Initial Characterization of the Hemolysin Stachylysin fromStachybotrys chartarum

Abstract

Stachybotrys chartarum is a toxigenic fungus that has been associated with human health concerns, including pulmonary hemorrhage and hemosiderosis. This fungus produces a hemolysin, stachylysin, which in its apparent monomeric form has a molecular mass of 11,920 Da as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry. However, it appears to form polydispersed aggregates, which confounds understanding of the actual hemolytically active form. Exhaustive dialysis or heat treatment at 60°C for 30 min inactivated stachylysin. Stachylysin is composed of about 40% nonpolar amino acids and contains two cysteine residues. Purified stachylysin required more than 6 h to begin lysing sheep erythrocytes, but by 48 h, lysis was complete. Stachylysin also formed pores in sheep erythrocyte membranes.Hemolysins are molecules that are designated as such because they have the ability to lyse erythrocytes (RBCs). It is now recognized that the biological significance of these toxins goes beyond their lysis of RBCs to their more general ability to form pores in many cells (4). Today, the consensus is that the majority of relevant bacterial pathogens produce pore-forming proteins (4). Hemolysins have been isolated and purified from many bacterial pathogens, and they are generally important virulence factors (3-45, 13, 17, 18, 22, 25).It is often thought that bacterial cytolysins act primarily by killing host cells, but nonlethal reactions in other cells, including endothelial and immune cells, occur as a result of exposure to these toxins (4, 6). Many bacterial hemolysins create pores not only in RBCs, but also in the membranes of nucleated cells (e.g., neutrophils, monocytes, and endothelial cells) (2,10,20), and can affect the aggregation of platelets (12). These hemolysin-mediated responses affect the pathophysiology of the host.A number of fungal pathogens produce hemolysins (11,19,26). Recently, production of a hemolysin by the toxigenic fungus Stachybotrys chartarum was described (23). S. chartarum is a toxigenic fungus that has been associated with human health concerns, including pulmonary hemorrhage and hemosiderosis (PH) in infants in Cleveland, Ohio (7). In this paper, we describe the isolation, purification, and characterization of a poreforming hemolysin, stachylysin, produced by S. chartarum. MATERIALS AND METHODSPurification of hemolysin. S. chartarum conidia of strain 58-06 were produced after 5 weeks of growth on sterile wall board, as previously described (23). Approximately 10 5 conidia in a 100-l suspension were used to inoculate 500 ml of tryptic soy broth (TSB) (Becton Dickinson, Sparks, Md.) in a 1-liter flask placed on an incubator shaker (LabLine, Inc. Melrose Park, Ill.) set at 36 Ϯ 1°C and mixed at 200 rpm/min. After 7 days of incubation, the cells and debris were removed from the culture by centrifugation for 15 min at 5,000 ϫ g in an RC5centrifuge (Dupont Instruments, Newton, Conn.), and the supernatant was recovered. The supernatant was centrifuged in a Millipore C...

Possible Role of Fungal Hemolysins in Sick Building Syndrome

“…So far, all of these hemolysins demonstrate a characteristic response on SBA-that is, there is initial darkening of the SBA medium beyond the colony. This darkened area turns bluish green and then clears over 24 to 48 h (Van Emon et al, 2003;Vesper et al, 2001). (This is similar to the slow reaction described by Luo et al [2001] for the Candida hemolysins.)…”

“…We showed that stachylysin caused pores in sheep 200 VESPER AND VESPER RBC membranes (Vesper et al, 2001), but L. terrestis has no oxygencarrying cells and a closed vascular system. The release of erythrocruorin hemoglobin from L. terrestis and the development of aneurism-like structures suggested that stachylysin caused cytolytic vascular leakage, resulting in an LD 50 of 1100 g/Kg in L. terrestis (Vesper and Vesper, 2002).…”

“…Not until 2001 was StachylysinÈ recognized as a proteinaceous hemolytic agent produced by the filamentous fungus Stachybotrys chartarum (Vesper et al, 1999(Vesper et al, , 2001. The monomeric form of stachylysin has a MW of 11.9 kDa, as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS), with 114 amino acids, two of which are cysteine (Vesper et al, 2001).…”

“…The monomeric form of stachylysin has a MW of 11.9 kDa, as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS), with 114 amino acids, two of which are cysteine (Vesper et al, 2001). More recently, chrysolysin was purified from Penicillium chrysogenum (Donohue et al, 2004).…”

Health hazard evaluation report: HETA #2005-0126 and #2005-0138-3004, International Marine Terminal, Scotia Prince Cruises and Department of Homeland Security, U.S. Customs and Border Protection, Portland, Maine.

“…2,3 After incubation, the coated plates w ere washed three times with phosphate-buffered saline-0.05% Tween 20 (PBST, Sigma). Standards of 11 different concentrations of stachy lysin-BSA (0 to 62.5 ng/ml) diluted in 1: 20 diluted (PBST) commercially available human sera o r diluted (1:20 in PBS T) subject sera (in triplicate) were mixed with affinit y-purified rabbit antistachylysin IgG ( Bethyl Larboratories, Montgomery, TX) diluted 1:20,000 in PBST) in test tubes and incubated at 4°C for 2 4 hours.…”