The serine peptidase inhibitor TPCK induces several morphophysiological changes in the opportunistic fungal pathogen Candida parapsilosis sensu stricto

Abstract

Candida parapsilosis sensu stricto (C. parapsilosis) has emerged as the second/third commonest Candida species isolated from hospitals worldwide. Candida spp. possess numerous virulence attributes, including peptidases that play multiple roles in both physiological and pathological events. So, fungal peptidases are valid targets for new drugs development. With this premise in mind, we have evaluated the effect of serine peptidase inhibitors (SPIs) on both cell biology and virulence aspects of C. parapsilosis. First, five different SPIs, phenylmethylsulfonyl fluoride, benzamidine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, N-α-tosyl-L-lysine chloromethyl ketone hydrochloride, and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) were tested, and TPCK showed the best efficacy to arrest fungal growth. Subsequently, the ability of TPCK to modulate physiopathological processes was investigated. Overall, TPCK was able to (i) inhibit the cell-associated serine peptidase activities, (ii) promote morphometric and ultrastructural alterations, (iii) induce an increase in the intracellular oxidation level, which culminates in a vigorous lipid peroxidation and accumulation of neutral lipids in cytoplasmic inclusions, (iv) modulate the expression/exposition of surface structures, such as mannose/glucose-rich glycoconjugates, N-acetylglucosamine-containing molecules, chitin, polypeptides and surface aspartic peptidases, (v) reduce the adhesion to either polystyrene or glass surfaces as well as to partially disarticulate the mature biofilm, (vi) block the fungal interaction with macrophages, and (vii) protect Galleria mellonella from fungal infection, enhancing larvae survivability. Altogether, these results demonstrated that TPCK induced several changes over fungal biology besides the interference with aspects associated to C. parapsilosis virulence and pathogenesis, which indicates that SPIs could be novel promising therapeutic agents in dealing with candidiasis.

Effects of a Serine Protease Inhibitor N-p-Tosyl-L-phenylalanine Chloromethyl Ketone (TPCK) on Leishmania amazonensis and Leishmania infantum

“…In fungal infections, it was reported that TPCK could prevent pathogen replication [ 11 ], while Trivedi and coworkers demonstrated that TPCK acts in a dose-dependent manner to prevent the viral replication of HIV-1 [ 30 ]. Furthermore, TPCK has also been shown to block the development of dipteran Oxysarcodexia thornax larvae.…”

“…In the present work, Leishmania parasites treated with TPCK presented mitochondrial alterations, oxidative stress, modifications in lipid content, flagellar alterations, and cytoplasmic vacuoles, factors that may be contributing to the death of the parasites. In Candida parapsilosis , TPCK also induced ROS production while increasing sterol and neutral lipid contents [ 11 ]. It is well known that ROS cause damage to lipids, proteins, nucleic acids, and other important biosystem components [ 37 ].…”

“…Furthermore, it also chemically modifies the side chain of His or Cys residues of the Nef protein critical for efficient viral replication and pathogenicity in HIV viruses [17]. TPCK is an irreversible serine protease inhibitor belonging to the chymotrypsin-like class that alkylates the His residue in the active site of serine proteases and has been widely applied in several parasite trials [11,18] and tested against other pathologies, such as cancer [19]. Although chymotrypsin is not present in the Leishmania genus [20], TPCK can inhibit other serine peptidases, as it has already been shown that TPCK can inhibit prolyl oligopeptidase of L. infantum [21].…”

“…Thus, studies concerning the development of drugs against therapeutic targets not homologous to those of mammals and exclusively present in Leishmania parasites are highly important for clinical treatment. Preliminary studies have investigated the role of serine proteases of several protozoan infections [ 10 ] and assessed the use of serine protease inhibitors against these parasites, resulting in both specific and general modes of action that have successfully impaired parasite viability and/or reduced parasite virulence [ 10 , 11 , 12 ].…”

Repositioning Lopinavir, an HIV Protease Inhibitor, as a Promising Antifungal Drug: Lessons Learned from Candida albicans—In Silico, In Vitro and In Vivo Approaches

“…The percentage of infected epithelial cells was determined by randomly counting at least 200 animal cells in each duplicate through microscopic examination using an immersion objective in a Zeiss Axioplan 2 microscopy. The association index was obtained by multiplying the percentage of infected epithelial cells by the number of fungi per each infected animal cell [46].…”