The complete genome and functional analysis of the Brazilian Purpuric Fever clone of Haemophilus influenzae biogroup Aegyptius

Abstract

The Brazilian Purpuric Fever (BPF) clone of Haemophilus influenzae biogroup Aegyptius (Hae) emerged in São Paulo in 1984, causing epidemic outbreaks of a life-threatening childhood infection characterised by shock and purpura fulminans. Strains of Hae have long been known to cause highly contagious and purulent conjunctivitis, but never previously implicated in invasive disease. Laboratory studies have revealed phenotypic and genetic differences between the BPF clone and other Hae strains, but failed to identify virulence factors responsible for the unusual virulence of this clone. This thesis describes the exhaustive annotation of the whole genome sequences of the invasive BPF clone isolate F3031 and non-invasive Hae conjunctivitis isolate F3047, inferring gene function through sequence homologies, and identifying insertions, deletions, pseudogenes and regulatory sites that may underlie phenotypic variation between these strains. Pan-genomic comparison of F3031 and F3047 to 5 other complete H. influenzae genomes allowed delineation of the 'Hae accessory genome', revealing a suite of novel adhesins not previously described for H. influenzae, presumably reflecting the conjunctival niche to which Hae has specialised. These include a striking ten-member family of trimeric autotransporter adhesins (TAAs) that share homology with TAAs established to play a role in virulence of other bacterial pathogens, and were selected for further study. Functional evaluation of variants of one of these genes, b/caaA1, through cloning and expression in E. coli, revealed differences in autoaggregation and in adherence of transformants to human epithelial cells in culture. Investigating gene function in Hae has been hampered by difficulties in genetically manipulating these strains. Competence for DNA uptake and transformation in Hae was investigated through in silico analysis of the genes involved in these processes, and the development of a plate transformation protocol that appears to reliably transform certain strains of Hae, providing a valuable tool for future work investigating the virulence functions of genes in their natural background.