We recently described the ability of PLD to reorganize host AZD5363 membrane lipid rafts, leading to enhanced bacterial adhesion [9]. Furthermore, A. haemolyticum was able to invade HeLa cells and once intracellular, PLD was able to kill host cells via direct necrosis [9]. These effects could potentially lead to bacterial dissemination to deeper tissues. It is thought that clinical microbiology laboratories often miss A.

haemolyticum in clinical specimens due to the organism’s weak hemolytic activity on the commonly-used sheep blood agar, and therefore it may be misinterpreted as commensal diphtheroids and the isolate discarded. However, this organism displays more pronounced hemolysis on human and rabbit blood [10, 11]. The organism has been known to have hemolytic activity since its initial discovery in 1946 [12], yet no bona fide hemolysin has been previously reported. PLD itself is not directly hemolytic, but causes synergistic hemolysis with bacteria that express cholesterol oxidase [13], prompting a search for the A. haemolyticum hemolysin. Possible clues to the identity of the

A. haemolyticum Bafilomycin A1 order hemolysin come from studies on the hemolytic bacterium T. pyogenes, which is closely related to A. haemolyticum. T. pyogenes expresses PLO, a member

of the cholesterol-dependent cytolysin (CDC) toxin family, as its primary virulence factor and this molecule is a hemolysin [14]. Thus, we hypothesized that the hemolytic activity expressed by A. haemolyticum was due to the Sitaxentan presence of an uncharacterized CDC. Here we report the identification and characterization of a CDC from A. haemolyticum, designated arcanolysin (ALN). We show that ALN has several distinct structural features among the CDC family and demonstrate that ALN is cholesterol-dependent and provide evidence that ALN has variable hemolytic and cytotoxic activity against mammalian cells from different species. We propose ALN is the long, sought-after hemolysin. Methods Bacteria and growth conditions ATCC 9345 is the A haemolyticum type strain. The other A. haemolyticum strains used in this study were archival isolates obtained from diverse human clinical cases (Table 1). A. haemolyticum and Escherichia coli strains were grown as previously described [9]. Table 1 Arcanobacterium strains used in this study. Strain (all A.