigenic heterogeneity exists among the cell surface-exposed antigenic determinants of the 39K outer membrane proteins among H. influenzae type b strains and that at least one internal antigenic determinant of the 39K protein is universally present in all H. influenzae type b strains. Radioimmunoprecipitation analysis also demonstrated that H. influenzae type b strains which lacked a 39K major outer membrane protein possessed a 38K major outer membrane protein which reacted with the anti-39K mabs, indicating that the 38K and 39K outer membrane proteins of different H. influenzae type b strains are antigenically related.     

DNA sequence analysis and restriction fragment length polymorphisms of the P1 gene of Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever.

Brazilian purpuric fever (BPF) is a fulminant pediatric disease caused by specific strains of Haemophilus influenzae biogroup aegyptius. A conserved epitope on the P1 protein of strains of H. influenzae biogroup aegyptius is seen on most virulent isolates. The P1 protein from a Brazilian case-clone strain of H. influenzae biogroup aegyptius was analyzed by cloning and sequencing the gene. Three major variable regions are present within the P1 gene of the BPF clone in an architecture similar to that of the previously sequenced P1 genes from H. influenzae. The DNA sequence data of the P1 gene provided information for restriction fragment length polymorphism analyses among strains of H. influenzae biogroup aegyptius. Using PCR for amplification of the P1 gene, we found that AlwI restriction of this gene allowed for a highly accurate segregation of virulent strains of H. influenzae biogroup aegyptius associated with BPF. The strong association of virulent phenotypes with specific AlwI restriction patterns of the P1 gene provides a basis for the convenient and accurate identification of strains of H. influenzae biogroup aegyptius which cause BPF.     

Comparative analysis of the structures of the outer membrane protein P1 genes from major clones of Haemophilus influenzae type b.

P1 outer membrane proteins from Haemophilus influenzae type b are heterogeneous antigenically and with respect to apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For determination of the molecular basis for the differences in the P1 proteins, the genes for the P1 proteins from strain 1613, representative of outer membrane protein subtype 3L, and strain 8358, representative of outer membrane protein subtype 6U, were cloned, sequenced, and compared with the previously reported gene for the P1 protein from strain MinnA, a strain with the outer membrane protein subtype 1H. These prototype strains are representatives of the three major clonal families of H. influenzae type b responsible for invasive disease in diverse areas of the world. The nucleotide sequences of the P1 genes from strains 1613 and 8358 were 94 and 90% identical to the MinnA sequence, respectively. The derived amino acid sequences were 91 and 86% identical, respectively. Heterogeneity between the MinnA and 1613 proteins was largely localized to two short variable regions; the protein from strain 8538 contained a third variable region not observed in the other P1 proteins. Thus, the outer membrane protein P1 genes are highly conserved; the variable regions may code for the previously demonstrated strain-specific antigenic determinants.