Life-threatening invasive fungal infections represent an urgent threat to human health worldwide. The limited set of antifungal drugs has critical constraints such as resistance development and/or adverse side effects. One approach to overcome these limitations is to mimic naturally occurring antifungal peptides called defensins. Inspired by their advantageous amphiphilic properties, a library of 35 synthetic, linear, ternary polyacrylamides was prepared by controlled/ living radical polymerization. The effect of the degree of polymerization (20, 40, and 100) and varying hydrophobic functionalities (branched, linear, cyclic, or aromatic differing in their number of carbons) on their antifungal activity was investigated. Short copolymers with a calculated log P of ∼1.5 revealed optimal activity against the major human fungal pathogen Candida albicans and other pathogenic fungal species with limited toxicity to mammalian host cells (red blood cells and fibroblasts). Remarkably, selected copolymers outperformed the commercial antifungal drug amphotericin B, with respect to the therapeutic index, highlighting their potential as novel antifungal compounds.

Mixed or parotid saliva from caries-active individuals consistently supported better growth of Streptococcus mutans (type c) than that from caries-free individuals. Electrophoretic studies revealed that certain proteins in caries-active salivas were susceptible to microbial attack, but similar proteins in caries-free salivas were refractory.

Mixed salivary proteins, obtained by fractional ultrafiltration of centrifuged human saliva were utilized by Streptococcus mutans or Streptococcus sanguis as sources of amino acids for growth, but these proteins supported only limited growth of Streptococcus salivarius or Streptococcus mitior. Comparative analyses of the isoelectric focused protein patterns before and following growth with these oral streptococci revealed a distinct pattern of salivary proteins which were susceptible to alteration by each species. Proteins with isoelectric points of 4.90, 5.33, 5.50, 5.58, 5.72, 5.80, 6.00 and 6.10 usually susceptible to alteration by S. mutans were seldom affected by the other oral streptococci. Growth attained by the oral streptococci in defined medium containing fractions of partially purified proteins was related to the presence or absence of proteins specifically altered by the organisms.

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Fungal infections represent a serious burden on human health. Increasing numbers of susceptible hosts, a limited set of approved antifungal drugs which frequently trigger undesired side effects, and the emergence of resistant strains highlight the urgent demand for novel antifungal drug formulations. However, the biological similarity of human and fungal cells hampers the development of new antifungals which do not also harm humans. In nature, organisms in almost all domains of life produce antimicrobial peptides to combat microbial pathogens. Those peptides share certain characteristics, such as being short, amphiphilic molecules with a positive net charge.1 We designed synthetic polyacrylamides which mimic the properties of naturally occurring antifungal peptides. These positively charged, amphiphilic polymers are advantageous over peptides because of their easy synthesis and stability against proteases. Initial structure-activity relationship studies revealed an optimal cLogP (the calculated hydrophobicity of a molecule) around 1.5 to ensure activity against C. albicans and simultaneous biocompatibility with host cells.2 Additionally, shorter polymers with a length of 20 subunits were more effective than their longer versions.2 In terms of their therapeutic index, certain compositions outperformed the broad-spectrum antifungal amphotericin B and were even effective against drug-resistant clinical isolates of C. albicans.2 Candida albicans strains with known antifungal drug-resistance mutations were not affected in their susceptibility to the polymers. Therefore, investigations were carried out to elucidate the mode of action of the polymers. The transcriptome of C. albicans cells treated with subinhibitory concentrations of the polymers revealed an increased expression of genes involved in general stress response and upregulation in protein processing in the endoplasmic reticulum, particularly glycosylation and degradation. These findings, together with electron microscopy observations, indicated damage to the mannoproteins in the cell wall of the fungus. Membrane damage was also observed utilizing a C. albicans strain expressing GFP intracellularly. The in vitro therapeutic potential of the most promising polymer was tested in a human epithelial cell (HEC) model simulating C. albicans infection. The polymer alone was not able to prevent C. albicans infection of HECs. However, the combination of polymer with caspofungin or fluconazole showed very strong synergistic effects at otherwise non-inhibitory concentrations of the individual antifungals, successfully stopping fungal infection in vitro without damaging the HECs. These results underline the potential of synthetic polymers as an alternative treatment for fungal infections with low toxicity to human cells and a novel mode of action. Sources: 1. Fernández de Ullivarri, M., Arbulu, S., Garcia-Gutierrez, E. and Cotter, P.D. Antifungal peptides as therapeutic agents. Front Cell Infect Microbiol 10, 00 105 (2020). 2. Schaefer, S. et al. Rational design of an antifungal polyacrylamide library with reduced host-cell toxicity. ACS Appl Mater Interfaces 13, 27430-27444 (2021).