Crystal structure of theHaemophilus influenzaeHap adhesin reveals an intercellular oligomerization mechanism for bacterial aggregation

Abstract

Bacterial biofilms are complex microbial communities that are common in nature and are being recognized increasingly as an important determinant of bacterial virulence. However, the structural determinants of bacterial aggregation and eventual biofilm formation have been poorly defined. In Gram-negative bacteria, a major subgroup of extracellular proteins called self-associating autotransporters (SAATs) can mediate cell-cell adhesion and facilitate biofilm formation. In this study, we used the Haemophilus influenzae Hap autotransporter as a prototype SAAT to understand how bacteria associate with each other. The crystal structure of the H. influenzae Hap S passenger domain (harbouring the SAAT domain) was determined to 2.2 Å by X-ray crystallography, revealing an unprecedented intercellular oligomerization mechanism for cell-cell interaction. The C-terminal SAAT domain folds into a triangular-prism-like structure that can mediate Hap-Hap dimerization and higher degrees of multimerization through its F1-F2 edge and F2 face. The intercellular multimerization can give rise to massive buried surfaces that are required for overcoming the repulsive force between cells, leading to bacterial cell-cell interaction and formation of complex microcolonies.

The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated bacterial clumping

“…2A). This α 43a crystallographic dimer resembles the trans oligomerization mechanism described for the Haemophilus influenzae Hap AT adhesin (18). However, whereas Hap-Hap assembly is only stabilized by weak van der Waals interactions, investigation of the α 43a dimer interfaces (interfaces 1 and 1′) using the web-based software PISA (19) identified hydrogen bonds and salt bridges stabilizing α 43a -α 43a assembly.…”

“…2 and 4) resembling Hap-Hap self-oligomerization (18). This allows us to propose a model for a universal mechanism of ATdriven bacterial aggregation, which resembles a modified molecular Fig.…”

“…Despite their abundance and the increasing amount of data linking AT proteins to bacterial virulence, very little structural information is available at a molecular level for these proteins. In fact, AT proteins are significantly underrepresented in the Protein Data Bank (PDB), with only 1 fulllength AT structure, 7 AT passenger domains, 5 AT β-domains, and 12 small domains of trimeric autotransporters deposited among the ∼87,500 structures currently available in the PDB (9,18,(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). None of these entries belong to the large family of AIDA-I-type AT proteins from Gamma-Proteobacteria (3,16).…”

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

“…Unlike the type I-IV secretory systems that involve multiple proteins, autotransporters are composed of a single protein consisting of an N-terminal signal peptide for secretion, followed by a passenger domain, and a C-terminal translocator (membrane anchoring) domain Leo et al, 2012). These autotransporters are multifunctional proteins ranging from monomeric to multimeric arrangements (Meng et al, 2011). Recently, the biological role of few autotransporters has been studied in H. influenzae.…”

“…Recently, the biological role of few autotransporters has been studied in H. influenzae. The monomeric autotransporter Haemophilus adhesion and penetration protein (Hap) is involved in bacterial aggregation and adherence to host cells (Meng et al, 2011;Hallström et al, 2011). A trimeric autotransporter adhesin (TAA) known as Hia has also been extensively studied for its functional and structural characteristics Spahich and St Geme, 2011).…”