Isolation, expression, and nucleotide sequencing of the pilin structural gene of the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius.

In this study we isolated the pilin gene from the Brazilian purpuric fever (BPF) clone of Haemophilus influenzae biogroup aegyptius, expressed the gene in Escherichia coli, and determined its nucleotide sequence. Comparison of the nucleotide sequence of the BPF pilin gene with the sequences of pilin genes from strains of H. influenzae sensu stricto demonstrated a high degree of identity. Consistent with this observation, hemagglutination inhibition studies performed with a series of glycoconjugates indicated that BPF pili and H. influenzae type b pili possess the same erythrocyte receptor specificity.     

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.     

Cloning and sequencing of a genomic island found in the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius

A genomic island was identified in the Haemophilus influenzae biogroup aegyptius Brazilian purpuric fever (BPF) strain F3031. This island, which was also found in other BPF isolates, could not be detected in non-BPF biogroup aegyptius strains or in nontypeable or typeable H. influenzae strains, with the exception of a region present in the type b Eagan strain. This 34,378-bp island is inserted, in reference to H. influenzae Rd KW20, within a choline transport gene and contains a mosaic structure of Mu-like prophage genes, several hypothetical genes, and genes potentially encoding an Erwinia carotovora carotovoricin Er-like bacteriocin. The product of the tail fiber ORF in the bacteriocin-like region shows a hybrid structure where the C terminus is similar to an H. influenzae phage HP1 tail protein implicating this open reading frame in altering host specificity for a putative bacteriocin. Significant synteny is seen in the entire genomic island with genomic regions from Salmonella enterica subsp. enterica serovar Typhi CT18, Photorhabdus luminescens subsp. laumondii TT01, Chromobacterium violaceum, and to a lesser extent Haemophilus ducreyi 35000HP. In a previous work, we isolated several BPF-specific DNA fragments through a genome subtraction procedure, and we have found that a majority of these fragments map to this locus. In addition, several subtracted fragments generated from an independent laboratory by using different but related strains also map to this island. These findings underscore the importance of this BPF-specific chromosomal region in explaining some of the genomic differences between highly invasive BPF strains and non-BPF isolates of biogroup aegyptius.     

Biochemical, genetic, and epidemiologic characterization of Haemophilus influenzae biogroup aegyptius (Haemophilus aegyptius) strains associated with Brazilian purpuric fever.

Brazilian purpuric fever (BPF) is a recently recognized fulminant pediatric disease characterized by fever, with rapid progression to purpura, hypotensive shock, and death. BPF is usually preceded by purulent conjunctivitis that has resolved before the onset of fever. Both the conjunctivitis and BPF are caused by Haemophilus influenzae biogroup aegyptius (formerly called H. aegyptius). Isolates from 15 BPF cases, mainly from blood or hemorrhagic cerebrospinal fluid, case-associated isolates from 42 persons in towns where BPF cases occurred, and control strains from 32 persons in towns without BPF cases were characterized biochemically, genetically, and epidemiologically. Results indicated that a single clone was responsible for all BPF cases identified in six Brazilian towns from 1984 through 1986. All of 15 (100%) case strains were the same clone as was 1 of 32 (3%) control strains (P = less than 10(-8). Isolates of the clone were preferentially intrarelated by DNA hybridization (99% relatedness, hydroxyapatite method at 60 and 75 degrees C) and were separable from other H. influenzae biogroup aegyptius strains (approximately 90% relatedness at 60 degrees C and 82% relatedness at 75 degrees C). All isolates of the BPF clone and no other strains contained a 24-megadalton plasmid of restriction endonuclease type 3031, were of a single multilocus enzyme mobility type, were of a single sodium dodecyl sulfate-polyacrylamide gel electrophoresis type, and were in one of two ribosomal DNA restriction patterns. All BPF clone isolates reacted with monoclonal antibodies produced from a case strain; only 3 of 62 (5%) other strains reacted with this monoclonal antibody. Ninety percent of BPF clone strains and 27% of other strains were relatively resistant to sulfamethoxazole-trimethoprim.     

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.     

Antibodies to lipooligosaccharide of a Brazilian purpuric fever isolate of Haemophilus influenzae biogroup aegyptius lack bactericidal and protective activity.

