Limited diversity of the immunoglobulin A1 protease gene (iga) among Haemophilus influenzae serotype b strains.

Immunoglobulin A1 (IgA1) proteases are thought to be important virulence factors in certain bacterial infections, including meningitis, and may have potential usage in vaccines. In this study, we compared the locations of EcoRI, BamHI, and PstI restriction endonuclease sites in the IgA1 protease gene (iga) region of whole-cell DNA from 76 Haemophilus influenzae strains. The analysis was performed by using isolated fragments of the cloned iga gene, which encodes the IgA1 protease originating from a H. influenzae serotype d strain, as probes in Southern blot experiments. All strains, including three without detectable IgA1 protease activity, had DNA sequences with a high degree of homology to the iga probes. The numbers and sizes of the DNA fragments hybridizing with the probes indicated that only three strains, none of which was of serotype b, had more than one iga gene. The iga restriction fragment length patterns of 60 clinical isolates of serotype b were of only four distinct types, which correlated with previously observed clusters of multilocus genotypes (electrophoretic types). This correlation supports the concept of the clonal population structure of H. influenzae. Three of the iga gene restriction types, which appear to represent 98% of the H. influenzae serotype b population, encode IgA1 proteases that were inhibited by antisera to any one of these types and therefore could form the basis for the development of a vaccine against H. influenzae meningitis.     

Characterization of the Streptococcus pneumoniae immunoglobulin A1 protease gene (iga) and its translation product.

Bacterial immunoglobulin A1 (IgA1) proteases constitute a very heterogenous group of extracellular endopeptidases which specifically cleave human IgA1 in the hinge region. Here we report that the IgA1 protease gene, iga, of Streptococcus pneumoniae is homologous to that of Streptococcus sanguis. By using the S. sanguis iga gene as hybridization probe, the corresponding gene from a clinical isolate of S. pneumoniae was isolated in an Escherichia coli lambda phage library. A lysate of E. coli infected with hybridization-positive recombinant phages possessed IgA1-cleaving activity. The complete sequence of the S. pneumoniae iga gene was determined. An open reading frame with a strongly biased codon usage and having the potential of encoding a protein of 1,927 amino acids with a molecular mass of 215,023 Da was preceded by a potential -10 promoter sequence and a putative Shine-Dalgarno sequence. A putative signal peptide was found in the N-terminal end of the protein. The amino acid sequence similarity to the S. sanguis IgA1 protease indicated that the pneumococcal IgA1 protease is a Zn-metalloproteinase. The primary structures of the two streptococcal IgA1 proteases were quite different in the N-terminal parts, and both proteins contained repeat structures in this region. Using a novel assay for IgA1 protease activity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we demonstrated that the secreted IgA1 protease was present in several different molecular forms ranging in size from approximately 135 to 220 kDa. In addition, interstrain differences in the sizes of the pneumococcal IgA1 proteases were detected. Southern blot analyses suggested that the S. pneumoniae iga gene is highly heterogenous within the species.     

Cleavage of chimpanzee secretory immunoglobulin A by Haemophilus influenzae IgA1 protease.

Immunoglobulin (Ig)A proteases synthesized by human mucosal pathogens have a unique specificity for human IgA and will not cleave IgA from other species. In contrast, animal pathogens have not reliably been shown to cleave IgA of the animals they infect. This lack of an animal model has prevented an understanding of the importance of IgA1 proteases as virulence factors. One strategy to develop an animal model would be to identify a species capable of infection by a human IgA-producing pathogen whose IgA was susceptible to cleavage by IgA1 protease of that bacterium. The chimpanzee can be infected with Haemophilus influenzae and is closely related immunologically to man. For these reasons it was sought to determine whether chimpanzee secretory IgA (SIgA) is susceptible to cleavage by IgA1 protease of H. influenzae. This report shows that chimpanzee SIgA can indeed be cleaved at the hinge region by H. influenzae IgA1 protease into Fab alpha and (Fc alpha)2.SC fragments. The susceptibility of chimpanzee SIgA to IgA1 protease of a human pathogen could serve as the basis of an animal model to determine the importance of IgA1 protease in pathogenesis.     

