Prevalent mutator genotype identified in fungal pathogen Candida glabrata promotes multi-drug resistance.

The fungal pathogen Candida glabrata has emerged as a major health threat since it readily acquires resistance to multiple drug classes, including triazoles and/or echinocandins. Thus far, cellular mechanisms promoting the emergence of resistance to multiple drug classes have not been described in this organism. Here we demonstrate that a mutator phenotype caused by a mismatch repair defect is prevalent in C. glabrata clinical isolates. Strains carrying alterations in mismatch repair gene MSH2 exhibit a higher propensity to breakthrough antifungal treatment in vitro and in mouse models of colonization, and are recovered at a high rate (55% of all C. glabrata recovered) from patients. This genetic mechanism promotes the acquisition of resistance to multiple antifungals, at least partially explaining the elevated rates of triazole and multi-drug resistance associated with C. glabrata. We anticipate that identifying MSH2 defects in infecting strains may influence the management of patients on antifungal drug therapy

Genetic Drivers of Multidrug Resistance in Candida glabrata

Both the incidence of invasive fungal infections and rates of multidrug resistance associated with fungal pathogen Candida glabrata have increased in recent years. In this perspective, we will discuss the mechanisms underlying the capacity of C. glabrata to rapidly develop resistance to multiple drug classes, including triazoles and echinocandins. We will focus on the extensive genetic diversity among clinical isolates of C. glabrata, which likely enables this yeast to survive multiple stressors, such as immune pressure and antifungal exposure. In particular, over half of C. glabrata clinical strains collected from U.S. and non-U.S. sites have mutations in the DNA mismatch repair gene MSH2, leading to a partial mutator phenotype and increased frequencies of drug-resistant mutants in vitro. Furthermore, recent studies and data presented here document extensive chromosomal rearrangements among C. glabrata strains, resulting in a large number of distinct karyotypes within a single species. By analyzing clonal, serial isolates derived from individual patients treated with antifungal drugs, we were able to document chromosomal changes occurring in C. glabrata in vivo during the course of antifungal treatment. Interestingly, we also show that both MSH2 genotypes and chromosomal patterns cluster consistently into specific strain types, indicating that C. glabrata has a complex population structure where genomic variants arise, perhaps during the process of adaptation to environmental changes, and persist over time

Differential Regulation of Echinocandin Targets Fks1 and Fks2 in Candida glabrata by the Post-Transcriptional Regulator Ssd1

Invasive infections caused by the opportunistic pathogen Candida glabrata are treated with echinocandin antifungals that target β-1,3-glucan synthase, an enzyme critical for fungal cell wall biosynthesis. Echinocandin resistance develops upon mutation of genes (FKS1 or FKS2) that encode the glucan synthase catalytic subunits. We have analyzed cellular factors that influence echinocandin susceptibility and here describe effects of the post-transcriptional regulator Ssd1, which in S. cerevisiae, can bind cell wall related gene transcripts. The SSD1 homolog in C. glabrata was disrupted in isogenic wild type and equivalent FKS1 and FKS2 mutant strains that demonstrate echinocandin resistance (MICs ˃ 0.5 µg/mL). A reversal of resistance (8- to 128-fold decrease in MICs) was observed in FKS1 mutants, but not in FKS2 mutants, following SSD1 deletion. Additionally, this phenotype was complemented upon expression of SSD1 from plasmid (pSSD1). All SSD1 disruptants displayed susceptibility to the calcineurin inhibitor FK506, similar to fks1∆. Decreases in relative gene expression ratios of FKS1 to FKS2 (2.6- to 4.5-fold) and in protein ratios of Fks1 to Fks2 (2.7- and 8.4-fold) were observed in FKS mutants upon SSD1 disruption. Additionally, a complementary increase in protein ratio was observed in the pSSD1 expressing strain. Overall, we describe a cellular factor that influences Fks1-specific mediated resistance and demonstrates further differential regulation of FKS1 and FKS2 in C. glabrata

Chitin Synthases with a Myosin Motor-Like Domain Control the Resistance of Aspergillus fumigatus to Echinocandins

Aspergillus fumigatus has two chitin synthases (CSMA and CSMB) with a myosin motor-like domain (MMD) arranged in a head-to-head configuration. To understand the function of these chitin synthases, single and double csm mutant strains were constructed and analyzed. Although there was a slight reduction in mycelial growth of the mutants, the total chitin synthase activity and the cell wall chitin content were similar in the mycelium of all of the mutants and the parental strain. In the conidia, chitin content in the ΔcsmA strain cell wall was less than half the amount found in the parental strain. In contrast, the ΔcsmB mutant strain and, unexpectedly, the ΔcsmA/ΔcsmB mutant strain did not show any modification of chitin content in their conidial cell walls. In contrast to the hydrophobic conidia of the parental strain, conidia of all of the csm mutants were hydrophilic due to the presence of an amorphous material covering the hydrophobic surface-rodlet layer. The deletion of CSM genes also resulted in an increased susceptibility of resting and germinating conidia to echinocandins. These results show that the deletion of the CSMA and CSMB genes induced a significant disorganization of the cell wall structure, even though they contribute only weakly to the overall cell wall chitin synthesis

Changes in In Vitro Susceptibility Patterns of Aspergillus to Triazoles and Correlation With Aspergillosis Outcome in a Tertiary Care Cancer Center, 1999-2015

