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. 2008 Oct;4(10):e1000190.
doi: 10.1371/journal.ppat.1000190. Epub 2008 Oct 31.

The Antimicrobial Peptide histatin-5 Causes a Spatially Restricted Disruption on the Candida Albicans Surface, Allowing Rapid Entry of the Peptide Into the Cytoplasm

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Free PMC article

The Antimicrobial Peptide histatin-5 Causes a Spatially Restricted Disruption on the Candida Albicans Surface, Allowing Rapid Entry of the Peptide Into the Cytoplasm

A Brian Mochon et al. PLoS Pathog. .
Free PMC article

Abstract

Antimicrobial peptides play an important role in host defense against microbial pathogens. Their high cationic charge and strong amphipathic structure allow them to bind to the anionic microbial cell membrane and disrupt the membrane bilayer by forming pores or channels. In contrast to the classical pore-forming peptides, studies on histatin-5 (Hst-5) have suggested that the peptide is transported into the cytoplasm of Candida albicans in a non-lytic manner, and cytoplasmic Hst-5 exerts its candicidal activities on various intracellular targets, consistent with its weak amphipathic structure. To understand how Hst-5 is internalized, we investigated the localization of FITC-conjugated Hst-5. We find that Hst-5 is internalized into the vacuole through receptor-mediated endocytosis at low extracellular Hst-5 concentrations, whereas under higher physiological concentrations, Hst-5 is translocated into the cytoplasm through a mechanism that requires a high cationic charge on Hst-5. At intermediate concentrations, two cell populations with distinct Hst-5 localizations were observed. By cell sorting, we show that cells with vacuolar localization of Hst-5 survived, while none of the cells with cytoplasmic Hst-5 formed colonies. Surprisingly, extracellular Hst-5, upon cell surface binding, induces a perturbation on the cell surface, as visualized by an immediate and rapid internalization of Hst-5 and propidium iodide or rhodamine B into the cytoplasm from the site using time-lapse microscopy, and a concurrent rapid expansion of the vacuole. Thus, the formation of a spatially restricted site in the plasma membrane causes the initial injury to C. albicans and offers a mechanism for its internalization into the cytoplasm. Our study suggests that, unlike classical channel-forming antimicrobial peptides, action of Hst-5 requires an energized membrane and causes localized disruptions on the plasma membrane of the yeast. This mechanism of cell membrane disruption may provide species-specific killing with minimal damage to microflora and the host and may be used by many other antimicrobial peptides.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Concentration-dependent localizations of FITC-Hst-5.
C. albicans cells were incubated for 30 minutes with 5, 10, 20, and 50 µM FITC-Hst-5. (A) Fluorescent and DIC images of cells with FITC-Hst-5 at the indicated concentrations and (B) flow cytometry analysis of the same cells. The concentration dependent localization is represented by the markers M1 (vacuole) and M2 (cytoplasm). (C) The bar graph represents the percentage of the population where Hst-5 is localized to the vacuole (M1) and to the cytoplasm (M2) for the varying concentrations.
Figure 2
Figure 2. Vacuolar localization of FITC-Hst-5 is dependent on receptor-mediated endocytic pathway.
(A) C. albicans wild-type, vps36, and snf7 cells were incubated for 30 minutes with 10 µM FITC-Hst-5 and 10 µM FM4-64 at 30°C. (B) C. albicans wild-type and myo5 cells were incubated for 10 minutes with 10 µM of FITC-Hst-5 and 10 µM FM4-64 at 30°C. The populations of plasma membrane and cytoplasmic localization, 80.99% and 19.01% as indicated, were determined by flow cytometry. (C) C. albicans wild-type cells were treated with either 50 µM latrunculin A or 5 µM cytochalasin A for 60 minutes at room temperature. The cells were then exposed to 5 µM FITC-Hst-5 for 30 minutes at 30°C.
Figure 3
Figure 3. The cationic charge of Hst-5 is important for its uptake into the cytoplasm.
(A) C. albicans cells were incubated with 50 µM and 200 µM FITC-Hst-5 m68 for 30 minutes at 30°C. (B) C. albicans yeast cells were treated with 20 µM FITC-Hst-5 for 30 minutes at 30°C under different pH conditions (4.5, 7.0, and 9.5) as indicated.
Figure 4
Figure 4. Cytoplasmic localization of Hst-5 is linked to its killing activity.
(A) C. albicans cells were incubated for 30 minutes with 10 µM FITC-Hst-5 at 30°C. The cells were sorted by gating the two peaks of the histogram representing vacuolar and cytoplasmic localization. The sorted cells were then plated onto YPD plates and incubated overnight at 30°C (data as a mean±1SD of triplicate cultures). (B) C. albicans, C. glabrata, and S. cerevisiae were incubated with either 50 µM FITC-Hst-5 or 50 µM FITC-Hst-5 and 10 mM NaN3 for 30 minutes at 30°C. The cells treated with and without sodium azide had equal fluorescence exposure times of 900 and 9 milliseconds, respectively.
Figure 5
Figure 5. Hst-5 causes a single perturbation on the cell surface of C. albicans.
(A) 50 µM FITC-Hst-5 was added to the buffer containing PI, and uptake of fluorescence was followed by time-lapse confocal microscopy at room temperature with frames recorded every 9 seconds for 7 minutes and 30 seconds. Six frames recorded at 9, 18, 27, 63, 90, and 297 seconds are shown. (B) 50 µM FITC-Hst-5 was added to the buffer containing RB, and uptake of fluorescence was followed by time-lapse confocal microscopy at room temperature with frames recorded every 8 seconds for a total of 6 minutes and 40 seconds. Six frames recorded at 8, 56, 64, 72, 112, and 128 seconds are shown.
Figure 6
Figure 6. The Hst-5 induced perturbation of the membrane does not co-localize with a specific extracellular region.
(A) 50 µM biotin-Hst-5 was added to C. albicans in buffer containing PI and calcofluor white. Uptake of PI was followed by time-lapse fluorescence microscopy at room temperature with frames recorded every 25 seconds for 10 minutes. The frame recorded at 400 seconds is shown. (B) C. albicans was incubated in water with 2% fetal calf serum and PI for 50 minutes at 37°C to induce Spa2-GFP to the site of polarized growth. Buffer containing 50 µM biotin-Hst-5 was added to the cells and uptake of PI was followed by time-lapse fluorescence microscopy at 37°C with frames recorded every twenty seconds. Frames recorded at 0, 20, 40, and 120 seconds are shown. The yellow arrows indicate the formation of a single breach site on the yeast.

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