Localization of the cleavage site specificity determinant of Haemophilus influenzae immunoglobulin A1 protease genes.

Immunoglobulin A1 (IgA1) proteases are produced by a number of different species of bacteria which cause infection at human mucosal surfaces. The sole substrate of these proteases is human IgA1. Cleavage is within the hinge region of IgA1, although there is variability in the exact peptide bond within the hinge region that is cut by a particular protease. The cleavage site of the Haemophilus influenzae type 1 protease is located four amino acids from the cleavage site of the type 2 enzyme. In this study, the region of the H. influenzae IgA1 protease gene (iga) that determines the cleavage site specificity was localized through the comparison of the type 1 and type 2 genes and the construction and analysis of type 1-type 2 hybrid genes. The hybrid genes were generated by in vivo and in vitro techniques which facilitated the selection and screening of randomly generated hybrids. The cleavage site determinant was found to be within a 370-base-pair region near the amino-terminal coding region, in one of two large areas of nonhomology between the two types of H. influenzae iga genes. DNA sequence analysis of the cleavage site determinant and surrounding regions did not reveal a simple mechanism whereby one enzyme type could be converted to the other type. Comparison of the type 2 gonococcal IgA1 protease gene to the two Haemophilus genes revealed a significant amount of homology around the cleavage site determinant, with the two type 2 genes showing greater homology.     

Genetic relationships of serologically nontypable and serotype b strains of Haemophilus influenzae.

A collection of 242 strains of Haemophilus influenzae, including 65 nontypable (unencapsulated) isolates and 177 encapsulated serotype b isolates recovered largely from children with invasive and noninvasive diseases in the United States, was characterized by the electrophoretic mobilities of 15 metabolic enzymes presumably encoded by chromosomal genes. All enzymes were polymorphic for three to seven electromorphs, and 94 distinctive multilocus genotypes (electrophoretic types [ETs]) were distinguished, among which mean genetic (allelic) diversity was 0.500. Isolates recovered from cases of invasive or noninvasive diseases did not differ significantly in level of genetic variation. The observation that 29 ETs were represented exclusively by serotype b isolates and that each of the 65 nontypable isolates was of a unique ET strongly confirmed the hypothesis that unencapsulated clinical isolates are not merely phenotypic variants of the common serotype b cell lines. Rather, the two types of isolates are distinctive subsets of the multilocus chromosomal genotypes of the species as a whole. Serotype b capsule occurred in three groups of isolates that are distantly related in multilocus enzyme genotype. Isolates of four closely related nontypable biotype IV ETs associated with obstetrical infections or neonatal bacteremia were highly divergent from all others examined and may be specifically distinct. A phylogenetic scenario was proposed in which the ancestor of H. influenzae was encapsulated and the nontypable clones arose by convergent evolutionary loss of the ability to synthesize or extracellularly express a polysaccharide capsule.     

Cloning of the gene encoding the major outer membrane protein of Haemophilus influenzae type b.

The major outer membrane protein (P2) of Haemophilus influenzae type b (Hib) with an apparent molecular weight of 37,000 to 40,000 has been previously shown to function as a porin and also as a target for antibodies protective against experimental Hib disease. The gene encoding the Hib P2 protein was cloned by using a shuttle vector capable of replication in both Escherichia coli and H. influenzae. The amino acid sequence of the amino terminus of the Hib P2 protein was determined and used to design an oligonucleotide probe corresponding to the first 20 amino acids of this protein. This oligonucleotide probe was used to identify Hib chromosomal DNA fragments containing the Hib P2 gene. These DNA fragments were ligated into the plasmid vector pGJB103 and then used to transform a rec-1 mutant of H. influenzae Rd. Recombinant clones expressing the Hib P2 protein were identified in a colony blot-radioimmunoassay by using a monoclonal antibody specific for a surface epitope of the Hib P2 protein. The gene encoding this Hib protein was present on a 10-kilobase Hib DNA insert in the recombinant plasmid. Transformation experiments involving the recombinant plasmid suggested that unregulated synthesis of Hib P2 is a lethal event in E. coli. The recombinant Hib P2 protein was exposed on the surface of the recombinant H. influenzae strain. This recombinant strain was used to develop a system for detecting polyclonal serum antibodies directed against surface determinants of the Hib P2 protein. The availability of the gene encoding the Hib P2 protein should facilitate investigation of both the immunogenicity and the structure-function relationship(s) of this major outer membrane protein.     

