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.

Non-typeable Haemophilus influenzae (NTHi) is the most common bacterial cause of infection of the lower airways in adults with chronic obstructive pulmonary disease (COPD). Infection of the COPD airways causes acute exacerbations, resulting in substantial morbidity and mortality. NTHi has evolved multiple mechanisms to establish infection in the hostile environment of the COPD airways, allowing the pathogen to persist in the airways for months to years. Persistent infection of the COPD airways contributes to chronic airway inflammation that increases symptoms and accelerates the progressive loss of pulmonary function, which is a hallmark of the disease. Persistence mechanisms of NTHi include the expression of multiple redundant adhesins that mediate binding to host cellular and extracellular matrix components. NTHi evades host immune recognition and clearance by invading host epithelial cells, forming biofilms, altering gene expression and displaying surface antigenic variation. NTHi also binds host serum factors that confer serum resistance. Here we discuss the burden of COPD and the role of NTHi infections in the course of the disease. We provide an overview of NTHi mechanisms of persistence that allow the pathogen to establish a niche in the hostile COPD airways.

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BackgroundReverse vaccinology accelerates the discovery of potential vaccine candidates (PVCs) prior to experimental validation. Current programs typically use one bacterial proteome to identify PVCs through a filtering architecture using feature prediction programs or a machine learning approach. Filtering approaches may eliminate potential antigens based on limitations in the accuracy of prediction tools used. Machine learning approaches are heavily dependent on the selection of training datasets with experimentally validated antigens (positive control) and non-protective-antigens (negative control). The use of one or few bacterial proteomes does not assess PVC conservation among strains, an important feature of vaccine antigens.ResultsWe present ReVac, which implements both a panoply of feature prediction programs without filtering out proteins, and scoring of candidates based on predictions made on curated positive and negative control PVCs datasets. ReVac surveys several genomes assessing protein conservation, as well as DNA and protein repeats, which may result in variable expression of PVCs. ReVac’s orthologous clustering of conserved genes, identifies core and dispensable genome components. This is useful for determining the degree of conservation of PVCs among the population of isolates for a given pathogen. Potential vaccine candidates are then prioritized based on conservation and overall feature-based scoring. We present the application of ReVac, applied to 69 Moraxella catarrhalis and 270 non-typeable Haemophilus influenzae genomes, prioritizing 64 and 29 proteins as PVCs, respectively.ConclusionReVac’s use of a scoring scheme ranks PVCs for subsequent experimental testing. It employs a redundancy-based approach in its predictions of features using several prediction tools. The protein’s features are collated, and each protein is ranked based on the scoring scheme. Multi-genome analyses performed in ReVac allow for a comprehensive overview of PVCs from a pan-genome perspective, as an essential pre-requisite for any bacterial subunit vaccine design. ReVac prioritized PVCs of two human respiratory pathogens, identifying both novel and previously validated PVCs.

Nontypeable Haemophilus influenzae (NTHi) is the primary cause of bacterially induced acute exacerbations of chronic obstructive pulmonary disease (COPD). NTHi adheres to and invades host respiratory epithelial cells as a means to persist in the lower airways of adults with COPD. Therefore, we mined the genomes of NTHi strains isolated from the airways of adults with COPD to identify novel proteins to investigate their role in adherence and invasion of human respiratory epithelial cells. An isogenic knockout mutant of the open reading frame NTHI1441 showed a 76.6% ± 5.5% reduction in invasion of human bronchial and alveolar epithelial cells at 1, 3, and 6 h postinfection. Decreased invasion of the NTHI1441 mutant was independent of either intracellular survival or adherence to cells. NTHI1441 is conserved among NTHi genomes. Results of whole-bacterial-cell enzyme-linked immunosorbent assay (ELISA) and flow cytometry experiments identified that NTHI1441 has epitopes expressed on the bacterial cell surface. Adults with COPD develop increased serum IgG against NTHI1441 after experiencing an exacerbation with NTHi. This study reveals NTHI1441 as a novel NTHi virulence factor expressed during infection of the COPD lower airways that contributes to invasion of host respiratory epithelial cells. The role in host cell invasion, conservation among strains, and expression of surface-exposed epitopes suggest that NTHI1441 is a potential target for preventative and therapeutic interventions for disease caused by NTHi.