An Antimicrobial Peptidomimetic Induces Mucorales Cell Death through Mitochondria-Mediated Apoptosis

The incidence of mucormycosis has dramatically increased in immunocompromised patients. Moreover, the array of cellular targets whose inhibition results in fungal cell death is rather limited. Mitochondria have been mechanistically identified as central regulators of detoxification and virulence in fungi. Our group has previously designed and developed a proteolytically-resistant peptidomimetic motif D(KLAKLAK)2 with pleiotropic action ranging from targeted (i.e., ligand-directed) activity against cancer and obesity to non-targeted activity against antibiotic resistant gram-negative rods. Here we evaluated whether this non-targeted peptidomimetic motif is active against Mucorales. We show that D(KLAKLAK)2 has marked fungicidal action, inhibits germination, and reduces hyphal viability. We have also observed cellular changes characteristic of apoptosis in D(KLAKLAK)2-treated Mucorales cells. Moreover, the fungicidal activity was directly correlated with vacuolar injury, mitochondrial swelling and mitochondrial membrane depolarization, intracellular reactive oxygen species accumulation (ROS), and increased caspase-like enzymatic activity. Finally, these apoptotic features were prevented by the addition of the ROS scavenger N-acetyl-cysteine indicating mechanistic pathway specificity. Together, these findings indicate that D(KLAKLAK)2 makes Mucorales exquisitely susceptible via mitochondrial injury-induced apoptosis. This prototype may serve as a candidate drug for the development of translational applications against mucormycosis and perhaps other fungal infections

Systemic combinatorial peptide selection yields a non-canonical iron-mimicry mechanism for targeting tumors in a mouse model of human glioblastoma

The management of CNS tumors is limited by the blood-brain barrier (BBB), a vascular interface that restricts the passage of most molecules from the blood into the brain. Here we show that phage particles targeted with certain ligand motifs selected in vivo from a combinatorial peptide library can cross the BBB under normal and pathological conditions. Specifically, we demonstrated that phage clones displaying an ironmimic peptide were able to target a protein complex of transferrin and transferrin receptor (TfR) through a non-canonical allosteric binding mechanism and that this functional protein complex mediated transport of the corresponding viral particles into the normal mouse brain. We also showed that, in an orthotopic mouse model of human glioblastoma, a combination of TfR overexpression plus extended vascular permeability and ligand retention resulted in remarkable brain tumor targeting of chimeric adeno-associated virus/ phage particles displaying the iron-mimic peptide and carrying a gene of interest. As a proof of concept, we delivered the HSV thymidine kinase gene for molecular-genetic imaging and targeted therapy of intracranial xenografted tumors. Finally, we established that these experimental findings might be clinically relevant by determining through human tissue microarrays that many primary astrocytic tumors strongly express TfR. Together, our combinatorial selection system and results may provide a translational avenue for the targeted detection and treatment of brain tumors

β-Neurexin Is a Ligand for the Staphylococcus aureus MSCRAMM SdrC

Gram-positive bacteria contain a family of surface proteins that are covalently anchored to the cell wall of the organism. These cell-wall anchored (CWA) proteins appear to play key roles in the interactions between pathogenic organisms and the host. A subfamily of the CWA has a common structural organization with multiple domains adopting characteristic IgG-like folds. The identified microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) belong to this subfamily, as does SdrC from S. aureus. However, an interactive host ligand for the putative MSCRAMM SdrC was not previously identified. We have screened a phage display peptide library and identified a peptide sequence found in β-neurexin that binds SdrC. A synthetic peptide corresponding to the identified sequence as well as a recombinant form of the β-neurexin 1 exodomain binds SdrC with high affinity and specificity. Furthermore, expression of SdrC on bacteria greatly enhances microbial adherence to cultured mammalian cells expressing β-neurexin on their surface. Taken together, our experimental results demonstrate that β-neurexin is a ligand for SdrC. This interaction involves a specific sequence located in the N-terminal region of the mammalian protein and the N(2)N(3) domain of the MSCRAMM. The fact that these two proteins interact when expressed on the appropriate cells demonstrates the functionality of the interaction. Possible implications of this interaction are discussed

_D(KLAKLAK)₂ has fungicidal activity against Mucorales.

<p>(<b>A</b>) Micrographs of <i>R. oryzae</i> and <i>M. circinelloides</i> spores after six hours of exposure to AMB (4 µg/ml), FLU (128 µg/ml), _D(KLAKLAK)₂ (150 µg/ml, 300 µg/ml) or a negative control peptidomimetic, _D(CVRAC)₂ (300 µg/ml), showing that germination was completely inhibited by _D(KLAKLAK)₂ at MIC (300 µg/ml)and by AMB (4 µg/ml). Scale bar, 200 µm. (<b>B</b> and <b>C</b>) Mycelia were incubated with AMB (4 µg/ml), FLU (128 µg/ml), _D(KLAKLAK)₂ (150 µg/ml, 300 µg/ml) or _D(CVRAC)₂ (300 µg/ml). The extent of hyphal damage was monitored over time by the XTT reduction assay, which indicated _D(KLAKLAK)₂-induced dose-dependent killing relative to control drugs (*p ≤ 0.0001). (<b>D</b> and <b>E</b>) Spores were exposed to AMB (4 µg/ml), FLU (128 µg/ml), _D(KLAKLAK)₂ (150 µg/ml, 300 µg/ml) or the negative control peptidomimetic (300 µg/ml) for one hour. _D(KLAKLAK)₂ created a post-antifungal effect demonstrated by a shift of the growth curve to the right compared to the control drugs. The lag period was increased by approximately three hours (**p ≤ 0.001), followed by a rapid recovery.</p

_D(KLAKLAK)₂ causes Mucorales apoptosis.