Immunoglobulin A1 protease production by Haemophilus influenzae and Streptococcus pneumoniae.

Bacterial strains of Haemophilus species and Streptococcus pneumoniae were examined for synthesis of the enzyme immunoglobulin A1 (IgA1) protease. Of 36 H. influenzae strains examined, 35 produced IgA1 protease; strains included all six capsular types, unencapsulated variants of types b and d, and untypable H. influenzae. Eight Haemophilus strains (non-H. influenzae) were studied, and two produced IgA1 protease. All 10 strains of S. pneumoniae produced IgA1 protease; these strains included 9 different capsular polysaccharide types and 1 untypable strain. Both IgA1 proteases cleaved myeloma IgA1 and secretory IgA but not myeloma IgA2, IgM, or IgG as determined by immunoelectrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that both enzymes cleaved IgA1 myeloma sera, but not IgA2, into two fragments. The apparent molecular weight of the cleaved fragments was dependent both on the apparent molecular weight of the cleaved fragments was dependent both on the specific IgA1 protease assayed and the specific IgA1 substrate utilized. It is postulated that both carbohydrate variation between the IgA1 substrates studied and the ability of S. pneumoniae glycosidases to cleave carbohydrates from glycoprotein offer an explanation for the different fragment sizes observed.     

Nucleotide sequence homology between the immunoglobulin A1 protease genes of Neisseria gonorrhoeae, Neisseria meningitidis, and Haemophilus influenzae.

Isolated DNA fragments encoding the immunoglobulin A1 (IgA1) protease of Neisseria gonorrhoeae were used as hybridization probes to search for homologous sequences in whole cell DNA from Neisseria meningitidis and Haemophilus influenzae. Significant homology was detected. That the detected homology represented IgA1 protease-specific sequences was confirmed by the cloning of these sequences in Escherichia coli HB101 and demonstrating the expression of IgA1 protease by these transformed cells. Molecular probing of commensal Neisseria and Haemophilus species, which do not elaborate IgA1 protease activity, revealed that they were devoid of sequence homology with the cloned IgA1 protease gene DNA.     

Identification, cloning, and sequencing of the immunoglobulin A1 protease gene of Streptococcus pneumoniae.

The pneumococcus expresses a protease that hydrolyzes human immunoglobulin A1 (IgA1). A gene for IgA1 protease was identified from a plasmid library of pneumococcal DNA because of the effect of its overexpression on the colony morphology of Streptococcus pneumoniae. The deduced 1,964-amino-acid sequence is highly homologous to that of the IgA1 protease from Streptococcus sanguis. The similarity to the S. sanguis enzyme and the presence of a putative zinc-binding site suggest that the pneumococcal enzyme is a metalloprotease. The two streptococcal sequences differ in a hydrophilic region with 10 tandem repeats of a 20-mer in S. sanguis, which is replaced by a similar but less repetitive sequence in S. pneumoniae. Antiserum reactive with the pneumococcal IgA1 protease was used to demonstrate that the majority of the protein is cell associated. The expression and function of this gene were confirmed by insertional mutagenesis. Interruption of the chromosomal gene resulted in loss of expression of an approximately 200-kDa protein and complete elimination of detectable IgA1 protease activity.     

Complete sequence of the cap locus of Haemophilus influenzae serotype b and nonencapsulated b capsule-negative variants

The complete capsule (cap) loci from three Haemophilus influenzae strains, one serotype b (Hib) and two nonencapsulated b capsule-negative variants, were sequenced. Two new open reading frames, hcsA and hcsB, were identified in region III and thought to be involved in postpolymerization modification of the capsule. The location of the cap locus in the Haemophilus influenzae chromosome was identified within section 97 of the Rd genome (chromosomal coordinates 1074542 to 1086327) and found to be the same for the Hib and two Hib(-) strains as well as some other encapsulated division I H. influenzae strains.     

