A clonal group of nontypeable Haemophilus influenzae with two IgA proteases is adapted to infection in chronic obstructive pulmonary disease

Abstract

Strains of nontypeable Haemophilus influenzae show enormous genetic heterogeneity and display differential virulence potential in different clinical settings. The igaB gene, which encodes a newly identified IgA protease, is more likely to be present in the genome of COPD strains of H. influenzae than in otitis media strains. Analysis of igaB and surrounding sequences in the present study showed that H. influenzae likely acquired igaB from Neisseria meningitidis and that the acquisition was accompanied by a ~20 kb genomic inversion that is present only in strains that have igaB. As part of a long running prospective study of COPD, molecular typing of H. influenzae strains identified a clonally related group of strains, a surprising observation given the genetic heterogeneity that characterizes strains of nontypeable H. influenzae. Analysis of strains by 5 independent methods (polyacrylamide gel electrophoresis, multilocus sequence typing, igaB gene sequences, P2 gene sequences, pulsed field gel electrophoresis) established the clonal relationship among the strains. Analysis of 134 independent strains collected prospectively from a cohort of adults with COPD demonstrated that ~10% belonged to the clonal group. We conclude that a clonally related group of strains of nontypeable H. influenzae that has two IgA1 protease genes (iga and igaB) is adapted for colonization and infection in COPD. This observation has important implications in understanding population dynamics of H. influenzae in human infection and in understanding virulence mechanisms specifically in the setting of COPD.

Figure 1. Genes in the region of the igaB insertion site.

A. Map of genes surrounding igaB (not to scale). Gene numbers correspond to numbers in strain Rd KW20 . Horizontal arrows indicate location of oligonucleotide primers used in PCR reactions. B. Ethidium bromide stained agarose gels showing results of PCR reactions with genomic DNA of H. influenzae strains that contained the igaB gene with the oligonucleotide primers noted in A. Lanes contain genomic DNA from strains a. 3P14H1, b. 6P5H, c. 6P18H1, d. 7P49H1, e. 11P6H, f. 12P37H1. Molecular size standards are noted in kilobases.

Figure 2. Genes in the region of the igaB insertion site.

A. Map of genes from ORFs HI0163 through HI0185 (not to scale) in H. influenzae strain Rd KW20 which does not contain the igaB gene. The dotted line represents ORFs HI0167 through HI0182. Horizontal arrows indicate location of oligonucleotide primers used in PCR reactions. B. Ethidium bromide stained agarose gels showing results of PCR reactions with genomic DNA of H. influenzae strains that do not contain the igaB gene with the oligonucleotide primers noted in A. Lanes contain genomic DNA from strains a. Rd KW20, b. 11P6H (which contains igaB), c. 5P28H1, d. 14P14H1, e. 31P7H1, f. 32P8H1, g. 48P28H1, h. 56P76H1, i. 1862, j. 3222B. Molecular size standards are noted in kilobases.

Figure 3. Diagram illustrating the location of the IS4 transposase sequences flanking the genomic inversion in igaB-containing strains.

The order of the ORFs is noted on the left (strain Rd nomenclature) and the box denotes the inversion. ORFs are noted as gray lines and IS4-like sequences are noted as black lines. Numbers denote base pairs.

Figure 4. Pulsed field gel electrophoresis of 12 strains of nontypeable H. influenzae isolated from the sputum of adults with COPD.

Lanes contain genomic DNA cut with SmaI from the strains a) 11P6H, b) 44P85H1, c) 87P37H1, d) 102P20H1, e) 105P9H1, f) 23P2H, g) 24P44H1, h) 59P3H1, i) 14P6H1, j) 14P26H1, k) 19P68H7, l) 19P70H8. Strains in lanes a through h belong to MLST 159 as noted.