Detection of phase variation in expression of proteins involved in hemoglobin and hemoglobin-haptoglobin binding by nontypeable Haemophilus influenzae

Haemophilus influenzae can utilize different protein-bound forms of heme fo r growth in vitro. A previous study (I. Maciver, J. L. Latimer, H. H. Liem, U. Muller-Eberhard, Z. Hrkal, and E. J. Hansen. Infect. Immun, 63:3703-371 2, 1996) indicated that nontypeable H. influenzae (NTHI) strain TN106 expre ssed a protein that bound hemoglobin-haptoglobin and was encoded by an open reading frame (ORF) that contained a CCAA nucleotide repeat, Southern blot analysis revealed that several NTHI strains contained between three and fi ve chromosomal DNA fragments that bound an oligonucleotide probe for CCAA r epeats. Three ORFs containing CCAA repeats were identified in NTHI strain N 182; two of these ORFs were arranged in tandem. The use of translational fu sions involving these three ORFs and the p-lactamase gene from pBR322 revea led that these three ORFs, designated hgbA, hgbB, and hgbC, encoded protein s that could bind hemoglobin, hemoglobin-haptoglobin, or both compounds. Mo noclonal antibodies (MAbs) specific for the HgbA, HgbB, and HgbC proteins w ere produced by immunizing mice with synthetic peptides unique to each prot ein. Both HgbA and HgbB were readily detected by Western blot analysis in N 182 cells grown in the presence of hemoglobin as the sole source of heme, w hereas expression of HgbC was found to be much less abundant then that of H gbA and HgbB, The use of these MAbs in a colony blot radioimmunoassay analy sis revealed that expression of both HgbA and HgbB was subject to phase var iation. PCR and nucleotide sequence analysis were used in conjunction with Western blot analyses to demonstrate that this phase variation involved the CCAA repeats in the hgbA and hgbB ORFs.