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

Abstract

Tonsils conduct immune surveillance of antigens entering the upper respiratory tract. Despite their immunological function, they are also sites of persistence and invasion of bacterial pathogens. Actinobacillus suis is a common resident of the tonsils of the soft palate in pigs, but under certain circumstances it can invade, causing septicemia and related sequelae. Twenty-four putative adhesins are predicted in the A. suis genome, but to date, little is known about how they might participate in colonization or invasion. To better understand these processes, swine tonsil lysates were characterized by mass spectrometry. Fifty-nine extracellular matrix (ECM) proteins were identified, including small leucine-rich proteoglycans, integrins, and other cell surface receptors. Additionally, attachment of the wild type and 3 adhesin mutants to 5 ECM components was evaluated. Exponential cultures of wild-type A. suis adhered significantly more than stationary cultures to all ECM components studied except collagen I. During exponential growth, the A. suis ⌬flp1 mutant attached less to collagen IV while the ⌬ompA mutant attached less to all ECMs. The ⌬comE1 strain attached less to collagen IV, fibronectin, and vitronectin during exponential growth and exhibited differential attachment to collagen I over short adherence time points. These results suggest that Flp1, OmpA, and ComE1 are important during early stages of attachment to ECM components found in tonsils, which supports the notion that other adhesins have compensatory effects during later stages of attachment.T onsils are secondary lymphoid organs that are an important part of the host immune response against antigens entering the body through the mouth and nose. Pigs have 5 sets of tonsils located around the oropharyngeal cavity in the so-called Waldeyer's ring, the largest being the tonsils of the soft palate (1), which are thought to be functionally equivalent to the palatine tonsils in humans (2). The tonsils of the soft palate of swine have as many as 200 branching crypts that end blindly within the lymphoid tissue beneath (3). The lumina of the crypts are covered by a layer of nonkeratinized squamous epithelium that is continuous with the epithelium of the oral cavity, while lymphoepithelium lines the deeper areas of the crypts (4). Lymphoepithelium has a thinner epithelial lining interspersed with goblet cells and M cells, the latter of which can be used by some pathogens to invade the tonsil (3, 5). The lymphoepithelium also allows passage of lymphocytes, plasma cells, and macrophages (2). The epithelium of the crypts overlays a basement membrane (sometimes fragmented), which in turn is supported by an interstitial extracellular matrix (ECM) of various thickness, depending on the location in the tonsil (3). Despite studies of the ultrastructure and fine structure of the tonsils of swine (1, 3) and studies into the ECM components and host cell receptors present in tonsils of other species (6-8), little has been done to characterize these components in the tonsi...