Energy Depletion Protects Candida albicans against Antimicrobial Peptides by Rigidifying Its Cell Membrane

Abstract

Inhibitors of the energy metabolism, such as sodium azide and valinomycin, render yeast cells completely resistant against the killing action of a number of cationic antimicrobial peptides, including the salivary antimicrobial peptide Histatin 5. In this study the Histatin 5-mediated killing of the opportunistic yeast Candida albicans was used as a model system to comprehensively investigate the molecular basis underlying this phenomenon. Using confocal and electron microscopy it was demonstrated that the energy poison azide reversibly blocked the entry of Histatin 5 at the level of the yeast cell wall. Azide treatment hardly induced depolarization of the yeast cell membrane potential, excluding it as a cause of the lowered sensitivity. In contrast, the diminished sensitivity to Histatin 5 of energy-depleted C. albicans was restored by increasing the fluidity of the membrane using the membrane fluidizer benzyl alcohol. Furthermore, rigidification of the membrane by incubation at low temperature or in the presence of the membrane rigidifier Me 2 SO increased the resistance against Histatin 5, while not affecting the energy charge of the cell. In line, azide induced alterations in the physical state of the interior of the lipid bilayer. These data demonstrate that changes in the physical state of the membrane underlie the increased resistance to antimicrobial peptides.In the last few decades an expanding number of antimicrobial peptides have been isolated from virtually all classes of organisms, where they play an important role in the innate defense against microbial and viral infections. Characterization of these peptides has revealed a wide diversity in amino acid sequences, yet they share characteristic features; they are usually polycationic and amphipathic, containing both a hydrophilic and a hydrophobic side. This promotes their insertion into and transmigration over the cytoplasmic membrane of the target cell, with killing of the cell as a final consequence. Interestingly, cellular sensitivity to cationic proteins and peptides such as salivary histatins and defensins is diminished by conditions that affect the energy status of the target cell (1-6). This effect is not restricted to cationic peptides, because azoles are also sensitive to the energy status of Candida glabrata (7). In Chlorella metabolic inhibition abolishes the membrane-disruptive effects of the polyene nystatin and even of the detergent Triton X-100 (8). As explanation for the desensitizing effects of energy depletion, it has been proposed that interaction of cationic peptides with the target cell would involve active transport systems, which for their activity are dependent on the energy charge of the cell (1, 9 -12). However, direct experimental proof corroborating this hypothesis is still lacking.The present study addresses the question of how on a molecular level the energy metabolism is linked with the sensitivity of the Candida cell to antimicrobial peptides. Thus far, many different types of mechanisms have been identified that contr...

Histatin 5 Uptake by Candida albicans Utilizes Polyamine Transporters Dur3 and Dur31 Proteins

“…Our finding that Hst 5 utilizes polyamine transporters for its intracellular uptake now explains the loss of Hst 5 uptake and killing following incubation of C. albicans with these agents. The requirement of ATPases for polyamine permease activity may also explain the finding that addition of the organic solvent dimethyl sulfoxide (10 -40%) to C. albicans cells reduced Hst 5 toxicity (20), because this solvent also inhibits ATPase activity (48). Thus, the observation that dimethyl sulfoxide reduces Hst 5 toxicity is likely not to be a result of increasing membrane rigidity as previously suggested (20), but rather due to loss of polyamine permease activity needed for Hst 5 uptake.…”

“…The requirement of ATPases for polyamine permease activity may also explain the finding that addition of the organic solvent dimethyl sulfoxide (10 -40%) to C. albicans cells reduced Hst 5 toxicity (20), because this solvent also inhibits ATPase activity (48). Thus, the observation that dimethyl sulfoxide reduces Hst 5 toxicity is likely not to be a result of increasing membrane rigidity as previously suggested (20), but rather due to loss of polyamine permease activity needed for Hst 5 uptake. A similar intracellular transport mechanism has been characterized for the antineoplastic drug bleomycin in human and S. cerevisaie cells.…”

“…Although it has been suggested that azide blocks Hst 5 internalization by cell membrane rigidification due to ATP depletion (20), two other energy-dependent mechanisms for intracellular transport are possible including endocytosis and active transport of histatin through a cellular transporter. Indeed, Hst 5 is internalized by endocytotic transport to the vacuole (21), although substantial levels of cytosolic Hst 5 are found prior to endocytotic uptake (17).…”

Bacillus globigii cell size is influenced by variants of the quorum sensing peptide extracellular death factor

“…Peptide synthesis and purification All peptides (Table 2, including all EDFs, D-EDFs and alanine substituted variants) were synthesized by solid phase peptide synthesis using Fmoc chemistry with a MilliGen 9050 peptide synthesizer (MilligenBiosearch, Bedford, MA, USA) and purification by reverse phase-HPLC was conducted as described previously (Veerman et al 2007). The purity of all peptides was [90 %.…”

Styraxjaponoside A and B, Antifungal Lignan Glycosides Isolated from Styrax japonica S. et Z.

“…The metabolic inhibitor regarding the ATP depletion can inhibit an imported system from extracellular environments into the cell interior and also disrupt the proton gradient, resulting in membrane depolarization (Veerman et al, 2007). Therefore, to examine the relationship between the cellular energy consumption and the transport pathway of styraxjaponoside A and B, an energy-dependence test was conducted.…”