Chromosomal Expression of the Haemophilus influenzae Hap Autotransporter Allows Fine-Tuned Regulation of Adhesive Potential via Inhibition of Intermolecular Autoproteolysis

Abstract

The Haemophilus influenzae Hap autotransporter is a nonpilus adhesin that promotes adherence to respiratory epithelial cells and selected extracellular matrix proteins and facilitates bacterial aggregation and microcolony formation. Hap consists of a 45-kDa outer membrane translocator domain called Hap ␤ and a 110-kDa extracellular passenger domain called Hap S . All adhesive activity resides within Hap S , which also contains protease activity and directs its own secretion from the bacterial cell surface via intermolecular autoproteolysis. In the present study, we sought to determine the relationship between the magnitude of Hap expression, the efficiency of Hap autoproteolysis, and the level of Hap-mediated adherence and aggregation. We found that a minimum threshold of Hap precursor was required for autoproteolysis and that this threshold approximated expression of Hap from a chromosomal allele, as occurs in H. influenzae clinical isolates. Chromosomal expression of wild-type Hap was sufficient to promote significant adherence to epithelial cells and extracellular matrix proteins, and adherence was enhanced substantially by inhibition of autoproteolysis. In contrast, chromosomal expression of Hap was sufficient to promote bacterial aggregation only when autoproteolysis was inhibited, indicating that the threshold for Hap-mediated aggregation is above the threshold for autoproteolysis. These results highlight the critical role of autoproteolysis and an intermolecular mechanism of cleavage in controlling the diverse adhesive activities of Hap.Most bacterial diseases begin with colonization of a particular mucosal surface. Successful colonization requires that bacteria overcome mechanical cleansing and evade the local immune response. A number of specific mechanisms exist to achieve these goals, including the expression of surface-exposed proteins called adhesins, which bind to receptors on the host epithelial surface, and the formation of structured communities called biofilms, which resist mechanical, chemical, and immunological attack (20).Nontypeable Haemophilus influenzae is a nonencapsulated, gram-negative bacterium that commonly causes human respiratory tract disease, including otitis media, sinusitis, bronchitis, and pneumonia (19). Infection by nontypeable H. influenzae begins with colonization of the nasopharynx (10). In most individuals, colonization persists for weeks to months without symptoms. However, under certain circumstances bacteria spread contiguously to the middle ear, the sinuses, or the lungs, resulting in disease. Studies of tympanostomy tubes from patients with recurrent otitis media and examination of experimentally infected chinchillas suggest that biofilm formation may represent an important mechanism of persistence by nontypeable H. influenzae (3, 11).To facilitate colonization, H. influenzae elaborates both pilus and nonpilus adhesins. The Hap protein is a nonpilus adhesin that was first identified based on its ability to promote intimate interaction with cultured epithelial cells ...

A Novel Autotransporter Adhesin Is Required for Efficient Colonization during Bubonic Plague

“…Conversely, cleavage may allow Y. pestis to release itself from YapE-mediated interactions with eukaryotic cells. A similar release mechanism has been proposed for the Hap adhesin of Haemophilus influenzae, although Hap relies on autoproteolysis for cleavage (24).…”

Proteolytic Processing Is Not Essential for Multiple Functions of the Escherichia coli Autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I)

“…To address this question, we evaluated the extent of autoproteolysis under different conditions of induction, in order to assess if AIDA-I cleavage varied with protein density. Similar experiments were used to characterize the intermolecular cleavage of Hap (14,15). We used plasmid pAg, which contains the aidA and aah genes in their original operon organization but under the control of an IPTG-inducible promoter.…”

“…The Haemophilus influenzae autotransporter adhesin Hap is also cleaved after translocation (52). Inhibition of the autoproteolytic cleavage of Hap increased bacterial autoaggregation and adhesion, suggesting that the cleavage acts as a regulation mechanism (15,23,24). These proteins, however, are significantly different from AIDA-I, in sequence and function; thus, the mechanism and function of the cleavage of AIDA-I remain obscure.…”

Type V Protein Secretion Pathway: the Autotransporter Story

“…Hendrixson and St Geme (207) found that the uncleaved cell-associated form of Hap mediates adherence to cultured epithelial cells and promotes bacterial aggregation and microcolony formation, whereas more recent data demonstrated an interaction between Hap and extracellular matrix proteins (135,137,138). Adherence and aggregation are augmented by secretory leukocyte protease inhibitor, a soluble protein component of respiratory secretions that inhibits Hap autoproteolysis.…”