Genetic Transformation of Candida auris via Homology-Directed Repair Using a Standard Lithium Acetate Protocol.

Reverse genetics is a particularly powerful tool in non-model organisms with known whole-genome sequences enabling the characterization of gene and, thus, protein function via a mutant phenotype. Reverse genetic approaches require genetic manipulation techniques which often need to be specifically developed for non-model organisms; this can be fraught with difficulties. Here, we describe a genetic transformation protocol for the recently emerged human pathogen Candida auris to target the integration of DNA constructs into genomic locations via homology-directed repair using long flanking homologous sequences (>1 kb). We detail the generation of DNA constructs for gene deletion with dominant drug resistance markers via fusion PCR, the transformation of these constructs into chemically competent C. auris cells, and the confirmation of correct integration by PCR. This strategy can be adapted to deliver DNA constructs other than deletion cassettes, including promoter exchanges and protein tags.

Control of Variant Surface Glycoprotein Expression by CFB2 in Trypanosoma brucei and Quantitative Proteomic Connections to Translation and Cytokinesis.

Variant surface glycoproteins (VSGs) coat parasitic African trypanosomes and underpin antigenic variation and immune evasion. These VSGs are superabundant virulence factors that are subject to posttranscriptional gene expression controls mediated via the VSG 3' untranslated region (UTR). To identify positive VSG regulators in bloodstream-form Trypanosoma brucei, we used genome-scale screening data to prioritize mRNA binding protein (mRBP) knockdowns that phenocopy VSG mRNA knockdown, displaying loss of fitness and precytokinesis accumulation. The top three candidates were CFB2 (cyclin F-box protein 2) (Tb927.1.4650), MKT1 (Tb927.6.4770), and PBP1 (polyadenylate binding protein 1) (Tb927.8.4540). Notably, CFB2 was recently found to regulate VSG transcript stability, and all three proteins were found to associate. We used data-independent acquisition for accurate label-free quantification and deep proteome coverage to quantify the expression profiles following the depletion of each mRBP. Only CFB2 knockdown significantly reduced VSG expression and the expression of a reporter under the control of the VSG 3' UTR. CFB2 knockdown also triggered the depletion of cytoplasmic ribosomal proteins, consistent with translation arrest observed when VSG synthesis is blocked. In contrast, PBP1 knockdown triggered the depletion of CFB2, MKT1, and other components of the PBP1 complex. Finally, all three knockdowns triggered the depletion of cytokinesis initiation factors, consistent with a cytokinesis defect, which was confirmed here for all three knockdowns. Thus, genome-scale knockdown data sets facilitate the triage and prioritization of candidate regulators. Quantitative proteomic analysis confirms the 3'-UTR-dependent positive control of VSG expression by CFB2 and interactions with additional mRBPs. Our results also reveal new insights into the connections between VSG expression control by CFB2, ribosomal protein expression, and cytokinesis. IMPORTANCE VSG expression represents a key parasite virulence mechanism and an example of extreme biology. Posttranscriptional gene expression controls in trypanosomatids also continue to be the subject of substantial research interest. We have identified three candidate VSG regulators and used knockdown and quantitative proteomics, in combination with other approaches, to assess their function. CFB2 is found to control VSG expression via the VSG 3' untranslated region, while other data support the view that MKT1 and PBP1 also form part of a CFB2 mRNA binding complex. Remarkably, we also find the depletion of cytoplasmic ribosomal proteins upon CFB2 knockdown, consistent with translation arrest observed when VSG synthesis is blocked. Proteomic profiles following knockdown further yield insights into cytokinesis defects. Taken together, our findings confirm and elaborate the role of CFB2 in controlling VSG expression and reveal new insights into connectivity with translation and cytokinesis controls.

What do we know about the biology of the emerging fungal pathogen of humans Candida auris?

