Localization of the Domains of the Haemophilus ducreyi Trimeric Autotransporter DsrA Involved in Serum Resistance and Binding to the Extracellular Matrix Proteins Fibronectin and Vitronectin

Abstract

Resisting the bactericidal activity of naturally occurring antibodies and complement of normal human serum is an important element in the evasion of innate immunity by bacteria. In the gram-negative mucosal pathogen Haemophilus ducreyi, serum resistance is mediated primarily by the trimeric autotransporter DsrA. DsrA also functions as an adhesin for the extracellular matrix proteins fibronectin and vitronectin and mediates attachment of H. ducreyi to keratinocytes. We sought to determine the domain(s) of the 236-residue DsrA protein required for serum resistance and extracellular matrix protein binding. A 140-amino-acid truncated protein containing only the C-terminal portion of the passenger domain and the entire translocator domain of DsrA exhibited binding to fibronectin and vitronectin and conferred serum resistance to an H. ducreyi serum-sensitive strain. A shorter DsrA construct consisting of only 128 amino acids was unable to bind to extracellular matrix proteins but was serum resistant. We concluded that neither fibronectin binding nor vitronectin binding is required for high-level serum resistance in H. ducreyi.

Trimeric Autotransporter DsrA Is a Major Mediator of Fibrinogen Binding in Haemophilus ducreyi

“…To complement a panel of truncated DsrA proteins already constructed (17), two more in-frame dsrA I deletion mutants were created in the N-terminal region of the passenger domain of DsrA I . Two forward primers, DsrAxH and DsrAxI (Table 2), were each used separately in combination with primer DsrAxE (17) and pUNCH1260 as the template to generate two separate PCR products using the following conditions: 95°C for 5 min for 1 cycle, followed by 30 cycles at 95°C for 1 min, 52°C for 1 min, and 72°C for 1 min, with a final polishing step at 72°C for 5 min. The products from these PCRs (DsrAxH-E and DsrAxI-E) were digested overnight with XmaI, cleaned, and ligated for 30 min at room temperature with XmaI-treated pUNCH1424, as previously described (17), to form pUNCH2105 and pUNCH2106, respectively.…”

“…DsrA does not bind collagen but interacts with Fn and vitronectin (Vn) (15,16). Fn and Vn binding are mediated by amino acids in the C-terminal region of the DsrA passenger domain (17).…”

Vitronectin in host pathogen interactions and antimicrobial therapeutic applications

“…Both Gram-positive and Gram-negative pathogens have, however, the capacity to bind Vn and utilize the molecule for inhibition of the MAC and/or adhesion to host tissues. The Gram-negative H. influenzae, for example, recruits Vn by the surface exposed adhesin protein E (PE), and Haemophilus surface fibrils (Hsf) [30][31][32], whereas H. ducreyi binds Vn by the protein DsrA [33]. On the other hand, Moraxella catarrhalis interacts with Vn by ubiquitous surface protein (Usp) A2 [7,34,35].…”

Attachment of Actinobacillus suis H91-0380 and Its Isogenic Adhesin Mutants to Extracellular Matrix Components of the Tonsils of the Soft Palate of Swine

“…The processes of attachment and colonization are mediated primarily by fimbrial and afimbrial bacterial adhesins and various host cell receptors, including ECM components. A limited number of studies has been done to identify host receptors in the oral cavity of swine, but among members of the family Pasteurellaceae that are human pathogens, the trimeric autotransporter adhesin DsrA of Haemophilus ducreyi has been shown to bind to fibronectin and vitronectin in vitro (16), and the EmaA autotransporter of Aggregatibacter actinomycetemcomitans binds to collagen V (17). In addition, the purified lipoprotein e (P4) of nontypeable Haemophilus influenzae binds with high affinity to laminin, fibronectin, and vitronectin (18), while the OmpA outer membrane protein (OMP) of the important veterinary pathogen Pasteurella multocida adheres to cell surface fibronectin (19), as does OmpA of Mannheimia haemolytica (20).…”