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.     

Examination of Haemophilus pleuropneumoniae for immunoglobulin A protease activity.

Haemophilus pleuropneumoniae, the etiological agent of porcine contagious pneumonia, was examined for the ability to produce an immunoglobulin A (IgA) protease specific for porcine IgA. No IgA protease activity against either porcine or human IgA was detected. Furthermore, no sequence homology was found between H. pleuropneumoniae chromosomal DNA and the gene which specifies IgA protease in Haemophilus influenzae.     

Cloning, expression, and sequence analysis of the Haemophilus influenzae type b strain M43p+ pilin gene.

By using antiserum against Haemophilus influenzae type b (Hib) strain M43p+ denatured pilin, we screened a genomic library of Hib strain M43p+ and identified a clone that expressed pilin, but not assembled pili, on its surface. Southern blot analysis revealed the presence of one structural gene, which was also present in strain M42p-, a nonpiliated variant. Five exonuclease III deletion mutants, two of which had deletions that extended into the structural gene and failed to express pilin, were used to obtain the nucleotide sequence of the structural gene. The amino acid sequence of the open reading frame agrees with 38 of 40 amino acids from the published sequence of purified Hib M43p+ pilin. The pilin gene coded for a mature protein of 193 amino acids, with a calculated molecular mass of 21,101 daltons. Comparison of the Hib M43p+ pilin amino acid sequence with those of pilins of other bacteria revealed strong conservation of amino- and carboxy-terminal regions in M43p+ and Escherichia coli F17, type 1C, and several members of the P pili family, as well as Klebsiella pneumoniae type 3 MR/K, Bordetella pertussis serotype 2, and Serratia marcescens US46 fimbriae.     

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.     

Cloning of the gene encoding streptococcal immunoglobulin A protease and its expression in Escherichia coli.

We have identified and cloned a 6-kilobase-pair segment of chromosomal DNA from Streptococcus sanguis ATCC 10556 that encodes immunoglobulin A (IgA) protease activity when cloned into Escherichia coli. The enzyme specified by the iga gene in plasmid pJG1 accumulates in the periplasm of E. coli MM294 cells and has a substrate specificity for human IgA1 identical to that of native S. sanguis protease. Hybridization experiments with probes from within the encoding DNA showed no detectable homology at the nucleotide sequence level with chromosomal DNA of gram-negative bacteria that excrete IgA protease. Moreover, the S. sanguis iga gene probes showed no detectable hybridization with chromosomal DNA of S. pneumoniae, although the IgA proteases of these two streptococcal species cleaved the identical peptide bond in the human IgA1 heavy-chain hinge region.     

Characterization of Haemophilus influenzae type b fimbriae.

We confirmed that the fimbriae of Haemophilus influenzae type b conferred hemagglutinating activity (HA) towards human erythrocytes, and erythrocytes of certain other species. Most (17/25) cerebrospinal fluid isolates lacked detectable HA on direct testing, but selective enrichment for fimbriation (f+) indicated that 22 of 25 strains could produce these surface structures. HA was unchanged from pH 4.5 to 9.5 and was not inhibited by mannose or certain other simple sugars. The HA titer of a suspension of three f+ strains was slightly decreased at 50 degrees C; HA was lost by heating at 60 degrees C for 3 min. Growth on a variety of solid and liquid media and under differing degrees of oxygenation did not change the HA titer of a suspension of three f+ strains. Fimbriation was not lost on repeated subculture. Wild-type fimbriated strains, and those derived by transformation, did not contain detectable plasmid DNA. Transformation of a strain lacking fimbriae to f+ was associated with the appearance of an outer membrane protein of 24 kilodaltons. This protein was purified from one strain to homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis by selective detergent solubilization and ammonium sulfate fractionation. Colonization capacity was equivalent with an isogenic untypable strain lacking or possessing fimbriae. Fimbriae of type b H. influenzae possess characteristics similar to those structures on other gram-negative bacteria; their role in cell physiology or pathogenesis of invasive disease is unknown.