Haemophilus influenzae surface fibril (Hsf) is a unique twisted hairpin-like trimeric autotransporter

Abstract

The Haemophilus surface fibril (Hsf) is an extraordinary large (2413 amino acids) trimeric autotransporter, present in all encapsulated Haemophilus influenzae. It contributes to virulence by directly functioning as an adhesin. Furthermore, Hsf recruits the host factor vitronectin thereby inhibiting the host innate immune response resulting in enhanced survival in serum. Here we observed by electron microscopy that Hsf appears as an 100 nm long fibril at the bacterial surface albeit the length is approximately 200 nm according to a bioinformatics based model. To unveil this discrepancy, we denaturated Hsf at the surface of Hib by using guanidine hydrochloride (GuHCl). Partial denaturation induced in the presence of GuHCl unfolded the Hsf molecules, and resulted in an increased length of fibres in comparison to the native trimeric form. Importantly, our findings were also verified by E. coli expressing Hsf at its surface. In addition, a set of Hsf-specific peptide antibodies also indicated that the N-terminal of Hsf is located near the C-terminal at the base of the fibril. Taken together, our results demonstrated that Hsf is not a straight molecule but is folded and doubled over. This is the first report that provides the unique structural features of the trimeric autotransporter Hsf. Highlights Trimeric autotransporters are important virulence factors of Gram negative bacteria, and have a "lollipop-shape" structure or straight trimeric strand.  Haemophilus surface fibril is involved in adherence to host epithelium and for recruitment of vitronectin.  Here we show for the first time that Haemophilus surface fibril has a twisted hairpin-like structure.

Host–pathogen interactions of nontypeable Haemophilus influenzae: from commensal to pathogen

“…However, recent evidence suggests increased incidence of infections related to H. influenzae type a, e, and f, although at a lesser number than NTHi . Notably, proteins such as protein H and Haemophilus surface fibrils (Hsf) are examples of virulence factors not detected in NTHi, that we have found to be important for encapsulated H. influenzae virulence.…”

New findings on the function and potential applications of the trimeric autotransporter adhesin

“…Most TAAs are nonlinear on the surface due to their specific structures, such as the ''hairpin-like'' twisted molecules model as an example, which results in shorter proteins than predicted based on their amino acid sequence (Singh et al 2014a).Though some previous studies have suggested some TAAs appear as straight, fibre-like structures on the cell surface, they become bent once they bind to host ligands (Agnew et al 2011;Ishikawa et al 2014). …”

“…Secondly, many TAAs contain more than one binding site. Daigneault et al found that AhsA from Mannheimia haemolytica A1 contains more than 21 collagen-binding motifs as identified by sequence analysis (Daigneault and Lo 2009); the Hia and Hif proteins contain 2 and 3 binding sites, respectively (Meng et al 2008;Singh et al 2014a). Finally, for interaction between collagen and TAAs, binding capacity can be affected by both collagen and state of TAAs.…”

Interactions between the Trimeric Autotransporter Adhesin EmaA and Collagen Revealed by Three-Dimensional Electron Tomography

“…Although sequence homology among the class members is low, the extracellular domains present a modular organization composed of different common motifs that have been annotated and are continuously updated (12). Structural information of a number of motifs has been obtained by X-ray crystallography of recombinantly expressed domains in combination with molecular modeling (11,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28) and of the native functional region of EmaA by electron tomography and subvolume averaging (29,30). Yet, despite all of the directly available wealth of information, no physical structural data is available for a TAA/substrate complex.…”