In Vitro Analysis of Tandem Peptides from Human CD5 and CD6 Scavenger Receptors as Potential Anti-Cryptococcal Agents

Abstract

Cryptococcus neoformans is included in the World Health Organization fungal priority pathogen list, complied to expedite improved research and public-health interventions. The limited number of available antifungal drugs, their associated toxicity, and the emergence of drug-resistant strains make the development of new therapeutic strategies mandatory. Pattern-recognition receptors (PRRs) from the host's innate immune system constitute a potential source of new antimicrobial agents. CD5 and CD6 are lymphoid members of the ancient scavenger receptor cysteine-rich superfamily (SRCR-SF) which bind pathogen-associated molecular patterns (PAMPs) of fungal and bacterial origin. Evidence supports the concept that such binding maps to 11-mer sequences present in each of their three SRCR extracellular domains. Herein, we have designed synthetic peptides containing tandems of such 11-mer sequences (namely CD5-T and CD6-T) and analyzed their C. neoformans-binding properties in vitro. Our results show both inhibitory effects on fungal growth and an ability to impact capsule formation and titanization, two critical virulence factors of C. neoformans involved in immune evasion. These effects hold promise for CD5-T and CD6-T peptides as single or adjuvant therapeutic agents against cryptococcosis.

Keywords: CD5; CD6; Cryptococcus; antifungal therapy; peptides; scavenger receptors.

Figure 1

Structural and physicochemical properties of CD5-T and CD6-T peptides. (A) Amino acid sequence and schematic 3D representations of CD5-T and CD6-T. (B) Physicochemical characteristics and properties of CD5-T and CD6-T peptides. MW (molecular weight), pI (isoelectric point).

Figure 2

Biocompatibility of CD5-T and CD6-T peptides. (A) Haemolytic activity of CD5-T and CD6-T peptides expressed as the percentage referred to saline and 10% SDS-induced haemolysis. The continuous line indicates the HA₅₀. (B) Cell viability determination by MTT after RAW264.7 cells’ incubation in the presence of CD5-T and CD6-T peptides. DMEM plus 10% (v/v) DMSO and DMEM alone were used as positive and negative cytotoxic controls, respectively. The continuous line indicates the CA₅₀. Results are represented as mean ± SEM of viable cell counts (quadruplicates). Statistical differences were assessed by Student’s t-test (*, p < 0.05).

Figure 3

Effect of CD5-T and CD6-T peptides on C. neoformans agglutination and viability. (A) C. neoformans suspensions (5 × 10⁸ CFU/mL) were incubated overnight with increasing concentrations of CD5-T and CD6-T peptides in TTC buffer. Fungal agglutination was arbitrarily scored as −, +/−, +, ++, or +++. Shown is a representative image of two experiments performed. (B) Absorbance measurements overtime of C. neoformans (1 × 10³ CFU/mL) cultured for 48 h in the presence of increasing concentrations of CD5-T or CD6-T peptides. (C) Number of viable fungal cells after culturing C. neoformans conidia (5 × 10⁵ CFU/mL) for 2 h with vehicle (control) or increasing concentrations of CD5-T and CD6-T peptides. Results are represented as mean ± SEM of CFU (triplicates). The dotted line indicates the control mean values ± SEM marked in grey. Statistical differences were assessed regarding control values by Student’s t-test (*, p < 0.05).

Figure 4

Effect of CD5-T and CD6-T peptides on C. neoformans capsule formation and titanization. (A) Quantification of fungal capsule width (top) and percentages of encapsulated C. neoformans cells (bottom) after C. neoformans were cultured in capsule-inducing conditions in the presence of increasing concentrations of CD5-T or CD6-T peptides. Sabouraud alone was used as negative control. (B) Quantification of C. neoformans diameter (top) and percentages of titan cells (bottom) assessed as in (A). Continuous variables were analyzed using ANOVA tests, while for contingency analyses, Fisher’s exact test was used. #, significant differences with respect to the positive control. *, significant differences between peptide concentrations. In all cases, differences with p < 0.05 were considered statistically significant.

Figure 5

Effect of CD5-T and CD6-T peptides on C. neoformans viability after capsule formation and titanization. (A) Number of viable C. neoformans cells (left) after capsule induction and proportion of encapsulated fungal cells after the killing assay (right) in the presence of vehicle (control) or increasing concentrations of CD5-T and CD6-T peptides. (B) Number of viable C. neoformans cells (left) after titanization induction and proportion of titan cells after the killing assay (right) in the presence of vehicle (control) or increasing concentrations of CD5-T and CD6-T peptides. The dotted line indicates the control mean values ± SEM marked in grey. Continuous variables were analyzed regarding control values using ANOVA tests, while for contingency analyses, Fisher’s exact test was used. *, significant differences with respect to the control condition. In all cases, differences with p < 0.05 were considered statistically significant.