Bio-available Zn2+ in the growth medium as a cue for Leishmania to express its protective surface protease

In the arthropod-vector phase of their digenetic life-cycle, the parasites that cause human leishmaniasis undergo a process termed metacyclogenesis, in which they acquire an enhanced ability to establish infection in mammalian macrophages (Chang et al., 1990; Handman and Bullen, 2002; Engwerda et al., 2004; Murray et al., 2005). This process is mimicked in axenic cultures of the parasites, when the multiplication of the Leishmania cells reaches its stationary phase (Da Silva and Sacks, 1987; Chang et al., 1990; Alexander et al., 1999; Ramamoorthy et al., 1992, 1995, 1996; Wilson et al., 1993; Roberts et al., 1995). The results of previous studies of Leishmania cultures (Kweider et al., 1987; Chaudhuri et al., 1989; Chang et al., 1990; Ramamoorthy et al., 1992; Wilson et al., 1993) indicate that the infectivity of the stationary-phase promastigotes is directly correlated with the increase in the surface density of a protease called leishmanolysin (also known as gp63 or Msp). The abundant expression of this protease on the surface of the promastigotes is also necessary for the establishment of infection inside macrophage phagolysosomes (Chaudhuri et al., 1989; Seay et al., 1996). In the present study, it was found that, in axenic cultures of L. amazonensis, high concentrations of bio-available Zn, but not of other similar metal ions that were investigated (data not shown), inhibit the expression of this protective protease during the early phases of growth. It appears to be the release from this Zn suppression, as the result of the secretion of metal-ion chelators by the parasite cells at the later phases of growth, that allows the infectivity of the parasites to increase (see Table). Promastigotes of L. amazonensis (MHOM/BR/73/LV78) were grown at 25uC in medium M199 supplemented with 10% heat-inactivated foetal bovine serum (Misra et al., 2005). The metallochromic indicator 4-(2-pyridylazo) resorcinol (PAR) was used to monitor the amount of ‘free’ Zn in the spent media (Shang et al., 1989). A 5 mM stock solution of PAR was prepared by dissolving the solid dye in deionized water and adjusting the pH to 8.8 by NaOH titration. Water or other stock solutions were de-ionized by routine passage through Chelex 100 (200to 400-mesh; BioRad, Hercules, CA). Total zinc in the medium was analysed (Shang et al., 1989) using an atomic absorption spectrometer (Model 2380; Perkin-Elmer, Norwalk, CT) containing a graphite furnace. Before use, all glassware employed in the study was soaked in 30% nitric acid and 10 mM EDTA and rinsed rigorously with de-ionized water. The Zn content was calculated from a standard curve constructed using a Zn standard solution (Fisher Scientific, Pittsburgh, PA) diluted with de-ionized water. For the purification and characterization of leishmanolysin, the Leishmania proteins were biosynthetically labelled (Chaudhuri et al., 1989; Seay et al., 1996) with Smethionine before the surface protease (SP) was immunoprecipitated and quantified (Chaudhuri et al., 1989; Seay et al., 1996). The proteinase activity of the purified SP (Chaudhuri et al., 1989; Seay et al., 1996) was assayed against I-bovine serum Annals of Tropical Medicine & Parasitology, Vol. 101, No. 1, 89–93 (2007)