Item does not contain fulltextBackground: Azole-resistant aspergillosis in high-risk patients with hematological malignancy or hematopoietic stem cell transplantation (HSCT) is a cause of concern. Methods: We examined changes over time in triazole minimum inhibitory concentrations (MICs) of 290 sequential Aspergillus isolates recovered from respiratory sources during 1999-2002 (before introduction of the Aspergillus-potent triazoles voriconazole and posaconazole) and 2003-2015 at MD Anderson Cancer Center. We also tested for polymorphisms in ergosterol biosynthetic genes (cyp51A, erg3C, erg1) in the 37 Aspergillus fumigatus isolates isolated from both periods that had non-wild-type (WT) MICs. For the 107 patients with hematologic cancer and/or HSCT with invasive pulmonary aspergillosis, we correlated in vitro susceptibility with 42-day mortality. Results: Non-WT MICs were found in 37 (13%) isolates and was only low level (MIC <8 mg/L) in all isolates. Higher-triazole MICs were more frequent in the second period and were Aspergillus-species specific, and only encountered in A. fumigatus. No polymorphisms in cyp51A, erg3C, erg1 genes were identified. There was no correlation between in vitro MICs with 42-day mortality in patients with invasive pulmonary aspergillosis, irrespective of antifungal treatment. Asian race (odds ratio [OR], 20.9; 95% confidence interval [CI], 2.5-173.5; P = .005) and azole exposure in the prior 3 months (OR, 9.6; 95% CI, 1.9-48.5; P = .006) were associated with azole resistance. Conclusions: Non-WT azole MICs in Aspergillus are increasing and this is associated with prior azole exposure in patients with hematologic cancer or HSCT. However, no correlation of MIC with outcome of aspergillosis was found in our patient cohort

Engineered fibroblast growth factor 19 protects from acetaminophen-induced liver injury and stimulates aged liver regeneration in mice

The liver displays a remarkable regenerative capacity triggered upon tissue injury or resection. However, liver regeneration can be overwhelmed by excessive parenchymal destruction or diminished by pre-existing conditions hampering repair. Fibroblast growth factor 19 (FGF19, rodent FGF15) is an enterokine that regulates liver bile acid and lipid metabolism, and stimulates hepatocellular protein synthesis and proliferation. FGF19/15 is also important for liver regeneration after partial hepatectomy (PH). Therefore recombinant FGF19 would be an ideal molecule to stimulate liver regeneration, but its applicability may be curtailed by its short half-life. We developed a chimaeric molecule termed Fibapo in which FGF19 is covalently coupled to apolipoprotein A-I. Fibapo retains FGF19 biological activities but has significantly increased half-life and hepatotropism. Here we evaluated the proregenerative activity of Fibapo in two clinically relevant models where liver regeneration may be impaired: acetaminophen (APAP) poisoning, and PH in aged mice. The only approved therapy for APAP intoxication is N-acetylcysteine (NAC) and no drugs are available to stimulate liver regeneration. We demonstrate that Fibapo reduced liver injury and boosted regeneration in APAPintoxicated mice. Fibapo improved survival of APAP-poisoned mice when given at later time points, when NAC is ineffective. Mechanistically, Fibapo accelerated recovery of hepatic glutathione levels, potentiated cell growth-related pathways and increased functional liver mass. When Fibapo was administered to old mice prior to PH, liver regeneration was markedly increased. The exacerbated injury developing in these mice upon PH was attenuated, and the hepatic biosynthetic capacity was enhanced. Fibapo reversed metabolic and molecular alterations that impede regeneration in aged livers. It reduced liver steatosis and downregulated p21 and hepatocyte nuclear factor 4 α (Hnf4α) levels, whereas it stimulated Foxm1b gene expression. Together our findings indicate that FGF19 variants retaining the metabolic and growth-promoting effects of this enterokine may be valuable for the stimulation of liver regeneration.Work in the authors' laboratory is supported by CIBERehd and Grants from Instituto de Salud Carlos III (ISCIII) co-financed by 'Fondo Europeo de Desarrollo Regional' (FEDER) 'Una manera de hacer Europa', numbers: FIS PI13/00359, PI13/00385 and PI16/01126. Grants SAF2015-66515-R, SAF201569944-R, SAF 2016-75972 R from Ministerio de Economía y Competitividad, and the center grant P50AA011999 funded by NIAAA. 'Ramón y Cajal-I3' contract to MUL, Mineco-FPI Fellowship to MB-V. Marie Curie EU contract to MGF-B. Fundación M Torres; Fundación Eugenio Rodríguez Pascual; Fundación Mario Losantos; Fundación Familia Puig-Infante and Fundación Bancaria La CaixaHepacare Project.Peer reviewe

The protein phosphatase PhzA of A. fumigatus is involved in oxidative stress tolerance and fungal virulence

Protein phosphatases Z that are unique to the fungal kingdom have been associated to resistance to high salt concentration, cell wall integrity, cell cycle regulation, and oxidative stress in fungi. In Aspergillus fumigatus it was shown that PHZA is under the control of the transcription factor Skn7 and is only involved in the control of the oxidative stress. Accordingly, the ΔphzA mutant showed a defect in virulence in an experimental model of corneal infection in immunocompetent animals and that the impact on susceptibility to cell wall drugs is only secondary