Impact of N -Chlorotaurine on Viability and Production of Secreted Aspartyl Proteinases of Candida spp

Abstract

N-Chlorotaurine (NCT) (Cl-HN-CH 2 -CH 2 -SO 3 Ϫ ), the Nchloro derivative of the amino acid taurine, is the main representative of long-lived oxidants produced by stimulated human leukocytes (19). Besides immune modulatory effects (11), NCT has bactericidal, virucidal, and vermicidal activities (12,14,15,20), including a lag of regrowth of bacteria (postantibiotic effect) after short, sublethal incubation times accompanied by a loss of virulence (13). It has been conceived to be useful as an antimicrobial agent in local treatment of, e.g., eye and urinary tract infections (16).The aim of this study was a quantitative evaluation of fungicidal and postantifungal effects of NCT against Candida albicans at a concentration of 1%, which is well tolerated by human tissue in vivo (16). In addition, the influence of NCT on the production of secreted aspartyl proteinases, a superfamily of enzymes important for cell adhesion and invasion and thus fungal virulence factors, was investigated (3, 10, 18).Pure NCT as a crystalline sodium salt (M r ϭ 181.57 g/mol; kindly prepared by Waldemar Gottardi, Institute of Hygiene and Social Medicine, Innsbruck, Austria) was dissolved in 0.01 M phosphate-buffered saline at pH 7.0 to 7.4.C. albicans strains CBS 5982 (Centraal Bureau voor Schimmelculturen, Baarn, The Netherlands), ATCC 5314, ATCC 15053, and five clinical Candida sp. isolates, C. krusei ATCC 6128 and a clinical isolate, C. glabrata ATCC 6425, C. tropicalis ATCC 13803, and C. dubliniensis (kindly provided by D. Coleman, University of Dublin, Ireland) were grown on Sabouraud dextrose agar (Oxoid, Basingstoke, United Kingdom) for 48 h. Yeast cells were suspended in saline, washed twice, adjusted to ca. 1 ϫ 10 8 CFU/ml, and diluted 50-fold (0.1 to 4.9 ml) to 2 ϫ 10 6 CFU/ml in buffered 1% NCT solutions. For testing of the fungicidal activity, aliquots of 250 l were removed after incubation times of 1 to 5 h at 20 or 37°C and mixed with 125 l of 6% sodium thiosulfate solution to inactivate NCT. Quantitative cultures of adequate dilutions on tryptic soy agar (Merck) were performed by using an automatic spiral plater (model WASP; Whitley, Shipley, United Kingdom), and CFU were counted after incubation at 37°C for 48 h (detection limit, 20 CFU/ml). Controls without NCT and controls with fungi added to NCT previously inactivated by sodium thiosulfate were performed in parallel.For testing of the postantifungal effect (PAFE), yeasts were incubated at 20°C in 1% NCT solution for sublethal incubation times of 1 to 45 min. Subsequent to inactivation of NCT and a washing step in saline, yeasts were diluted 100-fold in prewarmed tryptic soy broth (Merck) and regrown at 37°C. Quantitative cultures were performed hourly. The lag of regrowth was calculated as the difference between the test and control cultures in the time taken to increase 1 log 10 above the count at zero time (2).For testing of the effect of NCT on production of secreted aspartyl proteinases (Saps), yeasts (2 ϫ 10 6 CFU) were incubated at 20°C in 1 ml of Sap induction med...

N -Chlorotaurine Exhibits Fungicidal Activity against Therapy-Refractory Scedosporium Species and Lomentospora prolificans

“…Due to its low reactivity, which is the reason for its good tolerability in sensitive body regions (12), its killing activity against microorganisms is slower than that with highly reactive compounds, like hypochlorous acid (11,34). In previous studies (20,21,35), killing of different Candida species and molds needed incubation times of one or only few hours, and in the present study, that against Scedosporium spp. turned out to be similar.…”

“…However, already short and sublethal incubation times in NCT caused a lag in the regrowth of bacteria and fungi (20,30,43) and a reduction in the production of secretory aspartyl proteinases in Candida albicans (20). Moreover, this was connected with a loss of virulence in vivo in a staphylococcal and streptococcal mouse peritonitis model (30,43).…”

“…In previous studies, bacteria and yeasts treated for sublethal incubation times with NCT showed a lag in regrowth (postantibiotic effect) and a loss of virulence (20,28,30). This may also be important for a possible future in vivo application of NCT against Scedosporium.…”

“…Fungicidal activity has already been shown against other fungi (yeasts and molds [19][20][21]) without drug resistance, in contrast to the case with antifungals (22). Particularly for skin lesions, the addition of ammonium chloride (NH 4 Cl) with a formulation of monochloramine (NH 2 Cl), which kills fungi within minutes, has to be considered (21,23).…”

N -Chloramines, a Promising Class of Well-Tolerated Topical Anti-Infectives

“…Actually, the active chlorine compound NCT has been shown to remove the virulence of Staphylococcus aureus and Streptococcus pyogenes after sublethal incubation times in the mouse peritonitis model (30,55). This was connected with a lag of regrowth (30,55,56) and with chlorination of the surface of the pathogens (27). Moreover, secretory aspartyl proteinases of Candida albicans were downregulated by sublethal concentrations of NCT, and gliotoxin of Aspergillus fumigatus was obviously destroyed by this compound (56,57).…”