Understanding the molecular basis for adhesion by Haemophilus Surface Fibril

Invasive Haemophilus influenzae Infections have remained an epidemiological concern across
the globe even after 3 decades of the H. influenzae serotype b (Hib) vaccine use (Slack, Cripps,
Grimwood, Mackenzie, & Ulanova, 2021). With the emergence of severe invasive
H.influenzae infections in various parts of the world, it remains crucial to develop novel means
of diagnosis and intervention strategies. The Haemophilus surface fibril (Hsf) is a surface
adhesin expressed in all encapsulated strains of H. influenzae and is therefore believed to be
a critical virulence factor. As per the bioinformatic model, Hsf which is comprised of the passenger
domain and translocator domain is predicted to assume ~200 nm long homo-trimeric
morphology. Although the initial biochemical study recently proposed that Hsf assumes a
unique twisted hairpin-like architecture at the bacterial surface, this remains to be
established in the absence of its molecular structure. This thesis tested this hypothesis by
solving the high-resolution structure of the truncated Hsf888-1337 segment containing the
putative domain 3 (PD3) and the binding domain 3 (BD3). PD3 and BD3 are unique to Hsf and
have been implicated for the bend in the proposed hairpin architecture. Here I show that the
Hsf888-1337 assumes only a slight bend which is not significant enough to support the
hypothesis of a novel hairpin-like structure. Nevertheless, the work here renders the first-ever
structural evidence at atomic resolution for the presence of bend architecture in the
Trimeric Autotransporter Adhesins (TAA).
Altogether, the present finding doesn’t only refute the earlier proposition of Hsf forming a
hairpin structure but provides the first credible evidence for the presence of bending
morphology in the TAA protein. In particular, the Hsf888-1337 structure shows that the PD3 and BD3 domains are linked with a highly flexible neck. Interestingly, the PD3 and BD3 domains
are unique to Hsf but also show approximately 60% sequence similarity with the well-characterized
binding domain 1 (BD1) of the Haemophilus influenzae adhesin (Hia) protein.
The BD1 domain of Hia is responsible for its adhesion, hence implying the importance to study
the PD3 and BD3 in Hsf.
As the physical interaction between Hsf and the host factor- Vitronectin (abundantly available
in the blood) is already well established, for the second part of my thesis, I tested the adhesive
property of individual domains of the Hsf with Vitronectin. This part of the study was aimed
at demonstrating the utility of Hsf-vitronectin interaction in developing the point of care
testing for Invasive Haemophilus influenzae Infections. Using the Cyclic voltammetry, this
study contributes towards the development of improved, cost-effective diagnostic tools
against H. influenzae.