The immunological basis for protection against Brazilian purpuric fever (BPF), a fulminant infection of young children associated with bacteremia with Haemophilus influenzae biogroup aegyptius, is unknown. Candidate antigens to which protective antibodies may be directed include cell surface proteins and lipooligosaccharide (LOS). We studied the activity of antisera to LOS purified from a BPF H. influenzae biogroup aegyptius isolate. Anti-LOS antisera contained anti-LOS antibody by enzyme immunoassay and immunoblot and no detectable anti-outer membrane protein antibodies by immunoblot. Anti-LOS antisera had minimal bactericidal activity and were not protective against the homologous strain in an infant rat model of bacteremia. Antiserum to whole bacterial cells had a titer of anti-LOS antibody similar to that of anti-LOS antisera and was bactericidal and protective. Removal of anti-LOS antibodies from anti-whole cell antiserum by affinity chromatography did not result in a loss of bactericidal activity. Serum from a normal adult contained anti-LOS antibodies and had bactericidal activity. However, anti-LOS antibodies purified from this serum did not have detectable bactericidal activity. These studies suggest that anti-LOS antibodies produced in rats are not bactericidal and do not contribute to protection against experimental bacteremia with BPF strains of H. influenzae biogroup aegyptius.     

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.     

Comparison of lipopolysaccharides from Brazilian purpuric fever isolates and conjunctivitis isolates of Haemophilus influenzae biogroup aegyptius. Brazilian Purpuric Fever Study Group.

Haemophilus influenzae biogroup aegyptius (H. aegyptius) has been identified as the etiologic agent of the recently described disease Brazilian purpuric fever (BPF). Although there is heterogeneity among the strains associated with conjunctivitis, isolates from patients with BPF appear to be derived from a single clone. The clinical presentation of BPF suggests that bacterial lipopolysaccharides (LPS) are involved in its pathogenesis. We prepared LPS from H. influenzae biogroup aegyptius and found them to be similar to H. influenzae type b LPS in apparent size (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), biological activities, and fatty acid composition. We compared LPS from BPF clone isolates with LPS from non-BPF clone isolates in tests of Limulus lysate activation, spleen cell mitogenesis, promotion of neutrophil adherence to LPS-treated endothelial cells, and the dermal Shwartzman reaction. In none of these activities were LPS from the BPF clone isolates more potent. Because LPS shed from growing bacteria may be involved in the pathogenesis of purpura, we also measured the rate at which LPS were released into culture medium during bacterial growth and found no significant difference between BPF clone and non-BPF clone isolates.     

Role of the 145-kilodalton surface protein in virulence of the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius for infant rats.

Brazilian purpuric fever (BPF) is a fulminant infection associated with bacteremia with clonally related strains of Haemophilus influenzae biogroup aegyptius. Case-associated clone strains are more virulent for infant rats than are non-BPF case-associated H. influenzae biogroup aegyptius isolates. I sought to determine the possible role of P145, a 145-kDa surface protein of BPF case H. influenzae biogroup aegyptius clone isolates, in virulence. First, I compared the virulence of two case-associated clone isolates from the blood of children with BPF from Serrana, Brazil, which differed in P145 expression but were identical in all other phenotypic and genotypic characteristics studied. Twenty-four hours after intraperitoneal inoculation of infant rats, there was a significantly higher incidence (51 versus 26%; P = 0.035) and magnitude (2.9 +/- 5.8 versus 0.7 +/- 2.0 CFU/0.01 ml; P = 0.024) of bacteremia in rats inoculated with the P145-expressing strain. I next compared the virulence of a P145-expressing case-associated clone strain with two P145-nonexpressing phase variants of this strain. One variant exhibited a lower mean magnitude of bacteremia and one displayed a similar magnitude of bacteremia compared with that displayed the P145-expressing parental strain. P145-expressing revertants of the P145-nonexpressing strains exhibited the same virulence as the P145-negative variants from which they were derived. Colonies grown from blood cultures maintained the P145 phenotype of the inoculated strain. These results suggest that P145 expression does not contribute to the virulence of the BPF case clone strain for infant rats following intraperitoneal inoculation.     

Resistance to serum bactericidal activity distinguishes Brazilian purpuric fever (BPF) case strains of Haemophilus influenzae biogroup aegyptius (H. aegyptius) from non-BPF strains. Brazilian Purpuric Fever Study Group.

We studied the ability of normal human serum to lyse H. influenzae biogroup aegyptius (H. aegyptius) isolates recovered from patients with Brazilian purpuric fever (BPF clone) or non-BPF clone strains. BPF clone isolates, although similar to non-BPF clone isolates with regard to the ability to fix C3 to their surfaces, could be distinguished from non-BPF clone strains by their resistance to lysis in vitro following incubation with normal adult human serum.     

Cloning and sequence analysis of the structural pilin gene of Brazilian purpuric fever-associated Haemophilus influenzae biogroup aegyptius.