Candida auris is a worrisome fungal pathogen of humans which emerged merely about a decade ago. Ever since then the scientific community worked hard to understand clinically relevant traits, such as virulence factors, antifungal resistance mechanisms, and its ability to adhere to human skin and medical devices. Whole-genome sequencing of clinical isolates and epidemiological studies outlining the path of nosocomial outbreaks have been the focus of research into this pathogenic and multidrug-resistant yeast since its first description in 2009. More recently, work was started by several laboratories to explore the biology of C. auris. Here, we review the insights of studies characterizing the mechanisms underpinning antifungal drug resistance, biofilm formation, morphogenetic switching, cell aggregation, virulence, and pathogenicity of C. auris. We conclude that, although some progress has been made, there is still a long journey ahead of us, before we fully understand this novel pathogen. Critically important is the development of molecular tools for C. auris to make this fungus genetically tractable and traceable. This will allow an in-depth molecular dissection of the life cycle of C. auris, of its characteristics while interacting with the human host, and the mechanisms it employs to avoid being killed by antifungals and the immune system.

Pseudohyphal Growth of the Emerging Pathogen Candida auris Is Triggered by Genotoxic Stress through the S Phase Checkpoint.

The morphogenetic switching between yeast cells and filaments (true hyphae and pseudohyphae) is a key cellular feature required for full virulence in many polymorphic fungal pathogens, such as Candida albicans In the recently emerged yeast pathogen Candida auris, occasional elongation of cells has been reported. However, environmental conditions and genetic triggers for filament formation have remained elusive. Here, we report that induction of DNA damage and perturbation of replication forks by treatment with genotoxins, such as hydroxyurea, methyl methanesulfonate, and the clinically relevant fungistatic 5-fluorocytosine, cause filamentation in C. auris The filaments formed were characteristic of pseudohyphae and not parallel-sided true hyphae. Pseudohyphal growth is apparently signaled through the S phase checkpoint and, interestingly, is Tup1 independent in C. auris Intriguingly, the morphogenetic switching capability is strain specific in C. auris, highlighting the heterogenous nature of the species as a whole.IMPORTANCECandida auris is a newly emerged fungal pathogen of humans. This species was first reported in 2009 when it was identified in an ear infection of a patient in Japan. However, despite intense interest in this organism as an often multidrug-resistant fungus, there is little knowledge about its cellular biology. During infection of human patients, fungi are able to change cell shape from ellipsoidal yeast cells to elongated filaments to adapt to various conditions within the host organism. There are different types of filaments, which are triggered by reactions to different cues. Candida auris fails to form filaments when exposed to triggers that stimulate yeast filament morphogenesis in other fungi. Here, we show that it does form filaments when its DNA is damaged. These conditions might arise when Candida auris cells interact with host immune cells or during growth in certain host tissues (kidney or bladder) or during treatment with antifungal drugs.

ABC Transporter Genes Show Upregulated Expression in Drug-Resistant Clinical Isolates of Candida auris: A Genome-Wide Characterization of ATP-Binding Cassette (ABC) Transporter Genes.

ATP-binding cassette (ABC) superfamily members have a key role as nutrient importers and exporters in bacteria. However, their role as drug exporters in eukaryotes brought this superfamily member to even greater prominence. The capacity of ABC transporters to efflux a broad spectrum of xenobiotics represents one of the major mechanisms of clinical multidrug resistance in pathogenic fungi including Candida species. Candida auris, a newly emerged multidrug-resistant fungal pathogen of humans, has been responsible for multiple outbreaks of drug-resistant infections in hospitals around the globe. Our study has analyzed the entire complement of ABC superfamily transporters to assess whether these play a major role in drug resistance mechanisms of C. auris. Our bioinformatics analyses identified 28 putative ABC proteins encoded in the genome of the C. auris type-strain CBS 10913T; 20 of which contain transmembrane domains (TMDs). Quantitative real-time PCR confirmed the expression of all 20 TMD transporters, underlining their potential in contributing to the C. auris drug-resistant phenotype. Changes in transcript levels after short-term exposure of drugs and in drug-resistant C. auris isolates suggested their importance in the drug resistance phenotype of this pathogen. CAUR_02725 orthologous to CDR1, a major multidrug exporter in other yeasts, showed consistently higher expression in multidrug-resistant strains of C. auris. Homologs of other ABC transporter genes, such as CDR4, CDR6, and SNQ2, also displayed raised expression in a sub-set of clinical isolates. Together, our analysis supports the involvement of these transporters in multidrug resistance in C. auris.

