Antifungal resistance is an inevitable phenomenon when fungal pathogens are exposed to antifungal drugs. These drugs can be grouped in four distinct classes (azoles, candins, polyenes, and pyrimidine analogs) and are used in different clinical settings. Failures in therapy implicate the sequential or combined use of these different drug classes, which can result in some cases in the development of multidrug resistance (MDR). MDR is particularly challenging in the clinic since it drastically reduces possible treatment alternatives. In this study, we report the rapid development of MDR in Candida lusitaniae in a patient, which became resistant to all known antifungal agents used until now in medicine. To understand how MDR developed in C. lusitaniae, whole-genome sequencing followed by comparative genome analysis was undertaken in sequential MDR isolates. This helped to detect all specific mutations linked to drug resistance and explained the different MDR patterns exhibited by the clinical isolates.

Candida albicans is an important human pathogen and a major concern in intensive care units around the world. C. albicans infections are associated with a high mortality despite the use of antifungal treatments. One of the causes of therapeutic failures is the acquisition of antifungal resistance by mutations in the C. albicans genome. Fluconazole (FLC) is one of the most widely used antifungal and mechanisms of FLC resistance occurring by mutations have been extensively investigated. However, some clinical isolates are known to be able to survive at high FLC concentrations without acquiring resistance mutations, a phenotype known as tolerance. Mechanisms behind FLC tolerance are not well studied, mainly due to the lack of a proper way to identify and quantify tolerance in clinical isolates. We proposed here culture conditions to investigate FLC tolerance as well as an easy and efficient method to identity and quantify tolerance to FLC. The screening of C. albicans strain collections revealed that FLC tolerance is pH- and strain-dependent, suggesting the involvement of multiple mechanisms. Here, we addressed the identification of FLC tolerance mediators in C. albicans by an overexpression strategy focusing on 572 C. albicans genes. This strategy led to the identification of two transcription factors, CRZ1 and GZF3 . CRZ1 is a C2H2-type transcription factor that is part of the calcineurin-dependent pathway in C. albicans , while GZF3 is a GATA-type transcription factor of unknown function in C. albicans . Overexpression of each gene resulted in an increase of FLC tolerance, however, only the deletion of CRZ1 in clinical FLC-tolerant strains consistently decreased their FLC tolerance. Transcription profiling of clinical isolates with variable levels of FLC tolerance confirmed a calcineurin-dependent signature in these isolates when exposed to FLC.

Magnesium is one of the most abundant metal ions in living cells. Very specific and devoted transporters have evolved for transporting Mg 2+ ions across the membrane and maintain magnesium homeostasis. Using genetic screens, we were able to identify the main players in magnesium homeostasis in the opportunistic pathogen Staphylococcus aureus . Here, we show that import of magnesium relies on the redundant activity of either CorA2 or MgtE since in absence of these two importers, bacteria require increased amounts of magnesium in the medium. A third CorA-like importer seems to play a minor role, at least under laboratory conditions. For export of magnesium, we identified two proteins, MpfA and MpfB. MpfA, is the main actor since it is essential for growth in high magnesium concentrations. We show that gain of function mutations or overexpression of the minor factor, MpfB, which is part of a sigmaB controlled stress response regulon, can compensate for the absence of MpfA.

PublishedFIG 3 MRR1 and MFS7 mediate FLC resistance in C. lusitaniae. (A) Tenfold serial dilutions were performed, starting with an inoculum of about 10 5 cells. Mutants for MRR1 and MFS7 in P3 correspond to DSY5240 and DSY5246 and in P1 to isolates DSY5242 and DSY5248, respectively. MIC values were obtained by MIC measurements using the SYO system, as described in Materials and Methods. (B) Reversion of MRR1 deletion. Mutants for MRR1 correspond to DSY5416. Revertants for the MRR1 wild-type allele and MRR1 GOF allele (MRR1 V668G ) correspond to isolates DSY5437 and DSY5438, respectively (Table S1). The white line indicates removal of a yeast sample from the original agar plate.

1Multidrug resistance (MDR) has emerged in hospitals due to the use of several agents 2 administered in combination or sequentially to the same individual. We reported earlier MDR in 3Candida lusitaniae during therapy with amphotericin B (AmB), azoles and candins. We used here 4 comparative genomic approaches between the initial susceptible isolate and 4 other isolates with 5 different MDR profiles. From a total of 18 non-synonymous SNPs (NSS) in genome comparisons 6 with the initial isolate, six could be associated with MDR. One of the SNPs occurred in a putative 7 transcriptional activator (MRR1) resulting in a V668G substitution in isolates resistant to azoles 8 and 5-fluorocytosine (5-FC). We demonstrated by gene editing that MMR1 acted by upregulation 9 of MFS7 (a multidrug transporter) in the presence of the V668G substitution. MFS7 itself 10 mediated not only azole resistance but also 5-FC resistance, which represents a novel resistance 11 mechanism for this drug class. Three other distinct NSS occurred in FKS1 (a glucan synthase that 12 is targeted by candins) in three candin-resistant isolates. Lastly, two other NSS in ERG3 and ERG4 13