Candida species are a major cause of infection worldwide. The species associated with infection vary with geographical location and with patient population. Infection with Candida tropicalis is particularly common in South America and Asia, and Candida parapsilosis infections are more common in the very young. Molecular methods for manipulating the genomes of these species are still lacking. We describe a simple and efficient CRISPR-based gene editing system that can be applied in the C. parapsilosis species group, including the sister species Candida orthopsilosis and Candida metapsilosis . We have also constructed a separate system for gene editing in C. tropicalis .

Fungal pathogens represent a major human threat affecting more than a billion people worldwide. Invasive infections are on the rise, which is of considerable concern because they are accompanied by an escalation of antifungal resistance. Deciphering the mechanisms underlying virulence traits and drug resistance strongly relies on genetic manipulation techniques such as generating mutant strains carrying specific mutations, or gene deletions. However, these processes have often been time-consuming and cumbersome in fungi due to a number of complications, depending on the species (e.g., diploid genomes, lack of a sexual cycle, low efficiency of transformation and/or homologous recombination, lack of cloning vectors, nonconventional codon usage, and paucity of dominant selectable markers). These issues are increasingly being addressed by applying clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 mediated genetic manipulation to medically relevant fungi. Here, we summarize the state of the art of CRISPR–Cas9 applications in four major human fungal pathogen lineages: Candida spp., Cryptococcus neoformans, Aspergillus fumigatus, and Mucorales. We highlight the different ways in which CRISPR has been customized to address the critical issues in different species, including different strategies to deliver the CRISPR–Cas9 elements, their transient or permanent expression, use of codon-optimized CAS9, and methods of marker recycling and scarless editing. Some approaches facilitate a more efficient use of homology-directed repair in fungi in which nonhomologous end joining is more commonly used to repair double-strand breaks (DSBs). Moreover, we highlight the most promising future perspectives, including gene drives, programmable base editors, and nonediting applications, some of which are currently available only in model fungi but may be adapted for future applications in pathogenic species. Finally, this review discusses how the further evolution of CRISPR technology will allow mycologists to tackle the multifaceted issue of fungal pathogenesis.

The increasing frequency of multi-drug resistant microorganisms has driven research into alternative therapeutic strategies. In this respect, natural antimicrobial peptides (AMPs) hold much promise as candidates for the development of novel antibiotics. However, AMPs have some intrinsic drawbacks, such as partial degradation by host proteases or inhibition by host body fluid composition, potential toxicity, and high production costs. This review focuses on the hepcidins, which are peptides produced by the human liver with a known role in iron homeostasis, as well by numerous other organisms (including fish, reptiles, other mammals), and their potential as antibacterial and antifungal agents. Interestingly, the antimicrobial properties of human hepcidins are enhanced at acidic pH, rendering these peptides appealing for the design of new drugs targeting infections that occur in body areas with acidic physiological pH. This review not only considers current research on the direct killing activity of these peptides, but evaluates the potential application of these molecules as coating agents preventing biofilm formation and critically assesses technical obstacles preventing their therapeutic application.

Patients presenting with severe COVID-19 are predisposed to acquire secondary fungal infections such as COVID-19-associated candidemia (CAC), which are associated with poor clinical outcomes despite antifungal treatment. The extreme burden imposed on clinical facilities during the COVID-19 pandemic has provided a permissive environment for the emergence of clonal outbreaks of multiple Candida species, including C. auris and C. parapsilosis. Here we report the largest clonal CAC outbreak to date caused by fluconazole resistant (FLZR) and echinocandin tolerant (ECT) C. parapsilosis. Sixty C. parapsilosis strains were obtained from 57 patients at a tertiary care hospital in Brazil, 90% of them were FLZR and ECT. Although only 35.8% of FLZR isolates contained an ERG11 mutation, all of them contained the TAC1^L518F mutation and significantly overexpressed CDR1. Introduction of TAC1^L518F into a susceptible background increased the MIC of fluconazole and voriconazole 8-fold and resulted in significant basal overexpression of CDR1. Additionally, FLZR isolates exclusively harboured E1939G outside of Fks1 hotspot-2, which did not confer echinocandin resistance, but significantly increased ECT. Multilocus microsatellite typing showed that 51/60 (85%) of the FLZR isolates belonged to the same cluster, while the susceptible isolates each represented a distinct lineage. Finally, biofilm production in FLZR isolates was significantly lower than in susceptible counterparts Suggesting that it may not be an outbreak determinant. In summary, we show that TAC1^L518F and FKS1^E1393G confer FLZR and ECT, respectively, in CAC-associated C. parapsilosis. Our study underscores the importance of antifungal stewardship and effective infection control strategies to mitigate clonal C. parapsilosis outbreaks.

Candida parapsilosis is one of the most common causes of candidiasis, particularly in the very young and the very old. Studies of gene function are limited by the lack of a sexual cycle, the diploid genome, and a paucity of molecular tools. We describe here the development of a plasmid-based CRISPR-Cas9 system for gene editing in C. parapsilosis. A major advantage of the system is that it can be used in any genetic background, which we showed by editing genes in 20 different isolates. Gene editing is carried out in a single transformation step. The CAS9 gene is expressed only when the plasmid is present, and it can be removed easily from transformed strains. There is theoretically no limit to the number of genes that can be edited in any strain. Gene editing is increased by homology-directed repair in the presence of a repair template. Editing by non-homologous end joining (NHEJ) also occurs in some genetic backgrounds. Finally, we used the system to introduce unique tags at edited sites.

Riboswitches are non-coding RNA molecules that regulate gene expression by binding to specific ligands. They are primarily found in bacteria. However, one riboswitch type, the thiamin pyrophosphate (TPP) riboswitch, has also been described in some plants, marine protists and fungi. We find that riboswitches are widespread in the budding yeasts (Saccharomycotina), and they are most common in homologs of DUR31, originally described as a spermidine transporter. We show that DUR31 (an ortholog of N. crassa gene NCU01977) encodes a thiamin transporter in Candida species. Using an RFP/riboswitch expression system, we show that the functional elements of the riboswitch are contained within the native intron of DUR31 from Candida parapsilosis, and that the riboswitch regulates splicing in a thiamin-dependent manner when RFP is constitutively expressed. The DUR31 gene has been lost from Saccharomyces, and may have been displaced by an alternative thiamin transporter. TPP riboswitches are also present in other putative transporters in yeasts and filamentous fungi. However, they are rare in thiamin biosynthesis genes THI4 and THI5 in the Saccharomycotina, and have been lost from all genes in the sequenced species in the family Saccharomycetaceae, including S. cerevisiae. Thiamin, or Vitamin B1, is an essential requirement in all living organisms because it is a co-factor for many enzymes in metabolism. Unlike animals, many yeasts can synthesize thiamin, or they can import it from the environment. Expression of thiamin biosynthesis genes and of thiamin transporters is strictly regulated in response to the presence of thiamin. In many filamentous fungi, expression of thiamin biosynthesis genes is regulated by TPP riboswitches, RNA regulatory elements that are located within messenger RNA. TPP riboswitches are rare in yeasts. However, we find that TPP riboswitches are conserved in an ancient thiamin transporter, found in filamentous fungi, yeasts and other related organisms. There appears to be a high turnover of thiamin transporters in fungi, and there has been a gradual loss of TPP riboswitches in yeasts.