An Antimicrobial Peptide Induces FIG1-Dependent Cell Death During Cell Cycle Arrest in Yeast

Abstract

Although most antibiotics act on cells that are actively dividing and non-dividing cells such as in microbe sporulation or cancer stem cells represent a new paradigm for the control of disease. In addition to their relevance to health, such antibiotics may promote our understanding of the relationship between the cell cycle and cell death. No antibiotic specifically acting on microbial cells arrested in their cell cycle has been identified until the present time. In this study we used an antimicrobial peptide derived from α-pheromone, IP-1, targeted against MATa Saccharomyces cerevisiae cells in order to assess its dependence on cell cycle arrest to kill cells. Analysis by flow cytometry and fluorescence microscopy of various null mutations of genes involved in biological processes activated by the pheromone pathway (the mitogen-activated protein kinase pathway, cell cycle arrest, cell proliferation, autophagy, calcium influx) showed that IP-1 requires arrest in G0/G1 in order to kill yeast cells. Isolating cells in different cell cycle phases by elutriation provided further evidence that entry into cell cycle arrest, and not into G1 phase, is necessary if our peptide is to kill yeast cells. We also describe a variant of IP-1 that does not activate the pheromone pathway and consequently does not kill yeast cells that express the pheromone’s receptor; the use of this variant peptide in combination with different cell cycle inhibitors that induce cell cycle arrest independently of the pheromone pathway confirmed that it is cell cycle arrest that is required for the cell death induced by this peptide in yeast. We show that the cell death induced by IP-1 differs from that induced by α-pheromone and depends on FIG1 in a way independent of the cell cycle arrest induced by the pheromone. Thus, IP-1 is the first molecule described that specifically kills microbial cells during cell cycle arrest, a subject of interest beyond the process of mating in yeast cells. The experimental system described in this study should be useful in the study of the mechanisms at play in the communication between cell cycle arrest and cell death on other organisms, hence promoting the development of new antibiotics.

Antimicrobial Peptide against Mycobacterium Tuberculosis That Activates Autophagy Is an Effective Treatment for Tuberculosis

“…Thus, IP-1 toxicity does not involve mitochondrial function. In agreement with this idea, we have shown that IP-1 kills yeast cells lacking mitochondria DNA [ 27 ] and more recently reported that IP-1 kills yeast cells grown in either respiratory (mitochondria competent) or fermenting (mitochondria incompetent) media [ 51 ].…”

Selective Moonlighting Cell-Penetrating Peptides

“…However, if NLS-α-CE and chimera peptides would be disrupting the membrane potential of MATa cells as part of their toxicity, we would expect these peptides to penetrate more easily these cells than the α-NLS-C peptide, which is the opposite of what we observed, hence the observed reduced internalization of NLS-α-CE and chimera peptides may not be explained by the toxicity reported for these peptides. On the other hand, we have reported that modifying the C-terminus region of the pheromone reduces its activity in a moonlighting peptide [ 13 ]. The NLS-α-CE is the only peptide that did not modify the C-terminus region of the pheromone and consequently we would not expect this peptide to reduce its affinity for its receptor, while the α-NLS-C and chimera peptides did, providing a possible explanation for their reduced ability to selectively penetrate MATa cells.…”

“…We adapted a previous protocol reported to analyze fluorescent single yeast cells [ 13 ]. Briefly, cells were grown overnight and diluted to a final optical density measured at 600 nm of 1.0.…”

“…This procedure was adapted from a previously reported protocol [ 13 ]. Cells were incubated with TatNep, TatNepNoCPP or D-TatNepNoCPP peptides at a concentration of 10, 5 and 1 μM (see Table 1 ).…”

Cation Transporters of Candida albicans—New Targets to Fight Candidiasis?

“…To maintain the directionality of the growth, the influx of calcium ions is necessary [ 96 ], and this is where Fig1 is recruited for regulating calcium ion uptake at sites of polarized tip growth [ 90 ] to enhance membrane stability during morphological transitions. Deletion of Fig1 gene leads to significantly reduced ability of hyphal tips to reorient upon contact with ridges, but it does not affect Ca 2+ uptake during vegetative growth [ 97 , 98 ].…”