Study Provides New Insights for the Development of Novel Antifungal Drug Targets

This study was conducted by the research group of Professor Liao Guojian from the College of Pharmaceutical Sciences, Southwest University. Candidate genes potentially involved in lysine synthesis in C. neoformans were identified and knocked out. It was found that after the knockout of the genes CNAG_00992, CNAG_02565, CNAG_03588, CNAG_03476, and CNAG_06849, the strains exhibited lysine auxotrophy.

Subsequently, genetic complementation was performed on these knockout strains, and the complemented strains were able to restore growth in a lysine-free environment, further verifying that the products of these genes are functional enzymes involved in lysine synthesis. Based on their corresponding homologous proteins, the five genes were named LYS20, LYS4, LYS2, LYS9, and LYS1, respectively.

For the five identified lysine auxotrophic strains, the team examined their growth in media containing different concentrations of lysine (0, 10 μmol/L, 100 μmol/L, 1 mmol/L, 10 mmol/L). When the lysine concentration was lower than 100 μmol/L, the knockout strains were unable to grow, reflecting the high concentration demand for lysine in these fungi and suggesting that the relevant knockout strains struggle to grow normally in the micromolar concentration of lysine found in the host environment.

In addition, the team found that when cultivating the strains with lysine as the sole nitrogen source, lys1Δ exhibited a significant growth defect, inferring that LYS1 is also involved in the lysine catabolic process, indicating that C. neoformans possesses a more complex lysine metabolic regulatory system than Saccharomyces cerevisiae.

The team further examined the expression of virulence factors in each gene deletion strain. The results showed that the deletion of different genes had varying degrees of impact on capsule formation and melanin production. The ability of each strain to maintain dormancy under nutrient deprivation was significantly reduced, and their ability to cope with stresses such as temperatures, oxidative agents, and antifungal drugs also varied significantly. Among them, lys9Δ showed the most significant changes in all in vitro phenotypic results.

Therefore, they further analyzed the gene sequence information of LYS9 and found that this gene contains two domains involved in the synthesis of spermidine and lysine, respectively. Through processes such as protein site prediction, site-directed mutagenesis, and phenotypic verification of mutant strains, two key amino acid residues of this dual-domain protein were identified: Asp167 and Arg550. Both domains play crucial roles in the virulence of the fungus.

Further mouse nasal infection models were used to detect the in vivo pathogenicity of the strains. By examining the fungal loads in the lung and brain tissues of infected mice and the survival status within 60 days post-infection, the team verified the severely weakened pathogenicity of the five knockout strains. Among them, lys9Δ still showed the most prominent performance. They concluded that the simultaneous blockade of the two regulatory pathways-spermidine and lysine-is the key factor leading to the significant attenuation of the in vitro and in vivo virulence of lys9Δ, which provides new insights for the development of novel antifungal drug targets.