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Limited diversity of the protein D gene (hpd) among encapsulated and nonencapsulated Haemophilus influenzae strains.

ABSTRACT: Protein D is a surface-exposed lipoprotein of the gram-negative bacterium Haemophilus influenzae with affinity for human immunoglobulin D myeloma protein. The gene encoding protein D (hpd) in a serotype b strain of H. influenzae was cloned. Escherichia coli carrying the hpd gene bound human myeloma immunoglobulin D. Nucleotide sequence analysis identified an 1,092-bp open reading frame that was more than 99% identical to the hpd gene from a nontypeable H. influenzae strain. In the deduced amino acid sequences for protein D, only 2 of 364 amino acid residues differed. The restriction fragment length polymorphism of the hpd region in different strains was analyzed by Southern blot analyses of PstI- or EcoRI-digested genomic DNA from 100 H. influenzae strains. The analysis was performed by using isolated fragments of the cloned hpd gene, originating from the nontypeable H. influenzae 772, as probes. All strains tested had DNA sequences with a high degree of homology to the hpd probes. The analysis also showed that restriction endonuclease sites within the gene were more conserved than sites adjacent to the hpd gene. An interesting difference between type b strains and unencapsulated strains was observed. The majority of type b strains seem to have a 1.4-kbp DNA fragment upstream of the hpd gene that is absent in nontypeable strains. On the basis of the high degree of conservation of the hpd gene among H. influenzae strains, we conclude that protein D is a possible vaccine candidate.

SUBMITTER: Janson H

PROVIDER: S-EPMC281203 | biostudies-other | 1993 Nov

REPOSITORIES: biostudies-other

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Limited diversity of the protein D gene (hpd) among encapsulated and nonencapsulated Haemophilus influenzae strains.

Janson H H Ruan M M Forsgren A A

Infection and immunity 19931101 11

Protein D is a surface-exposed lipoprotein of the gram-negative bacterium Haemophilus influenzae with affinity for human immunoglobulin D myeloma protein. The gene encoding protein D (hpd) in a serotype b strain of H. influenzae was cloned. Escherichia coli carrying the hpd gene bound human myeloma immunoglobulin D. Nucleotide sequence analysis identified an 1,092-bp open reading frame that was more than 99% identical to the hpd gene from a nontypeable H. influenzae strain. In the deduced amino ...[more]

PMID: 8104899

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Project description:A subset of nontypeable Haemophilus influenzae (NTHI) biotype IV isolates from the human genital tract or from infected newborn infants forms a cryptic genospecies characterized by, among other features, the presence of peritrichous pili. The objective of this study was to determine the similarity of these pili to hemagglutinating, HifA- and HifE-containing pili expressed by respiratory H. influenzae isolates. For this analysis, the presence of hifA and hifE and their gene products in NTHI biotype IV strains was assessed, the binding of H. influenzae biotype IV strains to human epithelial cells was characterized, possible genital tissue tropism of these isolates was explored, and the role of HifA- and HifE-possessing pili in the adhesion of NTHI biotype IV strains to human epithelial cells was determined. None of the six biotype IV NTHI isolates tested agglutinated human red blood cells, nor could they be enriched for hemagglutinating variants. Although hifA, which encodes the major structural subunit of hemagglutinating pili, and hifE, which encodes the tip adhesin of hemagglutinating pili, were detected by PCR from six and five, respectively, of the six biotype IV strains tested, neither HifA nor HifE (the gene products of hifA and hifE) were detected in any of these strains by Western blot analysis using antisera that recognize HifA and HifE of respiratory strains. Transmission electron microscopy showed no surface pili on the two biotype IV H. influenzae isolates examined; strain 4162 containing an insertional mutation in hifA also showed no surface pili, whereas strain 1595 containing an insertional mutation in hifB showed pilus-like structures that were shorter and thicker than hemagglutinating pili of the respiratory strains AAr176 and M43. In enzyme-linked immunosorbent assays, biotype IV strains adhered to 16HBE14o(-) and HEp-2 cells of respiratory origin as well as to ME180 and HeLa cells of genital origin. This adherence was not pilus specific, however, as GM-1, a known pilus receptor analog, did not inhibit binding of biotype IV strains to ME180, HEp-2, or HeLa cells, and GM-1 inhibition of binding to 16HBE14o(-) cells did not correlate with the presence of hifE. While both nonpiliated variants and hifA and hifB (encoding the pilus chaperone) mutants of respiratory strain AAr176 showed reduced binding (64 to 87% of that of piliated AAr176) to 16HBE14o(-) and ME180 cells, hifA and hifB mutants of the biotype IV strains showed minimal reduction in binding to these cell lines (91 to 98% of that of wild-type strains). Thus, although biotype IV H. influenzae isolates of the cryptic genospecies possess the genes that code for HifA- and HifE-containing hemagglutinating pili, epithelial cell adherence exhibited by these strains is not mediated by expression of hemagglutinating pili.

Dataset Information

Limited diversity of the protein D gene (hpd) among encapsulated and nonencapsulated Haemophilus influenzae strains.

Publications

Limited diversity of the protein D gene (hpd) among encapsulated and nonencapsulated Haemophilus influenzae strains.

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