Molecular Mechanisms of Hemolysis by Brachyspira hampsonii

Abstract

The re-emergence of swine dysentery in Western Canadian swine herds has resulted in dramatic production losses for pork producers. The observation that pathogenic strains of the causative agents Brachyspira hyodysenteriae and Brachyspira hampsonii are strongly β-hemolytic in contrast to weakly β-hemolytic avirulent strains suggests that the production of one or more hemolysins by these bacteria may play a critical role in the pathogenesis of swine dysentery. Our research aimed to characterize the in vitro functionality of two hemolysins produced by Brachyspira hampsonii, termed TlyA and native hemolysin. TlyA protein from Brachyspira hampsonii was purified via heterologous expression from an E. coli host and found to possess both hemolytic and rRNA methyltransferase activities. Two amino acid residues, S9 and C80, were found to inhibit the oligomerization and hemolytic activity of TlyA partially or completely when mutated. These residues are conserved in TlyA proteins from both pathogenic Brachyspira species and unrelated pathogenic bacteria, suggesting a common mechanism of oligomerization for these proteins. Next, we purified a potent hemolysin from Brachyspira hampsonii broth cultures which we have termed native hemolysin. Although this toxin had previously been linked to a gene termed hlyA, the recent discovery of an operon encoding for a Streptolysin S homologue within the Brachyspira hampsonii strain 30446 genome has raised doubts on the identity of this toxin. The sagABCD genes from Brachyspira hampsonii strain 30446 were cloned and heterologously expressed in E. coli using a co-expression strategy, and the purified SagA peptide was found to possess hemolytic activity against pig erythrocytes, albeit with a much lower specific activity than the native hemolysin purified from Brachyspira hampsonii strain 30446 broth cultures. To assess the role native hemolysin plays in the pathogenesis of swine dysentery, we first exposed a colonic cell line to varying doses of native hemolysin to assess the cytotoxicity of this toxin. Surprisingly, no cytotoxic activity was observed in cells treated with 8 HU of native hemolysin for 24 hours. Rather, the observation that DIDS, a potent inhibitor of cellular anion-exchange proteins, could dramatically inhibit the hemolytic activity of native hemolysin suggested that the toxic effects of this peptide are not a result of direct pore-formation, but rather are mediated by the overactivation of Cl-/HCO3- exchangers such as band 3 or DRA. To test this hypothesis, Caco-2 monolayers were exposed to native hemolysin for 1 or 24 hours, and changes in electrogenic ion transport were assessed utilizing Ussing chamber studies. Remarkably, a significant decrease in electrogenic Na+ absorption through ENaC was observed within an hour of native hemolysin treatment, providing evidence that Brachyspira induced diarrhea initially occurs due to Na+ malabsorption within the colon of infected animals. Addition of DIDS to monolayers treated with native hemolysin ameliorated the decrease in electrogenic Na+ absorption, indicating that these effects are directly linked to the overactivation of the Cl-/HCO3- exchanger DRA by native hemolysin. Altogether, these results not only confirm that both TlyA and a Streptolysin S homologue produced by Brachyspira hampsonii strain 30446 are hemolytically active proteins, but also provide the first direct evidence of a link between the primary sequence of a TlyA protein and bacterial pathogenicity, along with providing a mechanistic explanation for the development of diarrhea during the acute stages of Brachyspira infection.