Uptake of the antifungal cationic peptide Histatin 5 by Candida albicans Ssa2p requires binding to non‐conventional sites within the ATPase domain

Abstract

Candida albicans Hsp70 Ssa1/2 proteins have been identified as cell wall binding partners for the antifungal cationic peptide Histatin 5 (Hst 5) in vivo. C. albicans Ssa2p plays a major role in binding and translocation of Hst 5 into fungal cells, as demonstrated by defective peptide uptake and killing in C. albicans SSA2 null mutants. Candidal Hsp70 proteins are classical chaperone proteins with two discrete functional domains consisting of peptide binding and ATP binding regions. Pull-down assays with full-length and truncated Ssa2 proteins found that the ATPase domain was required for Hst 5 binding. Further mapping of Ssa2p by limited digestion and peptide array analyses identified two discrete Hst 5-binding epitopes within the ATPase region. Expression of Ssa2p in C. albicans cells carrying mutations in the first epitope identified by thermolysin digestion (Ssa2128−132A3) significantly reduced intracellular transport and fungicidal activity of Hst 5, confirming its importance as a binding site for Hst 5 function in vivo. Since this Hst 5 binding site lies within the Ssa2p ATPase domain near the ATP-binding cleft, it is possible that ATP modulates Hst 5 binding to Ssa2p. Indeed, gel filtration assays demonstrated that although nucleotides are not required for Hst 5 binding, their presence improved binding affinity by 10-fold. Thus, C. albicans Ssa2p binds Hst 5 at a surface-localized epitope in a subunit of the ATPase domain; and this region is required for intracellular translocation and killing functions of Hst 5.

FLO11 Gene Is Involved in the Interaction of Flor Strains of Saccharomyces cerevisiae with a Biofilm-Promoting Synthetic Hexapeptide

“…As mentioned above, the glycosylated domain of the mucin-like Msb2p from C. albicans is capable of binding to histatin 5 and LL-37 and protects cells from the peptides (49). Also, in C. albicans, the CW chaperone Ssa2p has been shown to bind histatin 5/P113 (60,61). However, parental and ⌬ssa2 C. albicans cells bound similar amounts of P113 (34), in similarity to our findings determined with P113 and the ⌬flo11 mutant (Fig.…”

Interplay between Candida albicans and the Antimicrobial Peptide Armory

“…First, the peptide binds to the ATPase domain of cell envelope proteins Ssa1 and Ssa2 of C. albicans (15). Then, Hst 5 utilizes the C. albicans polyamine influx transporters Dur3 and Dur31 and accumulates intracellularly (16,17), where it induces formation of reactive oxygen species (ROS) and efflux of ions and ATP, resulting in cell death (18,19).…”

“…Recently, an Hst 5-spermidine conjugate has been developed, which enhanced fungicidal activity compared to nonconjugated Hst 5 (83). It is known that Hst 5 utilizes the polyamine transporters Dur3 and Dur31 for its uptake in C. albicans (16); therefore, the Hst 5 peptide (Hst 5 with amino acids 4 to 15 [Hst 5 [4][5][6][7][8][9][10][11][12][13][14][15] ]) conjugated with a GGG linker and spermidine was rapidly taken up, leading to higher in vitro and in vivo candicidal activity than with nonconjugated Hst 5 (83). Collectively, these results suggest that the development and investigation of AMP conjugates could lead to promising new antifungals.…”

How Does It Kill?: Understanding the Candidacidal Mechanism of Salivary Histatin 5

“…Transporter proteins and energy expenditure in the form of ATP utilization are needed to mediate passage of Hst 5 through this thick cell wall. Hst 5 binds both cell wall ␤-glucans (29) as well as fungal Ssa2 and Ssa1 proteins in the cell wall (6,(34)(35)(36)(37) in order to stabilize and perhaps localize itself with a transport complex. Ultimately, it is transported into the cell through the fungal polyamine transporters Dur3 and Dur31 in an energydependent process (38).…”