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The World Health Organization recently published the first list of priority fungal pathogens highlighting multipleCandidaspecies, includingCandida glabrata,Candida albicans, andCandida auris. However, prior studies in these pathogens have been mainly limited to the use of two drug resistance cassettes,NatMXandHphMX, limiting genetic manipulation capabilities in prototrophic laboratory strains and clinical isolates. In this study, we expanded the toolkit forC. glabrata,C. auris, andC. albicansto includeKanMXandBleMXwhen coupled with anin vitroassembled CRISPR-Cas9 ribonucleoprotein (RNP)-based system. Repurposing these drug resistance cassettes forCandida, we were able to make single gene deletions, sequential and simultaneous double gene deletions, epitope tags, and rescue constructs. We applied these drug resistance cassettes to interrogate the ergosterol pathway, a critical pathway for both the azole and polyene antifungal drug classes. Using our approach, we determined for the first time that the deletion ofERG3inC. glabrata,C. auris,andC. albicansprototrophic strains results in azole drug resistance, which further supports the conservation of the Erg3-dependent toxic sterol model. Furthermore, we show that anERG5deletion inC. glabratais azole susceptible at subinhibitory concentrations, suggesting that Erg5 could act as an azole buffer for Erg11. Finally, we identified a synthetic growth defect when bothERG3andERG5are deleted inC. glabrata,which suggests the possibility of another toxic sterol impacting growth. Overall, we have expanded the genetic tools available to interrogate complex pathways in prototrophic strains and clinical isolates.

The increasing problem of drug resistance and emerging pathogens is an urgent global health problem that necessitates the development and expansion of tools for studying fungal drug resistance and pathogenesis. Prior studies inCandida glabrata,Candida auris, andCandida albicanshave been mainly limited to the use ofNatMX/SAT1andHphMX/CaHygfor genetic manipulation in prototrophic strains and clinical isolates. In this study, we demonstrated thatNatMX/SAT1, HphMX, KanMX,and/orBleMXdrug resistance cassettes when coupled with a CRISPR-ribonucleoprotein (RNP)-based system can be efficiently utilized for deleting or modifying genes in the ergosterol pathway ofC. glabrata,C. auris, andC. albicans. Moreover, the utility of these tools has provided new insights intoERGgenes and their relationship to azole resistance inCandida. Overall, we have expanded the toolkit forCandidapathogens to increase the versatility of genetically modifying complex pathways involved in drug resistance and pathogenesis.

 

Fungi and bacteria are commonly found co-occurring both in natural and synthetic microbiomes, but our understanding of fungal–bacterial interactions is limited to a handful of species. Conserved mechanisms of interactions and evolutionary consequences of fungal–bacterial interactions are largely unknown. Our RNA sequencing and experimental evolution data with Penicillium species and the bacterium S. equorum demonstrate that divergent fungal species can elicit conserved transcriptional and genomic responses in co-occurring bacteria. Penicillium molds are integral to the discovery of novel antibiotics and production of certain foods. By understanding how Penicillium species affect bacteria, our work can further efforts to design and manage Penicillium -dominated microbial communities in industry and food production. 

ABSTRACT TUP1 is a well-characterized repressor of transcription in Saccharomyces cerevisiae and Candida albicans and is observed as a single-copy gene. We observe that most species that experienced a whole-genome duplication outside of the Saccharomyces genus have two copies of TUP1 in the Saccharomycotina yeast clade. We focused on Candida glabrata and demonstrated that the uncharacterized TUP1 homolog, C. glabrata TUP11 ( CgTUP11 ), is most like the S. cerevisiae TUP1 ( ScTUP1 ) gene through phenotypic assays and transcriptome sequencing (RNA-seq). Whereas CgTUP1 plays a role in gene repression, it is much less repressive in standard growth media. Through RNA-seq and reverse transcription-quantitative PCR (RT-qPCR), we observed that genes associated with pathogenicity ( YPS2 , YPS4 , and HBN1 ) are upregulated upon deletion of either paralog, and loss of both paralogs is synergistic. Loss of the corepressor CgCYC8 mimics the loss of both paralogs, but not to the same extent as the Cgtup1 Δ Cgtup11 Δ mutant for these pathogenesis-related genes. In contrast, genes involved in energy metabolism ( CgHXT2 , CgADY2 , and CgFBP1 ) exhibit similar behavior (dependence on both paralogs), but deletion of CgCYC8 is very similar to the Cgtup1 Δ Cgtup11 Δ mutant. Finally, some genes ( CgMFG1 and CgRIE1 ) appear to only be dependent on CgTUP11 and CgCYC8 and not CgTUP1 . These data indicate separable and overlapping roles for the two TUP1 paralogs and that other genes may function as the Cg Cyc8 corepressor. Through a comparison by RNA-seq of Sctup1 Δ, it was found that TUP1 homologs regulate similar genes in the two species. This work highlights that studies focused only on Saccharomyces may miss important biological processes because of paralog loss after genome duplication. IMPORTANCE Due to a whole-genome duplication, many yeast species related to C. glabrata have two copies of the well-characterized TUP1 gene, unlike most Saccharomyces species. This work identifies roles for the paralogs in C. glabrata , highlights the importance of the uncharacterized paralog, called TUP11 , and suggests that the two paralogs have both overlapping and unique functions. The TUP1 paralogs likely influence pathogenicity based on tup mutants upregulating genes that are associated with pathogenicity. 

Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A . fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A . fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A . fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis -thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A . nidulans . However, the A . nidulans genome does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A . fumigatus and A . nidulans , two distantly related Aspergillus species, and to identify additional components required for GT protection. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A . fumigatus and A . nidulans . However, we were able to observe homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A . fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae , was also essential for virulence and GT biosynthesis in A . fumigatus , and for GT protection in A . fumigatus , A . nidulans , and A . oryzae . KojR regulates rglT , gliT , gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species. 

ABSTRACT Microbe-microbe interactions can strongly influence growth and biofilm formation kinetics. For Pseudomonas aeruginosa and Candida albicans , which are found together in diverse clinical sites, including urinary and intravenous catheters and the lungs of individuals with cystic fibrosis (CF), we compared the kinetics of biofilm formation by each species in dual-species and single-species biofilms. We engineered fluorescent protein constructs for P. aeruginosa (producing mKO-κ ) and C. albicans (producing mKate2 ) that did not alter growth and enabled single-cell resolution imaging by live-sample microscopy. Using these strains in an optically clear derivative of synthetic CF sputum medium, we found that both P. aeruginosa and C. albicans displayed increased biovolume accumulation—by three- and sixfold, respectively—in dual-species biofilms relative to single-species biofilms. This result was specific to the biofilm environment, as enhanced growth was not observed in planktonic cocultures. Stimulation of C. albicans biofilm formation occurred regardless of whether P. aeruginosa was added at the time of fungal inoculation or 24 h after the initiation of biofilm development. P. aeruginosa biofilm increases in cocultures did not require the Pel extracellular polysaccharide, phenazines, and siderophores known to influence C. albicans . P. aeruginosa mutants lacking Anr, LasR, and BapA were not significantly stimulated by C. albicans , but they still promoted a significant enhancement of biofilm development of the fungus, suggesting a fungal response to the presence of bacteria. Last, we showed that a set of P. aeruginosa clinical isolates also prompted an increase of biovolume by C. albicans in coculture. IMPORTANCE There is an abundance of work on both P. aeruginosa and C. albicans in isolation, and quite some work as well on the way these two microbes interact. These studies do not, however, consider biofilm environments under flow, and our results here show that the expected outcome of interaction between these two pathogens can actually be reversed under flow, from pure antagonism to an increase in biomass on the part of both. Our work also highlights the importance of cellular-scale spatial structure in biofilms for understanding multispecies population dynamics. 

This article is to alert medical mycologists and infectious disease specialists of recent name changes of medically important species of the filamentous moldFusarium.Fusariumspecies can cause localized and life-threating infections in humans. Of the 70Fusariumspecies that have been reported to cause infections, close to one-third are members of theFusarium solanispecies complex (FSSC), and they collectively account for approximately two-thirds of all reportedFusariuminfections.

 

ABSTRACT Candida albicans is an opportunistic fungal pathogen of humans that is typically diploid yet has a highly labile genome tolerant of large-scale perturbations including chromosomal aneuploidy and loss-of-heterozygosity events. The ability to rapidly generate genetic variation is crucial for C. albicans to adapt to changing or stressful environments, like those encountered in the host. Genetic variation occurs via stress-induced mutagenesis or can be generated through its parasexual cycle, in which tetraploids arise via diploid mating or stress-induced mitotic defects and undergo nonmeiotic ploidy reduction. However, it remains largely unknown how genetic background contributes to C. albicans genome instability in vitro or in the host environment. Here, we tested how genetic background, ploidy, and the host environment impacts C. albicans genome stability. We found that host association induced both loss-of-heterozygosity events and genome size changes, regardless of genetic background or ploidy. However, the magnitude and types of genome changes varied across C. albicans strain background and ploidy state. We then assessed if host-induced genomic changes resulted in fitness consequences on growth rate and nonlethal virulence phenotypes and found that many host-derived isolates significantly changed relative to their parental strain. Interestingly, diploid host-associated C. albicans predominantly decreased host reproductive fitness, whereas tetraploid host-associated C. albicans increased host reproductive fitness. Together, these results are important for understanding how host-induced genomic changes in C. albicans alter its relationship with the host. IMPORTANCE Candida albicans is an opportunistic fungal pathogen of humans. The ability to generate genetic variation is essential for adaptation and is a strategy that C. albicans and other fungal pathogens use to change their genome size. Stressful environments, including the host, induce C. albicans genome instability. Here, we investigated how C. albicans genetic background and ploidy state impact genome instability, both in vitro and in a host environment. We show that the host environment induces genome instability, but the magnitude depends on C. albicans genetic background. Furthermore, we show that tetraploid C. albicans is highly unstable in host environments and rapidly reduces in genome size. These reductions in genome size often resulted in reduced virulence. In contrast, diploid C. albicans displayed modest host-induced genome size changes, yet these frequently resulted in increased virulence. Such studies are essential for understanding how opportunistic pathogens respond and potentially adapt to the host environment.