We have cloned and sequenced the Brazilian purpuric fever (BPF)-associated Haemophilus influenzae biogroup aegyptius (Hae) pilin gene. The sequence contained a 648-bp open reading frame encoding a mature pilin protein of 191 amino acids with a calculated mass of 20.5 kDa. There was 82% homology between the open reading frames of the BPF strain F3031 and H. influenzae type b (Hib) (strain M43) pilin genes and 71% homology at the amino acid level between the mature pilin proteins. However, areas of diversity were noted throughout the gene. A 17-bp probe corresponding to an area of diversity in the N-terminal region of the BPF-associated gene hybridized with other BPF strains but not with non-BPF Hae or Hib. In summary, the pilin protein of BPF-associated Hae is highly homologous to Hib pilin yet remains structurally distinct.     

Role of lipooligosaccharide in virulence of the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius for infant rats.

Clonally related strains of Haemophilus influenzae biogroup aegyptius have recently been associated with Brazilian purpuric fever (BPF), a fulminant, systemic disease in children. Using an infant rat bacteremia model for BPF, we found that a rat blood-passaged BPF isolate of H. influenzae biogroup aegyptius was more virulent than the original strain was. When compared with the original strain, the animal-passaged variant was found to display an altered lipooligosaccharide (LOS) phenotype and to lack pili. To examine the role of LOS phenotype and pili in virulence, we isolated isogenic variants differing in LOS phenotype or expression of pili. The virulence of variants was compared by examining the results of blood cultures obtained 24 h after intraperitoneal inoculation with 10(5) CFU. Our results indicate that the LOS phenotype is a critical determinant of BPF clone virulence for infant rats. To a lesser extent, the absence of piliation and an undefined additional factor(s) contribute to virulence.     

Surface structures and adherence properties of diverse strains of Haemophilus influenzae biogroup aegyptius.

Haemophilus influenzae biogroup aegyptius is an important cause of conjunctivitis and has recently been associated with Brazilian purpuric fever (BPF), a fulminant systemic disease of children. To gain insight into the bacterial factors involved in the pathogenesis of this disease, we investigated the surface structures and adherence properties of eight different strains of H. influenzae biogroup aegyptius, including both BPF and non-BPF isolates. All eight strains were able to express long peritrichous pili similar to those observed in H. influenzae. As in H. influenzae, piliation correlated with colony binding of human erythrocytes. However, two strains were capable of hemagglutination in the absence of pili; in these strains, hemagglutination was insensitive to protease treatment, suggesting a nonproteinaceous hemagglutinin. All strains possessed short, thin, surface fibers distinct from long pili and demonstrated efficient attachment to cultured human conjunctival cells. Attachment to conjunctival cells occurred independently of long pili or a capacity for hemagglutination.     

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.     

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.     

Characterization of P1-deficient isogenic mutant of Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever

Haemophilus influenzae biogroup aegyptius (formerly H. aegyptius) is the etiologic agent of Brazilian purpuric fever (BPF). A surface-exposed epitope on the outer membrane protein P1 is present on most strains of H. influenzae biogroup aegyptius associated with BPF but is absent in almost all non-disease associated strains. The role of the outer membrane protein P1 in the pathogenesis of this disease was evaluated by utilizing an isogenic P1-deficient mutant. We compared the ability of the wild type and P1 isogenic mutant to grow under various conditions. The P1-deficient strain grew at a similar rate to the wild type in both complex and chemically defined medium. The P1-deficient mutant also had a similar growth rate to the wild type under anaerobic conditions. Anaerobic growth, however, resulted in up-regulation of the P1 protein in the wild type strain. Three assays were used to examine the pathophysiologic role of the P1 protein in BPF: 1) serum resistance; 2) sustained bacteremia in the infant rat model; and 3) the human microvascular endothelial cell (HMEC) cytotoxicity assay. Both the mutant and wild-type strains were resistant to killing in 95% normal human serum. The P1-deficient strain was also as virulent as the wild type in both the infant rat model of bacteremia and in the HMEC-1 tissue culture model. These results demonstrate that serum resistance, sustained bacteremia in the infant rat, and cytotoxicity of HMEC cells occur in the absence of P1. The P1 protein is not essential for the pathogenic potential identified by these assays. However, these results demonstrate that an anaerobic environment is a potent physiologic regulator of P1 protein expression. The impact of anaerobiosis on protein expression and pathogenesis will require further investigations.     

Insertion sequence IS1016 and absence of Haemophilus capsulation genes in the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius.