<p>(<b>A</b>) Relative quantification indicated significantly higher levels (**p ≤ 0.001) of cytochrome <i>c</i> release from mitochondria of <i>R. oryzae</i> and <i>M. circinelloides</i> into the cytosol in the presence of _D(KLAKLAK)₂ (150 µg/ml) relative to FLU (128 µg/ml) or the negative control peptidomimetic (300 µg/ml). (<b>B</b>) Fluorescence and DIC micrographs of <i>R. oryzae</i> and <i>M. circinelloides</i> germlings stained with the CaspACE FITC-VAD-FMK In Situ Marker, indicating metacaspase activation upon incubation with _D(KLAKLAK)₂ (150 µg/ml) compared to FLU (128 µg/ml) or the negative control peptidomimetic (300 µg/ml). Scale bar, 200 µm. (<b>C</b>) Fluorescence quantitative analysis of <i>R. oryzae</i> and <i>M. circinelloides</i> germlings in the presence of drugs revealed significant levels of metacaspase activation in germlings treated with _D(KLAKLAK)₂ (**p ≤ 0.001).</p

_D(KLAKLAK)₂ induces ultrastructural cellular changes in <i>R. oryzae</i>.

<p>TEM of germlings (6,000-fold) incubated with _D(KLAKLAK)₂ (150 µg/ml, 300 µg/ml) revealed extensive vacuolization comparable to that induced by AMB (2 µg/ml). The increase in number and size of vacuoles was visualized with the vacuolar probe FM4-64 (red). staining of intact plasma membrane FM4-64 diffusion into the cytoplasm and staining of fragmented vacuoles in treated _D(KLAKLAK)₂ (150 µg/ml, 300 µg/ml) and AMB (2 µg/ml). MitoTracker staining (green) indicated morphological changes and swelling of mitochondria in _D(KLAKLAK)₂-exposed germlings as opposed to the punctate pattern observed in samples treated with control drugs. TEM scale bar, 2 µm. Fluorescent micrographs scale bar, 200 µm.</p

_D(KLAKLAK)₂ induces mitochondrial membrane depolarization (ΔΨ_m).

<p>(<b>A</b>) Fluorescence and DIC micrographs of <i>R. oryzae</i> and <i>M. circinelloides</i> germlings stained with the oxidation-sensitive dye RH-123, indicating membrane depolarization after incubation with _D(KLAKLAK)₂ (150 µg/ml) or AMB (2 µg/ml). Scale bar, 200 µm. (<b>B</b>) Quantitative analysis recorded with a microplate reader (excitation, 488 nm; emission, 525 nm), demonstrating significant membrane depolarization levels (**p ≤ 0.001), post-exposure to _D(KLAKLAK)₂ compared to FLU (128 µg/ml) or the negative control peptidomimetic (300 µg/ml).</p

Exposure to _D(KLAKLAK)₂ triggers intracellular ROS accumulation.

<p>(<b>A</b>) Fluorescence and DIC micrographs of <i>R. oryzae</i> and <i>M. circinelloides</i> germlings stained with the oxidation-sensitive dye DHR-123, showing ROS production after incubation with _D(KLAKLAK)₂ (150 µg/ml) or AMB (2 µg/ml). Scale bar, 200 µm. (<b>B</b>) Quantitative analysis recorded with a microplate reader (excitation, 488 nm; emission, 525 nm), demonstrating a significant increase in ROS release from mitochondria in the presence of _D(KLAKLAK)₂ (**p ≤ 0.001) relative to FLU (128 µg/ml) or negative control peptidomimetic (300 µg/ml).</p

_D(KLAKLAK)₂ alters plasma membrane homeostasis.

<p>(<b>A</b>) Fluorescence micrographs of <i>R. oryzae</i> and M. circinelloides mycelia post-exposure to AMB (2 µg/ml), FLU (128 µg/ml), _D(KLAKLAK)₂ (150 µg/ml, 300 µg/ml) or _D(CVRAC)₂ (300 µg/ml). The DiBAC bright green fluorescence is indicative of a loss of viability due increased membrane permeability. Differential interference contrast (DIC) images served as controls for the presence of germlings. Scale bar, 200 µm. (<b>B</b> and <b>C</b>) The dose-dependent cellular ATP release in the medium after <i>R. oryzae</i> and M. circinelloides hyphae treatment with _D(KLAKLAK)₂ (150 µg/ml, 300 µg/ml) was similar to that induced by colistin (32 µg/ml) (***p ≤ 0.0002).</p