Functional investigation of Haemophilus influenzae genes of unknown function implicated in pathogenesis
Authors:   , Tramonte Gabrielle , Sell Christian 
Published: August 2022

Background: Many bacteria are found in and on the human body as commensals, however, many can also transition to become pathogenic and cause disease. Haemophilus influenzae normally inhabits the nasopharynx of healthy children but it is also the major cause of morbidity in pediatric ear infections, chronic obstructive pulmonary disease in the elderly, and rarer but serious invasive infections. Although host factors contribute, differences in bacterial gene content are known to affect pathogenesis. In previous work, 1618 H. influenzae genomes were analyzed to identify genes whose presence or absence predicted bacterial isolates' clinical provenance. Many of the top ranked genes had no annotated function, prompting the current study, in which we sought to identify the unknown molecular function of four top-ranked genes whose presence varied among disease states. Materials and Methods: Two isolates, PA31 and PA43, contained all four candidate genes, so bioinformatic analyses using strain PA31 were conducted, including examining the function of flanking genes and applying de novo secondary structure prediction tools. Several databases were searched for homologs with known function or conserved domains, including BLAST, CDD, and EggNog. Protein structural predictions used SWISS-MODEL and AlphaFold, and structural homologs were identified using the DALI database. To produce new resources for investigating the candidates, the two strains and a laboratory strain, Rd, were characterized in assays for growth, biofilm formation, and natural competence. Finally, two knockout constructs were designed that target two candidates for replacement with a spectinomycin resistance cassette in the PA31 strain for future phenotypic characterization. Results: Gene cluster_5482 was enriched in carriage and lung isolates over others. Bioinformatic searches identified it as the toxin in a recently characterized competence- regulated toxin-antitoxin system. Since a previous study found that deletion of the antitoxin blocks expression of natural competence, a knockout construct of the antitoxin gene was pursued. Two candidates, cluster_1320 and cluster_339, were enriched in mucosal infections (ear, eye, and lung) over carriage or invasive disease. Both were found to be fragments of the large well-characterized adhesins HMW1 or HMW2. Artifacts of assembling these paralogs loci are likely responsible for the poor gene annotation, but the known role of HMW1/2 in modulating binding to different airway epithelial cell types indicates that the candidate list contains genes with a molecular role in pathogenesis. Finally, the function of cluster_229 remained a mystery after our analyses although a high-confidence structural prediction was made. A knockout was pursed, and the role of the gene in the function of replication of a phage was explored. Conclusion: Although the function of the four initial candidate genes was initially unknown based on automated annotation, bioinformatic analyses was able to identify that these represented three protein-coding genes, two of which could have a molecular function assigned. Completing the knockouts could aid in future studies of these genes' role in how H. influenzae cause different diseases, and this could help identify new potential diagnostics or therapeutic treatments.