Characterization of serum resistance in H. influenzae biogroup aegyptius associated with Brazilian purpuric fever.

摘要

H. influenzae biogroup aegyptius is the etiologic agent of Brazilian Purpuric Fever. Virulence factors for this organism have yet to be defined. This bacterium has been associated with non-virulent conjunctivitis for decades. However, a small subset of bacteria have acquired the ability to attach, invade and cause this overwhelming pediatric syndrome. A hallmark feature of BPF case-clone strains of H. influenzae biogroup aegyptius is their ability to evade serum bactericidal activity. The main focus of this thesis was to identify factors of this bacterium that enable it to be serum resistant.; The initial approach was to create isogenic mutants of H. influenzae biogroup aegyptius and then screen those mutants for serum sensitive transformants. However, repeated attempts with numerous mutagenesis systems, both in vitro and in vivo, failed to transform this bacterium. In order to gain insight into transformation of H. influenzae biogroup aegyptius we analyzed the genetic construction of the isogenic P1 deficient mutant of H. influenzae biogroup aegyptius created in this lab. This construct was used in the only successfully demonstrated transformation of this organism. Through DNA sequencing and systematic genetic deletions we have identified a 2.4 kilobase (kb) fragment of the original construction (∼11.5 kb), upstream from the P1 gene, that results in high efficiency transformation.; As a parallel approach to our genetic analysis, we examined the interaction of H. influenzae biogroup aegyptius with the complement cascade using flow cytometry. Our studies examined two crucial components of both the classic and alternative pathways of complement activation, C3b and membrane attack complex (MAC). Flow cytometry indicates a decrease of C3b deposition and MAC formation on the surface of BPF-associated strains of H. influenzae biogroup aegyptius as compared to non-BPF strains. This observation provides significant information on the interaction of BPF case-clone strains with critical complement components.