Rapid and extensive karyotype diversification in haploid clinical Candida auris isolates.

Candida auris is a newly emerged pathogenic microbe, having been identified as a medically relevant fungus as recently as 2009. It is one of the most drug-resistant yeast species known to date and its emergence and population structure are unusual. Because of its recent emergence, we are largely ignorant about fundamental aspects of its general biology, life cycle, and population dynamics. Here, we report the karyotype variability of 26 C. auris strains representing the four main clades. We demonstrate that all strains are haploid and have a highly plastic karyotype containing five to seven chromosomes, which can undergo marked alterations within a short time frame when the fungus is put under genotoxic, heat, or osmotic stress. No simple correlation was found between karyotype pattern, drug resistance, and clade affiliation indicating that karyotype heterogeneity is rapidly evolving. As with other Candida species, these marked karyotype differences between isolates are likely to have an important impact on pathogenic traits of C. auris.

The mycoparasitic yeast Saccharomycopsis schoenii predates and kills multi-drug resistant Candida auris.

Candida auris has recently emerged as a multi-drug resistant fungal pathogen that poses a serious global health threat, especially for patients in hospital intensive care units (ICUs). C. auris can colonize human skin and can spread by physical contact or contaminated surfaces and equipment. Here, we show that the mycoparasitic yeast Saccharomycopsis schoenii efficiently kills both sensitive and multi-drug resistant isolates of C. auris belonging to the same clade, as well as clinical isolates of other pathogenic species of the Candida genus suggesting novel approaches for biocontrol.

Nitrate assimilation pathway (NAP): role of structural (nit) and transporter (ntr1) genes in Fusarium oxysporum f.sp. lycopersici growth and pathogenicity.

The tomato pathogen Fusarium oxysporum f.sp. lycopersici possesses the capability to use nitrate as the only nitrogen source under aerobic and anaerobic conditions and to activate virulence-related functions when cultivated in the presence of nitrate, but not in ammonium. The genome of F. oxysporum f.sp. lycopersici encodes three paralogs nitrate reductase (NR) genes (nit1, nit2 and nit3) and one predicted ortholog of the Aspergillus nidulans high-affinity nitrate/nitrite transporters NtrA and NtrB, named ntr1. We set out to clarify the role of nit1, nit2, nit3 and ntr1 genes in nitrate assimilation and in the virulence of F. oxysporum f.sp. lycopersici. Quantitative RT-PCR analysis revealed that only nit1, nit2 and ntr1 are expressed at significant levels during growth in nitrate as the only nitrogen source. Targeted deletion of nit1 and ntr1, but not of nit2 or nit3, severely impaired growth of F. oxysporum on nitrate as nitrogen source, indicating that Nit1 and Ntr1 proteins are involved in nitrate assimilation by the fungus; biochemical analysis of nit mutants indicated that Nit1 and Nit2 enzymes contribute to about 50 and 30% of the total NR activity, respectively. In addition, a spontaneous chlorate-resistant mutant derived from F. oxysporum 4287, denoted NitFG, was characterized, showing inability to grow in nitrate under aerobic and anaerobic conditions and low levels of NR activity, in spite of its increased transcription levels of nit1 and nit2 genes. Tomato plant infection assays showed that NitFG and ∆ntr1 mutants induced an earlier death in tomato plants, whereas the single mutants ∆nit1, ∆nit2 and ∆nit1∆nit2 double mutant showed a mortality rate similar to the wild-type strain. Taken together, these results indicate that the Nit1 and Ntr1 proteins are important for nitrate assimilation of F. oxysporum f.sp. lycopersici incubated under aerobic and anaerobic conditions and that this metabolic process is not essential for the virulence of the fungus. These observations open new questions about the role of Nit1, Nit2, and Nit3 proteins in other routes of nitrate metabolism in this pathogenic fungus and in the possible regulatory role that can be exerted by the AreA protein in these routes.

