AasP autotransporter protein of Actinobacillus pleuropneumoniae does not protect pigs against homologous challenge.

Actinobacillus pleuropneumoniae is a major respiratory pathogen of pigs; current vaccines provide only limited protection. AasP, a subtilisin-like serine protease, is a conserved outer membrane-localised autotransporter protein. We hypothesized that AasP would induce protective immunity and may thus constitute a useful component of a vaccine against A. pleuropneumoniae infection. Here we confirm experimentally that AasP is an antigenic in vivo-expressed protein. In pig protection studies, a detectable specific antibody response was induced in response to recombinant AasP. However, the vaccine was not capable of protecting pigs from colonization, infection or severe clinical disease resulting from challenge with the homologous A. pleuropneumoniae strain.

Functional characterization of AasP, a maturation protease autotransporter protein of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a highly contagious respiratory infection in pigs. AasP, a putative subtilisin-like serine protease autotransporter, has recently been identified in A. pleuropneumoniae. We hypothesized that, similarly to other autotransporters of this type, AasP may undergo autocatalytic cleavage resulting in release of the passenger domain of the protein. Furthermore, AasP may be responsible for cleavage of other A. pleuropneumoniae outer membrane proteins. To address these hypotheses, the aasP gene was cloned and the expressed recombinant AasP protein used to raise monospecific rabbit antiserum. Immunoblot analysis of whole-cell lysates and secreted proteins demonstrated that AasP does not undergo proteolytic cleavage. Immunoblot analysis also confirmed that AasP is universally expressed by A. pleuropneumoniae. Confirmation of the maturation protease function of AasP was obtained through phenotypic analysis of an A. pleuropneumoniae aasP deletion mutant and by functional complementation. Comparison of the secreted proteins of the wild type, an aasP mutant derivative, and an aasP mutant complemented in trans led to the identification of OmlA protein fragments that were present only in the secreted-protein preparations of the wild-type and complemented strains, indicating that AasP is involved in modification of OmlA. This is the first demonstration of a function for any autotransporter protein in Actinobacillus pleuropneumoniae.

Helicobacter pylori lipopolysaccharide interacts with TFF1 in a pH-dependent manner.

BACKGROUND & AIMS: Little is known about how bacteria establish chronic infections of mucosal surfaces. Helicobacter pylori (H. pylori), a chronic pathogen that lives in the gastric mucosa of humans, interacts with the trefoil factor family (TFF) protein TFF1, which is found in gastric mucus. We aimed to characterize the interaction of H. pylori with TFF1 and to assess the role of this interaction in mediating colonization. METHODS: Subcellular fractions of H. pylori were immobilized and then probed with TFF1, TFF2, or TFF3. The effect of glycosidases and preincubation with monosaccharides on the interaction and binding of TFF1 to a H. pylori adhesin was assessed. The interaction between H. pylori adhesin and TFF1 was characterized using surface plasmon resonance, flow cytometry, nondenaturing polyacrylamide gel electrophoresis, coimmunofluoresence, and incubation with tissue sections. RESULTS: The H. pylori core oligosaccharide portion (rough form) of lipopolysaccharide (RF-LPS) bound to TFF1 and to a lesser extent TFF3; this interaction was inhibited by incubation of RF-LPS with mannosidase, glucosidase, or mixed monosaccharides. TFF1 also bound to human serum albumin-conjugated mannose and glucose. The optimum pH for binding was 5.0-6.0 for TFF1 and 7.0 for TFF3. H. pylori bound TFF1 in gastric mucus ex vivo; binding of LPS-coated latex beads to human antral gastric tissue was inhibited by TFF1. CONCLUSIONS: TFF1 interacts specifically with H. pylori RF-LPS. The pH dependence of this interaction indicates that binding of H. pylori to TFF1 in the stomach could promote colonization of the mucus layer adjacent to the gastric epithelial surface.