Prevalence and distribution of the hmw and hia genes and the HMW and Hia adhesins among genetically diverse strains of nontypeable Haemophilus influenzae

Abstract

Nontypeable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. In previous work we identified high-molecular-weight adhesins referred to as HMW1 and HMW2, expressed by nontypeable strain 12, and determined that most strains of nontypeable H. influenzae express one or two antigenically related proteins. More recently, we determined that some strains lack HMW1- and HMW2-like proteins and instead express an adhesin called Hia. In the present study, we determined the prevalence and distribution of the hmw and hia genes in a collection of 59 nontypeable strains previously characterized in terms of genetic relatedness. Based on Southern analysis, 47 strains contained sequences homologous to the hmw1 and hmw2 genes and nine strains contained homologs to hia. No strain harbored both hmw and hia, and three strains harbored neither. Although the hmw and hia genes failed to define distinct genetic divisions, the hmw-deficient strains formed small clusters or lineages within the larger population structure. Additional analysis established that the IS1016 insertion element was uniformly absent from strains containing hmw sequences but was present in two-thirds of the hmw-deficient strains. As IS1016 is associated with the capsule locus (cap) in most encapsulated strains of H. influenzae, we speculate that hmw-deficient nontypeable strains evolved more recently from an encapsulated ancestor.

FIG. 1

Genetic relationships between the 59 strains examined in this study. The dendrogram was generated previously by Musser et al. (8) by the average-linkage method of clustering from a matrix of coefficients of genetic distance, based on 15 metabolic enzymes. Circles indicate strains that hybridized with hmw but not hia, squares indicate strains that hybridized with hia but not hmw, and triangles indicate strains that failed to hybridize with either hia or hmw. Blackened symbols indicate strains that hybridized with pUO38 and IS1016. The numbers and letters to the right of the symbols are strain designations. Numbers in parentheses refer to level of adherence.

FIG. 2

Southern blotting performed with probes for hmw sequences and hia. Chromosomal DNA was digested with BglII, separated by agarose electrophoresis, and subjected to Southern analysis. (A) Southern analysis with a probe corresponding to the promoter and 5′ coding sequence of hmw1A. Strains by lane: 1, 12; 2, 11; 3, 3219B; 4, 3686; 5, 3242A; 6, 3219C; 7, 3655; 8, 1667; 9, 3246A; 10, 1484A; 11, 1674. Less chromosomal DNA was inadvertently loaded in lanes 7 and 9. (B) Southern analysis with a probe that represents an intragenic fragment of hia. Strains by lane: 1, 12; 2, 11; 3, 1862; 4, 3179B; 5, 3640; 6, 3A; 7, 1860; 8, 3230B; 9, 3248A; 10, 3230A; 11, 1396B. Comparable quantities of DNA were loaded in each lane.

FIG. 3

Southern blotting performed with radiolabeled pUO38. Chromosomal DNA was digested with EcoRI, separated by agarose electrophoresis, and subjected to Southern analysis. Strains by lane: 1, Eagan; 2, 1862; 3, 3179B; 4, 3640; 5, 3A; 6, 1860; 7, 3230B; 8, 3248A; 9, 3230A; 10, 1396B; 11, 3232A; 12, 3639.

FIG. 4

Western immunoblotting performed with serum 25G against HMW1 and HMW2 or serum 36B against Hia. Whole-cell lysates were prepared, and proteins were separated on a 7.5% polyacrylamide gel. (A) Western analysis with serum 25G. Strains by lane: 1, 12; 2, 11; 3, 3219B; 4, 3686; 5, 3242A; 6, 3219C; 7, 3655; 8, 1667; 9, 3246A; 10, 1484A; 11, 1674; 12, 3894. (B) Western analysis with serum 36B. Strains by lane: 1, 12; 2, 11; 3, 11hia::kan; 4, 1862; 5, 3179B; 6, 3640; 7, 3A; 8, 1860; 9, 3230B; 10, 3248A; 11, 3230A; 12, 1396B.