Brazilian purpuric fever (BPF) strains of Haemophilus influenzae biogroup aegyptius form a clone of organisms distinct from more innocuous, conjunctivitis-associated isolates. There has been controversy over whether the virulence of BPF strains might derive from the presence of a polysaccharide capsule analogous to that found in conventional invasive H. influenzae, a controversy fuelled by the observation (G. M. Carlone, L. Gorelkin, L. L. Gheesling, A. L. Erwin, S. K. Hoiseth, M. H. O. Mulks, S. P. Connor, R. S. Weyant, J. Myrick, L. Rubin, R. S. Mumford III, E. H. White, R. J. Arko, B. Swaminathan, L. M. Graves, L. W. Mayer, M. K. Robinson, S. P. Caudill, and the Brazilian Purpuric Fever Study Group, J. Clin, Microbiol. 27:609-614, 1989) that a capsulation DNA probe from H. influenzae type b hybridized uniquely to BPF strains. In this work, the basis for this hybridization has been established as the possession by BPF strains, but not by non-BPF strains, of the Haemophilus insertion element IS1016. Although IS1016 is associated with the capsulation locus in some Haemophilus spp., a Southern hybridization study suggests that in BPF strains there are no capsulation genes.     

Genomic analysis of the F3031 Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius by PCR-based subtractive hybridization

PCR-based subtractive genome hybridization produced clones harboring inserts present in Brazilian purpuric fever (BPF) prototype strain F3031 but absent in noninvasive Haemophilus influenzae biogroup aegyptius isolate F1947. Some of these inserts have no matches in the GenBank database, while others are similar to genes encoding either known or hypothetical proteins. One insert represents a 2.3-kb locus with similarity to a Thermotoga maritima hypothetical protein, while another is part of a 7.6-kb locus that contains predicted genes encoding hypothetical, phage-related, and carotovoricin Er-like proteins. The presence of DNA related to these loci is variable among BPF isolates and nontypeable H. influenzae strains, while neither of them was detected in strains of types a to f. The data indicate that BPF-causing strain F3031 harbors unique chromosomal regions, most of which appear to be acquired from unrelated microbial sources.     

Lipooligosaccharides (LOS) of some Haemophilus species mimic human glycosphingolipids, and some LOS are sialylated.

The lipooligosaccharides (LOS) of strains of Haemophilus ducreyi, Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica contain epitopes that are antigenically and structurally similar to carbohydrates present in human glycosphingolipids. LOS from strains of Haemophilus influenzae and H. influenzae biogroup aegyptius were tested for the binding of monoclonal antibodies (MAbs) that bind to human glycosphingolipids possessing Gal beta 1-4GlcNAc (MAb 3F11) and Gal alpha 1-4Gal beta 1-4Glc (MAb anti-Pk). In solid-phase radioimmunoassays, the LOS of 18 of 19 H. influenzae type b (Hib), 8 of 19 nontypeable H. influenzae, and 10 of 20 H. influenzae biogroup aegyptius strains bound MAb anti-Pk. The LOS of 13 of 19 Hib, 10 of 16 nontypeable H. influenzae, and 2 of 18 H. influenzae biogroup aegyptius strains bound MAb 3F11. Neuraminidase treatment of the strains increased the binding of MAb 3F11 by more than twofold in 47% of the H. influenzae strains, suggesting that sialic acid occluded the LOS structure recognized by MAb 3F11. The material released from neuraminidase-treated Hib LOS was confirmed to be sialic acid by high-performance anion-exchange chromatography. A recombinant plasmid containing genes involved in Hib LOS biosynthesis directed the expression (assembly) of the 3F11 epitope in Escherichia coli. These studies demonstrate that H. influenzae and H. influenzae biogroup aegyptius express at least two LOS epitopes that are similar to those present in human glycosphingolipids. Sialic acid was present on the LOS of some H. influenzae strains and prevented the binding of MAb 3F11 to its epitope. The oligosaccharide portion of sialylated LOS may also resemble sialylated oligosaccharides present in human glycosphingolipids (gangliosides).     

Comparison of outer membrane protein and biochemical profiles of Haemophilus aegyptius and Haemophilus influenzae biotype III.

Haemophilus aegyptius and Haemophilus influenzae biotype III are morphologically and biochemically similar; however, their outer membrane protein (Sarkosyl insoluble) profiles are distinct. Of 18 strains of H. aegyptius examined, 15 had a type 1 protein profile, and 3 had a type 2 profile, whereas the 5 strains of H. influenzae biotype III examined had three other protein profile types. All Haemophilus strains examined had 31- and 76-kilodalton (kDa) proteins and minor proteins with molecular masses between 20 and 100 kDa. H. aegyptius, with a type 1 protein profile, had major outer membrane proteins with apparent molecular masses of 27, 35.5, and 41.5 kDa, and H. aegyptius, with a type 2 protein profile, had 26-, 29-, 39.5-, and 41-kDa proteins. The type strain of H. influenzae biotype III had three major outer membrane proteins with apparent molecular masses of 29, 38.5 and 40 kDa. Four other strains designated as H. influenzae biotype III had major outer membrane proteins between 27 and 41.5 kDa representing two additional protein profiles.