Identification of a novel trimeric autotransporter adhesin in the cryptic genospecies of Haemophilus

Abstract

Haemophilus biotype IV strains belonging to the recently recognized Haemophilus cryptic genospecies are an important cause of maternal genital tract and neonatal systemic infections and initiate infection by colonizing the genital or respiratory epithelium. To gain insight into the mechanism of Haemophilus cryptic genospecies colonization, we began by examining prototype strain 1595 and three other strains for adherence to genital and respiratory epithelial cell lines. Strain 1595 and two of the three other strains demonstrated efficient adherence to all of the cell lines tested. With a stably adherent variant of strain 1595, we generated a Mariner transposon library and identified 16 nonadherent mutants. All of these mutants lacked surface fibers and contained an insertion in the same open reading frame, which encodes a 157-kDa protein designated Cha for cryptic haemophilus adhesin. Analysis of the predicted amino acid sequence of Cha revealed the presence of an N-terminal signal peptide and a C-terminal domain bearing homology to YadA-like and Hia-like trimeric autotransporters. Examination of the C-terminal 120 amino acids of Cha demonstrated mobility as a trimer on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the capacity to present the passenger domain of the Hia trimeric autotransporter on the bacterial surface. Southern analysis revealed that the gene that encodes Cha is conserved among clinical isolates of the Haemophilus cryptic genospecies and is absent from the closely related species Haemophilus influenzae. We speculate that Cha is important in the pathogenesis of disease due to the Haemophilus cryptic genospecies and is in part responsible for the apparent tissue tropism of this organism.

FIG. 1.

Adherence of Haemophilus cryptic genospecies strains 420, 422, 1595, and 1673 to Chang, Detroit 562, HEC-1-B, and HeLa epithelial cells. Adherence was measured after incubating bacteria with epithelial cell monolayers for 30 min and calculated by dividing the number of adherent CFU per epithelial cell monolayer by the number of inoculated CFU. Adherence values represent the mean of measurements from representative experiments performed in triplicate. Error bars represent standard errors.

FIG. 2.

Genomic organization of the cha locus and domain architecture of the Cha protein. (A) Arrows indicate the locations of transposon insertions among the nonadherent mutants (1595cha). The disrupted open reading frame, which was designated cha for cryptic haemophilus adhesin, is flanked upstream by putative thioredoxin and cytochrome oxidase genes and is flanked downstream by two genes of unknown function. (B) The domain organization of Cha from strain 1595 was evaluated with SignalP (3) and Pfam (2) analyses.

FIG. 3.

Western analysis of Haemophilus cryptic genospecies strains 1595-A9. 1595cha, 420, and 420/pCha and H. influenzae strains DB117/pLS88P and DB117/pCha for expression of Cha. Outer membrane samples were denatured with formic acid and examined by Western analysis with an antiserum that was raised against Cha residues 70 to 473 and diluted 1:1,000. Samples were loaded as follows: lane 1, 1595-A9; lane 2, 1595cha; lane 3, 420; lane 4, 420/pCha; lane 5, DB117/pLS88P; lane 6, DB117/pCha. Arrowheads indicate monomeric Cha protein.

FIG. 4.

Evidence that Cha is a trimeric autotransporter. (A) Western blot assay of outer membrane fractions of E. coli DH5α/pChaC′ without (−) or with (+) formic acid denaturation by probing with anti-HAT antibody. (B) Quantitative adherence assay with Chang cells examining the ability of the Cha C terminus to present the Hia passenger domain on the bacterial cell surface in a functional form. Plasmid pHiaPD-HiaC′ encodes the Hia passenger domain fused to the Hia C terminus, plasmid pHiaPD-ChaC′ encodes a chimeric protein with the Hia passenger domain fused to the Cha C terminus, plasmid pChaC′ encodes only the C terminus of Cha, and plasmid pHiaPD encodes only the passenger domain of Hia. Error bars represent standard errors.

FIG. 5.

Southern hybridization analysis by probing for the cha gene among Haemophilus cryptic genospecies isolates. Chromosomal DNA was isolated from nine cryptic genospecies strains and two H. influenzae strains and digested to completion with BglII. An ECL-labeled probe consisting of the full-length cha PCR amplicon from strain 1595 was used for hybridization.