Haemophilus influenzae genome evolution during persistence in the human airways in chronic obstructive pulmonary disease

Abstract

Nontypeable (NTHi) exclusively colonize and infect humans and are critical to the pathogenesis of chronic obstructive pulmonary disease (COPD). In vitro and animal models do not accurately capture the complex environments encountered by NTHi during human infection. We conducted whole-genome sequencing of 269 longitudinally collected cleared and persistent NTHi from a 15-y prospective study of adults with COPD. Genome sequences were used to elucidate the phylogeny of NTHi isolates, identify genomic changes that occur with persistence in the human airways, and evaluate the effect of selective pressure on 12 candidate vaccine antigens. Strains persisted in individuals with COPD for as long as 1,422 d. Slipped-strand mispairing, mediated by changes in simple sequence repeats in multiple genes during persistence, regulates expression of critical virulence functions, including adherence, nutrient uptake, and modification of surface molecules, and is a major mechanism for survival in the hostile environment of the human airways. A subset of strains underwent a large 400-kb inversion during persistence. NTHi does not undergo significant gene gain or loss during persistence, in contrast to other persistent respiratory tract pathogens. Amino acid sequence changes occurred in 8 of 12 candidate vaccine antigens during persistence, an observation with important implications for vaccine development. These results indicate that NTHi alters its genome during persistence by regulation of critical virulence functions primarily by slipped-strand mispairing, advancing our understanding of how a bacterial pathogen that plays a critical role in COPD adapts to survival in the human respiratory tract.

Molecular signatures of non-typeable Haemophilus influenzae lung adaptation in paediatric chronic lung disease

“…Phylogenomic analysis 166 confirmed that pairs from individual patients clustered closely together yet were distinct from 167 other NP-BAL pairs. Consistent with previous studies (17,22,27), there was no evidence of 168 NP-or BAL-specific clades. Isolate pairs were identical sequence types (STs), with 5 of the 12 169 pairs possessing novel STs (ST-1906 to ST-1910 ; Table S2).…”

“…The remaining SSR indels occurred in the haemoglobin-haptoglobin binding protein B-encoding gene hgpB, 316 which is involved in NP colonisation and iron acquisition (51), and lic3A, which encodes a 317 lipooligosaccharide (LOS) sialyltransferase that facilitates NTHi colonisation in different 318 anatomical sites (52,53). 319 Our genetic findings concur with the recent studies, which found that phase variation in lic3A, 320 hsdM3 and hgpB are common in NTHi isolates retrieved from COPD airways (17,46). In 321 addition to the hgpB SSR variant in 60068 BAL Hi1 that truncates the protein from 992 to 12 322 residues, a non-synonymous SNP (HgpBArg407Lys) was seen in 60295 BAL Hi1.…”

“…In this study, we aimed to identify signals of NTHi 297 pathoadaptation to the lower airways using 12 paired isogenic strains retrieved from the 298 upper (NP) and the lower (BAL fluid) airways of 11 paediatric patients with CLSD or 299 bronchiectasis. Similar studies have employed WGS to document the genome-wide changes 300 leading to chronic adaptation and persistence of NTHi retrieved from COPD sputum in adults 301 (17,46). The current work expands on these prior studies by examining both the genomic and 302 transcriptomic profiles of NTHi adaptation in children's airways.…”

“…Five pairs were genetically 310 identical, with the remaining seven encoding a total of two SNPs and seven indels. Notably, 311 four of these indels occurred in tandem repeat regions, or SSR tracts, of phase-variable genes, 312 which are abundant in the NTHi genome (17,48). SSRs provide a rapid mechanism for gene 313 expression modulation and adaptation (49,50).…”

Selective and non‐selective bottlenecks as drivers of the evolution of hypermutable bacterial loci

“…Using WGS of multiple longitudinal non‐typeable Haemophilus influenzae (NTHi) isolates from 61 persistently chronic obstructive pulmonary disease (COPD) patients, Pettigrew et al . found that increases in repeat number were positively correlated with length of persistence (Pettigrew et al, ). A more detailed analysis of closed genome sequences of four isolates revealed 22 SSR‐containing genes, with variations in repeat number of the HMW1A/HMW2A‐related adhesins and the haemoglobin‐haptoglobin‐binding proteins occurring in all four isolates.…”