Pentamidine Inhibition of Group I Intron Splicing in Candida albicans Correlates with Growth Inhibition

Abstract

We previously demonstrated that pentamidine, which has been clinically used against Pneumocystis carinii, inhibits in vitro a group I intron ribozyme from that organism. Another fungal pathogen, Candida albicans, also harbors a group I intron ribozyme (Ca.LSU) in the essential rRNA genes in almost half of the clinical isolates analyzed. To determine whether pentamidine inhibits Ca.LSU in vitro and in cells, phylogenetically closely related intron-containing (4-1) and intronless (62-1) strains were studied. Splicing in vitro of the Ca.LSU group I intron ribozyme was completely inhibited by pentamidine at 200 M. On rich glucose medium, the intron-containing strain was more sensitive to growth inhibition by pentamidine than was the intronless strain, as measured by disk or broth microdilution assays. On rich glycerol medium, they were equally susceptible to pentamidine. At pentamidine levels selectively inhibiting the intron-containing strain (1 M) in glucose liquid cultures, inhibition of splicing and rRNA maturation was detected by quantitative reverse transcription-PCR within 1 min with a 10-to 15-fold accumulation of precursor rRNA. No comparable effect was seen in the intronless strain. These results correlate the cellular splicing inhibition of Ca.LSU with the growth inhibition of strain 4-1 harboring Ca.LSU. Broth microdilution assays of 13 Candida strains showed that introncontaining strains were generally more susceptible to pentamidine than the intronless strains. Our data suggest that ribozymes found in pathogenic microorganisms but absent in mammals may be targets for antimicrobial therapy.

Fast formation of the P3-P7 pseudoknot: A strategy for efficient folding of the catalytically active ribozyme

“…This magnesium requirement is similar to that required for the optimal self-splicing activity of the ribozyme (Miletti and Leibowitz 2000;Zhang and Leibowitz 2001), suggesting that the folding of the native P3-P7 correlates with the natural catalytic activity of the ribozyme. However, this magnesium requirement is much lower than that required for the maximal activity of the first transesterification reaction mimicked by the Ca.L-11 catalyzed substrate cleavage (Xiao et al 2003), indicating that the tertiary interactions stabilized by high magnesium concentrations accelerate the first transesterification reaction, but hinder the self-splicing reaction, which involves two reactions at a single guanosine binding site (Cech 1990).…”

Combination Treatment of Invasive Fungal Infections

“…The aromatic diamidine pentamidine (PN) displays multiples effects against protozoa, bacteria and fungi (17, 53, 58, 132, 137, 217a, 237). Among the fungi, its effect has been mostly studied against Pneumocystis carinii (95,228), C. neoformans (10), C. albicans (150,162), and A. fumigatus (3). The in vitro effect of PN-plus-ITRA and PN-plus-KETO combinations on 11 C. albicans strains (including one azole-resistant isolate) has also been studied (217a).…”

Slow formation of a pseudoknot structure is rate limiting in the productive co-transcriptional folding of the self-splicing Candida intron

“…It has previously been demonstrated that inhibitors of group I introns that alter the ribozyme folding can act as antimicrobial agents in Candida strains in which the function of such ribozymes is essential for viability (Miletti and Leibowitz 2000;Zhang et al 2002). This report shows that the splicing activity of the C. albicans group I ribozyme is effectively compromised by AONs interrupting the folding of functionally important pseudoknot structures.…”