Regulatory Mechanisms of a Highly Pectinolytic Mutant of Penicillium occitanis and Functional Analysis of a Candidate Gene in the Plant Pathogen Fusarium oxysporum.

Penicillium occitanis is a model system for enzymatic regulation. A mutant strain exhibiting constitutive overproduction of different pectinolytic enzymes both under inducing (pectin) or repressing conditions (glucose) was previously isolated after chemical mutagenesis. In order to identify the molecular basis of this regulatory mechanism, the genomes of the wild type and the derived mutant strain were sequenced and compared, providing the first reference genome for this species. We used a phylogenomic approach to compare P. occitanis with other pectinolytic fungi and to trace expansions of gene families involved in carbohydrate degradation. Genome comparison between wild type and mutant identified seven mutations associated with predicted proteins. The most likely candidate was a mutation in a highly conserved serine residue of a conserved fungal protein containing a GAL4-like Zn2Cys6 binuclear cluster DNA-binding domain and a fungus-specific transcription factor regulatory middle homology region. To functionally characterize the role of this candidate gene, the mutation was recapitulated in the predicted orthologue Fusarium oxysporum, a vascular wilt pathogen which secretes a wide array of plant cell wall degrading enzymes, including polygalacturonases, pectate lyases, xylanases and proteases, all of which contribute to infection. However, neither the null mutant nor a mutant carrying the analogous point mutation exhibited a deregulation of pectinolytic enzymes. The availability, annotation and phylogenomic analysis of the P. occitanis genome sequence represents an important resource for understanding the evolution and biology of this species, and sets the basis for the discovery of new genes of biotechnological interest for the degradation of complex polysaccharides.

Combined action of the major secreted exo- and endopolygalacturonases is required for full virulence of Fusarium oxysporum.

The genome of the tomato pathogen Fusarium oxysporum f. sp. lycopersici encodes eight different polygalacturonases (PGs): four endoPGs and four exoPGs. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed that endoPGs pg1 and pg5 and exoPGs pgx4 and pgx6 are expressed at significant levels during growth on citrus pectin, polygalacturonic acid or the monomer galacturonic acid, as well as during the infection of tomato plants. The remaining PG genes exhibit low expression levels under all the conditions tested. Secreted PG activity was decreased significantly during growth on pectin in the single deletion mutants lacking either pg1 or pgx6, as well as in the double mutant. Although the single deletion mutants did not display a significant virulence reduction on tomato plants, the Δpg1Δpgx6 double mutant was significantly attenuated in virulence. The combined action of exoPGs and endoPGs is thus essential for plant infection by the vascular wilt fungus F. oxysporum.

Lipolytic system of the tomato pathogen Fusarium oxysporum f. sp. lycopersici.

The lipolytic profile of Fusarium oxysporum f. sp lycopersici was studied by in silico search and biochemical enzyme activity analyses. Twenty-five structural secreted lipases were predicted based on the conserved pentapeptide Gly-X-Ser-X-Gly-, characteristic of fungal lipases, and secretion signal sequences. Moreover, a predicted lipase regulatory gene was identified in addition to the previously characterized ctf1. The transcription profile of thirteen lipase genes during tomato plant colonization revealed that lip1, lip3, and lip22 were highly induced between 21 and 96 h after inoculation. Deletion mutants in five lipase genes (lip1, lip2, lip3, lip5, and lip22) and in the regulatory genes ctf1 and ctf2 as well as a Δctf1Δctf2 double mutant were generated. Quantitative reverse transcription-polymerase chain reaction expression analyses of structural lipase genes in the Δctf1, Δctf2, and Δctf1Δctf2 mutants indicated the existence of a complex lipase regulation network in F. oxysporum. The reduction of total lipase activity, as well as the severely reduced virulence of the Δctf1, Δctf2, and Δctf1Δctf2 mutants, provides evidence for an important role of the lipolytic system of this fungus in pathogenicity.