Data_Sheet_1_Evaluation of proline-rich antimicrobial peptides as potential lead structures for novel antimycotics against Cryptococcus neoformans.docx

BackgroundCryptococcosis and cryptococcal meningitis, caused by Cryptococcus neoformans infections, lead to approximately 180,000 deaths per year, primarily in developing countries. Individuals with compromised immune systems, e.g., due to HIV infection (AIDS) or chemotherapy, are particularly vulnerable. Conventional treatment options are often limited and can cause severe side effects. Therefore, this study aimed to investigate the antifungal effect of insect-derived proline-rich antimicrobial peptides (PrAMPs) against C. neoformans. These peptides are known for their low toxicity and their high efficacy in murine infection models, making them a promising alternative for treatment.ResultsA preliminary screening of the minimal inhibitory concentrations (MICs) of 20 AMPs, including the well-known PrAMPs Onc112, Api137, and Chex1Arg20 as well as the cathelicidin CRAMP against the C. neoformans strains 1841, H99, and KN99α revealed promising results, with MICs as low as 1.6 μmol/L. Subsequent investigations of selected peptides, determining their influence on fungal colony-forming units, confirmed their strong activity. The antifungal activity was affected by factors such as peptide net charge and sequence, with stronger effects at higher net charges probably due to better intracellular uptake confirmed by confocal laser scanning microscopy. Inactive scrambled peptides suggest a specific intracellular target, although scanning electron microscopy showed that PrAMPs also damaged the cell exterior for a low proportion of the cells. Possible pore formation could facilitate entry into the cytosol.</p

Methylation status of <i>ZIP4</i> gene regions and expression in jejunal epithelium after different zinc treatments.

<p>Methylation status of <i>ZIP4</i> gene regions and expression in jejunal epithelium after different zinc treatments.</p

Influence of zinc on <i>ZIP4</i> expression in jejunal epithelium after one and four weeks treatment.

<p>Influence of zinc on <i>ZIP4</i> expression in jejunal epithelium after one and four weeks treatment.</p

Zinc concentration in piglet’s jejunal digesta fed different zinc diets for one and four weeks.

<p>Abbreviations: LZn, low dietary zinc = 57 mg zinc/kg feed; NZn, normal dietary zinc = 164 mg zinc/kg feed; HZn, high dietary zinc = 2,425 mg zinc/kg feed.</p><p>^a,bLabelled means with different letters in a row are significantly different (<i>p</i> < 0.01).</p><p>^{<i>A</i>,<i>B</i>}Labelled means with different letters in a column are different by trend (<i>p</i> < 0.1).</p><p>Zinc concentration in piglet’s jejunal digesta fed different zinc diets for one and four weeks.</p

Influence of methylation of <i>ZIP4</i> gene regions on expression of long transcripts in jejunal epithelium.

<p>Influence of methylation of <i>ZIP4</i> gene regions on expression of long transcripts in jejunal epithelium.</p

List of RT-qPCR primers.

<p>¹ Both transcripts ZIP4-201 and Zip4-001 include all 12 exons of the gene, but they differ in their transcription start sites; the given primer pair amplifies both transcripts and does not discriminate them.</p><p>² Genome assembly: Sscrofa10.2 (GCA_000003025.4).</p><p>³<a href="http://www.ensembl.org/Sus_scrofa/Transcript/Summary?db=core;g=ENSSSCG00000005905;r=4:464087-469224;t=ENSSSCT00000006485" target="_blank">http://www.ensembl.org/Sus_scrofa/Transcript/Summary?db=core;g=ENSSSCG00000005905;r=4:464087-469224;t=ENSSSCT00000006485</a></p><p>List of RT-qPCR primers.</p

Correlation of zinc concentration in jejunal digesta with respective <i>ZIP4</i> expression in jejunal epithelium.

<p>Correlation of zinc concentration in jejunal digesta with respective <i>ZIP4</i> expression in jejunal epithelium.</p

Amplicons, assays, and primers used for methylation status analysis at the <i>ZIP4</i> gene locus.

<p>Abbreviations: # CpGs, number of CpGs analysed in this fragment; TSS, transcriptional start site; Bio, biotinylated; Seq, sequencing primer.</p><p>Amplicons, assays, and primers used for methylation status analysis at the <i>ZIP4</i> gene locus.</p

Early fungal growth control in pulmonary infection with <i>C. neoformans</i> in the presence of IL-4Rα signaling.

<p>Wild-type (WT, open circle) and IL-4Rα^−/− (gray circle) mice on C57BL/6J background were infected intranasally with <i>C. neoformans</i>. Analysis of fungal burdens in the lung was done at different days <i>post infectionem</i> (dpi) as indicated. Shown is data from n = 7 mice per group from one representative of three independent experiments (14 dpi) or from two independent experiments (7; 21 and 42 dpi). Statistical analysis was done using the unpaired Student's t-test (7 dpi) or Mann-Whitney test. *P<0.05; **P<0.01.</p

Stronger pulmonary inflammation, eosinophilia, and mucus production in WT as compared with IL-4Rα^−/− mice.

<p>Lung slices from WT and IL-4Rα^−/− mice infected for 14 days were stained with H&E (A-F) and periodic acid Schiff reagent (G, H). Leukocyte infiltration and fungal load are depicted in panels A, C and B, D. Sites of inflammation contain eosinophils (arrowheads) and large, multinucleated macrophages (E) or lymphocytes (F). Mucus production by bronchial epithelial cells is depicted in G and H. One representative experiment out of three with n = 6–7 animals per group is shown.</p